CN101210244A - Neutrokine-alpha and Neutrokine-alpha splice variants - Google Patents

Neutrokine-alpha and Neutrokine-alpha splice variants Download PDF

Info

Publication number
CN101210244A
CN101210244A CNA2007101537856A CN200710153785A CN101210244A CN 101210244 A CN101210244 A CN 101210244A CN A2007101537856 A CNA2007101537856 A CN A2007101537856A CN 200710153785 A CN200710153785 A CN 200710153785A CN 101210244 A CN101210244 A CN 101210244A
Authority
CN
China
Prior art keywords
polypeptide
neutrokine
sequence
seq
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CNA2007101537856A
Other languages
Chinese (zh)
Other versions
CN101210244B (en
Inventor
克雷格·A·罗森
倪健
莱因哈德·埃布纳
余国良
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Human Genome Sciences Inc
Original Assignee
Human Genome Sciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Publication of CN101210244A publication Critical patent/CN101210244A/en
Application granted granted Critical
Publication of CN101210244B publication Critical patent/CN101210244B/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to a novel cytokine which has been designated Neutrokine-alpha (''Neutrokine-alpha''). In addition, an apparent splicing variant of Neutrokine-alpha has been identified and designated Neutrokine-alphaSV. In specific embodiments, the present invention provides nucleic acid molecules encoding Neutrokine-alpha and Neutrokine-alphaSV polypeptides. In additional embodiments, Neutrokine-alpha and Neutrokine-alphaSV polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same.

Description

Neutrokine-α and Neutrokine-alpha splice variant
The application is February 22, application number in 2000 the dividing an application for the application for a patent for invention of " Neutrokine-α and Neutrokine-alpha splice variant " that be 00806541.1 (international application no is PCT/US00/04336), denomination of invention for the applying date.
The present invention relates to a kind of new cytokine that is called as Neutrokine-α.In addition, differentiate the apparent splice variant of a kind of Neutrokine-α, be called Neutrokine-α SV.In specific embodiment, the invention provides the nucleic acid molecule of coding Neutrokine-α and Neutrokine-α SV polypeptide.In other embodiments, Neutrokine-α and Neutrokine-α SV polypeptide also are provided, and carrier, host cell, and the recombination method of producing them.
Correlation technique
Human tumor necrosis factor (TNF-α) and (TNF-β, or lymphotoxin) is the relevant member of the polypeptide amboceptor of a big classification, this classification comprises Interferon, rabbit, interleukin-and somatomedin, be referred to as cytokine (Beutler, B. and Cerami, A. immune analysis research 7:625-655 (1989)).The pair cell factor acceptor carries out the subfamily that some membranins have been set forth in sequential analysis: (1) Ig superfamily, (2) hematopoietin (cytokine receptor superfamily), (3) (TNF/ nerve growth factor (NGF) receptor superfamily is (with reference to the TNF superfamily for tumour necrosis factor, see Gruss and Dower, blood 85 (12): 3378-3404 (1995) and Aggarwal and Natarajan, Eur.Cytokine Netw.7(2)：93-124(1996))。The TNF/NGF receptor superfamily contains at least 10 kinds of different protein (Gruss and Dower are as preceding).The part of these acceptors has been differentiated and has been belonged at least two kinds of cytokine superfamilies (Gruss and Dower are as preceding).
Tumour necrosis factor (mixture of TNF-α and TNF-β) is owing to their anti-tumor activity is found at first, yet discerning them now is to have multiple bioactive multiple-effect cytokine, comprise having and make some cell transformed system/program death, the activation of mediated cell and propagation, and in immunomodulatory and inflammation, also play an important role.
The member of known tnf ligand superfamily comprises TNF-α, and TNF-β (lymphotoxin-α), LT-β, OX40L, Fas part, CD30L, CD27L, CD40L, and 4-IBBL.The part of tnf ligand superfamily is a tart, has the class TNF molecule of about 20% (12%-36% scope) sequence homology in extracellular domain, and mainly presents and have tripolymer/the more film combining form of the biologically active form of aggressiveness mixture.The soluble form of tnf ligand superfamily is differentiated at present only TNF, and β-LT and Fas part (referring to Gruss, H and Dower, S.k. blood 85 (12): 3378-3404 (1995) incorporates reference into they whole.These protein are contained in cell proliferation, in the adjusting of activation and differentiation, comprise survival or death (Armstage, R.J.Curr.Opin by apoptosis or cytotoxicity control cell.Immund.b:407 (1994) and Smith, C.A. cell 75:959 (1994).
(TNF-α is also referred to as cachectin to tumor necrosis factor-alpha; Hereinafter be called " TNF ") be extracellular toxin or other stimulon to be replied and excretory at first by monocyte and scavenger cell, be a kind of homotrimer (Smith of soluble 17 KD protein subunits, R.A. etc., journal of biological chemistry 262:6951-6954 (1987)). also set forth precursor forms (kriegIer, the M of membrane-bound 26 KD of a kind of TNF.Deng, cell 53:45-53 (1988)).
Institute's cumulative result of study shows that TNF is a kind of active regulatory cell factor of multiple-effect biological that has.These activity comprise: suppress synthetic (" cachectin " active (Beutler, B. etc., natural 316:552 (1985), activation polymorphonuclear leukocyte (Klebanoff, S.J. etc., the Journal of Immunology 136:4220 (1986) of lipoprotein lipase; Perussia, B.Deng, Journal of Immunology 138:765 (1987)), cell growth inhibiting or stimulate cell growth (Vilcek, J. etc., J.Exp.Med.163:632 (1986); Sugarman, B.J. etc., science 230:943 (1985); Lachman.L.B. etc., Journal of Immunology 138:2913 (1987)), to the cytotoxic effect of some cell transformed (Laehman, L.B. etc. are as preceding; Darzynkiewicz, Z. etc., cancer research 44:83 (1984), antiviral activity (Kohase, M. etc., cell 45:659 (1986); Wong, G.H.W. etc., natural 323:819 (1986) stimulates bone resorption (Bertolini, D.R. etc., natural 319:516 (1986); Saklatvala, J. nature 322:547 (1986) stimulates collagenase and prostaglandin F2 to produce (Dayer, J.-M. etc., J.Exp.Med.162:2163 (1985); And immunoregulatory activity, comprise activated T cell (Yokota, S. etc., Journal of Immunology 140:531 (1988), activate B cell (Kehrl.J.H. etc., J.Exp.Med.160:786 (1987)), activated mononuclear cell (Philip, R. etc., natural 323:86 (1986), activate thymocyte (Ranges, G.E. etc., J.Exp.Med.167:1472 (1988)), and stimulate major histocompatibility complex (MHC) I class and II quasi-molecule at cell surface expression (Couins, T. etc., institute of American Academy of Sciences reports 83:446 (1986); Pujol-Borrel, R. etc., natural 326:304 (1987)).
Notice that TNF has short inflammatory effect, cause tissue injury, as blood coagulation enhancing effect (Pober, J.S. etc. on the induction of vascular endothelial cell, Journal of Immunology 136:1680 (1986), improve neutrophil and lymphocytic sticking (Pober, J.S. etc., Journal of Immunology 138:3319 (1987)), and stimulate from scavenger cell, discharge platelet activating factor (Camussi, G. etc., J.Exp.Med.166 in neutrophil and the vascular endothelial cell; 1390 (1987). nearest result of study is at many infection (Cerami.A etc. about TNF, modern immunology 9:28 (1988)), immune disorder, tumorigenicity pathology, as follow cachexy (Oliff, the A etc. of some malignant tumours, cell 50:555 (1987)), and autoimmunization pathology and graft to the pathological effect in the host disease Neo-Confucianism (Piguet, P.F. etc., J Exp.Med.166:1280 (1987)).TNF and cancer and the pathological relation of infection are usually directed to host's katabolism state.Cancer patients's a subject matter is weight loss, and is general relevant with apocleisis.The result that lasting consumption is gone down causes known " emaciation " (Kern, K.A. etc., J.Parent.Enter.Nutr.12:286-298 (1988)). and emaciation comprises that carrying out gonosome heavily descends, and apocleisis and malignant growth cause corroding lastingly physique.Therefore the emaciation state is with significantly morbid state is relevant, and relevant with most cancer mortalities, and many researchs have inferred that TNF is neutralize cachectic a kind of important amboceptor in other katabolism state of cancer infectivity pathology.
TNF be considered in the pathologic, physiologic result of Gram-negative septicopyemia and interior toxicogenic shock, work (Michie, H.R. etc., Br.J.Surg.76:670-671 (1989); Debets, J.M.H etc., Second Vienna Shock Forum, P 463-466 (1989); Simpson, Crit.Care.Clin 5:27-47 (1989) such as S.Q. comprise heating, and are uncomfortable, apocleisis and evil liquid.Intracellular toxin is a kind of potential monocyte/macrophage activator, stimulates TNF (Kornbluth, S.K. etc., Journal of Immunology 137:2585-2591 (1986)) and other production of cytokines and secretion.Because TNF can simulate many endotoxic biological activitys, is considered to the main amboceptor relevant with the clinical picture of endotoxin-related diseases.TNF and the cytokine mediated of other monocyte derived are replied (Micdhie, H.R. etc., N.Eng.J.Med.318:1481-1486 (1988)) to endotoxic metabolic and neurohormone.Intracellular toxin is applied to people volunteer, produces the acute illness with similar influenza symptom, these symptoms comprise heating, tachycardia, and metabolic rate and stress hormone discharge increases (Revhauy, A. etc., Arch.Surg.123:162-170 (1988)).Also found to improve (Waage, A. etc., Lancetl:355-357 (1987)) in patient's body-internal-circulation TNF level of suffering from the Gram-negative septicopyemia; Hammerle, A.F etc., Second Vienna Shock Forum is (1989) p.715-718; Debets, Crit.Core.Med.17:489-497 such as J.M.H. (1989); Colandra T. etc., J.Infec.Dis.161:982-987 (1990)).
Passive immunotherapy with the TNF level in being intended to has gratifying effect to Gram-negative septicopyemia and inner toxemia, increases and the raising of TNF level as above-mentioned the generation based on TNF in these pathological states.Cerami etc. (EPO patent disclosure 0,212 489, on March 4th, 1987) have disclosed at the antibody of differentiating " conditioning agent " (the finding identical with TNF after a while) for cachectin.This antibody can be used for the shock in diagnostic immunoassay and the treatment infectation of bacteria.Rubrn etc. have disclosed the monoclonal antibody of people TNF in (EPO patent disclosure 0,218 868, on April 22nd, 1987), secrete the hybridoma of this antibody, and the method for producing this antibody reaches the use of this antibody in the immunoassay of TNF.Yone etc. (Epo patent disclosure on October 26th, 0288088,1988) have disclosed anti-TNF antibodies and have comprised mAbs, and their effects in the immunoassay diagnosis of pathology especially Kawasaki ' s pathology and cell infection.Has Kawasakis pathology (the mucocutaneous lymph node syndrome of children's acute heat generation; Kawasaki, T.Auergg 16:178 (1967); Kawasaki, the TNF level that patient's body fluid of T.Shonica (Pediatrics) 26:935 (1985) contains improves, this relevant with the pathology process (Yone etc. are as preceding).
Other researchist has set forth the mAbs that is specific to recombinant human TNF, and it has neutralization active (Liang, C-M. etc., a biological chemistry biological physiology Review Study 137:847-854 (1986) external; Medger, A. etc., hybridoma 6:305-311 (1987); Hirai, M. etc., immunological method magazine 96:57-62 (1987); Mouer, A. etc., cytokine 2:162-169 (1990)).There are some to be used for that (Fendly etc. are as preceding to the epitope mapping of people TNF and exploitation enzyme immunoassay among these mAbs; Hirai etc. are as preceding; Mouer etc. are as preceding), and help purification of Recombinant TNF (Bringman etc., as preceding) yet, the basis that these researchs can not provide production that the TNF neutralizing antibody of diagnosis or therapeutic action is arranged in vivo is because immunogenicity lacks due to specificity and/or the medicine adaptability.
Among the TNF and antiserum(antisera) or mAbs to be illustrated in the Mammals except the man, can eliminate disadvantageous physiological change and prevent death carrying out inner toxemia and bacteremic experimental deadly research after.This effect for example confirms (Mathison, Journal of Clinical Investigation 81:1925-1937 (1988) such as J.C. in analysis of rodent lethality and primate pathological model system; Beutler, B. etc., science 229:869-871 (1985); Tracey, K.J. etc., natural 330:662-664 (1987); Shimamoto, Y. etc., immunology communication 17:311-318 (1988); Silva, A.T. etc., infectious diseases magazine 162:421-427 (1990); Opal, S.M. etc., infectious diseases magazine 161:1148-1152 (1990); Hinshaw, L.B. etc., Circ.Shock 30:279-292 (1990)).
Should note being restricted in the test that the anti-TNF mAb of used inside human body treats, but gratifying treatment result is shown, for example in treatment of arthritis and septicopyemia. for example referring to Elliott, M.J. etc., Baillieres Clin.Rheumotol 9:633-52 (1995); Feldmann M. etc., Ann.N.Y.Acad.Sci.USA 766:272-8 (1995); Poll, T. etc., shock 3:1-12 (1995); Wheny etc., Crit.Care.Med.21:s436-40 (1993); Tyacey, K.J. etc., Crit.Care.Med.21:S 415-22 (1993).
Mammals grows and to depend on the propagation and the differentiation of cell, and passes through apoptosis program necrocytosis L.Walker etc., Methods Achiev.Exp.Pathol.13:18 (1988) take place.Apoptosis plays a crucial role in the destruction of the immune thymocyte of identification autoantigen.The not normal autoimmune disorder (Gammon etc., modern immunology 12:193 (1991)) that causes of this normal elimination process.
Itoh etc. (cell 66:233 (1991)) have set forth a kind of cell-surface antigens, are that the mediated cell program is dead and be contained in Fas/CD95 in the T cell clone disappearance.Fas is at the activated T cell, and the B cell is expressed in the neutrophilic granulocyte, and except at the activated T cell, at the thymus gland of adult rats, liver is expressed in the heart and lung and the ovary outside B cell and the neutrophilic granulocyte.In the crosslinked test of mono-clonal Ab and Fas, programmed cell death is inductive (Yonehare etc., experimental technique magazine 169:1747 (1989); Trauth etc., science 245:301 (1989)), in addition, having mono-clonal Ab to combine with Fas stimulates the such example of T cell (Alderson etc., experimental technique magazine 178:2231 (1993)) under certain condition.
Fas antigen is the cell surface protein of the relevant MW of 45 Kd.The Fas gene of people and mouse is by clone such as Watanabe-Fukunaga etc. (Journal of Immunology 148:1274 (1992)) and Itoh (cell 66:233 (1991)).Protein by these genes encodings all is the transmembrane proteins that have structural homology with nerve growth factor/tumor necrosis factor receptor super family, this superfamily comprises two kinds of TNF acceptors, low affinity trk C and CD40, CD27, CD30 and OX40.
Set forth Fas part (Suda etc., cell 75:1169 (1993)) recently.Aminoacid sequence shows that the Fas part is the II type transmembrane protein that belongs to TNF family.Therefore, the FAS ligand polypeptide comprises 3 primary structure territories: at aminoterminal short born of the same parents' internal area, at the longer extracellular domain of C-terminal, connect the wetting ability membrane-spanning domain of these two structural domains.The Fas part is expressed in splenocyte and thymocyte, and is consistent with the cellular toxicity that T is cell-mediated.The Fas part of purifying has the MW of 40 Kd.
Confirmed that in recent years interaction between the Fas/Fas part is programmed cell death required (Ju etc., the natural 373:444 (1995) after the T cell activation; Brunner etc., natural 373:441 (1995)) two kinds of protein on the activation inducing cell surface of .T cell.Interaction between part and the acceptor subsequently causes programmed cell death.This has supported during normal immunne response this possible regulating effect of inductive programmed cell death by the interaction between the Fas/Fas part.
Therefore, need provide the cytokine that participates in being similar in the pathological symptom TNF.The cytokine that this class is new can be used for producing new antibodies or other antagonist in conjunction with these classes TNF cytokine, with diagnosis and treatment and the relevant disease of class TNF cytokine.
Summary of the invention
According to one embodiment of the invention, a kind of extracellular domain of new Neutrokine-α polypeptide is provided, with a kind of extracellular domain of new Neutrokine-α SV polypeptide, with and fragment, analogue and derivative bioactive and diagnosis or therepic use arranged.
According to another embodiment of the present invention, the isolated nucleic acid molecule of coding people Neutrokine-α or Neutrokine-α SV is provided, comprise mRNA, DNA, cDNA, genomic dna, with and the fragment and the derivative of bioactive and diagnosis or therepic use arranged.
Isolated nucleic acid molecule provided by the invention comprises or is made up of a kind of polynucleotide, and is similar with relevant cytokine with TNF on this polynucleotide encoding structure, and has similar biological action and active cytokine and apparent splice variant thereof.This cytokine is called Neutrokine-α, and the Neutrokine-α polypeptide that the present invention includes has aminoacid sequence shown at least a portion Figure 1A and the 1B (SEQ ID NO:2), or having at least a portion by being deposited among the ATCC on October 22nd, 1996, preserving number is cDNA clone (HNEDU15) amino acid sequence coded of 97768.By the nucleotide sequence that the Neutrokine-α cloning and sequencing of preservation is determined, be shown in Figure 1A and 1B (SEQ ID NO:1), it contains the open reading frame of the complete polypeptide of 285 amino-acid residues of encoding, these 285 amino acid comprise the N-terminal methionine(Met), born of the same parents' internal area of the derivation of about 46 amino-acid residues, the membrane-spanning domain of the derivation of about 26 amino-acid residues, the extracellular domain of about 213 amino acid whose derivations, and this complete proteinic molecular weight of deriving is approximately 31KDa.As other II type transmembrane protein, the Neutrokine-α of soluble form comprises all or part extracellular domain of cracking from membrane-spanning domain, and comprises the polypeptide of the complete Neutrokine-α polypeptide that lacks membrane-spanning domain, promptly is connected in the extracellular domain of born of the same parents' internal area.
The apparent splice variant of Neutrokine-α is called Neutrokine-α SV, and the Neutrokine-α SV that the present invention includes comprises or by at least a portion of aminoacid sequence shown in Fig. 5 A and the 5B (SEQ ID NO:19), or is that at least a portion of 203518 cDNA clone HDPMC52 amino acid sequence coded is formed by being deposited in preserving number among the ATCC on December 10th, 1998.Clone definite nucleotides sequence that checks order by Neutrokine-α SV and be shown in Fig. 5 A and 5B (SEQ ID NO:18) preservation, it contains the open reading frame of 266 amino acid whose complete polypeptide of a coding, these 266 amino acid comprise the N-terminal methionine(Met), about 46 amino acid whose born of the same parents' internal areas of inferring, about 26 amino acid whose membrane-spanning domains of inferring, about 194 amino acid whose extracellular domains of inferring, and this complete about 29KDa of proteinic molecular weight that derives.The same with other II type transmembrane protein, the Neutrokine-α SV of soluble form comprises that cracking from all or a part of extracellular domain of membrane-spanning domain with comprise the polypeptide of the complete Neutrokine-α SV polypeptide that lacks membrane-spanning domain, promptly is connected in the extracellular domain of born of the same parents' internal area.
Therefore one embodiment of the invention provide a kind of isolated nucleic acid molecule, it comprises or forms by having the polynucleotide that are selected from next group nucleotide sequence: the nucleotide sequence of the total length of (a) encoding Neutrokine-α polypeptide, this polypeptide have (SEQ ID NO:2) shown in Figure 1A and the 1B or by the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768; (b) nucleotide sequence of the coding Neutrokine-α polypeptide extracellular domain of inferring, this extracellular domain has the 73-285 amino acids sequence shown in Figure 1A and the 1B (SEQ ID NO:2), or by the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768; (c) coding has the segmental nucleotide sequence of (b) polypeptide of Neutrokine-α functionally active (being biological activity); (d) nucleotide sequence of coding one peptide species, this polypeptide comprises Neutrokine-α born of the same parents internal area (being estimated as about 1-46 position continuous amino acid residue of Figure 1A and 1B (SEQ ID NO:2)), or by the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768; (e) nucleotide sequence of coding one peptide species, this polypeptide comprises Neutrokine-α membrane-spanning domain (being estimated as about 47-72 position continuous amino acid residue of Figure 1A and 1B (SEQ ID NO:2)), or by the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768; (f) coding has extracellular domain and born of the same parents' internal area but the nucleotide sequence of the solubility Neutrokine-α polypeptide of disappearance membrane-spanning domain; (g) be complementary to above (a), (b), (c), (d), the nucleotide sequence of any nucleotide sequence (e) or (f).
Other embodiment of the present invention comprises isolated nucleic acid molecule, it comprises or is made up of polynucleotide, these polynucleotide have and above (a), (b), (c), (d), (e) (f) or (g) in any nucleotides sequence show at least 80%, 85% or 90% homogeny, preferably at least 95%, 96%, 97%, the nucleotide sequence of 98% or 99% homogeny, perhaps these polynucleotide under stringent hybridization condition with above (a), (b), (c), (d), (e) (f) or (g) in multi-nucleotide hybrid.The polynucleotide of its hybridization under stringent condition not with the multi-nucleotide hybrid that only has by A residue or the nucleotide sequence only formed by the T residue.
Another embodiment of the present invention provides a kind of isolated nucleic acid molecule, and it comprises or forms by having the polynucleotide that are selected from next group nucleotide sequence; (a) nucleotide sequence of coding total length Neutrokine-α SV polypeptide, this polypeptide has complete amino acid sequence shown in Fig. 5 A and the 5B (SEQID NO:19), or have by the contained cDNA of preservation thing at the ATCC preserving number 203518 of preservation on December 10 in 1998, clones coding; (b) nucleotide sequence of the coding Neutrokine-α SV polypeptide extracellular domain of inferring, this extracellular domain have 73-266 amino acids sequence shown in Figure 1A and the 1B (SEQ ID NO:2) or by the contained cDNA clones coding of preservation thing at the ATCC preserving number 203518 of preservation on December 10 in 1998; (c) coding comprises the polynucleotide sequence of polypeptide of Neutrokine-α SV born of the same parents internal area (being estimated as about 1-46 position continuous amino acid residue of Fig. 5 A and 5B (SEQ ID NO:19)), born of the same parents' internal area or by the contained cDNA clones coding of preservation thing of the ATCC preserving number 203518 of preservation on December 10 in 1998; (d) coding comprises the polynucleotide sequence of polypeptide of Neutrokine-α SV membrane-spanning domain (being estimated as about 47-72 position continuous amino acid residue of Fig. 5 A and 5B (SEQ ID NO:19)), membrane-spanning domain or by the contained cDNA clones coding of preservation thing of the ATCC preserving number 203518 of preservation on December 10 in 1998; (e) nucleotide sequence of coding solubility Neutrokine-α SV polypeptide, this polypeptide have extracellular domain and born of the same parents' internal area but do not have membrane-spanning domain; (f) be complementary to above (a), (b), (c), (d), or the nucleotide sequence of any nucleotide sequence (e).
Other embodiment of the present invention comprises isolated nucleic acid molecule, and it comprises or is made up of polynucleotide, and these polynucleotide have and above (a), (b), (c), (d), (e) any nucleotide sequence has at least 80%, 85% or 90% homogeny or (f), and preferably at least 95%, 96%, 97%, 98% or the nucleotide sequence of 99% homogeny, perhaps this multinuclear glycosides under stringent hybridization condition with above (a), (b), (c), (d), (e) or the multi-nucleotide hybrid (f).The polynucleotide of its hybridization under stringent hybridization condition not with the multi-nucleotide hybrid that only has by A residue or the nucleotide sequence only formed by the T residue.
In one embodiment, the apparent splice variant of Neutrokine-α comprises or is amino acid whose by sequence Gly142-Leu266 shown in Fig. 5 A and the 5B (SEQ ID NO:19), or is that at least a portion of 203518 cDNA clone HDPMC52 amino acid sequence coded is formed by being deposited in preserving number among the ATCC on December 10th, 1998.
In other embodiments, nucleic acid molecule of the present invention comprises or is made up of polynucleotide, this polynucleotide encoding has above (a), (b), (c), (d), (e) (f) or (g) in the aminoacid sequence that carries the epi-position part of the Neutrokine-α of aminoacid sequence or Neutrokine-α SV.Another nucleic acid embodiment of the present invention relates to a kind of isolated nucleic acid molecule, it comprises or is made up of polynucleotide, this polynucleotide encoding Neutrokine-α or Neutrokine-α SV amino acid sequence of polypeptide, described polypeptide has and contains at least 1 but be no more than 50, preferably be no more than 40, more preferably no more than 30, be most preferably not exceeding 20 aminoacid addition, the aminoacid sequence that replaces and/or lack.Certainly, particularly preferably the encode aminoacid sequence of polynucleotide of Neutrokine-α or Neutrokine-α SV amino acid sequence of polypeptide contains and is no more than 10,9,8,7,6,5,4,3,2, or 1 or 1-100,1-50,1-25,1-20,1-15, a 1-10 or 1-5 aminoacid addition replaces and/or disappearance, preferred conservative the replacement.
The invention still further relates to recombinant vectors, it comprises isolated nucleic acid molecule of the present invention, also relate to the host cell that contains recombinant vectors, and the method for producing this carrier and host cell, and produce the method for Neutrokine-α polypeptide by recombinant technology with them.
According to another embodiment of the present invention, a kind of method of producing this peptide species by recombinant technology is provided, be included under the condition that promotes described expression of polypeptides, reorganization protokaryon and/or eukaryotic host cell that cultivation contains Neutrokine-α of the present invention or Neutrokine-α SV nucleotide sequence reclaim described polypeptide subsequently.
The present invention also provides isolating Neutrokine-α polypeptide, it comprises or is made up of the aminoacid sequence that is selected from next group: (a) have complete amino acid sequence shown in Figure 1A and the 1B (being the 1-285 position of SEQ ID NO:2), or by the plasmid-encoded total length Neutrokine-α amino acid sequence of polypeptide of the contained cDNA of the preservation thing of ATCC preserving number 97768; (b) has shown in the SEQ ID NO:2 total length Neutrokine-α amino acid sequence of polypeptide of N-terminal methionine(Met) (being the 2-285 amino acids sequence of SEQ ID NO:2) except the complete amino acid sequence; (c) has the fragment of (b) polypeptide of Neutrokine-α functionally active (being biological activity); (d) aminoacid sequence of the Neutrokine-α polypeptide extracellular domain of inferring, this extracellular domain has 73-285 amino acids sequence shown in Figure 1A and the 1B (SEQ ID NO:2), or by the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768; (e) nucleotide sequence of coding Neutrokine-α polypeptide has 134-285 amino acids sequence shown in Figure 1A and the 1B (SEQ ID NO:2); (f) aminoacid sequence of Neutrokine-α polypeptide born of the same parents internal area (about 1-46 position continuous amino acid of Figure 1A that infers and 1B (SEQ ID NO:2)), this born of the same parents' internal area or plasmid-encoded by the contained cDNA of the preservation thing of ATCC preserving number 97768; (g) Neutrokine-α membrane-spanning domain aminoacid sequence (about 47-72 position continuous amino acid residue among Figure 1A that infers and the 1B (SEQ ID NO:2)), this membrane-spanning domain or plasmid-encoded by the contained cDNA of the preservation thing of ATCC preserving number 97768; (h) have extracellular domain and born of the same parents' internal area but do not have the solubility Neutrokine-α amino acid sequence of polypeptide of membrane-spanning domain, wherein said these structural domains as above limit; (i), (a), (b), (c), (d), (e), (f), (g) or (h) fragment of polypeptide.Polypeptide of the present invention also comprise with above (a), (b), (c), (d), (e), (f), (g), (h) or (i) aminoacid sequence has at least 80%, and preferably at least 85% or 90%, more preferably at least 95%, 96%, 97%, the polypeptide of the aminoacid sequence of 98% or 99% homogeny, and have with above aminoacid sequence and have at least 80%, 85%, or 90%, the more preferably polypeptide of the aminoacid sequence of at least 95% similarity.Other embodiments of the present invention relate to a peptide species, and it comprises or above by having (a), (b), (c), (d), (e), (f), (g), (h) or the aminoacid sequence that carries epi-position part of the Neutrokine-α polypeptide of aminoacid sequence (i) form.Neutrokine-α polypeptide of the present invention comprises have at least 4 of this peptide species, at least 5, at least 6, at least 7, at least 8, preferably at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, a more preferably about at least 30-50 amino acid whose part, the polypeptide that carries epi-position until comprising the whole aminoacid sequences of polypeptide of the present invention of certain any length is also included within the present invention.
The especially preferred embodiment of the present invention relates to nucleic acid molecule, it comprises or is made up of a kind of polynucleotide, and the nucleotide sequence that these polynucleotide have has at least 80%, 85% with the polynucleotide sequence of coding Neutrokine-α polypeptide, 90% homogeny, preferably have at least 95%, 96%, 97%, 98%, 99%, or 100% homogeny, Neutrokine-α polypeptide has 134-285 amino acids sequence shown in Figure 1A and the 1B (SEQ ID NO:2).The preferred embodiment of the invention relates to the nucleic acid molecule that comprises or be made up of polynucleotide, the nucleotide sequence that these polynucleotide have has at least 90% homogeny with the polynucleotide sequence of coding Neutrokine-α polypeptide, and Neutrokine-α polypeptide has 134-285 amino acids sequence shown in Figure 1A and the 1B (SEQ IDNO:2).The present invention more embodiment preferred relates to the nucleic acid molecule that comprises or be made up of polynucleotide, the nucleotide sequence that these polynucleotide have has at least 95% homogeny with the polynucleotide sequence of coding Neutrokine-α polypeptide, and Neutrokine-α polypeptide has 134-285 amino acids sequence shown in Figure 1A and the 1B (SEQ ID NO:2).The present invention more embodiment preferred relates to the nucleic acid molecule that comprises or be made up of polynucleotide, the nucleotide sequence that these polynucleotide have has at least 96% homogeny with the polynucleotide sequence of coding Neutrokine-α polypeptide, and Neutrokine-α polypeptide has 134-285 amino acids sequence shown in Figure 1A and the 1B (SEQ ID NO:2).
In addition, the present invention more embodiment preferred relates to the nucleic acid molecule that comprises or be made up of polynucleotide, and the polynucleotide sequence that nucleotide sequence that these polynucleotide have and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 97% homogeny.In addition, the present invention more embodiment preferred relates to the nucleic acid molecule that comprises or be made up of polynucleotide, and the polynucleotide sequence that nucleotide sequence that these polynucleotide have and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 98% homogeny.In addition, the embodiment preferred nucleic acid branch that relates to comprise or be made up of polynucleotide more, the polynucleotide sequence that nucleotide sequence that these polynucleotide have and coding have the polypeptide of 134-285 amino acids sequence of N eutrokine-α shown in Figure 1A and the 1B (SEQID NO:2) has at least 99% homogeny.
The above polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these polynucleotide and nucleic acid molecule encoding is also contained in the present invention.
The present invention further provides isolating Neutrokine-α SV polypeptide, it comprises or is made up of the aminoacid sequence that is selected from next group: (a) total length Neutrokine-α SV amino acid sequence of polypeptide, this polypeptide has complete amino acid sequence shown in Fig. 5 A and the 5B (being the 1-266 amino acids of SEQ ID NO:19), or is 203518 cDNA clones coding by being deposited in preserving number among the ATCC on December 10th, 1998; (b) total length Neutrokine-α SV amino acid sequence of polypeptide, this polypeptide have the complete amino acid sequence except that the N-terminal methionine(Met) shown in the SEQ ID NO:19 (being the 2-266 amino acids of SEQ ID NO:19); (c) aminoacid sequence of inferring extracellular domain of Neutrokine-α SV polypeptide, this extracellular domain has 73-266 amino acids sequence shown in Fig. 5 A and the 5B (SEQ ID NO:19), or is 203518 cDNA clones coding by being deposited in preserving number among the ATCC on December 10th, 1998; (d) aminoacid sequence of Neutrokine-α SV polypeptide born of the same parents internal area, it was the continuous amino acid residue of about 1-46 position shown in Fig. 5 A and the 5B (SEQ ID NO:19) that this born of the same parents' internal area is inferred, or was deposited in the cDNA clones coding of ATCC No.203518 by on December 10th, 1998; (e) aminoacid sequence of Neutrokine-α SV membrane-spanning domain, it was the continuous amino acid residue of about 47-72 position shown in Fig. 5 A and the 5B (SEQ ID NO:19) that this membrane-spanning domain is inferred, or was deposited in the cDNA clones coding of ATCC No.203518 by on December 10th, 1998; (f) solubility Neutrokine-α SV amino acid sequence of polypeptide, this polypeptide have extracellular domain and born of the same parents' internal area but do not have membrane-spanning domain, and wherein each structural domain as above limits; (g) above (a), (b), (c), (d), (e), or (f) fragment of polypeptide.Polypeptide of the present invention also comprises having and above (a), (b), and (c), (d), (e), (f) or (g) described sequence has at least 80%, preferably at least 85% or 90%, more preferably at least 95%, 96%, 97%, the polypeptide of the aminoacid sequence of 98% or 99% homogeny, and have with above-mentioned sequence at least 80%, 85% or 90% similarity is arranged, the polypeptide of the aminoacid sequence of preferred at least 95% similarity.Another embodiment of the present invention relates to a peptide species, and this polypeptide comprises or is made up of the aminoacid sequence that carries the epi-position part of Neutrokine-α SV polypeptide, and this polypeptide has above (a), (b), (c), (d), (e), (f) or aminoacid sequence (g).Peptide or polypeptide with aminoacid sequence that carries the epi-position part of Neutrokine-α SV polypeptide of the present invention, have at least 4 that comprise this peptide species, at least 5, at least 6, at least 7, at least 8, preferably at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, a more preferably 30-50 amino acid whose part at least, the polypeptide that carries epi-position until comprising the whole aminoacid sequences of polypeptide of the present invention of certain any length is also included within the present invention.
Nonrestrictive embodiments more of the present invention relate to a peptide species, it has the aminoacid sequence that carries the epi-position part of Neutrokine-α or Neutrokine-α SV polypeptide, this Neutrokine-α or Neutrokine-α SV polypeptide have above (a), (b), (c), (d), (e), (f), (g), (h) or aminoacid sequence (i).In other embodiments, the invention provides isolated antibody, its specificity (promptly uniquely) is in conjunction with having above (a), (b), (c), (d), (e), (f), (g), (h) or the Neutrokine-α of aminoacid sequence (i) or Neutrokine-α SV polypeptide.
The present invention also provides the method for separation antibody, and (promptly uniquely) combination specifically of this antibody has the Neutrokine-α or the Neutrokine-α SV polypeptide of the above aminoacid sequence.This antibody is as described below to be used for diagnosing or treatment.
The present invention also provides pharmaceutical composition, it comprises soluble Neutrokine-α and/or Neutrokine-α SV polypeptide, especially people Neutrokine-α and/or Neutrokine-α SV polypeptide, and/or anti-Neutrokine-Alpha antibodies and/or anti-Neutrokine-α SV antibody, they can be used for, for example treatment, prevention, prognosis and/or diagnosing tumour and metastases, infectation of bacteria, virus and other parasitic infection, immune deficiency, inflammation, the lymph gland disease, autoimmune disease, graft versus host disease, stimulate peripheral tolerance, destroy some cell transformed systems, mediated cell activation, survival and propagation, with mediation immunomodulatory and inflammatory response, and strengthen or the inhibition immunne response.
In some embodiments; use Neutrokine-α and/or Neutrokine-α SV polypeptide or its agonist of solubility of the present invention; with treatment; prevention; prognosis and/or diagnosis immune deficiency disorder (the chain severe combined immunodeficiency of X (SCID) for example; euchromosome SCID; adenosine deaminase damaged (ADA is damaged); gamma-globulin very few (XLA) in the chain blood of X; Bruton ' s disease; gamma-globulin is very few in the congenital blood; gamma-globulin is very few in the chain children's's blood of X; gamma-globulin is very few in the acquired blood; gamma-globulin is very few in the adult outbreak blood; gamma-globulin is very few in the late onset blood; gamma-globulin is very few in the unusual blood; hypogammaglobulinemia; the temporary hypogammaglobulinemia of children's; gamma-globulin is very few in the non-specific blood; gamma-globulin is very few in the blood; common mutability immune deficiency (CVID) (acquired); Wiskott-Aldrich syndrome (WAS); the chain high IgM immune deficiency of X; the chain high IgM immune deficiency of non-X; selective IgA deficiency; IgG subclass defective (have or do not have IgA defective); have normal or increase the antibody defective of horizontal IgS; the thymoma immune deficiency; the Ig heavy chain deletion; the K chain is damaged; B cell lymphocytic hyperplasia disease (BLPD), selective IgM immune deficiency, gamma-globulin very few (Swiss type) in the recessive blood; reticular dysgenesis; newborn infant's neutropenia, the congenital oligoleukocythemia of severe, thymic alymphoplasia-underdevelopment or heteroplasia immune deficiency; the ataxia telangiectasis; short-limb dwarfism, the chain lymphocytic hyperplasia syndrome of X (XLP), Nezolf associating IgS immunodeficiency syndrome; (PNP) is damaged for purine nucleoside phosphorylase; MHC II class damaged (Bare lymphocyte syndrome), and severe combined immunodeficiency), or the disease relevant with immune deficiency.
In a special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or their agonist, with treatment, prevention, prognosis and/or diagnose common mutability immune deficiency.
In a special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or their agonist, with treatment, prevention, the very few disease of gamma-globulin in prognosis and/or the chain blood of diagnosis X.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or their agonist, with treatment, prevention, prognosis and/or diagnosis severe severe combined immunodeficiency (SCID).
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or their agonist, with treatment, prevention, prognosis and/or diagnosis Wiskott-Aldrich syndrome.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or their agonist, with treatment, prevention, the Ig defective of prognosis and/or the chain high IgM level of diagnosis X.
In another embodiment, use Neutrokine-alpha-2 antagonists and/or Neutrokine-α SV antagonist (for example anti-Neutrokine-Alpha antibodies), with treatment, prevention, prognosis and/or diagnosis autoimmune disease (rheumatic arthritis for example, systemic lupus erythematous, primary thrombocytopenic purpura, autoimmune hemolytic anemia, anti-phosphatide (antiphospholipid) syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (for example IgA nephropathy), multiple sclerosis, neuritis, uveitis illness in eye, purpura (as the Henloch-Scoenlein purpura), Reiter ' s disease, Stiff-Man syndrome, autoimmune pulmonary inflammation, Guillain Barre syndrome, insulin-dependent diabetes mellitus, with the autoimmunity ophthalmia, autoimmune thyroid disease, hypothyroidism (being Hashimoto ' s thyroid disease), Goodpasture ' s syndrome, pemphigus, the receptor autophosphorylation immunity is as (a) Grave ' s disease, (b) Myasthenia Grave and (c) insulin resistance, autoimmune hemolytic anemia, autoimmunity thrombocyte purpura, anticol original antibody schleroderma, connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, primary Addison ' s disease, Infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, BP, Siogren ' s syndrome, diabetes, with class suprarenin medicine resistance (the adrenin drug resistance that comprises asthma or gallbladder cystic fibrosis), chronic active hepatitis, the sclerosis of primary gall-bladder, other incretory gland imbalance, vitiligo, vasculitis, behind the MI, cardiotomy syndrome, rubella, atopic dermatitis, asthma, inflammatory myositis, with other inflammation, granulamatous degenerates, with atrophy) or the disease relevant with autoimmune disease.In a special preferred embodiment, with anti-Neutrokine-Alpha antibodies of the present invention and/or anti-Neutrokine-α SV antibody and/or other antagonist for treating, prevention, prognosis and/or diagnosticating rheumatic sacroiliitis.In another special preferred embodiment, with anti-Neutrokine-Alpha antibodies and/or anti-Neutrokine-α SV antibody and/or anti-Neutrokine-α SV antibody and/or other antagonist for treating, prevention, prognosis and/or diagnositc system lupus erythematosus.In another preferred embodiment, with anti-Neutrokine-Alpha antibodies and/or anti-Neutrokine-α SV antibody and/or other antagonist for treating, prevention, prognosis and/or diagnosing primary thrombocyte purpura.In another embodiment preferred, with anti-Neutrokine-Alpha antibodies of the present invention and/or anti-Neutrokine-α SV antibody and/or other antagonist for treating, prevention, prognosis and/or diagnosis of iga ephrosis.In an embodiment preferred, with anti-Neutrokine-Alpha antibodies and/or anti-Neutrokine-α SV Antybody therapy, prevention, autoimmune disorder and functional disorder and/or pathology that prognosis and/or diagnosis are relevant with functional disorder with above disease.
The present invention further provides a kind of composition, it comprises Neutrokine-α or Neutrokine-α SV polynucleotide, Neutrokine-α or Neutrokine-α SV polypeptide, and/or anti-Neutrokine-Alpha antibodies or anti-Neutrokine-α SV antibody, be applied to external cell, the cell of ex vivo, intravital cell, or multicellular organisms.In preferred embodiments, composition of the present invention comprises Neutrokine-α and/or Neutrokine-α SV polynucleotide, with at host expression in vivo Neutrokine-α polypeptide and/or Neutrokine-α SV polypeptide, thus the treatment disease.In a preferred embodiment, composition of the present invention comprises Neutrokine-α and/or Neutrokine-α SV polynucleotide, with at host expression in vivo Neutrokine-α and/or Neutrokine-α SV polypeptide, thus treatment immunodeficiency diseases or the disease relevant with immune deficiency.Particularly preferably at patient's expression in vivo with the active relevant functional disorder of treatment and Neutrokine-α or Neutrokine-α SV gene unconventionality endogenous, (for example strengthening the expression of unusual B cell function) by increase B cell count or raising B cell survival.
The present invention also provides a kind of screening method, to differentiate the compound that can strengthen or suppress by Neutrokine-α and/or Neutrokine-α SV inductive cell response, this method comprises that the cell of expressing Neutrokine-α and/or Neutrokine-α SV contacts with the compound of candidate, analysis of cells is replied, and reply with standard cell lines and to compare, it is to analyze under the situation of candidate compound not having that standard is replied; Thereby high the replying with standard of cell response shows that this compound is an agonist, and cell response is lower than standard and replys and show that this compound is an antagonist.
In another embodiment, provide the method for a kind of Neutrokine-of discriminating α and/or Neutrokine-α SV acceptor, and the method for analyzing agonist and antagonist with this acceptor screening.This analysis comprises determines that candidate compound is to Neutrokine-α and/or the Neutrokine-α SV influence in conjunction with Neutrokine-α and/or Neutrokine-α SV acceptor.Especially, this method comprises Neutrokine-α and/or Neutrokine-α SV acceptor and Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide and candidate compound is contacted, and determine that Neutrokine-α and/or Neutrokine-α SV polypeptide improve or reduce with combining of Neutrokine-α and/or Neutrokine-α SV acceptor owing to there is candidate compound.Antagonist can be used for preventing septic shock, inflammation, the brain malaria, the activation of HIV virus, graft/host repels, bone resorption, rheumatic arthritis, emaciation (expendable or dystrophic), function of immune system imbalance, lymphoma and autoimmunity functional disorder (for example rheumatic arthritis and systemic lupus erythematous).
The inventor has also disclosed Neutrokine-α and has not only expressed at monocytic series, also at kidney, and lung, peripheral blood cells, marrow, t cell lymphoma, B cell lymphoma, the activated T cell, osteocarcinoma, unstriated muscle is expressed in scavenger cell and the Cord blood tissue.The inventor further discloses Neutrokine-α SV and presents only high expression level in original dendritic cell.Some imbalances of these tissues and cell are as tumour and metastases, bacterium, virus and other parasitic infection, immune deficiency (as the chronic variable immune deficiency), septic shock, inflammation, brain is cruel, the activation of HIV virus, graft/host repels, bone resorption, rheumatic arthritis, autoimmune disorder is (as rheumatic arthritis, and cachexy (expendable or dystrophic) and systemic lupus erythematous).Can be sure of taking from some tissues of individuality (as marrow) with this imbalance or body fluid (as serum, blood plasma, urine, immersion liquid or cerebrospinal fluid) in can detect the Neutrokine-α and/or the Neutrokine-α SV genetic expression of obviously higher or lower level, " standard " Neutrokine-α or Neutrokine-α SV gene expression dose promptly are at the tissue of the individuality of taking from no this imbalance or the expression level in the body fluid.Therefore, the invention provides diagnostic method used during diagnosing the illness, comprise that (a) analyzes Neutrokine-α and/or Neutrokine-α SV gene expression dose in individual cells or body fluid; (b) this expression level and standard Neutrokine-α and/or Neutrokine-α SV gene expression dose are compared, comparing result is than the high or low existence that shows disease of standard level.
Other embodiment of the present invention relates to a kind of method that needs improve or keep the individuality of interior Neutrokine-α of body and/or Neutrokine-α SV activity level for the treatment of, comprise this individuality used a kind of composition that said composition comprises of the present invention isolating Neutrokine-α and/or the Neutrokine-α SV polypeptide or their agonist for the treatment of significant quantity.
Another embodiment of the present invention relates to a kind of method that needs reduce the individuality of interior Neutrokine-α of body and/or Neutrokine-α SV activity level for the treatment of, comprise this individuality used a kind of composition that said composition comprises the Neutrokine-α and/or the Neutrokine-α SV antagonist for the treatment of significant quantity.Being used for preferred antagonist of the present invention is Neutrokine-alpha specific antibody and/or Neutrokine-α SV specific antibody.
The accompanying drawing summary
The following drawings illustrates embodiment of the present invention, and does not limit claim scope of the present invention.
Figure 1A and 1B illustrate the Nucleotide (SEQ ID NO:1) of Neutrokine-α and amino acid (the SEQ ID NO:2) sequence of deriving.Born of the same parents' internal area that the representative of 1-46 amino acids is inferred, the membrane-spanning domain (following stroke of two-wire place) that the representative of 47-72 amino acids is inferred, the extracellular domain (remaining sequence) that the representative of 73-285 amino acids is inferred.Glycosylation site l-asparagine symbol (N) with black matrix in Figure 1A and 1B that the potential l-asparagine connects represents that in Neutrokine-alpha amino acid sequence # number of black matrix arranged on first Nucleotide of asparagine residue in the coding Neutrokine-α nucleotide sequence.The glycosylation sequences that potential N connects find Neutrokine-alpha amino acid sequence with upper/lower positions: N124-Q127 (N-124, S-125, S-126, Q-127) and N242-C245 (N-242, N-243, S-244, C-245).
Neutrokine-α, Neutrokine-α SV, TNF-α, TNF-β, LT-β, and highly identical zone (Fig. 2 illustrates the contrast of these sequences) underscore in Figure 1A and 1B between the closely-related Fas part.These zones are not restrictive, structural domain (CD)-I that mark is gone bail for and kept in Figure 1A and 1B, CD-II, CD-III, CD-IV, CD-V, CD-VI, CD-VII, CD-VIII, CD-IX, CD-X, and CD-XI.
Fig. 2 A and 2B illustrate Neutrokine-α (SEQ ID NO:2) and Neutrokine-α SV (SEQ ID NO:19) and TNF-α (among Fig. 2 A and the 2B " TNF-α ", GenBank No.Z15026; (SEQ ID NO:3), and TNF-β (among Fig. 2 A and the 2B " TNF-β ", GenBank No.Z15026; SEQ ID NO:4), and lymphotoxin-β (among Fig. 2 A and the 2B " LT-β ", GenBankNo.L11016; SEQ ID NO:5) and the FAS part (among Fig. 2 A and the 2B " FASL ", GenBankNo.U1182I; SEQ IDNO:6) identical zone between the aminoacid sequence, it is established by " MegAlign " program, and this program is a part that is called " DAN*STAR " computer program.Residue shadow representation with the consensus sequence coupling.
Fig. 3 illustrates the sequential analysis of Neutrokine-alpha amino acid.Default parameters with described computer program is inferred the aminoacid sequence of SEQ ID NO:2, and the α district is shown, β district, the corner regions and the district of curling; Wetting ability and hydrophobicity; The amphiphilic district; Flex region; Antigenic index and surperficial probability.Among the figure, indicated the position in the high antigenicity zone of Neutrokine-α at " antigenic index-Jameson-Wolf ", this district promptly therefrom can obtain the zone that the present invention carries epitope peptide.Antigenic polypeptide comprises about Phe115～Leu147 of SEQ ID NO:2, Ile150～Tyr163, Ser171～Phe194, Glu223～Tyr246, Ser271～Phe278.
Data among Fig. 3 also with sheet form shown in the table 1.Row " residue ", " position " and Roman number I-XIV mark.The mark of row refers to the following feature of aminoacid sequence shown in Fig. 3 and the table 1: " residue " refers to the amino-acid residue of SEQ ID NO:2 and Figure 1A and 1B; " position " refers to the position of corresponding residue in SEQ ID NO:2 and Figure 1A and the 1B; I: α district-Garnier-Robson; II: α district-Chou-Fasman; IH: β district-Garnier-Robson; IV: β district-Chou-Fasman; V: corner regions-Garnier-Robson; VI: corner regions-Chou-Fasman; VII: district-Garnier-Robson curls; VIII: hydropathic profile-Kyte-Doolittle; IX: hydrophobicity profile-Hopp-Woods; X: α amphiphilic district-Eisenberg; XI: β amphiphilic district-Eisenberg; XII: flex region-Karplus-Schulz; XIII: antigenic index-Jameson-Wolf; And XIV: surperficial probability graph-Emini.
Fig. 4 A, 4B and 4C illustrate and are called as HSOAD55 (SEQ ID NO:7), the people cDNA clone that the present invention of HSLAH84 (SEQ ID NO:8) and HLTBM08 (SEQ ID NO:9) is relevant is with the sequence contrast of the Neutrokine-α nucleotide sequence of determining from the people cDNA clone who is deposited in ATCC No.97768.
Fig. 5 A and 5B illustrate proteic nucleotide sequence of Neutrokine-α SV (SEQ IDNO:18) and deduced amino acid (SEQ ID NO:19).Born of the same parents' internal area that the representative of 1-46 amino acids is inferred, the membrane-spanning domain (following stroke two-wire) that the representative of 47-72 amino acids is inferred, the extracellular domain (rest part sequence) that the representative of 73-266 amino acids is inferred.The glycosylation site that the potential l-asparagine connects with l-asparagine symbol (N) mark in Neutrokine-α SV aminoacid sequence of black matrix, marks with black matrix # number on first Nucleotide of asparagine residue in coding Neutrokine-α SV nucleotide sequence in Fig. 5 A and 5B.The glycosylation sequences that potential N connects find in Neutrokine-α SV aminoacid sequence with upper/lower positions: N124～Q127 (N124, S125, S126, Q127), and N223～C226 (N223, N224, S225, C226).Antigenic polypeptide comprises about Pro32～Leu47 of SEQ ID NO:19 aminoacid sequence, Glu116～Ser143, Phe153-Tyr173, Pro218-Tyr227, Ala232-Gln241, Ile244-Ala249, and Ser252-Val257.
Neutrokine-α, Neutrokine-α SV, TNF-α, TNF-β, LT-β, and height underscore place in Figure 1A and 1B, homogeny zone (contrast of these sequences is shown in Fig. 2) between the closely-related Fas part.(Neutrokine-α and Neutrokine-α SV's) these conservative zones are labeled as conservative territory (CD)-I, CD-II, CD-III, CD-V, CD-VI, CD-VII, CD-VIII, CD-IX, CD-X, and CD-XI in Fig. 5 A and 5B.Neutrokine-α SV does not contain the CD-IV sequence described in Figure 1A and the 1B description of drawings.
Neutrokine-α peptide sequence (SEQ ID NO:2) and APRIL, TNF-α, the sequence contrast of LT-α is shown in Fig. 7 A.In Fig. 7 A, the βZhe Die district is shown, it is described in Fig. 7 A description of drawings as following.
Fig. 6 illustrates Neutrokine-α SV amino acid sequence analysis, infers the α district of the aminoacid sequence of SEQ ID NO:19 with the default parameters of described computer program, β district, the corner regions and the district of curling; Wetting ability and hydrophobicity; The amphiphilic district; Flex region; Antigenic index and surperficial probability.The position in the proteic high antigenicity of Neutrokine-α zone is shown in " antigenicity index-Jameson-Wolf " figure, and this zone promptly is therefrom can obtain the zone that the present invention carries epitope peptide.Antigenic polypeptide comprises but the non-following amino acid whose polypeptide that is limited to the aminoacid sequence that comprises SEQ ID NO:19: about Pro32-Leu47, about Glu116-Ser143, about Phe153-Tyr173, about Pro218-Tyr227, about Ser252-Thr258, about Ala232-Gln241, about Ile244-Ala249, about Ser252-Val257.
Data shown in Figure 6 can be similar to the tabulated form representative of data shown in the table 1.The form of this representative precise information shown in Figure 6 can be with the MegAligh component of DNA*STAR computer sequential analysis routine package, sets up default parameters and produces.This is identical with the program that is used to produce Fig. 3 and Fig. 6.
The aminoacid sequence of Fig. 7 A:Neutrokine-α, and ligand binding domain of inferring and APRIL, TNF-α, sequence contrast (people APRIL polypeptide (the SEQ ID NO:20 particularly, of the ligand binding domain of LT-α; ATCC preserving number No.AF046888) 115-250 amino acids residue, TNF-α (SEQ ID NO:3; GenBankNo.Z15026) the 62-205 amino acids residue of 88-233 amino acids residue and LT-α (being also referred to as TNF-β, SEQ IDNO:4); GenBankNo.Z15026).Illustrate Neutrokine-α infer stride the film district, and the cracking site of Neutrokine-α is represented with italics.The βZhe Die zone that the sequence of underscore (A-H) representative is inferred.
Fig. 7 B:Neutrokine-α mRNA expresses.Make probe with Neutrokine-α open reading frame,, carry out the Northern hybridization analysis deriving from poly-(A)+RNA trace (Clonetech) of a series of people types of organizations and selected cancerous cell line.The Neutrokine-α mRNA that detects a 2.6Kb is at placenta, heart, lung, tire liver, high level expression in thymus gland and the pancreas.The Neutrokine-α mRNA of this 2.6Kb also detects in HL-60 and K562 clone.
Fig. 8 A and 8B: after activating the person monocytic cell by IFN-γ, the expression of Neutrokine-α improves.Fig. 8 A: on the monocyte of vitro culture, the fluidic cell quantitative analysis of Neutrokine-α protein expression.The cell of purifying is having or is not having under the situation of IFN-γ (100U/ml) and cultivated 3 days.Then with contrast (IgGl) (long and short dash line) dyeing of cell with Neutrokine-alpha specific mAb (2E5) (solid line) or isotype coupling.Be used in three independently the test in purifying obtain comparable result from the monocyte of three different donors.Fig. 8 B: preparation Neutrokine-alpha specific TagMan primer, and be used for determining in monocyte that do not stimulate and IFN-γ (100U/ml) processing, relative expression's level of Neutrokine-α mRNA.The nucleotide sequence of TagMan primer is as follows: (a) probe: 5 '-CCA CCA GCT CCA GGA GAA GGC AAC TC-3 ' (SEQ ID NO:24); (b) 5 ' amplimer: 5 '-ACC GCG GGA CTG AAA ATC T-3 ' (SEQID NO:25); (c) 3 ' amplimer: 5 '-CAC GCT TAT TTC TGC TGTTCT GA-3 ' (SEQ ID NO:26).
Fig. 9 A and 9B:Neutrokine-α are a kind of potential bone-marrow-derived lymphocyte stimulants.Fig. 9 A: in standard bone-marrow-derived lymphocyte combined stimulation is analyzed, utilize streptococcus aureus cowan 1 (SAC) to make guiding agent, determine the biological activity of Neutrokine-α.Single background count with SAC generation 1427 ± 316.The numerical value of being reported is the mean value ± deviation of gained result in three parts of holes.Reorganization Neutrokine-α with the HEK293T cell of the CHO transfection body of purifying self stabilization and transient transfection obtains analog result.Fig. 9 B: with the propagation of Neutrokine-α and itself and anti-IgM combined stimulation tonsilla B cell.Carry out bioanalysis as described in analyzing with SAC, except wrapping quilt in advance with the anti-human IgM antibody of goat in each hole, antibody concentration is 10 μ g/ml PBS.
Figure 10 A and the expression of 10B:Neutrokine-α acceptor in normal people's peripheral blood lymphocytes and tumor cell line.Figure 10 A: the human peripheral karyoblast derives from normal volunteer, and separates by density gradient centrifugation.Cell dyes with biotinylated Neutrokine-α, and streptavidin of puting together with PE and FITC or PerCP link coupled are specific to CD3 subsequently, CD20, CD14, the mAbs dyeing of CD56 and CD66b.On Becton DickinsonFACScan, use CellQuest software analysis cell.One of data represented 4 independent experiments.Figure 10 B:Neutrokine-α combines with histocyte clone U-937 and myeloma cell line IM-9's.
Figure 11 A, 11B, and 11C: the vivo effect of in BALB/cAn NCR mouse body, using Neutrokine-α.Figure 11 A: with the spleen paraffin embedding of formalin fixed, and with 5 microns section with h and E dyeing (top one group).Below one group be the section of taking from same animals, this animal is with anti-CD45R (B220) mAb dyeing, and develops the color with anti-mouse Ig of the rabbit of horseradish peroxidase (mouse absorption) and substrate four diaminobenzidine hydrochlorides (DAB).Slide Mayer ' s phenodin negative staining.CD45R (B220) express cell is brown.Figure 11 B: use the fluidic cell quantitative analysis on (one group on the right side) of PE-CD45R (B220) and FITC-ThB (LybD) painted normal (one group in left side) and Neutrokine-α processing.Figure 11 C: serum IgM in the mouse of normal and Neutrokine-α processing, the level of IgG and IgA.
Describe in detail
The invention provides isolated nucleic acid molecule, it comprises the polynucleotide of coding Neutrokine-α polypeptide, and this polypeptide has aminoacid sequence shown in Figure 1A and the 1B (SEQ ID NO:2), and this sequence is by to the cDNA cloning and sequencing and definite.Nucleotide sequence shown in Figure 1A and the 1B (SEQ ID NO:1) is by getting the HNEDU15 cloning and sequencing, this is cloned in and was deposited in American type culture collection (ATCC) on October 22nd, 1996, preserving number ATCC No.97768, ATCC is positioned at the road 20110-2209 of Manassas, Virginia city university, postcode 10801.The clone of preservation be contained in pBluescriptSK (-) plasmid (Stratagene, La Jolla, CA) in.
The present invention also provides isolated nucleic acid molecule, and it comprises the polynucleotide of coding Neutrokine-α SV polypeptide, and this polypeptide has aminoacid sequence shown in Fig. 5 A and the 5B (SEQ ID NO:19), and this sequence is by to the cDNA cloning and sequencing and definite.Nucleotide sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18) is by getting the HDPMC52 cloning and sequencing, and this is cloned in and was deposited in American type culture collection (ATCC), preserving number ATCC No.203518 on December 10th, 1998.The clone of preservation be contained in pBluescript SK (-) plasmid (Stratagene, La Jolla, CA) in.
Neutrokine-α of the present invention and Neutrokine-α SV polypeptide and TNF-α, TNF-β, LT-β, Fas part, the translation product of the people mRNAs of APRIL and LT-α are sequence homology (seeing Fig. 2 A, 2B and 7A).As implied above, TNF-α is considered to be in cytotoxicity, necrosis, programmed cell death, the common stimulation, propagation, lymphatic vessel forms, immunoglobulin class conversion, differentiation, an important cytokine that works in antiviral activity and adjusting adhesion molecule and other cytokine and the somatomedin.
Nucleic acid molecule
Unless otherwise indicated, all are (as Model 373 types with the automated DNA sequenator by the nucleotide sequence that dna molecular order-checking is herein determined, Applied Brosystems, Inc, Foster City, CA) determine, and all aminoacid sequences of the polypeptide of the dna molecule encode of being determined by this paper are to infer by translating above definite dna sequence dna.Therefore, known in the art as any dna sequence dna of determining by this automated method, can there be some errors at this any nucleotide sequence of determining.The nucleotide sequence that automated method is determined is typically shown approximately at least 90% with the real nucleotides sequence of the dna molecular of order-checking, and about at least 95%～99.9% homogeny is more typically arranged.True sequence can be determined by other method more accurately, comprise artificial DNA sequencing well known in the art.Also known in the art, compare with real sequence, single insertion or disappearance in the nucleotide sequence of determining, to cause frameshit in the Nucleotide translation, thus from that of this insertion or disappearance, will be different from true aminoacid sequence fully by the dna molecule encode of order-checking by the nucleotide sequence coded aminoacid sequence of determining of inferring.
" nucleotide sequence " of nucleic acid molecule or polynucleotide, it with regard to dna molecular or polynucleotide the deoxyribonucleotide sequence, with regard to RNA molecule or polynucleotide corresponding ribonucleoside acid sequence (A, G, C and U), each thymidine (T) deoxyribonucleotide in the wherein special deoxyribonucleotide sequence is replaced by ribonucleotide uridine (U).
The information of utilizing this paper to provide, as the nucleotide sequence among Figure 1A and the 1B, the nucleic acid molecule available standards clone of the present invention and the screening method of coding Neutrokine-α polypeptide obtain, as make the method for initiator clone eDNA with mRNA.As shown in the present, the described nucleic acid molecule of Figure 1A and 1B (SEQ ID NO:1) is found in the cDNA library derived from neutrophilic granulocyte.Expressed sequence mark corresponding to a Neutrokine-α cDNA part is also found at kidney, lung, and peripheral blood leucocyte, marrow, t cell lymphoma, B cell lymphoma, the activated T cell, cancer of the stomach, unstriated muscle is in scavenger cell and the Cord blood tissue.In addition, with the Nucleotide information that provides among Fig. 5 A and the 5B, the nucleic acid molecule available standards clone of the present invention and the screening method of coding Neutrokine-α SV polypeptide obtain, as make the method for initiator clone cDNA with mRNA.As shown in the present, nucleic acid molecule shown in Fig. 5 A and the 5B (SEQ ID NO:18) is found in the cDNA library derived from original dendritic cell.
The Neutrokine-α plasmid HNEDU15 of ATCC preserving number 97768 contains the proteinic open reading frame that a coding has about 285 amino-acid residues, about 46 amino acid whose born of the same parents' internal areas of inferring (about 1-46 amino acids residue of Figure 1A and 1B (SEQ ID NO:2)), about 26 amino acid whose membrane-spanning domains of inferring (amino-acid residue of about 47-72 position underscore among Figure 1A and the 1B (SEQ ID NO:2)), about 213 amino acid whose extracellular domains of inferring (about 73-285 amino acids among Figure 1A and the 1B (SEQ ID NO:2)); The molecular weight of deriving is about 31KDa.(Neutrokine-α polypeptide and humanTNF-have about 20% similarity and about 10% homogeny to figure shown in 1A and the 1B (SEQ ID NO:2), and TNF-α registration number in GenBank is No.339764.
The Neutrokine-α SV plasmid HDPMC52 of ATCC preserving number 203518 contains the proteinic open reading frame of nearly 266 amino-acid residues of coding, about 46 amino acid whose born of the same parents' internal areas of inferring (about 1-46 amino acids residue among Fig. 5 A and the 5B (SEQ ID NO:19)), about 26 amino acid whose membrane-spanning domains of inferring (amino-acid residue of about 47-72 position underscore among Fig. 5 A and the 5B (SEQ ID NO:19)), about 194 amino acid whose extracellular domains of inferring (about 73-266 amino acids residue among Fig. 5 A and the 5B (SEQ ID NO:19)); The molecular weight of deriving is about 29KDa.
Neutrokine-α SV polypeptide shown in Fig. 5 A and the 5B (SEQ ID NO:19) is that 339764 humanTNF-has about 33.9% similarity and about 22.0% homogeny with registration number in GenBank.
Those of skill in the art know because may there be error in order-checking as mentioned above, comprise respectively about 285 and 266 amino acid whose, sometimes can be short slightly by the true complete Neutrokine-α and/or the Neutrokine-α SV polypeptide of the cDNA coding of preservation.Especially, Neutrokine-α and the Neutrokine-α SV encoding sequence determined contain second common methionine(Met) codon, it can be used as the initiator codon of open reading frame translation, and this codon is positioned at 64-66 position Nucleotide shown in Figure 1A and the 1B (SEQ ID NO:18).More generally, real open reading frame can infer from N-terminal shown in Figure 1A and 1B (SEQ ID NO:1) and Fig. 5 A and the 5B (SEQ ID NO:18) first or second methionine(Met) codon ± 20 amino acid, especially ± the 10 interior any position of an amino acid whose scope.Should further recognize, polypeptide structure described herein territory is to be determined by Computer Analysis, therefore, and according to the analytical standard that is used to differentiate various structural domains, the extracellular domain of Neutrokine-α and Neutrokine-α SV polypeptide, the accurate position of born of the same parents' internal area and membrane-spanning domain can be different.For example, (for example can there be slight variation the accurate position of Neutrokine-α and Neutrokine-α SV extracellular domain according to the standard in used limiting structure territory among Figure 1A and 1B (SEQ ID NO:2) and Fig. 5 A and the 5B (SEQID NO:19), approximately 1-20 residue, especially approximately 1-5 residue can " be moved " in the position).In this case, the terminal of membrane-spanning domain and the starting point of extracellular domain are inferred on the basis of hydrophobic amino acid sequence in the position shown in more than differentiating, as Fig. 3 and 6 and table 1 shown in.As described below, the present invention further provides and had the polypeptide of various disappearances from the various residues of complete polypeptide N-terminal and/or C-terminal, comprise the amino acid whose polypeptide of one or more extracellular domain N-terminal described herein of disappearance, it constitutes the Neutrokine-α of soluble form and the extracellular domain of Neutrokine-α SV polypeptide.
Nucleic acid molecule of the present invention and polynucleotide can be rna forms, as mRNA, or dna form, comprise by clone or synthetic cDNA and the genomic dna that obtains that produce.This DNA can be two strands or strand.Single stranded DNA or RNA can be coding strands, are also referred to as sense strand, can be noncoding strands maybe, are also referred to as antisense strand.
The nucleic acid molecule of " separation " is meant isolated nucleic acid molecule from its natural surroundings (DNA or RNA).For example, the recombinant DNA molecules that comprises in the carrier of the present invention is considered to isolating.Isolated DNA molecule for example comprises the recombinant DNA molecules that remains in the heterologous host cell in addition, or purifying (part or basic purifying) dna molecular in the solution.Isolating RNA molecule comprises the interior or external rna transcription thing of the body of dna molecular of the present invention.Yet, be included in the nucleic acid among the clone, or separate or the karyomit(e) (as caryogram " karyomit(e) smear ") that moves from cell or cell lysate is not isolating in the present invention, described clone be not with the library in other member (as contain this clone and other library member homology solution form) isolating library (as genome or cDNA library) member.As described in the present invention further, can be natural according to isolated nucleic acid molecule of the present invention, reorganization or synthetic generation.
Isolated nucleic acid molecule of the present invention comprises dna molecular, and this dna molecular comprises or is made up of the open reading frame (ORF) with initiator codon of the 147-149 position of nucleotide sequence shown in Figure 1A and the 1B (SEQ ID NO:1).In addition, the dna molecular that isolated nucleic acid molecule of the present invention comprises comprises or by being different from the above sequence substantially, but because the degeneracy of genetic code, the proteic sequence of the Neutrokine-α that still encodes is formed.Certainly, genetic code is well known in the art.Therefore, those skilled in the art can the conventional variant that produces above-mentioned degeneracy.In another embodiment, isolated nucleic acid molecule provided by the invention comprises or is made up of the sequence of coding Neutrokine-α polypeptide, and Neutrokine-α polypeptide has the cDNA amino acid sequence coded that comprises in the plasmid by ATCC preserving number 97768.Preferably, this nucleic acid molecule comprises or is made up of the extracellular domain of coded polypeptide or the ripe or solvable peptide sequence of polypeptide, and this polypeptide is encoded by the cDNA in the plasmid of ATCC preserving number 97768.
Isolated nucleic acid molecule of the present invention also comprises such dna molecular, and it comprises the open reading frame (ORF) with initiator codon of the 1-3 position of nucleotide sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18).In addition, the dna molecular that isolated nucleic acid molecule of the present invention comprises comprises or by being different from the above sequence substantially, but because the degeneracy of genetic code, the sequence of the Neutrokine-α SV polypeptide of still encoding is formed.Certainly, genetic code is well known in the art.Therefore, those skilled in the art can conventionally produce above-mentioned degeneracy variant.In another embodiment, isolating nucleic acid molecule provided by the invention comprises or is made up of the sequence of coding Neutrokine-α SV polypeptide, and Neutrokine-α SV polypeptide has the cDNA amino acids coding that comprises in the plasmid by ATCC preserving number 203518.Preferably, this nucleic acid molecule comprises or is made up of the sequence of coded polypeptide extracellular domain or ripe solvable peptide sequence, the cDNA coding that comprises in the plasmid of this polypeptide by ATCC preserving number 203518.
The nucleic acid molecule that the present invention further provides comprises or by nucleotide sequence shown in Figure 1A and the 1B (SEQ IDNO:1), or be contained in the nucleotide sequence of the Neutrokine-α cDNA in the plasmid of ATCC No.97768, or the nucleotide sequence with the sequence that is complementary to one of above sequence is formed.In addition, isolated nucleic acid molecule provided by the invention comprises or by nucleotide sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18), or be contained in the nucleotide sequence of the Neutrokine-α SV cDNA in the plasmid of ATCC No.203518, or the nucleotide sequence with the sequence that is complementary to one of above sequence is formed.This isolating molecule is dna molecular especially, have and comprise but non-being limited to as by carrying out the probe of gene mapping with Chromosomal in situ hybridization, and for example by Northern or Western engram analysis, the effects such as expression of Neutrokine-α and Neutrokine-α SV in the detection people tissue.
In one embodiment, polynucleotide of the present invention comprise or are made up of sequence shown in the SEQ ID NO:22.Sequence shown in the SEQ ID NO:22 makes up from some the overlapping mouse est sequences (AI 182472, and AA 422749, AA 25047 and AI 122485) that derive from GenBank.Est sequence is compared class Neutrokine-α polynucleotide sequence shown in the generation SEQ ID NO:22.The aminoacid sequence that derives from SEQ ID NO:22 translation is shown in SEQ IDNO:23.The fragment of sequence shown in SEQ ID NO:22 and the SEQ ID NO:23, variant and derivative also are encompassed in the present invention.
In another embodiment, polynucleotide of the present invention comprise or by sequence shown in the SEQ ID NO:27, and/or the sequence of aminoacid sequence shown in the coding SEQ ID NO:28, its fragment, and variant and derivative are formed.These polynucleotide also are encompassed in the present invention.For example, the polynucleotide that embodiments more of the present invention relate to comprise or are made up of the sequence of coding one peptide sequence, and the 68-219 amino acids of this peptide sequence and SEQ ID NO:28 has at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny.The aminoacid sequence that derives from SEQ ID NO:27 translation is shown in SEQ ID NO:28.The fragment of sequence shown in polypeptide that comprises or be made up of the aminoacid sequence of SEQ ID NO:28 and the SEQ ID NO:28, variant and derivative are also forgiven in the present invention.For example, the polypeptide that embodiments more of the present invention relate to comprises or has at least 80%, 85% by the 68-219 amino acids with SEQ ID NO:28, and the peptide sequence of 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny is formed.Nucleic acid molecule with sequence shown in the SEQ ID NO:27 is to obtain from cyanomologous monkey (being Macaea irus) PBMC through two kinds of degenerated primers by RT-PCR.In brief, total RNA by with Trizol (available from Life Technologies, Inc, Rockville, MD) according to manufacturer's guidance preparation from cyanomologous monkey PBMC.Then, from cyanomologous monkey PBMC prepared product, use the synthesizing single-stranded cDNA of standard method with widow-dT primer.Neutrokine-alpha specific primer designs based on the conservative region between mouse and the people Neutrokine-alpha molecule (being respectively SEQ ID NO:22 and 1).Then by the antisense oligonucleotide primer deposits yields cyanomologous monkey Neutrokine-'alpha ' nucleic acids molecule of PCR with two kinds of degeneracys below the cDNA form assembly.5 ' primer: 5 '-TAC CAG ITG GCI GCC ITG CAA G-3 ' (SEQID NO:35) and 3 ' primer: 5 '-GTI ACA GCA GTT TIA IIG CAC C-3 ' (SEQ ID NO:36).In the primer sequence of degeneracy (SEQ IDNO:35 and 36), " I " represents Hypoxanthine deoxyriboside or didanosine.
In another embodiment, polynucleotide of the present invention comprise or by sequence shown in the SEQ ID NO:29, and/or the sequence of aminoacid sequence shown in the coding SEQ ID NO:30, its fragment, and variant and derivative are formed.These polynucleotide are also contained within the scope of the present invention.For example, the polynucleotide that relate to of embodiments more of the present invention comprise or have at least 80%, 85% by coding and sequence 68-219 amino acid shown in the SEQ IDNO:30,90%, 92%, 95%, 96%, 97%, 98% or the sequence of the peptide sequence of 99% homogeny form.The aminoacid sequence that derives from SEQ IDNO:29 translation is shown in SEQ ID NO:30.Comprise or by the fragment of sequence shown in aminoacid sequence shown in the SEQ IDNO:30 and SEQ ID NO:29 and the SEQ ID NO:30, variant and derivative also are encompassed in the present invention.For example, the polypeptide that embodiments more of the present invention relate to comprises or has at least 80%, 85% by the 68-219 amino acids with SEQ ID NO:30, and 90%, 92%, 95%, 96%, the peptide sequence of 97%, 98% or 99% homogeny.Nucleic acid molecule with sequence shown in the SEQ ID NO:29 is by RT-PCR, obtains from macaque PBMC with two kinds of degenerated primers.In brief, total RNA by with Trizol (available from Life Technologies, Inc, Rockviue MD), according to manufacturer's guidance, prepares from macaque PBMC.Then, from macaque PBMC prepared product, use the synthesizing single-stranded cDNA of standard method with oligo dT primer.The conservative region that Neutrokine-alpha specific primer is based between mouse and the people Neutrokine-alpha molecule (being respectively SEQ ID NO:22 and 1) designs.Then, macaque Neutrokine-'alpha ' nucleic acids molecule is by PCR, with the antisense oligonucleotide primer deposits yields of the following two kinds of degeneracys of cDNA form assembly.5 ' primer: 5 '-TAC CAG ITG GCI GCC ITG CAA G-3 ' (SEQ IDNO:35) and 3 ' primer: 5 '-GTI ACA GCA GTT TIA IIG CAC C-3 ' (SEQID NO:36).In the sequence of the primer of degeneracy (SEQ ID NO:35 and 36), " I " represents Hypoxanthine deoxyriboside or didanosine.
The present invention also provides a kind of nucleic acid molecule, it has the relevant nucleotide sequence in extension with SEQ ID NO:1 and SEQID NO:18, described nucleotide sequence is determined from following relevant cDNA clone: HSOAD 55 (SEQ ID NO:7), HSLAH84 (SEQ ID NO:8), and HLTBM08 (SEQ ID NO:9).
The invention further relates to the nucleic acid molecule of a coding nucleotide sequence part described herein, and the fragment of this isolated nucleic acid molecule.In one embodiment, the invention provides the polynucleotide of the nucleotide sequence with part of representing SEQ ID NO:1, this part is made up of the 1-1001 position Nucleotide of SEQ ID NO:1.In another embodiment, the invention provides the polynucleotide of the nucleotide sequence with part of representing SEQ ID NO:18, this part is made up of the 1-798 position Nucleotide of SEQ ID NO:18.
The invention further relates to the fragment of nucleic acid molecule described herein (being polynucleotide).For example having, the nucleic acid molecule fragment of following nucleotide sequence is meant that length is at least 15, preferably at least 20 or 25, more preferably at least 30, most preferably at least 40,50,100,150,200,250,300,325,350,375,400,450, or 500 Nucleotide, described nucleotide sequence for example is: the nucleotide sequence of the cDNA that comprises in the plasmid of ATCC preserving number 97768, the nucleotide sequence of the cDNA encoded polypeptides sequence that comprises in the plasmid of coding by ATCC preserving number 97768, the nucleotide sequence of SEQ ID NO:1, the nucleotide sequence of the peptide sequence of coding SEQ ID NO:2, the nucleotide sequence of the cDNA that comprises in the plasmid of ATCC preserving number 203518, the nucleotide sequence of SEQ ID NO:18, the nucleotide sequence of coding SEQ IDNO:20 peptide sequence, or their complementary strand.These fragments have many purposes, comprise but non-diagnostic probe as described herein and the primer of being limited to.Certainly, bigger fragment such as length are that the fragment of 501-1500 Nucleotide also is useful according to the present invention, this fragment is equivalent to the following nucleotide sequence of great majority (if not all): the nucleotide sequence of the cDNA that comprises in the plasmid of ATCC preserving number 97768, the nucleotide sequence of SEQ ID NO:1, the nucleotide sequence of the nucleotide sequence of the cDNA that comprises in the plasmid of ATCC preserving number 203518 and SEQ ID NO:18.The preferred nucleic acid fragment of the present invention comprises the nucleic acid molecule of the peptide species of encoding, this polypeptide comprises or partly is made up of the Neutrokine-α that illustrates respectively among Figure 1A and 1B (SEQ IDNO:2) and Fig. 5 A and the 5B (SEQ ID NO:19) and/or the epi-position of carrying of Neutrokine-α SV polypeptide, and sees the following detailed.Also be encompassed in the present invention by these polynucleotide passage encoded polypeptides.
For example having, the nucleic acid molecule fragment of following nucleotide sequence is meant that length is at least 15, preferably at least 20 or 25, more preferably at least 30, most preferably at least 40,50,100,150,200,250,300,325,350,375,400,450 or 500 Nucleotide, described nucleotide sequence for example is: the nucleotide sequence of SEQ ID NO:21, the nucleotide sequence of SEQ ID NO:22, the nucleotide sequence of SEQ ID NO:27, the nucleotide sequence of SEQ ID NO:29, the nucleotide sequence of the peptide sequence of coding SEQ ID NO:23, the nucleotide sequence of the peptide sequence of coding SEQ ID NO:28, the nucleotide sequence of the peptide sequence of coding SEQ ID NO:30 or their complementary strand.These fragments have many purposes, comprise but non-be limited to as described herein as diagnostic probe and primer.Certainly, bigger fragment such as length are that the fragment of 501-1500 Nucleotide also is useful according to the present invention, this fragment is equivalent to following great majority (if not all) nucleotide sequence: the nucleotide sequence of SEQ ID NO:21, the nucleotide sequence of SEQ ID NO:22, the nucleotide sequence of SEQ ID NO:27, the nucleotide sequence of SEQ ID NO:29, the nucleotide sequence of the peptide sequence of coding SEQ ID NO:23, the nucleotide sequence of the peptide sequence of coding SEQ ID NO:28, the nucleotide sequence of the peptide sequence of coding SEQ ID NO:30, or their complementary strand.Also be encompassed in the present invention by these polynucleotide passage encoded polypeptides.
The representative segment of Neutrokine-α polynucleotide passage of the present invention for example comprises: comprise or by about 1-50 of SEQ ID NO:1's or its complementary strand or ATCC preserving number 97768 cDNA, 51-100,101-146,147-200,201-250,251-300,301-350,351-400,401-450,451-500,501-550,551-600,600-650,651-700,701-750,751-800,800-850,851-900,901-950,951-1000, the fragment that 1001-1050 and/or 1051-1082 position nucleotide sequence are formed." approximately " comprises the scope that particularly points out in the literary composition, and Duo slightly than this scope in one or both ends in office or the scope of few slightly several (5,4,3,2, or 1) Nucleotide.
The representative segment of Neutrokine-α SV polynucleotide passage of the present invention for example comprises: comprise or by SEQ ID NO:18's or its complementary strand, or about 1-50 of the cDNA of ATCC preserving number 203518,51-100,101-146,147-200,201-250,251-300,301-350,351-400,401-450,451-500,501-550,551-600,601-650,651-700,701-750,751-800,801-850, and/or 851-900 position nucleotide sequence is formed." approximately " comprises the scope of refering in particular in the literary composition, and Duo slightly than this scope in one or both ends in office or the scope of few slightly several (5,4,3,2, or 1) Nucleotide.
In certain preferred aspects, polynucleotide of the present invention comprise or by the 571-627 of SEQ IDNO:1,580-627,590-627,600-627,610-627,571-620,580-620,590-620,600-620,571-610,580-610,590-610,571-600,580-600 and/or 571-590 position nucleotide residue are formed.
In some other embodiment preferred, polynucleotide of the present invention comprise or are made up of the following nucleotide residue of SEQID NO:18: 1-879,25-879,50-879,75-879,100-879,125-879,150-879,175-879,200-879,225-879,250-879,275-879,300-879,325-879,350-879,375-879,400-879,425-879,450-879,475-879,500-879,525-879,550-879,575-879,600-879,625-879,650-879,675-879,700-879,725-879,750-879,775-879,800-879,825-879,850-879,1-850,25-850,50-850,75-850,100-850,125-850,150-850,175-850,200-850,225-850,250-850,275-850,300-850,325-850,350-850,375-850,400-850,425-850,450-850,475-850,500-850,525-850,550-850,575-850,600-850,625-850,650-850,675-850,700-850,725-850,750-850,775-850,800-850,825-850,1-825,25-825,50-825,75-825,100-825,125-825,150-825,175-825,200-825,225-825,250-825,275-825,300-825,325-825,350-825,375-825,400-825,425-825,450-825,475-825,00-825,525-825,550-825,575-825,600-825,625-825,650-825,675-825,700-825,725-825,750-825,775-825,800-825,1-800,25-800,50-800,75-800,100-800,125-800,150-800,175-800,200-800,225-800,250-800,275-800,300-800,325-800,350-800,375-800,400-800,425-800,450-800,475-800,500-800,525-800,550-800,575-800,600-800,625-800,650-800,675-800,700-800,725-800,750-800,775-800,1-775,25-775,50-775,75-775,100-775,125-775,150-775,175-775,200-775,225-775,250-775,275-775,300-775,325-775,350-775,375-775,400-775,425-775,450-775,475-775,500-775,525-775,550-775,575-775,600-775,625-775,650-775,675-775,700-775,725-775,750-775,1-750,25-750,50-750,75-750,100-750,125-750,150-750,175-750,200-750,225-750,250-750,275-750,300-750,325-750,350-750,375-750,400-750,425-750,450-750,475-750,500-750,525-750,550-750,575-750,600-750,625-750,650-750,675-750,700-750,725-750,1-725,25-725,50-725,75-725,100-725,125-725,150-725,175-725,200-725,225-725,250-725,275-725,300-725,325-725,350-725,375-725,400-725,425-725,450-725,475-725,500725,525-725,550-725,575-725,600-725,625-725,650-725,675-725,700-725,1-700,25-700,50-700,75-700,100-700,125-700,150-700,175-700,200-700,225-700,250-700,275-700,300-700,325-700,350-700,375-700,400-700,425-700,450-700,475-700,500-700,525-700,550-700,575-700,600-700,625-700,650-700,675-700,1-675,25-675,50-675,75-675,100-675,125-675,150-675,175-675,200-675,225-675,250-675,275-675,300-675,325-675,350-675,375-675,400-675,425-675,450-675,475-675,500-675,525-675,550-675,575-675,600-675,625-675,650-675,1-650,25-650,50-650,75-650,100-650,125-650,150-650,175-650,200-650,225-650,250-650,275-650,300-650,325-650,350-650,375-650,400-650,425-650,450-650,475-650,500-650,525-650,550-650,575-650,600-650,625-650,1-625,25-625,50-625,75-625,100-625,125-625,150-625,175-625,200-625,225-625,250-625,275-625,300-625,325-625,350-625,375-625,400-625,425-625,450-625,475-625,500-625,525-625,550-625,575-625,600-625,1-600,25-600,50-600,75-600,100-600,125-600,150-600,175-600,200-600,225-600,250-600,275-600,300-600,325-600,350-600,375-600,400-600,425-600,450-600,475-600,500-600,525-600,550-600,575-600,1-575,25-575,50-575,75-575,100-575,125-575,150-575,175-575,200-575,225-575,250-575,275-575,300-575,325-575,350-575,375-575,400-575,425-575,450-575,475-575,500-575,525-575,550-575,1-550,25-550,50-550,75-550,100-550,125-550,150-550,175-550,200-550,225-550,250-550,275-550,300-550,325-550,350-550,375-550,400-550,425-550,450-550,475-550,500-550,525-550,1-525,25-525,50-525,75-525,100-525,125-525,150-525,175-525,200-525,225-525,250-525,275-525,300-525,325-525,350-525,375-525,400-525,425-525,450-525,475-525,500-525,1-500,25-500,50-500,75-500,100-500,125-500,150-500,175-500,200-500,225-500,250-500,275-500,300-500,325-500,350-500,375-500,400-500,425-500,450-500,475-500,1-475,25-475,50-475,75-475,100-475,125-475,150-475,175-475,200-475,225-475,250-475,275-475,300-475,325-475,350-475,375-475,400-475,425-475,450-475,1-450,25-450,50-450,75-450,100-450,125-450,150-450,175-450,200-450,225-450,250-450,275-450,300-450,325-450,350-450,375-450,400-450,425-450,1-425,25-425,50-425,75-425,100-425,125-425,150-425,175-425,200-425,225-425,250-425,275-425,300-425,325-425,350-425,375-425,400-425,1-400,25-400,50-400,75-400,100-400,125-400,150-400,175-400,200-400,225-400,250-400,275-400,300-400,325-400,350-400,375-400,1-375,25-375,50-375,75-375,100-375,125-375,150-375,175-375,200-375,225-375,250-375,275-375,300-375,325-375,350-375,1-350,25-350,50-350,75-350,100-350,125-350,150-350,175-350,200-350,225-350,250-350,275-350,300-350,325-350,1-325,25-325,50-325,75-325,100-325,125-325,150-325,175-325,200-325,225-325,250-325,275-325,300-325,1-300,25-300,50-300,75-300,100-300,125-300,150-300,175-300,200-300,225-300,250-300,275-300,1-275,25-275,50-275,75-275,100-275,125-275,150-275,175-275,200-275,225-275,250-275,1-250,25-250,50-250,75-250,100-250,125-250,150-250,175-250,200-250,225-250,1-225,25-225,50-225,75-225,100-225,125-225,150-225,175-225,200-225,1-200,25-200,50-200,75-200,100-200,125-200,150-200,175-200,1-175,25-175,50-175,75-175,100-175,125-175,150-175,1-150,25-150,50-150,75-150,100-150,125-150,1-125,25-125,50-125,75-125,100-125,1-100,25-100,50-100,75-100,1-75,25-75,50-75,1-50,25-50, and/or 1-25
In the other embodiment preferred, polynucleotide of the present invention comprise or are made up of the following nucleotide residue of SEQID NO:1: 400-627,425-627,450-627,475-627,500-627,525-627,550-627,575-627,600-627,400-600,425-600,450-600,475-600,500-600,525-600,550-600,575-600,400-575,425-575,450-575,475-575,500-575,525-575,550-575,400-550,425-550,450-550,475-550,500-550,525-550,400-500,425-500,450-500,475-500,400-475,425-475,450-475,400-450,425-450,571-800,600-800,625-800,650-800,675-800,700-800,725-800,750-800,775-800,571-775,600-775,625-775,650-775,675-775,700-775,725-775,750-775,571-750,600-750,625-750,650-750,675-750,700-750,725-750,571-725,600-725,625-725,650-725,675-725,700-725,571-700,600-700,625-700,650-700,675-700,571-675,600-675,625-675,650-675,571-650,600-650,625-650,571-625,600-625, and/or 571-600
In other embodiment preferred, polynucleotide of the present invention comprise or are made up of the following nucleotide residue of SEQID NO:1: 147-500,147-450,147-400,147-350,200-500,200-450,200-400,200-350,250-500,250-450,250-400,250-350,300-500,300-450,300-400,300-350,350-750,350-700,350-650,350-600,350-550,400-750,400-700,400-650,400-600,400-550,425-750,425-700,425-650,425-600,425-550,450-1020,450-1001,450-950,450-900,450-850,450-800,450-775,500-1001,500-950,500-900,500-850,500-800,500-775,550-1001,550-950,550-900,550-850,550-800,550-775,600-1001,600-950,600-900,600-850,600-800,600-775,650-1001,650-950,650-900,650-850,650-800,650-775,700-1001,700-950,700-900,700-850,700-800,700-775,825-1082,850-1082,875-1082,900-1082,925-1082,950-1082,975-1082,1000-1082,1025-1082, and/or 1050-1082
Preferably, polynucleotide passage coding of the present invention shows the polypeptide of Neutrokine-α and/or Neutrokine-α SV functionally active.Polypeptide with " functionally active " is meant and can shows one or more and total length and/or excretory Neutrokine-α polypeptide and/or the relevant active polypeptide of known function of Neutrokine-α SV polypeptide.This functionally active comprise but the non-biological activity that is limited to (as stimulating B cell proliferation, survival differentiation and/or activated ability), antigenicity (in conjunction with the ability of (or combine with Neutrokine-α and/or Neutrokine-α SV polypeptide are competitive) anti-Neutrokine-α and/or anti-Neutrokine-α SV antibody), immunogenicity (producing ability) in conjunction with the antibody of Neutrokine-α and/or Neutrokine-α SV polypeptide, form polymeric ability with Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide, reach ability in conjunction with Neutrokine-α and/or Neutrokine-α SV polypeptide receptor or part.
In other special embodiment, born of the same parents' internal area (the 1-46 amino acids of SEQ ID NO:2) that polynucleotide passage encoded polypeptides of the present invention comprises or inferred by Neutrokine-α, the membrane-spanning domain of inferring (the 47-72 amino acids of SEQ ID NO:2), the extracellular domain of inferring (the 73-284 amino acids of SEQ ID NO:2), the TNF that infers are guarded territory (the 191-284 amino acids of SEQ ID NO:2) and are formed.In another embodiment, polynucleotide passage of the present invention comprises or by 1,2,3 or the arbitrary combination in whole 4 said structure territories form.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
In another special embodiment, polynucleotide passage encoded polypeptides of the present invention comprises or by born of the same parents' internal area of inferring (the 1-46 amino acids of SEQ ID NO:19) of Neutrokine-α SV, the membrane-spanning domain of inferring (the 47-72 amino acids of SEQ ID NO:19), the extracellular domain of inferring (the 73-266 amino acids of SEQ ID NO:19), or the conservative territory (the 172-265 amino acids of SEQ ID NO:19) of the TNF that infers is formed.In other embodiments, polynucleotide passage encoded polypeptides of the present invention comprises or by 1,2,3 or the arbitrary combination in whole 4 said structure territories form.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
In another embodiment, polynucleotide passage of the present invention comprises or is made up of a kind of polynucleotide, this polynucleotide encoding is selected from the Met 1-Lys113 of SEQ ID NO:2, Leu114-Thr141, Ile142-Lus160, Gly161-Gln198, the aminoacid sequence of Val199-Ala248 and Gly250-Leu285.In addition, the polynucleotide of the arbitrary combination of coding 2,3,4,5 or more a plurality of these aminoacid sequences also are encompassed in the present invention.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
In another embodiment, polynucleotide passage of the present invention comprises or is made up of a kind of polynucleotide, this polynucleotide encoding is selected from the Met 1-Lys113 of SEQ ID NO:19, Leu114-Thr141, Gly142-Gln179, Val180-Ala229, the aminoacid sequence of Gly230-Leu266 residue.In addition, the polynucleotide of the arbitrary combination of coding 2,3,4,5 or more a plurality of these aminoacid sequences also are encompassed in the present invention.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
In another embodiment, polynucleotide passage of the present invention comprises or is made up of a kind of polynucleotide, this polynucleotide encoding is selected from the Met 1-Lys106 of SEQ ID NO:23, Leu107-Thr134, Glu135-Asn165, Ile167-Lys184, Gly185-Gln224, the aminoacid sequence of Val225-Ala272 and Gly273-Leu309 residue.In addition, the polynucleotide of the arbitrary combination of coding 2,3,4,5 or more a plurality of these aminoacid sequences also are encompassed in the present invention.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
In another embodiment, polynucleotide passage of the present invention comprises or is made up of a kind of polynucleotide, this polynucleotide encoding is selected from the Tyr 1-Lys47 of SEQ ID NO:28, Leu48-Thr75, Ile76-Lys94, Gly95-Gln132, the aminoacid sequence of Val133-Ala182 and Gly183-Ala219 residue.In addition, the polynucleotide of the arbitrary combination of coding 2,3,4,5 or more a plurality of these aminoacid sequences also are encompassed in the present invention.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
In another embodiment, polynucleotide passage of the present invention comprises or is made up of a kind of polynucleotide, this polynucleotide encoding is selected from the Tyr 1-Lys47 of SEQ ID NO:30, Leu48-Thr75, Ile76-Lys94, Gly95-Gln132, the aminoacid sequence of Val 133-Ala182 and Gly183-Ala219 residue.In addition, the polynucleotide of the arbitrary combination of coding 2,3,4,5 or more a plurality of these aminoacid sequences also are encompassed in the present invention.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
In another embodiment, polynucleotide of the present invention comprise or are made up of sequence shown in the SEQ ID NO:21.Sequence encoding one peptide species shown in the SEQ ID NO:21, this polypeptide initial methionine residues reaches the composition of the Ala134-Leu285 residue of Neutrokine-α peptide sequence shown in the SEQ ID NO:2 that links to each other.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
In other the preferred embodiment, polynucleotide of the present invention comprise or are made up of the following nucleotide residue of SEQID NO:21: 1-459,15-459 at some, 30-459,45-459,60-459,75-459,90-459,105-459,120-459,135-459,150-459,165-459,180-459,195-459,210-459,225-459,240-459,255-459,270-459,285-459,300-459,315-459,330-459,345-459,360-459,375-459,390-459,405-459,420-459,435-459,450-459,1-450,15-450,30-450,45-450,60-450,75-450,90-450,105-450,120-450,135-450,150-450,165-450,180-450,195-450,210-450,225-450,240-450,255-450,270-450,285-450,300-450,315-450,330-450,345-450,360-450,375-450,390-450,405-450,420-450,435-450,1-435,15-435,30-435,45-435,60-435,75-435,90-435,105-435,120-435,135-435,150-435,165-435,180-435,195-435,210-435,225-435,240-435,255-435,270-435,285-435,300-435,315-435,330-435,345-435,360-435,375-435,390-435,405-435,420-435,1-420,15-420,30-420,45-420,60-420,75-420,90-420,105-420,120-420,135-420,150-420,165-420,180-420,195-420,210-420,225-420,240-420,255-420,270-420,285-420,300-420,315-420,330-420,345-420,360-420,375-420,390-420,405-420,1-405,15-405,30-405,45-405,60-405,75-405,90-405,105-405,120-405,135-405,150-405,165-405,180-405,195-405,210-405,225-405,240-405,255-405,270-405,285-405,300-405,315-405,330-405,345-405,360-405,375-405,390-405,1-390,15-390,30-390,45-390,60-390,75-390,90-390,105-390,120-390,135-390,150-390,165-390,180-390,195-390,210-390,225-390,240-390,255-390,270-390,285-390,300-390,315-390,330-390,345-390,360-390,375-390,1-375,15-375,30-375,45-375,60-375,75-375,90-375,105-375,120-375,135-375,150-375,165-375,180-375,195-375,210-375,225-375,240-375,255-375,270-375,285-375,300-375,315-375,330-375,345-375,360-375,1-360,15-360,30-360,45-360,60-360,75-360,90-360,105-360,120-360,135-360,150-360,165-360,180-360,195-360,210-360,225-360,240-360,255-360,270-360,285-360,300-360,315-360,330-360,345-360,1-345,15-345,30-345,45-345,60-345,75-345,90-345,105-345,120-345,135-345,150-345,165-345,180-345,195-345,210-345,225-345,240-345,255-345,270-345,285-345,300-345,315-345,330-345,1-330,15-330,30-330,45-330,60-330,75-330,90-330,105-330,120-330,135-330,150-330,165-330,180-330,195-330,210-330,225-330,240-330,255-330,270-330,285-330,300-330,315-330,1-315,15-315,30-315,45-315,60-315,75-315,90-315,105-315,120-315,135-315,150-315,165-315,180-315,195-315,210-315,225-315,240-315,255-315,270-315,285-315,300-315,1-300,15-300,30-300,45-300,60-300,75-300,90-300,105-300,120-300,135-300,150-300,165-300,180-300,195-300,210-300,225-300,240-300,255-300,270-300,285-300,1-285,15-285,30-285,45-285,60-285,75-285,90-285,105-285,120-285,135-285,150-285,165-285,180-285,195-285,210-285,225-285,240-285,255-285,270-285,1-270,15-270,30-270,45-270,60-270,75-270,90-270,105-270,120-270,135-270,150-270,165-270,180-270,195-270,210-270,225-270,240-270,255-270,1-255,15-255,30-255,45-255,60-255,75-255,90-255,105-255,120-255,135-255,150-255,165-255,180-255,195-255,210-255,225-255,240-255,1-240,15-240,30-240,45-240,60-240,75-240,90-240,105-240,120-240,135-240,150-240,165-240,180-240,195-240,210-240,225-240,1-225,15-225,30-225,45-225,60-225,75-225,90-225,105-225,120-225,135-225,150-225,165-225,180-225,195-225,210-225,1-210,15-210,30-210,45-210,60-210,75-210,90-210,105-210,120-210,135-210,150-210,165-210,180-210,195-210,1-195,15-195,30-195,45-195,60-195,75-195,90-195,105-195,120-195,135-195,150-195,165-195,180-195,1-180,15-180,30-180,45-180,60-180,75-180,90-180,105-180,120-180,135-180,150-180,165-180,1-165,15-165,30-165,45-165,60-165,75-165,90-165,105-165,120-165,135-165,150-165,1-150,15-150,30-150,45-150,60-150,75-150,90-150,105-150,120-150,135-150,1-135,15-135,30-135,45-135,60-135,75-135,90-135,105-135,120-135,1-120,15-120,30-120,45-120,60-120,75-120,90-120,105-120,1-105,15-105,30-105,45-105,60-105,75-105,90-105,1-90,15-90,30-90,45-90,60-90,75-90,1-75,15-75,30-75,45-75,60-75,1-60,15-60,30-60,45-60,1-45,15-45,30-45,1-30, and/or 15-30.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
Therefore, special embodiment of the present invention relates to the polynucleotide of the peptide species of encoding, and this polypeptide comprises or by βZhe Die district A shown in Fig. 7 A and the embodiment 6, A ', and B, B ', C, D, E, F, the aminoacid sequence of G or H is formed.Other embodiment of the present invention relates to the polynucleotide of coding Neutrokine-α polypeptide, and this polypeptide comprises or by 1,2,3,4,5,6,7,8,9 or all 10 Fig. 7 A and embodiment 6 shown in the arbitrary combination of βZhe Die district A-H form.The other embodiment preferred of the present invention relates to a peptide species, and this polypeptide comprises or by βZhe Die district A shown in Fig. 7 A and the embodiment 6, A ', and B, B ', C, D, E, F, the Neutrokine-alpha amino acid sequence of G or H is formed.Other embodiment of the present invention relates to Neutrokine-α polypeptide, its comprise or by shown in Fig. 7 A and the embodiment 61,2,3,4,5,6,7,8,9 or the arbitrary combination of all 10 βZhe Die district A-H form.
At some in other the preferred embodiment, polynucleotide of the present invention comprise or by the 34-57 of SEQID NO:21,118-123,133-141,151-159,175-216,232-255,280-315,328-357,370-393 and/or 430-456 position nucleotide residue are formed.These polynucleotide and polypeptide are equivalent to the βZhe Die district of inferring shown in Fig. 7 A.In certain embodiments, polynucleotide of the present invention comprise or by with coding 1,2,3,4,5,6,7,8, the polynucleotide sequence in 9 or 10 above-mentioned βZhe Die districts has at least 90%95%, 96%, the polynucleotide of 97%, 98% or 99% homogeny are formed.The above-mentioned polynucleotide sequence that merges the heterologous polynucleotide sequence is also contained in the present invention.Polypeptide by these polynucleotide sequence codings also is encompassed in the present invention.In another embodiment, the invention provides isolated nucleic acid molecule, it is included under the stringent hybridization condition and 1,2,3,4,5,6,7,8, the polynucleotide of the βZhe Die multi-nucleotide hybrid of 9 or 10 the invention described above.At this used " stringent condition " as previously mentioned.
In other embodiment preferred, polynucleotide of the present invention comprise or by the 576-599 of SEQ IDNO:1,660-665,675-683,693-701,717-758,744-803,822-857,870-899,912-935, and/or 972-998 position nucleotide residue is formed.Also be encompassed in the present invention by these polynucleotide passage encoded polypeptides.These polynucleotide and polypeptide fragment are equivalent to the βZhe Die district of inferring shown in Fig. 7 A.
In other embodiment preferred, polynucleotide of the present invention comprise or by the 457-462 of SEQ IDNO:18,472-480, and 490-498,514-555,571-600,619-654,667-696,699-732 and/or 769-795 position nucleotide residue are formed.Also be encompassed in the present invention by these polynucleotide passage encoded polypeptides.These polynucleotide and polypeptide fragment are equivalent to the βZhe Die district of inferring shown in Fig. 7 A.
In other embodiment preferred, polynucleotide of the present invention comprise or by the 124-129 of SEQ IDNO:22,139-147, and 157-165,181-222,238-267,286-321,334-363,376-399 and/or 436-462 position nucleotide residue are formed.Also be encompassed in the present invention by these polynucleotide passage encoded polypeptides.These polynucleotide and polypeptide fragment are equivalent to the βZhe Die district of inferring shown in Fig. 7 A.Comprise or by 1,2,3,4,5,6,7,8,9,10 or all the polypeptide formed of the aminoacid sequence of these regional arbitrary combination also be encompassed in the present invention.
The relative position on several introns/exon border of mouse Neutrokine-α (SEQ ID NO:22 and SEQ ID NO:23) is based on the sequential analysis of musculus cdna group DNA and determines.Apparent second exon (being called " exon 2 " in advance) of mouse Neutrokine-α genomic clone 5 ' end is made up of sequence Tyr187-Gln222 shown in the SEQ ID NO:23.Mouse Neutrokine-α genomic clone 5 ' apparent the 3rd exon (being called " exon 3 " in advance) of end comprises the Val 223-Gly273 of sequence shown in the SEQ ID NO:23.
Therefore, in one embodiment, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or is made up of the aminoacid sequence of the Tyr187-Gln222 of SEQ ID NO:23.The invention still further relates to a kind of nucleic acid molecule, it comprises or has at least 80%, 85% by the polynucleotide sequence with the above-mentioned mouse Neutrokine-α polypeptide of coding, and the polynucleotide sequence of 90%, 95%, 96%, 97%, 98% or 99% homogeny is formed.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention.
In another embodiment, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or is made up of the aminoacid sequence of the Val223-Gly273 residue of SEQ ID NO:23.The invention still further relates to and comprise or have at least 80%, 85% by polynucleotide sequence, the nucleic acid molecule that the polynucleotide sequence of 90%, 95%, 96%, 97%, 98% or 99% homogeny is formed with the above-mentioned mouse Neutrokine-α polypeptide of coding.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention.
In addition, the relative position on corresponding intron/exon border is based on that the sequence contrast of mouse and people Neutrokine-α polypeptide determines among the people Neutrokine-α (SEQ ID NO:1 and SEQ ID NO:2).Apparent second exon (also being called " exon 2 " in advance) of people Neutrokine-α 5 ' end is made up of the Tyr163-Gln198 of sequence shown in the SEQ ID NO:2.Apparent the 3rd exon (also being called " exon 3 " in advance) of people Neutrokine-α 5 ' end is made up of the Val 199-Gly249 of sequence shown in the SEQ IDNO:2.
Therefore, in one embodiment, the invention provides the polynucleotide of the polypeptide that coding comprises or be made up of Tyr163-Gln198 aminoacid sequence shown in the SEQ IDNO:2.The invention still further relates to a kind of nucleic acid molecule, it comprises or has at least 80%, 85% by the polynucleotide sequence with the above-mentioned Neutrokine-α polypeptide of coding, and the polynucleotide sequence of 90%, 95%, 96%, 97%, 98% or 99% homogeny is formed.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention.
In another embodiment, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or is made up of the Val199-Gly249 aminoacid sequence of SEQ ID NO:2.The invention still further relates to a kind of nucleic acid molecule, it comprises or has at least 80%, 85% by the polynucleotide sequence with the above-mentioned Neutrokine-α polypeptide of coding, and the polynucleotide sequence of 90%, 95%, 96%, 97%, 98% or 99% homogeny is formed.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention.
Neutrokine-α and/or Neutrokine-α SV polypeptide, and fragment, the functionally active of variant derivative and analogue can be by the whole bag of tricks analysis as herein described and well known in the art.
For example, in one embodiment, just analyze combination or combine anti-Neutrokine-α and/or anti-Neutrokine-α SV antibody with total length Neutrokine-α and/or Neutrokine-α SV polypeptide competitiveness, or in conjunction with the ability of Neutrokine-α acceptor on the B cell and/or Neutrokine-α SV acceptor, can use various immunoassay known in the art, comprise but non-being limited to: radioimmunoassay in order to the competitiveness and the noncompetitive analysis of method down, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoradiometric assay(IRMA), gel diffusion precipitation reaction, immunodiffusion(ID) analysis, the original position immunoassay is (with for example Radioactive colloidal gold, enzyme or labelled with radioisotope), Westem trace, precipitin reaction, aggegation is analyzed and (is analyzed as the gel aggegation, blood coagulation analysis), complement fixation(CF) analysis, immunofluorescence analysis, A analysis of protein and IEA etc.In one embodiment, antibodies is to detect by the mark that detects on the original antibody.In another embodiment, one anti-be anti-and reagent and one resists combines and detect by detecting two.In yet another embodiment, two anti-be mark.Bonded method in many detection immunoassays known in the art, and within the scope of the present invention.
In another embodiment, when differentiating Neutrokine-α and/or Neutrokine-α SV part, or evaluation polypeptide fragment of the present invention, during the ability of variant or derivative polymerization, can analyze combination, for example by well known method as the reduction or non-reduced gel chromatography, protein affinity chromatography and affine trace.See Phizicky, E. etc., 1995, microbiological research 59:94-123.In another embodiment, Neutrokine-α and/or Neutrokine-α SV are analyzed in conjunction with the physiological correlations of its substrate.
In addition, analysis as herein described (as seeing embodiment 6 and 7) and other analysis known in the art can be used to measure Neutrokine-α and/or Neutrokine-α SV polypeptide and fragment thereof routinely, the variant derivative excites Neutrokine-α and/or the relevant bioactive ability (as stimulating or suppressing (under the situation of Neutrokine-α and/or Neutrokine-α SV antagonist) B cell proliferation, differentiation and/or activation and/or prolongation B cell are in external or intravital survival) of Neutrokine-α SV with analogue.
Those skilled in the art know other method and are also included within the present invention.
In other embodiments, polynucleotide encoding of the present invention has the polypeptide of the functional characteristics of Neutrokine-α and Neutrokine-α SV.The preferred embodiment of the invention for this purpose comprises and comprising or by the fragment of forming with lower area of Neutrokine-α and Neutrokine-α SV polypeptide: α spiral and α spiralization district (" α district "), βZhe Die and βZhe Die form district (" β district "), corner and corner form district's (" corner regions "), curl and curl into district (" curl and distinguish "), hydrophilic area, hydrophobic region, α amphiphilic district, β amphiphilic district, flex region, the surface forms district and high antigenic index region.
One or more βZhe Die district that can be sure of Neutrokine-α shown in Fig. 7 A is important to Dimerized and Neutrokine-α and ligand-ligand interaction thereof.
The preferred zone of for this purpose some is shown in Fig. 3 (table 1).Data shown in Fig. 3 and the table 1 are only represented the multi-form of the identical result that obtains when analyzing the aminoacid sequence of SEQ IDNO:2 with the default parameters of DNA*STAR computer program.
Above-mentioned preferred zone shown in Fig. 3 and the table 1 comprises but non-being limited to by analyzing the aforementioned all types of zones that aminoacid sequence is differentiated shown in Figure 1A and the 1B.Shown in Fig. 3 and table 1, this preferred zone comprises Garnier-Robson α district, β district, corner regions, with the district of curling, Chou-Fasman α district, the β district and the district of curling, Kyte-Doolittle hydrophilic area and hydrophobic region, Eisenberg α and β amphiphilic district, the Karplus-Schulz flex region, the Emini surface forms district and Jameson-Wolf high antigenic index region.Polynucleotide very preferably are polynucleotide of the polypeptide be made up of the zone of Neutrokine-α and/or Neutrokine-α SV of those codings, that has made up in described zone is more above-mentioned (as 1,2,3, or 4 kinds) characteristics.
In addition, the VIII of table 1, IX, the data in XIII and the XIV row can conventional be used to determine to be the zone of highly potential antigenic Neutrokine-α, and (the VIII row representative of table 1 is according to the wetting ability of Kyte-DooLittle; The IX row representative of table 1 is according to the hydrophobicity of Hopp-Woods; The XIII row representative of table 1 is according to the antigenic index of Jameson-Wolf; The XIV row representative of table 1 is according to the surperficial probability of Emini).High antigenic zone is from VIII, IX, XIII, and/or shown in the XIV in the data, by selecting representative in certain environment, to be easy to determine that at the numerical value in the surperficial polypeptide zone that exposes of polypeptide described environment is the environment that just begins to take place antigen recognition in immunne response.Data shown in Figure 6 also can be routinely with similar sheet form representative, and aminoacid sequence gets shown in Fig. 6 (SEQ ID NO:19) by detecting easily with the DNA*STAR pattern of setting default parameters and program.As mentioned above, aminoacid sequence shown in Fig. 6 also can be used for determining to be the zone of highly potential antigenic Neutrokine-α, can be to scheme (as Fig. 6) or to represent with table (as table 1) form.
Table 1
Figure S2007101537856D00481
Table 1 (continuing)
Figure S2007101537856D00491
Table 1 (continuing)
Figure S2007101537856D00501
Table 1 (continuing)
Figure S2007101537856D00511
Table 1 (continuing)
Other preferred nucleic acid fragment of the present invention comprises the nucleic acid molecule that comprises or be made up of the sequence of carrying epi-position part of one or more Neutrokine-α of coding.Especially this nucleic acid fragment of the present invention comprises and comprises or be selected from the nucleic acid molecule that the sequence of following polypeptide is formed by coding: about Phe115-Leu147 of SEQ ID NO:2 aminoacid sequence, Ile150-Tyr163, Ser170-Phe194, Glu223-Tyr246, Ser271-Phe278." approximately " refers to specific scope and amino and the carboxyl terminal one or both ends many or less 5,4,3,2 in the literary composition, or this scope of 1 amino-acid residue.Polypeptide by these nucleic acid molecule encodings also is encompassed in the present invention.Carry the polypeptide fragment of the antigenic epitopes of Neutrokine-α, available above-mentioned Jameson-Wolf antigenic index is analyzed by those skilled in the art and is determined easily, as shown in Figure 3.The epi-position method partly of carrying of determining other this Neutrokine-α sees for details following.
Other preferred nucleic acid fragment of the present invention comprises the nucleic acid molecule formed of sequence that comprises or carried part by the epi-position of one or more Neutrokine-α SV of coding.Especially this nucleic acid fragment of the present invention comprises and comprises or be selected from the nucleic acid molecule that the sequence of following polypeptide is formed by coding: about Pro32-Leu47 of SEQ ID NO:19 aminoacid sequence, Glu116-Ser143, Phe153-Tyr173, Pro218-Tyr227, Ser252-Thr258, Ala232-Gln241, Ile244-Ala249, Ser252-Val257." approximately " refers to specific scope in the literary composition, and many or few 5,4,3,2 in aminoterminal and carboxyl terminal one or both ends, or this scope of 1 amino-acid residue.Carrying the polypeptide fragment of Neutrokine-α antigenic epitopes can be determined by those skilled in the art easily with the analysis of above-mentioned Jameson-Wolf antigenic index.The method of carrying the epi-position part of determining other this Neutrokine-α SV sees for details following.
In special embodiment, polynucleotide length of the present invention is less than 100,000kb, 50,000kb, 10,000kb, 1,000kb, 500kb, 400kb, 350kb, 300kb, 250kb, 200kb, 175kb, 150kb, 125kb, 100kb, 75kb, 50kb, 40kb, 30kb, 25kb, 20kb, 15kb, 10kb, 7.5kb, or 5kb.
In another embodiment, polynucleotide of the present invention comprise at least 15, at least 30, at least 50 of Neutrokine-α encoding sequence, at least 100 or at least 250, at least 500, at least 1000 continuous nucleotides, but by shown in Figure 1A and 1B (SEQ ID NO:1) or Fig. 5 A and the 5B (SEQ ID NO:18) 5 ' or being less than or equaling 1000kb of 3 ' coding nucleotide side, 500kb, 250kb, 200kb, 150kb, 100kb, 75kb, 50kb, 30kb, 25kb, 20kb, 15kb, the genomic dna of 10kb or 5kb is formed.In another embodiment, polynucleotide of the present invention comprise at least 15, at least 30 of Neutrokine-α encoding sequence, at least 50, at least 100 or at least 250, at least 500, or at least 1000 continuous nucleotides, but do not comprise all or part of of any Neutrokine-α intron.In another embodiment, the nucleic acid that comprises Neutrokine-α encoding sequence do not contain genomic flanking gene encoding sequence (be in the genome Neutrokine-α gene 5 ' or 3 ' sequence).In other embodiments, polynucleotide of the present invention do not contain and surpass 1000,500,250,100,50,25,20,15,10,5,4,3, the encoding sequence of 2 or 1 genomic flanking genes.
In another embodiment, the invention provides isolated nucleic acid molecule, its be included under the stringent condition with the invention described above nucleic acid molecule in the polynucleotide of part hybridization of polynucleotide, for example: be complementary to coding and/or the non-coding sequence shown in Figure 1A and Figure 1B (SEQ ID NO:1), the cDNA clone's of ATCC preserving number 97768 sequence, be complementary to coding and/or the non-coding sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18), be deposited in the cDNA clone's of ATCC preserving number 203518 sequence, be complementary to coding and/or the non-coding sequence shown in the SEQ ID NO:21, (promptly transcribe, untranslated) sequence, be complementary to the coding shown in the SEQ ID NO:22 and/or the sequence of non-coding sequence, be complementary to the coding shown in the SEQ ID NO:27 and/or the sequence of non-coding sequence, be complementary to the sequence of coding shown in the SEQ IDNO:29 and/or non-coding sequence, or the fragment of these sequences (for example its open reading frame or fragment)." stringent hybridization condition " is meant in containing the solution of following material and is incubated overnight at 42 ℃, wash filter membrane at about 65 ℃ with 0.1xSSC subsequently, described solution comprises: 50% methane amide, 5xSSC (750mM NaCl, the 75mM trisodium citrate), 50mM sodium phosphate (PH7.6), 5X Denhard ' s liquid, salmon sperm DNA is sheared in 10% dextran sulfate and 20 μ g/ml sex change
Be meant with length with the polynucleotide of polynucleotide " part " hybridization and be at least 15, preferably at least 20, more preferably at least 30, most preferably at least 30～70 (as 40,50, or 60), any integer Nucleotide in preferred especially 30～70 or 80～150 scopes, even preferred total length is with reference to the polynucleotide (DNA or RNA) of multi-nucleotide hybrid.The purposes of these polynucleotide comprises but non-being limited to as diagnostic probe and primer, and sees for details following.The part of the polynucleotide that length " is at least about 20 Nucleotide " for example is meant to comprise specific scope.Be greater than or less than with reference to 5 of the one or both ends of the nucleotide sequence of polynucleotide, 4,3,2,1 or 0 amino acid is (as arbitrary or two sequences of the cDNA of two preservations, the complementary strand of nucleotide sequence shown in Figure 1A and the 1B (SEQ ID NO:1), the complementary strand of nucleotide sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18), the complementary strand of nucleotide sequence shown in the SEQ IDNO:21, the complementary strand of nucleotide sequence shown in the SEQ ID NO:22, the complementary strand of nucleotide sequence shown in the SEQ ID NO:27, and/or the complementary strand of nucleotide sequence shown in the SEQ IDNO:29).Certainly, only be not included in the polynucleotide of a part that the present invention is used to hybridize nucleic acid of the present invention with the polynucleotide of following sequence hybridization, because this polynucleotide can hybridize any nucleic acid molecule of containing poly-(A) sequence or its complementary sequence (as, in fact do any double-stranded cDNA clone that primer produces with few dT), described sequence is poly-(A) sequence (3 of Neutrokine-α cDNA ' terminal poly-(A) tract shown in Figure 1A and 1B (SEQ ID NO:1), 3 of Neutrokine-α SV cDNA ' terminal poly-(A) tract shown in Fig. 5 A and the 5B (SEQ ID NO:18), or 3 ' end of the SV of Neutrokine-α shown in SEQ IDNO:22 cDNA gathers (A) tract), or one section T (or U) residue complementary sequence.
As implied above, the nucleic acid molecule of the present invention of coding Neutrokine-α or Neutrokine-α SV polypeptide can comprise but non-being limited to: the polynucleotide of the aminoacid sequence of the corresponding polypeptide extracellular domain of encoding itself; The encoding sequence of the extracellular domain of corresponding polypeptide and additional sequences, as the sequence of coding born of the same parents' internal area and membrane-spanning domain sequence, or preceding albumen, proteinogen or preproprotein sequence; There is or do not have the encoding sequence of the corresponding polypeptide extracellular domain of aforementioned extra encoding sequence.
Nucleic acid of the present invention is also encoded and is had the above-mentioned protein sequence of extra non-coding sequence, for example comprise but non-intron and noncoding 5 ' and the 3 ' sequence that is limited to, as transcribing, mRNA processing comprise in montage and the polyadenylation signal karyomit(e) for example in conjunction with and mRNA stability aspect that transcribe, the untranslated sequence that work; Extra encoding sequence, the extra amino acid of encoding provides the amino acid of additional functionality as those.
Therefore, the sequence of coded polypeptide can be blended in flag sequence, promotes the sequence of the peptide of fusion polypeptide purifying as coding.In embodiment preferred more of the present invention, marker amino acid sequence is a 6-Histidine peptide, and is as the label that provides in the pQE carrier (QIAGEN, Inc, 9259 Eton, Averue, Chatsworth, CA, 91311), wherein many commercially available.As Gentz etc., to report described in the 86:821-824 (1989) in institute of American Academy of Sciences, the 6-Histidine makes the conventional purifying of fusion rotein." HA " label is the peptide that another kind is used for purifying, and it is equivalent to the epi-position derived from influenza hemagglutinin protein, with by elaborations such as Wilson, and cell 37:767 (1984).As described below, other this fusion rotein is included in Neutrokine-α or the Neutrokine-α SV polypeptide that N or C-terminal are blended in Fc.
The invention still further relates to the variant of nucleic acid molecule of the present invention, the Neutrokine-α of its coding SEQ ID NO:2 or a part, analogue or the derivative of Neutrokine-α SV polypeptide.Variant can be natural generation, as natural allele variant." allelotrope " variant is meant one of some versions that occupy the gene of given locus on the body karyomit(e).See gene II, Lewin, B.ed, John Wiley ﹠amp; Sons, New York (1985).The variant that non-natural takes place can produce with mutafacient system known in the art, comprises but non-ly is limited to oligonucleotide mediated mutagenesis, and L-Ala scans, PCR mutagenesis, and site-directed mutagenesis (is for example seen Carter etc., nucleic acids research 13:4331 (1986); With Zoller etc., nucleic acids research 10:6487 (1982), box mutagenesis (for example seeing Wells etc., gene 34:315 (1985)), restricted selection mutagenesis (for example seeing Wells etc., Philos.Trans.R.Soc.London SerA 317:415 (1986)).
This variant comprises that those replace by Nucleotide, disappearance or add and the variant that produces.This replacement, disappearance or interpolation can comprise one or more amino acid.Variant can be in the coding region, changes in non-coding region or this two district.Variation can produce conservative or non-conserved amino acid replacement, disappearance or interpolation in the coding region.Wherein particularly preferably reticent the replacement, add and disappearance, it does not change Neutrokine-α and/or Neutrokine-α SV polypeptide or its a part of character and activity.Equally particularly preferably conserved amino acid replaces.
Other embodiment of the present invention relates to isolated nucleic acid molecule, it comprises the polynucleotide of following Neutrokine-α of coding and/or Neutrokine-α SV amino acid sequence of polypeptide, this amino acid sequence of polypeptide contains at least 1 but be no more than 50, preferably is no more than 40, more preferably no more than 30, especially preferably be no more than 20,10-20,5-10,1-5,3-5, or 1-3 conserved amino acid replaces.Certainly, preferred order is coding Neutrokine-α and/or containing of Neutrokine-α SV polypeptide to be no more than 10,9,8,7,6,5,4,3,2 successively, or the polynucleotide of the aminoacid sequence of 1 conserved amino acid replacement.
Other embodiment comprises isolated nucleic acid molecule, its comprise or by be selected from next the group polynucleotide have at least 80%, 85% or 90% homogeny, preferably at least 95%, 96%, 97%, 98%, or the polynucleotide of the nucleotide sequence of 99% homogeny are formed: and (a) coding has the nucleotide sequence of the Neutrokine-α polypeptide of complete amino acid sequence shown in Figure 1A and the 1B (being the 1-285 position of SEQ ID NO:2); (b) coding has shown in Figure 1A and the 1B nucleotide sequence of the Neutrokine-α polypeptide of complete amino acid sequence (being the 2-285 amino acids of SEQ ID NO:2) except that the N-terminal methionine(Met); (c) has the fragment of (b) polypeptide of Neutrokine-α functionally active (as antigenicity or biological activity); (d) coding has the nucleotide sequence of the extracellular domain of inferring of 73-285 amino acids sequence of N eutrokine-α polypeptide among Figure 1A and the 1B (SEQ ID NO:2); (e) coding has the nucleotide sequence of 134-285 amino acids sequence of N eutrokine-α polypeptide among Figure 1A and the 1B (SEQ ID NO:2); (f) nucleotide sequence of coding Neutrokine-α polypeptide, this polypeptide has the complete amino acid sequence by the cDNA clones coding that is deposited in ATCC preserving number 97768; (g) nucleotide sequence of coding Neutrokine-α polypeptide extracellular domain, this polypeptide has by the cDNA amino acid sequence coded that is deposited in ATCC preserving number 97768; (h) be complementary to above (a), (b), (c), (d), (e), (f), (g) or the nucleotide sequence of any nucleotide sequence (h).The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these polynucleotide and nucleic acid molecule encoding also is encompassed in the present invention.
The present invention particularly embodiment preferred relates to nucleic acid molecule, it comprises or has at least 80% by the polynucleotide sequence that has with coding Neutrokine-α polypeptide, 85%, 90% homogeny, preferably at least 95%, 96%, 97%, the polynucleotide of the nucleotide sequence of 98%, 99% or 100% homogeny are formed, and described Neutrokine-α polypeptide has 134-285 amino acids sequence among Figure 1A and the 1B (SEQ ID NO:2).The preferred embodiment of the invention relates to nucleic acid molecule, it comprises or is made up of the polynucleotide of the nucleotide sequence of at least 90% homogeny the polynucleotide sequence that has with coding Neutrokine-α polypeptide, and described Neutrokine-α polypeptide has 134-285 amino acids sequence among Figure 1A and the 1B (SEQ ID NO:2).The preferred embodiment of the present invention relates to nucleic acid molecule, it comprises or is made up of the polynucleotide of the nucleotide sequence of at least 95% homogeny the polynucleotide sequence that has with coding Neutrokine-α polypeptide, and described Neutrokine-α polypeptide has 134-285 amino acids sequence among Figure 1A and the 1B (SEQ ID NO:2).The preferred embodiment of the present invention relates to nucleic acid molecule, it comprises or is made up of the polynucleotide of the nucleotide sequence of at least 96% homogeny the polynucleotide sequence that has with coding Neutrokine-α polypeptide, and described Neutrokine-α polypeptide has 134-285 amino acids sequence among Figure 1A and the 1B (SEQ ID NO:2).
In addition, the preferred embodiment of the present invention relates to nucleic acid molecule, it comprises or is made up of the polynucleotide of the nucleotide sequence of at least 97% homogeny the polynucleotide sequence that has with coding Neutrokine-α polypeptide, and described Neutrokine-α polypeptide has 134-285 amino acids sequence among Figure 1A and the 1B (SEQ ID NO:2).The preferred embodiment of the present invention relates to nucleic acid molecule, it comprises or is made up of the polynucleotide of the nucleotide sequence of at least 98% homogeny the polynucleotide sequence that has with coding Neutrokine-α polypeptide, and described Neutrokine-α polypeptide has 134-285 amino acids sequence among Figure 1A and the 1B (SEQ ID NO:2).The preferred embodiment of the present invention relates to nucleic acid molecule, it comprises or is made up of the polynucleotide of the nucleotide sequence of at least 99% homogeny the polynucleotide sequence that has with coding Neutrokine-α polypeptide, and described Neutrokine-α polypeptide has 134-285 amino acids sequence among Figure 1A and the 1B (SEQ ID NO:2).
Another embodiment of the present invention relates to the isolated nucleic acid molecule that comprises polynucleotide, this polynucleotide encoding Neutrokine-α SV amino acid sequence of polypeptide (Neutrokine-α SV polypeptide fragment as described herein) contains at least 1 but be no more than 50, preferably be no more than 40, more preferably no more than 30, be most preferably not exceeding 20 conserved amino acids and replace.Certainly, particularly preferred polynucleotide are the Neutrokine-α polypeptid acid sequences of encoding, have to contain to be no more than 7-10, and 5-10,3-7,3-5,2-5,1-5,1-3,10,9,8,7,6,5,4,3,2, or 1 aminoacid sequence that conserved amino acid replaces.
Other embodiment comprises the isolated nucleic acid molecule that comprises or be made up of polynucleotide, these polynucleotide have and are selected from next group polynucleotide at least 80%, 85%, 90% homogeny, preferably at least 95%, 96%, 97%, 98%, or the nucleotide sequence of 99% homogeny: (a) coding has the nucleotide sequence of the Neutrokine-α SV polypeptide of complete amino acid sequence shown in Fig. 5 A and the 5B (being the 1-266 position of SEQ IDNO:19); (b) coding has shown in Fig. 5 A and the 5B nucleotide sequence of the Neutrokine-α SV polypeptide of complete amino acid sequence (being the 2-266 position of SEQ ID NO:19) except that the N-terminal methionine(Met); (c) coding have 73-285 amino acids sequence among 5A and the 5B (SEQ ID NO:19) the nucleotide sequence of the extracellular domain of inferring of Neutrokine-α SV polypeptide; (d) nucleotide sequence of coding Neutrokine-α SV polypeptide, this Neutrokine-α SV polypeptide has the complete amino acid sequence by the cDNA clones coding that is deposited in ATCC preserving number 203518; (e) nucleotide sequence of coding Neutrokine-α SV polypeptide extracellular domain, this extracellular domain has the aminoacid sequence by the cDNA clones coding that is deposited in ATCC preserving number 203518; (f) be complementary to above-mentioned (a), (b), (c), the nucleotide sequence of any nucleotide sequence (d) or (e).
In addition, the polynucleotide that the present invention includes comprise or are equal to about at least 10 of Figure 1A and the middle 1-1082 nucleotide sequence of 1B (SEQ ID NO:1) by at least 90% or 95%, 20,25 or 30 continuous nucleotides, the sequence of any part of preferred about at least 40 or 50 continuous amino acids is formed, preferred nucleotide sequence and 797-1082,810-1082 and the 346-542 position nucleotide sequence of from above-mentioned 4 cDNA clone, determining that do not comprise.The present invention also comprises a kind of polynucleotide, it comprises or is equal to about 10 of sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18) by at least 90% or 95%, 20,25 or 30 continuous nucleotides, the sequence of any part of preferred about at least 40 or 50 Nucleotide is formed, and does not preferably comprise the nucleotide sequence of determining from above-mentioned 4 cDNA clone.The present invention also comprises a kind of polynucleotide, it comprises or is equal to about 10 of sequence shown in the SEQID NO:21 by at least 90% or 95%, 20,25 or 30 continuous nucleotides, the sequence of any part of preferred about at least 40 or 50 continuous nucleotides is formed, and does not preferably comprise the nucleotide sequence of determining from above-mentioned 4 cDNA clone.The present invention also comprises a kind of polynucleotide, it comprises or is equal to about 10 of sequence shown in the SEQ ID NO:22 by at least 90% or 95%, 20,25 or 30 continuous nucleotides, the sequence of any part of preferred about at least 40 or 50 continuous nucleotides is formed, and does not preferably comprise the nucleotide sequence of determining from above-mentioned 4 cDNA clone.The present invention also comprises a kind of polynucleotide, it comprises or is equal to about at least 10 of sequence shown in the SEQ ID NO:27 by at least 90% or 95%, 20,25 or 30 continuous nucleotides, the sequence of any part of preferred about at least 40 or 50 continuous nucleotides is formed, and does not preferably comprise the nucleotide sequence of determining from above-mentioned 4 cDNA clone.The present invention also comprises a kind of polynucleotide, it comprises or is equal to about at least 10 of sequence shown in the SEQ ID NO:29 by at least 90% or 95%, 20,25 or 30 continuous nucleotides, the sequence of any part of preferred about at least 40 or 50 continuous nucleotides is formed, and does not preferably comprise the nucleotide sequence of determining from above-mentioned 4 cDNA clone." approximately " comprises specific scope in the literary composition, and at one end or two ends are many or few several (promptly 5,4,3,2 or 1) amino acid.
Have with coding Neutrokine-α and/or Neutrokine-α SV polypeptide show for example polynucleotide of the nucleotide sequence of at least 95% " homogeny " with reference to nucleotides sequence, the nucleotide sequence that is meant these polynucleotide is identical with canonical sequence, can comprise the mispairing that has in per 100 Nucleotide of canonical sequence of coding Neutrokine-α and/or Neutrokine-α SV polypeptide more than 5 except this polynucleotide sequence.In other words, for obtaining to have the polynucleotide that the nucleotide sequence of at least 95% homogeny is arranged with canonical sequence, can lack near 5% Nucleotide in the canonical sequence or replace with other Nucleotide, the Nucleotide that perhaps accounts for canonical sequence total nucleotide number 5% can insert in the canonical sequence.These sudden changes in the canonical sequence can occur in reference to 5 of nucleotide sequence ' or 3 ' terminal position, or any position between these terminal positions, are dispersed in the canonical sequence each Nucleotide or one or more is continuously between the group.With reference to (reference) sequence can be the complete nucleotide sequence of coding Neutrokine-α and/or Neutrokine-α SV, respectively shown in Figure 1A and 1B (SEQ ID NO:1) and Fig. 5 A and 5B (SEQ ID NO:18), or any Neutrokine-α SEQ ID NO:21 for example, 22,27, the α of Neutrokine-shown in 28 polynucleotide, or the fragment of any Neutrokine-α as herein described or Neutrokine-α SV polynucleotide.
In fact, any specific nucleic acid molecule whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% is equal to for example nucleotide sequence shown in Figure 1A and 1B, or nucleotide sequence shown in Fig. 5 A and the 5B, or the cDNA of preservation clone's nucleotide sequence, or any Neutrokine-α polynucleotide such as SEQ ID NO:21,22, Neutrokine-α polynucleotide shown in 27 or 28 or its fragment, can determine (Wisconsin sequential analysis routine package, Version 8 for Unix, genetics computer group with the known computer program routinely, the university research garden, 575 Seience Drive, Madison, WI 53711).The local homologous sequence contrast program of Bestfit use Smith and Waterman is found the best homology sections (applied mathematics progress 2:482-489 (1981)) between two sequences.When using Bestfit or any other sequence contrast program when determining that whether particular sequence for example has 95% homogeny with canonical sequence of the present invention, certainly want setup parameter, thereby calculate the homogeny percentage in total length on reference to nucleotide sequence, and allow in the canonical sequence homology breach that accounts for total nucleotide several 5% is arranged.
In a special embodiment, with reference to the homogeny between (reference) sequence (sequence of the present invention) and the target sequence, being also referred to as whole sequence contrast, is to use (Comp.App.Biosci 6:237-245 (1990)) determined based on the correlated FAS TDB of Brutlag and colleague's thereof sequence computer program.In sequence contrast, reference sequence and target sequence all are dna sequence dnas.The RNA sequence can be by being converted to T with U and being contrasted.Described whole sequence is correlated result represent with the homogeny percentage.The FASTDB sequence contrast that is used for dna sequence dna to calculate the percentile preferred parameter of homogeny is: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or target nucleotide sequence length are got shorter.According to this embodiment, if target sequence because 5 ' or 3 ' disappearance but not shorter than reference sequence because of inherent disappearance, to manually adjust the result, because the FASTDB program when calculating the homogeny percentage, reckons without target sequence 5 ' and 3 ' brachymemma.With regard to respect to reference sequence 5 ' and the target sequence of 3 ' terminal brachymemma with regard to, by calculate 5 of target sequence ' and 3 ' the not coupling/correlated base number of the reference sequence percentage that accounts for reference sequence total alkali radix recently adjust the homogeny percentage.Whether the definite kernel thuja acid is that coupling/contrast is to determine by FASTDB sequence comparing result.Then this percentage is deducted from the homogeny percentage that calculates by above-mentioned FASTDB program with special parameter, homogeny percentage to the end.The result of this adjustment is used for the purpose of this embodiment.Have only by FASTDB sequence contrast be presented at 5 of target sequence ' and 3 ' base outside, not with reference sequence coupling/correlated base, need calculating with artificial adjustment homogeny percentage result.For example, the homogeny percentage is determined in the contrast of the reference sequence of the target sequence of 90 bases and 100 bases.Disappearance occurs in 5 of target sequence ' flavor end, thereby the not shown coupling/contrast in 5 ' terminal preceding 10 bases of FASTDB sequence contrast.These 10 unpaired bases are represented 10% sequence (5 ' and 3 ' end base number/reference sequence total alkali radix of coupling not), so this 10% should deduct from the homogeny percentage result who calculates by the FASTDB program.If remaining 90 bases are fully mated, then final homogeny percentage is 90%.In another embodiment, the reference sequence of the target sequence of 90 bases and 100 bases compares.Current disappearance is inherent disappearance, therefore 5 of target sequence ' and 3 ' end be provided with not and reference sequence coupling/correlated base.In this case, the homogeny percentage that is calculated by FASTDB does not need artificial adjustment.Equally, have only 5 of target sequence ' and 3 ' base not with reference sequence coupling/contrast, just manually adjustment of needs.This embodiment does not need to carry out other artificial adjustment.
The nucleotide sequence (being polynucleotide) that the present invention relates to disclose with this paper has at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, the nucleic acid molecule of 98% or 99% homogeny has the polypeptide of Neutrokine-α and/or Neutrokine-α SV functionally active (as biological activity) no matter whether they encode, and nucleotide sequence disclosed herein for example is the nucleotide sequence of the cDNA of sequence shown in Figure 1A and the 1B (SEQ ID NO:1) or preservation.In addition, the nucleotide sequence that the invention still further relates to the cDNA of nucleotide sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18) or preservation has at least 80%, 85%, 90%, 92%, 95%, 96%, the nucleic acid molecule of 97%, 98% or 99% homogeny has the active polypeptide of Neutrokine-α SV no matter whether they encode.In addition, the invention still further relates to the NO:21 with SEQ ID, nucleotide sequence has at least 80% shown in 22,27 or 28,85%, 90%, 92%, 95%, 96%, 97%, 98% or the nucleic acid molecule of 99% homogeny, have the polypeptide of Neutrokine-alpha active no matter whether they encode.Even this is that those skilled in the art also know how to use this nucleic acid molecule for example as hybridization probe or polymerase chain reaction (PCR) primer because a specific nucleic acid molecule is not encoded and had Neutrokine-α and/or the active polypeptide of Neutrokine-α SV.The purposes of the nucleic acid molecule with Neutrokine-α and/or the active polypeptide of Neutrokine-α SV of not encoding of the present invention comprises: (1) separates Neutrokine-α and/or Neutrokine-α SV gene or its allele variant in the cDNA library; (2) with Metaphase Chromosome smear in situ hybridization (as " FISH "), so that the accurate chromosome position of Neutrokine-α and/or Neutrokine-α SV gene to be provided, as Verma etc., human chromosome basic skills handbook, Pergamon press is described in New York (1988); And be used for the Northern engram analysis to detect Neutrokine-α and/or Neutrokine-α SVmRNT expression in specific tissue.
Yet, nucleotide sequence preferred and disclosed herein (nucleotide sequence shown in Figure 1A and 1B (SEQ IDNO:1), or and the nucleotide sequence of the cDNA of preservation, or their fragment) have at least 80%, 85%, 90%, 92%, 95%, 96%, the nucleic acid molecule of 97%, 98% or 99% homogeny, and it is in fact encoded and has the polypeptide of Neutrokine-α and/or Neutrokine-α SV polypeptide functionally active (as biological activity).Equally preferably have and the nucleotide sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18), or the nucleotide sequence of the cDNA of preservation has at least 80%, 85%, 90%, 92%, 95%, 96%, the nucleic acid molecule of 97%, 98% or 99% homogeny, and it is in fact encoded and has the polypeptide of Neutrokine-α and/or Neutrokine-α SV polypeptide functionally active (as biological activity).Equally preferably has NO:21 with SEQ ID, 22,27, or nucleotide sequence has at least 80% shown in 28,85%, 90%, 92%, 95%, 96%, 97%, or the nucleic acid molecule of the sequence of 98% homogeny, and it is in fact encoded and has the polypeptide of Neutrokine-α and/or Neutrokine-α SV polypeptide functionally active (as biological activity).
" polypeptide " and " polypeptide " with Neutrokine-α SV polypeptide functionally active (as biological activity) with Neutrokine-α polypeptide functionally active (as biological activity), be meant in specific functional analysis (as immunology or biological analysis) and measure, to the activity of the extracellular domain of Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or full-length polypeptide present similar substantially, but active polypeptide that needn't be identical.For example, the functionally active of Neutrokine-α and/or Neutrokine-α SV polypeptide can form the ability of polymer (as homology dimer and homotrimer) by the extracellular domain of described peptide sequence and complete Neutrokine-α and/or Neutrokine-α SV or Neutrokine-α and/or Neutrokine-α SV, and measures in conjunction with the ability of Neutrokine-α and/or Neutrokine-α SV polypeptide ligand.Neutrokine-α and/or Neutrokine-α SV polypeptide functionally active also can be by determining polypeptid induction lymphocyte of the present invention (as the B cell) propagation, differentiation or activation and/or prolong the ability of B cell survival time and measure.These functional analyses can be carried out with methods described herein (for example seeing embodiment 6) and other method routine known in the art.In addition, Neutrokine-α of the present invention or Neutrokine-α SV polypeptides for modulating cell proliferation, cellular toxicity, cell survival and necrocytosis.Can carry out body outer cell proliferation by using detection cellular replication well known in the art and that can buy usually and/or dead reagent, cellular toxicity, cell survival and necrocytosis analysis are to measure the effect of protein to some cell.It is for example, many that active this analysis sees in the various reference to the TNF associated protein.In brief, this analysis for example comprises collects human or animal (as mouse) cell, and they and (1) contained the host cell supernatant of the transfection of Neutrokine-α albumen (or candidate's polypeptide), or the host cell supernatant of the untransfected of (2) contrast mixes, after the incubation certain hour, measure the influence of pair cell number or viability.This cell proliferation that can measure in this alanysis and/or survival are regulated activity and be can be used for treating tumour, and metastases infects, autoimmune disease, inflammation and other Ia disease.
Neutrokine-α regulates cell proliferation and differentiation in the dose-dependently mode in above-mentioned analysis.Therefore, preferred " polypeptide with Neutrokine-α polypeptide functionally active (as biological activity) " is included in and also presents more any cells in the dose-dependently mode in the above-mentioned analysis and regulate (especially immunomodulatory) active polypeptide.Although the active degree of dose-dependently does not need to be equal to Neutrokine-α polypeptide, but preferably " polypeptide with Neutrokine-α polypeptide functionally active " compares with Neutrokine-α polypeptide, in given activity, present similar dose-dependently (to be candidate's polypeptide present greater activity than the Neutrokine-α polypeptide of reference, or be not less than 25 times preferably be not less than 10 times of activity) substantially.
In certain preferred aspects, " polypeptide " and " polypeptide " with Neutrokine-α SV polypeptide functionally active (as biological activity) with Neutrokine-α polypeptide functionally active (as biological activity), comprise and also present Fig. 8 A, 8B, 9A, 9B, 11,10,12A regulates (especially immunomodulatory) active polypeptide with 12B with any identical B cell (or other cell) described in the embodiment 6.
Similar to other member of TNF family, Neutrokine-α presents hemocyte is for example comprised monocyte, and lymphocyte (as the B cell) and neutrophil leucocyte work.Therefore, Neutrokine-α is inducing these cell proliferation, is active in differentiation and the migration.This activity is used for immunostimulant or inhibition, marrow protection, stem cell migration, control acute and chronic inflammation, and treatment leukemia.This active method of mensuration known in the art.For example see Peters etc., modern immunology 17:273 (1996); Young etc., experimental technique magazine 182:1111 (1995); Caux etc., natural 390:258 (1992); With Santiago-Schwarz etc., biological experimental method progress 378:7 (1995) is described.
Certainly, because the degeneracy of genetic code, those skilled in the art will recognize will the encode polypeptide of " having Neutrokine-α polypeptide functionally active (as biological activity) " of a large amount of nucleic acid molecule immediately, described nucleic acid molecule has at least 80%, 85%, 90%, 92%, 95%, the nucleotide sequence that 96%, 97%, the 98% or 99% contained cDNA clone of preservation thing who is equal to ATCC preserving number 97768 comprises, or nucleotide sequence shown in Figure 1A and the 1B (SEQ ID NO:1), or their fragments sequence.Those skilled in the art also will recognize a large amount of nucleic acid molecule encodings " polypeptide with Neutrokine-α SV polypeptide functionally active (as biological activity) " immediately, described nucleic acid molecule have with the cDNA of ATCC preserving number 203518 clone in the nucleotide sequence that comprises, or nucleotide sequence shown in Fig. 5 A and the 5B (SEQ ID NO:18), have at least 80%, 85%, 90%, 92%, 95%, 96%, the sequence of 97%, 98% or 99% homogeny.In fact, the phase homopolypeptide because the degeneracy variant of these nucleotide sequences is all encoded, even do not carry out above-mentioned comparative analysis, those skilled in the art also know this point.This nucleic acid molecule that is not the degeneracy variant is further recognized in this area, has some also to encode and has Neutrokine-α and/or the active polypeptide of Neutrokine-α SV.This is because those skilled in the art recognize that fully aminoacid replacement obviously influences hardly or the not obvious protein function (as replacing an aliphatic amino acid with another aliphatic amino acid) that influences, and sees for details following.
Similarly, coding contains the polynucleotide of polypeptide in the V142-K160 zone of all or part of SEQ ID NO:2, seemingly detects and/or changes the polynucleotide that diagnosis and therapeutic value are arranged that Neutrokine-α or Neutrokine-α SV polynucleotide are expressed.In addition, cross over Neutrokine-α SV peptide T 141 shown in the SEQ ID NO:19 and G142 amino-acid residue joint portion (between insert the V142-K160 aminoacid sequence of Neutrokine-α significantly) polynucleotide, as if also have and diagnose and therapeutic action.This T141/G142 crosses over polynucleotide and presents and Neutrokine-α SV multi-nucleotide hybrid ratio and the higher possibility of Neutrokine-α polynucleotide.The non-limiting non-exclusive examples of part by this Neutrokine-α SV of polynucleotide encoding of the present invention comprises or forms by being selected from following aminoacid sequence: the G121-E163 of SEQ ID NO:19; E122-E163; G123-E163; N124-E163; S125-E163; S126-E163; Q127-E163; N128-E163; S129-E163; R130-E163; N131-E163; K132-E163; R133-E163; A134-E163; V135-E163; Q136-E163; G137-E163; P138-E163; E139-E163; E140-E163; T141-E163; G142-E163; S143-E163; Y144-E163; T145-E163; F146-E163; U147-E163; P148-E163; W149-E163; L150-E163; L151-E163; S152-E163; F153-E163; K154-E163; R155-E163; G156-E163; S157-E163; A158-E163; L159-E163; E160-E163; E161-E163; K162-E163; G121-K162; G121-E161; G121-E160; G121-L159; G121-A158; G121-S157; G121-G156; G121-R155; G121-K154; G121-F153; G121-S152; G121-L151; G121-L150; G121-W149; G121-P148; G121-V147; G121-F146; G121-T145; G121-Y144; G121-S143; G121-G142; G121-T141; G121-E140; G121-E139; G121-P138; G121-G137; G121-Q136; G121-V135; G121-A134; G121-R133; G121-K132; G121-N131; G121-R130; G121-S129; G121-N128; G121-Q127; G121-S126; G121-S125; G121-N124; G121-G123; And G121-E122.
Polypeptide by these polynucleotide encodings also is encompassed in the present invention.
Carrier and host cell
The invention still further relates to a kind of carrier that comprises isolated DNA molecule of the present invention, organize the host cell of the generation polypeptide of the present invention vector gene through engineering approaches or the alternate manner through engineering approaches with the host of this recombinant vectors generation polypeptide of the present invention genetically engineered or the alternate manner through engineering approaches, and pass through the method for reorganization or synthetic method production Neutrokine-α and/or Neutrokine-α SV polypeptide.
In one embodiment, polynucleotide of the present invention are connected with carrier (as clone or expression vector).Carrier for example can be phage, plasmid, virus or retroviral vector.Retroviral vector can be reproducible or replication defective.In the latter case, virus multiplication generally only occurs in the complementary host cell.Polynucleotide can be connected to breed in the host with the carrier that contains selectable mark.The vector construction body is imported host cell can be undertaken by technology known in the art, comprise but the non-calcium phosphate transfection that is limited to, and the transfection of DEAE-dextran mediation, cationic-liposome-mediated transfection, electroporation, transduction is infected or other method.These methods see described in many standard test handbooks, as Davis etc., molecular biology basic skills (1986).
Usually, recombinant expression vector comprises replication orgin and the selective marker that allows host cell to transform, as colibacillary ampicillin resistance gene and Saccharomyces cerevisiae TRP1 gene, reach the promotor of transcribing that instructs the downstream configurations sequence derived from cance high-expression gene.This promotor can be derived from coding glycolytic ferment such as glycerol 3-phosphate acid kinase (PGK), alpha factor, acid phosphatase, or operon such as heat shock protein(HSP).The allos structure sequence preferably can directly be gone into the protein secreting of translation the appropriate state of the leader sequence in the outer substratum of periplasmic space or born of the same parents and assemble to be had translation initiation and terminator sequence.Randomly, a kind of fusion rotein of heterologous sequence codified, it comprises that the N-terminal with required feature differentiates peptide, for example stable or purifying recombinant products of expressing easily.
In one embodiment, DNA of the present invention combines with suitable allos regulatory factor (as promotor or enhanser), as lambda phage PL promotor, intestinal bacteria Lac, Trp, PhoA and tac promotor, the promotor of SV40 early stage and late promoter and retrovirus LTRs etc.Other suitable promotor also known in the art.
As mentioned above, expression vector preferably includes at least one selective marker.This mark comprises the Tetrahydrofolate dehydrogenase of cultivating at eukaryotic cell, G418 or neomycin resistance gene and at tsiklomitsin, kantlex or the ammonia benzyl hydrogen mycin resistant gene of intestinal bacteria and other microbial culture.Suitably the host for example comprises but non-bacterial cell such as intestinal bacteria, streptomyces, the salmonella typhimurium of being limited to; Fungal cell such as yeast cell (as yeast saccharomyces cerevisiae or Pichia pastoris (ATCC No.201178)); Insect cell such as fruit bat S2 and fall army worm Sf 9 cells; Zooblast such as CHO, COS, 293 and the Bowes melanoma cells; And vegetable cell.The suitable substratum and the culture condition of above-mentioned host cell known in the art.
Host cell can be a higher eucaryotic cells, as mammalian cell (for example people's deutero-cell), or eukaryotic cell such as yeast cell such as low, perhaps host cell can be prokaryotic cell prokaryocyte such as bacterial cell.Can select to regulate the expression of the gene order of inserting with required particular form, or the host strain of modification and processed gene product.Can under the situation that has some inductor, improve the expression from some promotor; Therefore genetically engineered polypeptide expression can be controlled.In addition, different host cells has translation and translation post-treatment and modification (as phosphorylation, cracking) proteic feature and special mechanism.Can select suitable clone to strengthen the proteinic required modification and the processing of heterogenous expression.The suitable carrier of expressing in host cell and the selection of promotor are known in the art, and expression vector establishment, carrier is imported the host and essential method those skilled in the art of in the host, expressing also known.
The effectively expressing carrier that is used for bacterium is the structural DNA sequence by the desirable proteins of will encoding, and inserts the reading handled with functional promotor together with suitable translation initiation and termination signal and makes up mutually.Carrier comprises one or more Phenotypic Selection mark and replication orgin keeping carrier, and if desired, then is provided at amplification in the host.The suitable prokaryotic cell prokaryocyte host who transforms comprises intestinal bacteria, subtilis, and bacillus typhi murium, and Rhodopseudomonas, the various bacterial classifications in streptomyces and the Staphylococcus are although other bacterial classification also can be selected to use.Be used for the expression vector representativeness of cell and can comprise a selective marker and bacterium replication orgin without limitation, the bacterium replication orgin is derived from the genic plasmid that comprises the cloning vector pBR 322 (ATCC 37017) that knows that is purchased.The carrier of this purchase for example comprise pKK 223-3 (Pharmacia Fine Chemicds, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA).These pBR322 " skeleton " part and suitable promotor and the structure sequence combination of being expressed.The carrier that preferably is used for bacterium comprises the pHE4-5 (ATCCNO.209311 available from QIAGEN company; And varient), pQE70, pQE60, and pQE-9; Available from the pBS carrier of Stratagene, Phagescript carrier, Bluescript vector, pNH8A, pNH16A, pNH18A, pNH46A; Available from the ptrc 99a of Pharmacia, pKK223-3, pKK233-3, pDR540, pRIT5.The preferred expression carrier that is used for Yeast system comprises but the non-pYES2 of being limited to pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZ α, pPIC 9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC 3.5K, pPIC9K, and PAO815 (all can be available from Invitrogen, Carlsbad, CA).Preferred eukaryotic cell carrier is the pWLNEO available from Stratagene, pSV2CAT, pOG44, pXT1 and pSG; With the pSVK3 available from Pharmaeia, pBPV, pMSG, and pSVL.
Other suitable carrier those skilled in the art are also known.
After suitable host strain transforms and grows to suitable cell density, induce the promotor (as temperature inversion or chemical induction) of selection by proper method, and cell is cultivated for some time in addition.Cell destroys by physics or chemical process by centrifugal results, and keeps the gained crude extract to be further purified.
The microorganism cells that is used for protein expression can destroy by any ordinary method, comprises the freeze thaw circulation, supersound process, and physical disturbance, or use the lysis agent, these methods those skilled in the art know.
In an above-mentioned embodiment, Pichia pastoris is used for eukaryotic cell system and expresses Neutrokine-α albumen.Pichia pastoris is a kind of methylotrophy yeast, and energy metabolism is as the methyl alcohol of its unique carbon source.A key step in the methanol metabolic pathway is methyl alcohol and O 2Be oxidized to formaldehyde.This reaction is by alcohol oxidase catalysis.For metabolism as its unique carbon source methyl alcohol, Pichia pastoris must produce high-caliber alcohol oxidase, because alcohol oxidase and O 2Affinity lower.So in the growth medium of methyl alcohol as main carbon source, the promoter region of one or two alcohol oxidase gene (AOX 1) is highly active.Exist under the situation of methyl alcohol, the alcohol oxidase that originates from AOX 1 gene comprises the about more than 30% of total soluble protein in the Pichia pastoris.See Ellis, S.B. etc., cellular elements biology 5:1111-21 (1985); Koutz, P.J. etc., yeast 5:167-77 (1989); Tschopp, J.F. etc., nucleic acids research 15:3859-76 (1987).Therefore, allogeneic coding sequence such as Neutrokine-α of the present invention or Neutrokine-α SV polypeptide are regulated under the transcriptional regulatory of sequence at all or part of AOX 1, in the pichia of in having the situation of methyl alcohol, growing with rare high level expression.
In one embodiment, plasmid vector pPIC 9K is used for expressing in the pichia system DNA of coding Neutrokine-α or Neutrokine-α SV polypeptide, pichia Yeast system basic as " pichia scheme: molecular biology method ", D.R.Higgins and J.Cregg edit, Humana press, Totowa, NJ, 1998 is described.This expression vector makes Neutrokine-α of the present invention or Neutrokine-α SV protein expression and secretion by means of strong AOX 1 promotor that is connected with Pichia pastoris alkaline phosphatase (PHO) secreting signal peptide (being homing sequence) that is positioned at the multiple clone site upstream.
Those skilled in the art recognize that many other yeast vectors can be used for replacing pPIC9K, as pYES2, and pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZ α, pPIC 9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC 3.5K, and PAO815 are as long as proposed expression construct provides and transcribed, translation, the suitable signal for locating of secretion (if desired) etc. comprises AUG in the required frame.
In one embodiment, the high level expression of allogeneic coding sequence such as Neutrokine-α of the present invention or Neutrokine-α SV polynucleotide, can be by heterologous polynucleotide of the present invention be cloned among expression vector such as pGAPZ or the pGAPZ α, and under no first ferment situation culturing yeast thing and obtaining.
The DNA of code book invention polypeptide transcribes by higher eucaryote, is improved by enhancer sequence is inserted carrier.Enhanser is the cis acting factor of DNA, and normal length is 10-300bp, and it acts on promotor and transcribes to improve it.For example be included in the SV40 enhanser of replication orgin one a side 100-270 bp in late period, the sub-enhanser of cytomegalovirus early promoter is at the polyomavirus enhanser and the adenovirus enhanser of replication orgin side in late period one.
Various mammalian cell culture systems also can be used for express recombinant protein.Mammals is expressed system and for example comprises by the fibroblastic COS-7 clone of the described monkey kidney of Gluzman (cell 23:175 (1981)) and can express other clone of consistency carrier, for example C127,3T3, CHO, Hela and bhk cell system.Mammalian expression vector comprises a replication orgin, suitable promotor and enhanser, and any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, the sequence that transcription termination sequence and 5 ' flank are not transcribed.Derived from the dna sequence dna of SV40 spliceosome, and the polyadenylation site also can be used for the gene of not transcribing that provides required.
In a special embodiment, the construct of the part of the extracellular domain of preferred expression Neutrokine-α (as Ala 134-Leu285 amino-acid residue).Those skilled in the art can use respectively and design the polynucleotide primer to produce this expression construct by polynucleotide shown in SEQ ID NO:1 and SEQ ID NO:2 or SEQ ID NO:18 and the SEQ ID NO:19 and peptide sequence.
In another embodiment, the construct of whole extracellular domains of inferring of preferred expression Neutrokine-α (being the Gln73-Leu285 amino-acid residue).Those skilled in the art can use respectively and design the polynucleotide primer to produce this expression construct by polynucleotide shown in SEQ ID NO:1 and SEQ ID NO:2 or SEQ ID NO:18 and the SEQID NO:19 and peptide sequence.
Except containing the host cell that contains described vector construction body, in former generation, also contained in the present invention, subculture, with the vertebrates of infinite multiplication source, especially the host cell in Mammals source, this host cell is through engineering approaches disappearance or displacement endogenous genetic material (as Neutrokine-α encoding sequence), and/or comprise genetic material (as the heterologous polynucleotide sequence), this genetic material is relevant with Neutrokine-α polynucleotide of the present invention, and activate, change and/or amplification Neutrokine-α polynucleotide.For example, means known in the art can be used for operably (for example seeing the U.S. Patent No. 5641670 that on June 24th, 1997 issued in conjunction with allos control region (as promotor and/or enhanser) and endogenous Neutrokine-α polynucleotide sequence by consanguinity association; The international publication No.WO 96/29411 that on September 26th, 1996 announced; The international publication No.WO 94/12650 that on August 4th, 1994 announced; Koller etc., institute of American Academy of Sciences report 86:8932-8935 (1989); With Zijtstra etc., natural 342:435-438 (1989), with shown in document whole incorporate into reference to).
Aforementioned host cell can use in a usual manner, to produce the gene product by the recombination sequence coding.Perhaps, the also available RNA derived from DNA construct of the present invention of cell free translation system produces polypeptide of the present invention.
The formal representation that polypeptide of the present invention can be modified or synthetic as fusion rotein (comprising by the heterologous protein sequence bonded polypeptide of peptide bond with (different proteins)), and can not only comprise secretion signal, also comprises other allos functional zone.This fusion rotein can suitably be connected to each other in the frame by the required nucleotide sequence with polynucleotide of the present invention and the amino acid needed sequence of coding, and by means known in the art expressed fusion protein product.Perhaps, this fusion rotein can be produced by the protein synthesis method, as using peptide synthesizer.Therefore, for example extra amino acid region, polare Aminosaeren especially, the N-terminal that can add polypeptide be with during purifying, or in subsequently maintenance and improved stability and persistence between the shelf lives.Equally, the peptide moiety can add in the polypeptide to promote purifying.This zone can be removed before final preparation polypeptide.In polypeptide, add peptide composition and secrete or drainage to produce, or improved stability and promotion purifying etc., be method well known and commonly used.
In one embodiment, the polynucleotide of code book invention Neutrokine-α and/or Neutrokine-α SV polypeptide can be blended in pelB pectate lyase signal sequence, to improve this polypeptide expression and purification efficacy in gram negative bacterium.See U.S. Patent No. 5576195 and 5846818, incorporate reference into they whole.
One preferred fusion protein comprises an allos zone of immunoglobulin (Ig), and it is used for stable and protein purification.For example EP-A-0464533 (the corresponding application 2045869 of Canada) has disclosed the each several part of the constant region that comprises immunoglobulin molecules and the fusion rotein of other human body protein or its part.In many cases, the Fc in the fusion rotein partly is used for the treatment of and diagnosis is very favorable, thereby for example improves pharmacokinetic property (EP-A 0232262).On the other hand, express, after detection and the purifying, need and this Fc partly can be removed with above-mentioned advantageous manner at fusion rotein.This is in the situation of the obstacle in partly becoming treatment as Fc and diagnosing, for example when fusion rotein is used as the antigen of immunization.In drug development, for example human body protein such as hIL-5 with the Fc meromixis, be used for the high throughput screening analysis to differentiate the antagonist of hIL-5.See D.Bennett etc., molecular recognition magazine 8:52-58 (1995) and K.Johanson etc., journal of biological chemistry 270:9459-9471 (1995).
Polypeptide of the present invention comprises the product of natural purifying, the product of chemosynthesis and the product that from protokaryon or eucaryon host, produces by recombination method, and the host for example comprises bacterium, yeast, higher plant, insect and mammalian cell.According to the host who uses in the recombinant method for production, polypeptide of the present invention can be glycosylation or nonglycosylated.In addition, polypeptide of the present invention also can comprise the methionine residues by the initial modification due to the processing of host's mediation.
Polypeptide of the present invention can be with technology chemosynthesis known in the art (for example: Creighton, 1983, protein structure and molecules principle, W.H.Freeman; Co.N.y. and Hunkapiller, M. etc., 1984, natural 310:105-111).For example, be equivalent to the segmental peptide of complete Neutrokine-α of the present invention or Neutrokine-α SV polypeptide, can use peptide synthesizer synthetic.In addition, if desired, non-classical amino acid or chemical amino acid analogue can be used as substituent or additive imports Neutrokine-α or Neutrokine-α SV polynucleotide sequence.Non-classical amino acid comprises but the non-D isomer that is limited to common amino acid, 2,4-diamino-butanoic, a-aminoisobutyric acid, the 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminocaprolc acid, Aib, 2-aminoisobutyric acid, 3-alanine, ornithine, nor-leucine, norvaline, oxyproline, sarkosine, citrulline, positive citrulline, Gelucystine, tertiary butyl glycine, tertiary butyl L-Ala, phenylglycine, Cyclohexylalanine, the b-L-Ala, fluoroamino acid, planner's (designer) amino acid such as b-methylamino acid, Ca-methylamino acid, Na-methylamino acid and amino acid whose general analogue.In addition, amino acid can be D (dextral) or L (left-handed).
The present invention has been contained at the Neutrokine-α of translate duration or special modification afterwards or Neutrokine-α SV polypeptide; for example by glycosylation; acetylize; phosphorylation; amidation; by the derivatize of known protection/blocking group, proteolytic cleavage is connected etc. with antibody molecule or other cell ligand.Any chemically modified can be undertaken by currently known methods, comprises but non-being limited to by cyanogen bromide trypsinase, Alphachymdean Catarasce, papoid, V8 proteolytic enzyme, NaBH 4Chemical cracking, acetylize, formylation, oxidation, reduction exists under the situation of tunicamycin metabolism synthetic etc.
Other posttranslational modification that the present invention is contained for example comprises carbohydrate chain that N-connects or that O-connects, the processing of N or C-terminal, chemical composition is attached to amino acid backbone, N connects or the chemically modified of the carbohydrate chain that O connects, and because the interpolation or the disappearance of N-terminal methionine residues due to the prokaryotic organism host cell expression.The also available detectable label of polypeptide is modified, and is fluorescein-labelled as detecting and the enzyme labelling of isolated protein, isotropic substance or affinity marker.In addition, polypeptide of the present invention can be modified by iodization.
In one embodiment, Neutrokine-α of the present invention and/or the also available biotin labeling of Neutrokine-α SV polypeptide.In other one embodiment of being correlated with; biotinylated Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide for example can be used as photographic developer or the instrument of differentiating one or more Neutrokine-α and/or Neutrokine-α SV acceptor or other coreceptor or common ligands molecule.
The present invention also provides the derivative of the chemically modified of Neutrokine-α or Neutrokine-α SV, it has extra advantage as increasing solubility, interior or the cardiopulmonary bypass time of the body of stability and polypeptide, or reduce immunogenicity (seeing U.S. Patent No. 4179337).The chemical composition that is used for derivatize can be selected from water-soluble polymers such as polyoxyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol etc.Polypeptide can be modified at intramolecular random site, or modifies in the intramolecularly predetermined position, and can comprise 1,2,3, or a plurality of chemical composition of adhering to.
Polymkeric substance can be any molecular weight, and branch can be arranged or do not have branch.With regard to polyoxyethylene glycol, preferred molecular weight used and produces being easy in approximately between the 1-100KDa (term " approximately " refers in the preparation at polyoxyethylene glycol, some molecules weights or be lighter than standard molecular weight).According to required principle of reatment (as the time length of required lasting release, to bioactive influence, be easy to use, antigenic degree or forfeiture reach the known action of polyoxyethylene glycol to therapeutic protein or analogue), can use other big or small polyoxyethylene glycol.For example the molecular-weight average of polyoxyethylene glycol can be about 200,500,1000,1500,2000,2500,3000,3500,4000,4500,5000,5500,6000,6500,7000,7500,8000,8500,9000,9500,10,000,10,500,11,000,11,500,12000,12500,13000,13500,14000,14500,15000,15500,16000,16500,17000,17500,18000,18500,19000,19500,20000,25000,30000,35000,40000,50000,55000,60000,65000,70000,75000,80000,85000,90000,95000 or 100,000KDa.
As implied above, polyoxyethylene glycol can have branched structure.The ramose polyoxyethylene glycol is for example by U.S. Patent No. 5,643 575; Morpurgo etc., biological chemistry biotechnology applications 56:59-72 (1996); Vorobjev etc., nucleoside nucleotide 18:2745-2750 (1999); With Caliceti etc., it is described that biology is puted together chemical 10:638-646 (1996), and described document is all incorporated reference into it.
Peg molecule (or other chemical composition) should be considered its influence to protein functional domain or antigenicity structural domain when being attached on the protein.Many attachment meanss known in the art as EPO 401 384 (PEG and G-CSF coupling), also see Malik etc., and Exp.Hematol.20:1028-1035 (1992) (GM-CSF being carried out pegylation with tresyl chloride) is described.For example, polyoxyethylene glycol can be by reactive group such as free amine group or carboxyl covalent attachment amino-acid residue.Reactive group is the combinable groups of those activated polyglycol molecules.Amino-acid residue with free amine group can for example comprise lysine residue and N-terminal amino-acid residue; Amino-acid residue with free carboxy can comprise asparagicacid residue, glutaminic acid residue and C-terminal amino-acid residue.Sulfydryl also can be used as the reactive group that adheres to peg molecule.Therapeutic purpose are preferably adhered at amino, as attached to N-terminal or Methionin group.
As implied above, polyoxyethylene glycol can be attached to protein by being connected with any amino acid.For example, polyoxyethylene glycol can by with Methionin, histamine, aspartic acid, the covalent linkage of L-glutamic acid or halfcystine and being connected on the protein.One or more reactive chemistry can be used for polyoxyethylene glycol and is attached to proteinic special amino-acid residue (as Methionin, histamine, aspartic acid, L-glutamic acid or halfcystine), or proteinic more than one amino-acid residues are (as Methionin, histamine, aspartic acid, L-glutamic acid or halfcystine and combination thereof)
People especially demand at the protein of N-terminal chemically modified.With the polyoxyethylene glycol is example, can select various peg molecules (by molecular weight, branch etc.), the ratio of peg molecule and protein (or peptide) molecule in reaction mixture, carry out the type of PEGization (pegylation) reaction, obtain the N-terminal PEGization method of protein of selecting.The method (promptly this component of separation from the component of other single PEGization) if desired that obtains N-terminal PEGization prepared product can be by with N-terminal PEGization material purifying from the protein molecule group of PEGization.The protein of the selection of the chemically modified of modifying at N-terminal can reach by the reduction alkylation reaction, and this reaction utilization is suitable for the differential responses of the dissimilar original amine groups (Methionin of relative N-terminal) of derivatize in specific proteins.Under appropriate reaction conditions, reach protein substantially in the selective derivatizationization of N-terminal with the carbonyl group of containing polymkeric substance.
As implied above, PEGYLATION OF PROTEINS of the present invention can reach by many methods.For example, polyoxyethylene glycol can directly or by a bayonet joint be attached to protein.Polyoxyethylene glycol is attached to proteinic adapter system sees Delgado etc., Crit.Rev.Thera.Drug CarrierSys, 9:249-304 (1992); Francis etc., Intern.J of Hematol.68:1-18 (1998); U.S. Patent No. 4002531; U.S. Patent No. 5349052; WO 95/,060 58; Described with WO98/32466.Above-mentioned document is all incorporated reference into it.
One with polyoxyethylene glycol need not between bayonet joint and the system that directly is attached to proteinic amino-acid residue utilizes the MPEG of tresylated, the MPEG of tresylated is with 2,2,2-Halothane SULPHURYL CHLORIDE (ClSO 2CH 2CF 3) modify mono methoxy polyethylene glycol (MPEG) and produce.Based on the reaction of protein and tresylated MPEG, polyoxyethylene glycol directly is attached to proteinic amine groups.Therefore, the present invention includes by protein of the present invention with have 2,2, the reaction of the peg molecule of 2-Halothane sulfonyl group and protein-polyoxyethylene glycol conjugate of producing.
Polyoxyethylene glycol also available many different between bayonet joint be attached to protein.For example U.S. Patent No. 5612460 (with its incorporate in full with reference to) disclosed the urethane joint that polyoxyethylene glycol and albumen are coupled together.Polyoxyethylene glycol is to be attached to proteinic protein-polyoxyethylene glycol conjugate by joint therein, also can produce by protein and compound reaction, described compound such as MPEG-succinimido succinate, with 1,1 '-carbonyl dimidazoles activatory MPEG, MPEG-2,4,5-trichlorophenyl carbonic ether, MPEG-p-NP carbonic ether and various MPEG-succinate derivative.The poly-ethanol of other is attached to proteinic polyethyleneglycol derivative and the reactive chemistry process sees that WO 98/32466 is described, incorporates reference in full into it.The PEGization protein that produces with chemical reaction process described herein is included in the present invention.
The number (promptly replacing degree) that is attached to every kind of proteinic polyoxyethylene glycol moiety of the present invention also can change.For example, the protein of PEGization of the present invention on average can connect 1,2,3,4,5,6,7,8,9,10,12,15,17,20, or more a plurality of peg molecule.Similarly, on average replace degree as 1-3,2-4,3-5,4-6,5-7,6-8,7-9,8-10,9-11,10-12,11-13,12-14,13-15,14-16,15-17,16-18,17-19, or 18-20 polyoxyethylene glycol component/protein molecule.Determine to replace degree methods and see Delgado etc., Crit.Rev.Thera.Drug Carrier Sys.9:249-304 (1992) is described.
Neutrokine-α and/or Neutrokine-α SV polypeptide can reclaim and purifying by currently known methods, described method comprises but non-ammonium sulfate or the ethanol sedimentation of being limited to, acid extraction, negatively charged ion or cation-exchange chromatography, the phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite and lectin chromatography.Preferred high performance liquid chromatography (" HPLC ") is used for purifying.
Neutrokine-α polypeptide
Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide can be monomer or polymer (being dimer, tripolymer, the tetramer, and higher polymer).Therefore, the present invention relates to the monomer and the polymer of Neutrokine-α of the present invention and Neutrokine-α SV polypeptide, their preparation and comprise their composition (preferred pharmaceutical compositions).In special embodiment, polypeptide of the present invention is a monomer, dimer, tripolymer or the tetramer.In other embodiments, polymer of the present invention is dimer, tripolymer or the tetramer at least.
The polymer that the present invention is contained can be homologous polymerization thing or allos polymkeric substance.Term used herein " homologous polymerization thing " is meant the polymer that only contains Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide (fragment that comprises Neutrokine-α and/or Neutrokine-α SV, variant and fusion rotein).These homologous polymerization things can contain Neutrokine-α and/or the Neutrokine-α SV polypeptide with identical or different aminoacid sequence.In a special embodiment, homologous polymerization thing of the present invention is only to contain the Neutrokine-α with same acid sequence and/or the polymer of Neutrokine-α SV polypeptide.In another special embodiment, homologous polymerization thing of the present invention is to contain the polymer with different aminoacids sequence of N eutrokine-α and/or Neutrokine-α SV polypeptide.In special embodiment, polymer of the present invention is homodimer (as containing Neutrokine-α and/or the Neutrokine-α SV polypeptide with identical or different aminoacid sequence), or homotrimer (as containing Neutrokine-α and/or the Neutrokine-α SV polypeptide with identical or different aminoacid sequence).In an embodiment preferred, polymer of the present invention is a homotrimer.In another embodiment, homology polymer of the present invention is homodimer, homotrimer or homotetramer at least.
Used term in the literary composition " allos polymkeric substance " is meant and contains except that Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide, contains the polymer of heterologous polypeptide (being the polypeptide of different proteins).In a special embodiment, polymer of the present invention is a heterodimer, the heterotrimer or the allos tetramer.In other embodiments, allos polymkeric substance of the present invention is heterodimer at least, the heterotrimer or the allos tetramer.In another embodiment except not, allos polymkeric substance of the present invention contains CD40 ligand polypeptide sequence, or it has bioactive fragment or variant.
Polymer of the present invention can be hydrophobic, hydrophilic, and ionic and/or covalency associate and/or can be to connect indirectly, for example forms by liposome to connect.Therefore in one embodiment, polymer of the present invention such as homology dimer, or homotrimer are in formation when polypeptide of the present invention contacts with another polypeptide in solution.In another embodiment, allos polymkeric substance of the present invention such as heterotrimer or the allos tetramer are to form when contacting with the antibody (antibody that comprises allogeneic polypeptide sequence in the fusion rotein of the present invention) of polypeptide of the present invention in solution when polypeptide of the present invention.In other embodiments, polymer of the present invention be by and and/or Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide between the covalency relation form.This covalency relation can comprise one or more and be contained in amino-acid residue in the peptide sequence (peptide sequence is the sequence of the polypeptide of the cDNA clones coding of sequence shown in SEQ ID NO:2 or the SEQID NO:19 or preservation related to the present invention in this way).In one case, to associate be to be arranged in peptide sequence crosslinked between the interactional cysteine residues of polypeptide of natural (being natural generation) to covalency.In another case, the covalency association is the result of chemistry or reorganization operation.Perhaps, this covalency relation can comprise one or more amino-acid residue that allogeneic polypeptide sequence comprised in Neutrokine-α and/or the Neutrokine-α SV fusion rotein.In one embodiment, the covalency association is the covalency association (seeing that for example U.S. Patent No. 5478925 is described) between the heterologous sequence that comprises in the fusion rotein of the present invention.In a special embodiment, it is that covalency between the heterologous sequence that comprises in Neutrokine-α-Fc of the present invention and/or the Neutrokine-α SV-Fc fusion rotein associates that covalency associates.In another special embodiment, it is to associate from the covalency between the ligand/receptor member's of another TNF family allogeneic polypeptide sequence that the covalency of fusion rotein of the present invention associates, described allogeneic polypeptide sequence can form the associating polymer of covalency, oseteoprotegerin (see that International Publication No. WO 98/49305 is described, its content all incorporate into reference to) for example.In another special embodiment, it is to associate from the covalency between the allogeneic polypeptide sequence of CD40L or its soluble fragments that the covalency of fusion rotein of the present invention associates.In another embodiment, two or a plurality of Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide be to connect by synthetic linker (as peptide, carbohydrate or polymer soluble joint).These peptide linkers are for example by U.S. Patent No. 5073627 described (incorporating reference into).The protein that comprises by isolating a plurality of Neutrokine-α of peptide linker and/or Neutrokine-α SV polypeptide can produce with conventional recombinant DNA method.
The polymeric other method for preparing Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide comprises and uses Neutrokine-α and/or the Neutrokine-α SV polypeptide that is blended in leucine zipper or isoleucine zipper peptide sequence.Leucine zipper or isoleucine zipper structural domain are the polypeptide that promotes to find therein the protein polymerization of zipper.Leucine zipper is differentiated (Landschulz etc., science 240:1759 (1988)) at first in some DNA are conjugated protein, and has found in many different protein.Known leucine zipper or isoleucine zipper are the peptide and the derivatives thereof of natural generation dimer or tripolymerization.It is for example described by the PCT application wo 94/10308 that incorporates reference into to be suitable for producing soluble polymeric Neutrokine-α and/or the proteinic leucine zipper of Neutrokine-α SV.Comprise the soluble Neutrokine-α of the peptide that is blended in solution dimer or tripolymerization and/or the recombination fusion protein of Neutrokine-α SV and in appropriate host cell, express, and from the culture supernatant, reclaim the Neutrokine-α and/or the Neutrokine-α SV of the soluble polymerization of gained with means known in the art.
Some members of protein TNF family be sure of to be trimeric form (Beutler and Huffel, science 264:667,1994; Banner etc., cell 73:431,1993).Therefore, the Neutrokine-α of tripolymerization and/or Neutrokine-α SV have enhanced biological activity.Preferred leucine zipper component is preferential trimerical those components that form.(the FEBS 344:191 that communicates by letter, No.08/446 922 is described for (1994) and U.S. Patent application series, has disclosed a leucine zipper derived from pulmonary surfactant protein D (SPD) as Hoppe etc.Other peptide derived from the trimer protein of natural generation can be used for preparing the Neutrokine-α and/or the Neutrokine-α SV of tripolymerization.
In another embodiment, albumen of the present invention is to get in touch by the interaction that is contained between the Flag  peptide sequence in Flag -Neutrokine-α or Flag -Neutrokine-α SV fusion rotein.In another embodiment, albumen of the present invention is allogeneic polypeptide sequence by being contained in Flag -Neutrokine-α or Flag -Neutrokine-α SV fusion rotein and the interaction between the anti-Flag  antibody and associating.
Polymer of the present invention can produce with chemical process known in the art.For example, the polypeptide that need be included in the polymer of the present invention can be with linkers known in the art and linkers length optimization method and chemically crosslinked (seeing the U.S. Patent No. 5478925 of incorporating reference into).In addition, polymer of the present invention can produce with methods known in the art, and described method is between the half Guang amino residue in the sequence that is contained in the polypeptide in the polymer at need, forms one or more intramolecular crosslinking.(seeing the U.S. Patent No. 5478925 of incorporating reference into).In addition, polypeptide of the present invention can be by add halfcystine or vitamin H at the C of polypeptide or N-terminal conventional the modification, and available methods known in the art produce the polymer (seeing the U.S. Patent No. 5478925 of incorporating reference into) that contains one or more these modified polypeptides.In addition, available methods known in the art produce and contain the liposome (seeing the U.S. Patent No. 5478925 of incorporating reference into) that need be included in the polypeptide fraction in the polymer of the present invention.
Perhaps, polymer of the present invention can produce with gene engineering method known in the art.In one embodiment, the polypeptide that is included in the polymer of the present invention produces (seeing the U.S. Patent No. 5478925 of incorporating reference into) with fusion rotein method as herein described or other method reorganization known in the art.In a special embodiment, the polynucleotide of code book invention homodimer are by the polynucleotide sequence of the code book invention polypeptide sequence with coding joint polypeptide is connected, and opposite direction (disappearance homing sequence) is connected in the synthetic polynucleotide (seeing the U.S. Patent No. 5478925 of incorporating reference into) of coded polypeptide translation product from the starting point C-terminal to N-terminal then.In another embodiment, produce recombinant polypeptide of the present invention with recombination method as herein described or other method known in the art, this polypeptide contains membrane-spanning domain and can mix in the liposome by the membrane reconstitution method (sees the U.S. Patent No. 5478925 of incorporating reference into).
In one embodiment, the invention provides isolating Neutrokine-α polypeptide, this polypeptide has the aminoacid sequence by the cDNA clones coding of ATCC preserving number 97768, or have aminoacid sequence shown in Figure 1A and the 1B (SEQ ID NO:2), perhaps the invention provides the isolating polypeptide that comprises an aforementioned polypeptides part (being fragment).In another embodiment, the invention provides isolating Neutrokine-α SV polypeptide, this polypeptide has the aminoacid sequence by the cDNA clones coding of ATCC preserving number 203518, or have aminoacid sequence shown in Fig. 5 A and the 5B (SEQ ID NO:19), the polypeptide that comprises an aforementioned polypeptides part and (being fragment) perhaps is provided.
Polypeptide fragment of the present invention comprises a peptide species, this polypeptide comprises or by aminoacid sequence shown in (SEQ IDNO:2), by cDNA amino acid sequence coded in the plasmid of ATCC preserving number 97768, or by with the clone of preservation in the aminoacid sequence of nucleic acid encoding of nucleotide sequence complementary strand hybridization (as under) shown in the nucleotide sequence that comprises or Figure 1A and the 1B (SEQ ID NO:1) at stringent hybridization condition form.
In addition, polypeptide fragment of the present invention comprises a peptide species, this polypeptide comprises or by the aminoacid sequence shown in (SEQID NO:19), the cDNA amino acid sequence coded that comprises in the plasmid by ATCC preserving number 203518, or by with the clone of preservation in the aminoacid sequence of nucleic acid encoding of nucleotide sequence complementary strand hybridization (as under) shown in the nucleotide sequence that comprises or Fig. 5 A and the 5B (SEQ ID NO:18) at stringent hybridization condition form.
In addition, polypeptide fragment of the present invention comprises a peptide species, and this polypeptide comprises or by forming with the aminoacid sequence of the nucleic acid encoding of nucleotide sequence complementary strand shown in SEQID NO:21 hybridization (under at stringent hybridization condition).
Polypeptide fragment of the present invention also comprises a peptide species, this polypeptide comprise or by with aminoacid sequence shown in the SEQ IDNO:23, or by forming with the aminoacid sequence of the nucleic acid encoding of nucleotide sequence complementary strand shown in SEQ ID NO:22 hybridization (as under) at stringent hybridization condition.
In addition, polypeptide fragment of the present invention comprises a peptide species, this polypeptide comprises or by aminoacid sequence shown in the SEQ IDNO:28, or by forming with the aminoacid sequence of the nucleic acid encoding of the hybridization of nucleotide sequence complementary strand shown in the SEQ ID NO:27 (under at stringent hybridization condition).
In addition, polypeptide fragment of the present invention comprises a peptide species, this polypeptide comprises or by aminoacid sequence shown in the SEQ IDNO:30, or by forming with the aminoacid sequence of the nucleic acid encoding of the hybridization of nucleotide sequence complementary strand shown in the SEQ ID NO:29 (under at stringent hybridization condition).
Polypeptide fragment of the present invention comprises a peptide species, this polypeptide comprises or by aminoacid sequence shown in the SEQ IDNO:2, the cDNA amino acid sequence coded that comprises among the clone by preservation, or by with the clone of preservation in comprise Figure 1A and 1B (SEQ ID NO:1) shown in or the aminoacid sequence of the nucleic acid encoding of its complementary nucleotide sequence hybridization (as under) at stringent hybridization condition.Protein fragments can be " freely existing ", or is included in the bigger polypeptide, and fragment forms a part or zone of polypeptide, preferably as an independent successive zone.Polypeptide fragment of the present invention for example comprises typically and comprising or by the 1-50 of SEQ ID NO:2,51-100,101-150,151-200,201-250, and/or the fragment formed of 251-285 amino acids residue.In addition, the length of polypeptide fragment can be at least 10,20,30,40,50,60,70,80,90,100,110,120, and 130,140,150,175 or 200 amino acid.
In special embodiment, polypeptide fragment of the present invention comprises or by the 1-46 of sequence shown in Figure 1A and the 1B (SEQ ID NO:2), 31-44,47-72,73-285,73-83,94-102,148-152,166-181,185-209,210-221,226-237,244-249,253-265 and/or 277-284 amino acids residue are formed.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Those skilled in the art will recognize that the sudden change in a zone that is oriented to Neutrokine-α polypeptide of the present invention can have influence on the biological activity that is observed of Neutrokine-α polypeptide, undiscovered 19 amino acid whose insertion bodies in Neutrokine-α SV peptide sequence (being Val 142-Lys 160 amino-acid residues of sequence shown in Figure 1A and 1B and the SEQ ID NO:2) have been contained in described zone.More particularly, this residue of the Neutrokine-α peptide sequence of orientable mutagenesis comprises the following amino-acid residue of Neutrokine-α polypeptide shown in the SEQ ID NO:2: V142 without limitation; T143; Q144; D145; C146; L147; Q148; L149; I150; A151; D152; S153; E154; T155; P156; T157; I158; Q159 and K160.Be coded in that in the V142-K160 zone of SEQ ID NO:2 the polynucleotide of the Neutrokine-α polypeptide of one or more sudden change to be arranged be desired.Polypeptide by these polynucleotide encodings is also contained within the scope of the present invention.
Polypeptide fragment can be " freely existing ", or is included in the big polypeptide, and fragment forms a part or the zone of polypeptide, preferably as an independent successive zone.Polypeptide fragment of the present invention for example comprises typically and comprising or by about 1-15 of aminoacid sequence shown in the SEQ ID NO:2,16-30,31-46,47-55,56-72,73-104,105-163,163-188, the fragment that 186-210 and 210-284 amino acids residue are formed.Polypeptide fragment of the present invention for example comprises in addition and comprising or by about 1-143 of aminoacid sequence shown in the SEQ ID NO:19,1-150,47-143,47-150,73-143,73-150,100-150,140-145,142-148,140-150,140-200, the fragment that 140-225 and 140-266 amino acids residue are formed.The length of polypeptide fragment can be at least 10,20,30,40 in addition, 50,60,70,80,90,100,110,120, and 130,140,150,175 or 200 amino acid." approximately " is meant specific scope in the literary composition, and in the many of aminoterminal or carboxyl terminal or aminoterminal and carboxyl terminal or few, several, 5,4,3,2, this scope of 1 amino-acid residue.The polynucleotide of these polypeptide fragments of encoding also are encompassed in the present invention
The polypeptide fragment that other embodiment preferred contains comprises or is made up of following structure: born of the same parents' internal area that Neutrokine-α infers (the 1-46 amino acids residue of SEQ ID NO:2), the membrane-spanning domain of inferring of Neutrokine-α (the 47-72 amino acids residue of SEQ ID NO:2), the extracellular domain of inferring of Neutrokine-α (the 73-285 amino acids residue of SEQ ID NO:2), the structural domain (the 191-284 amino acids residue of SEQ ID NO:2) that the TNF that infers of Neutrokine-α is conservative and comprise or infer the polypeptide that born of the same parents' internal area (the 1-46 amino acids residue that is SEQ ID NO:2 is blended in 73-285 amino acids residue) is formed of inferring of extracellular domain by being blended in of Neutrokine-α.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
The polypeptide fragment contained of embodiment preferred comprises or is made up of following structure in addition: born of the same parents' internal area of inferring of Neutrokine-α SV (the 1-46 amino acids residue of SEQ ID NO:19), the membrane-spanning domain of inferring of Neutrokine-α SV (the 47-72 amino acids residue of SEQ ID NO:19), the extracellular domain of inferring of Neutrokine-α SV (the 73-266 amino acids residue of SEQ IDNO:19), structural domain (the 172-265 amino acids residue of SEQ ID NO:19) that the TNF that infers of Neutrokine-α SV is conservative and the polypeptide that comprises or form by the syzygy of inferring born of the same parents' internal area and extracellular domain (the 1-46 amino acids residue of SEQ IDNO:19 and the fusion of 73-266 amino acids residue) of α SV-Neutrokine.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
The polypeptide fragment that other embodiments of the present invention contain comprises or is made up of the βZhe Die of the inferring zone of differentiating among Fig. 7 A.These polypeptide fragments of the present invention comprise or by the Gln144-Ala151 of SEQ IDNO:2, Phe172-Lys173, Ala177-Glu179, Asn183-Ile185, Gly191-Lys204, His210-Val219, Leu226-Pro237, Asn242-Ala251, Gly256-Ile263 and/or Val276-Leu284 amino acids residue are formed.In some other embodiments, these polypeptide fragments of the present invention also comprise or by the Phe153-Lys154 of SEQID NO:19, Ala158-Glu160, Asn164-Ile166, Gly172-Lys185, His191-Val200, Leu207-Pro218, Asn223-Ala232, Gly237-Ile244, and/or Val257-Leu256 amino acids residue is formed, reach Phe42-Lys43 by SEQ ID NO:23, Ala47-Glu49, Asn53-Ile55, Gly61-Pro74, His80-Val89, Leu96-Pro107, Asn112-Ala121, Gly126-Ile133 and/or Asp146-Leu154 amino acids residue are formed.In other embodiments, these polypeptide fragments of the present invention also comprise or comprise the Gln78-Ala85 of SEQ ID NO:28, Phe106-Lys107, Ala111-Glu113, Asn117-Ile119, Gly125-Lys138, His144-Val153, Leu160-Pro171, Asn176-Ala185, Gly190-Ile197, and/or Val210-Leu218 amino acids residue is formed, reach Gln78-Ala85 by SEQ ID NO:30, Phe106-Lys107, Ala111-Glu113, Asn117-Ile119, Gly125-Lys138, His144-Val153, Leu160-Pro171, Asn176-Ala185, Gly190-Ile197, and/or Val210-Leu218 amino acids residue is formed.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
The non-sex-limited ground of polypeptide more of the present invention for example comprises or is made up of the combination of aminoacid sequence of the present invention, for example comprises [Met1-Lys113] and [Leu114-Thr141] and [Ile142-Lys160] and [Gly161-Gln198] and the syzygy of [Val199-Ala248] and [Gly250-Leu285] of SEQ ID NO:2; The syzygy of [the Met 1-Lys113] of SEQ ID NO:2 and [Ile142-Lys160] and [Gly161-Gln198] and [Val199-Ala248] and [Gly250-Leu285]; Or the syzygy of SEQ ID NO:2 [Met1-Lys113] and [Leu114-Thr141] and [Ile142-Lys160] and [Gly161-Gln198] and [Val199-Ala248] and [Gly250-Leu285].Or the syzygy of SEQ ID NO:2 [Met1-Lys113] and [Leu114-Thr141] and [Ile142-Lys160] and [Gly161-Gln198] and [Gly250-Leu285].Other combination can comprise polypeptide fragment except that aforesaid combination (as the syzygy of [Leu114-Thr141] and [Val199-Ala248] and [Gly250-Leu285] and [Ile142-Lys160] of SEQ ID NO:2).Other combination also can comprise heterologous polypeptide fragment as herein described and/or other polypeptide of the present invention or polypeptide fragment (as the syzygy of [Met1-Lys113] and [Leu114-Thr141] and [Ile142-Lys160] and [Gly161-Gln198] and [Gly250-Leu285] Yu the FLAG mark of SEQ ID NO:2).The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
In addition, the non-sex-limited ground of polypeptide of the present invention for example comprises or is made up of aminoacid sequence, for example comprises the syzygy of [Met1-Lys113] and [Leu114-Thr141] and [Gly142-Gln179] and [Val180-Ala229] and [Gly230-Leu266] of SEQ ID NO:19; The syzygy of [Met1-Lys113] of SEQ ID NO:19 and [Gly142-Gln179] and [Val180-Ala229] and [Gly230-Leu266]; Or the syzygy of SEQ ID NO:19 [Met1-Lys113] and [Leu114-Thr141] and [Gly142-Gln179] and [Gly230-Leu266].Other combination can comprise polypeptide fragment except that aforesaid combination (as the syzygy of [Leu114-Thr141] and [Val180-Ala229] and [Gly230-Leu266] and [Gly142-Gln179] of SEQ ID NO:19).Other combination also can comprise heterologous polypeptide fragment as herein described and/or other polypeptide of the present invention or polypeptide fragment (as the syzygy of [Met1-Lys113] and [Leu114-Thr141] and [Gly142-Gln179] and [Gly230-Leu266] Yu the FLAG mark of SEQ IDNO:19).The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
The in addition non-sex-limited ground of polypeptide of the present invention for example comprises or is made up of aminoacid sequence, for example comprises [Met1-Lys106] and [Leu107-Thr134] and [Ile167-Lys184] and [Gly185-Gln224] and [Val225-Ala272] and the Gly273-Leu309 of SEQ ID NO:23] syzygy; The syzygy of [Met1-Lys106] of SEQ ID NO:23 and [Glu135-Asn165] and [Ile167-Lys184] and [Gly185-Gln224] and [Val225-Ala272] and [Gly273-Leu309], or the syzygy of SEQ ID NO:23 [Met1-Lys106] and [Leu107-Thr134] and [Glu135-Asn165] and [Ile167-Lys184] and [Gly185-Gln224] and [Gly273-Leu309].Other combination can comprise the polypeptide fragment (as [Met1-Lys106] and [Gly185-Gln224] and [Ile167-Lys184] and [Val225-Ala272] and the syzygy of [Leu107-Thr134] and [Gly273-Leu309] of SEQ ID NO:23) except that aforesaid combination.Other combination also can comprise heterologous polypeptide fragment as herein described and/or other polypeptide of the present invention or polypeptide fragment (as the syzygy of [Met1-Lys106] and [Glu135-Asn165] and [Ile167-Lys184] and [Gly185-Gln224] and [Val225-Ala272] and [Gly273-Leu309] Yu the FLAG mark of SEQ IDNO:23).The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
The non-in addition sex-limited ground of polypeptide of the present invention for example comprises or is made up of aminoacid sequence, for example comprises [Tyr1-Lys47] and [Leu48-Thr75] and [Ile76-Lys94] and [Gly95-Gln132] and the syzygy of [Val133-Ala182] and [Gly183-Ala219] of SEQ ID NO:28; The syzygy of [Tyr1-Lys47] of SEQ ID NO:28 and [Leu48-Thr75] and [Ile76-Lys94] and [Val133-Ala182]; Or the syzygy of SEQ ID NO:28 [Tyr1-Lys47] and [Leu48-Thr75] and [Ile76-Lys94] and [Val133-Ala182] and [Gly183-Ala219].Other combination can comprise polypeptide fragment except that aforesaid combination (as the syzygy of [Tyr1-Lys47] and [Gly183-Ala219] and [Val133-Ala182] and [Leu48-Thr75] of SEQ ID NO:28).Other combination also can comprise heterologous polypeptide fragment as herein described and/or other polypeptide of the present invention or polypeptide fragment (as [Leu48-Thr75] and [Ile76-Lys94] and [Gly95-Gln132] and [Val133-Ala182] of SEQ ID NO:28 and the syzygy of Fc receptor marker).The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
The non-in addition sex-limited ground of polypeptide of the present invention for example comprises or is made up of aminoacid sequence, for example comprises [Tyr1-Lys47] and [Leu48-Thr75] and [Ile76-Lys94] and [Gly95-Gln132] and the syzygy of [Val133-Ala182] and [Gly183-Ala219] of SEQ ID NO:30; The syzygy of [Tyr1-Lys47] of SEQ ID NO:30 and [Leu48-Thr75] and [Ile76-Lys94] and [Val133-Ala182]; Or the syzygy of SEQ ID NO:30 [Tyr1-Lys47] and [Ile76-Lys94] and [Val133-Ala182] and [Gly183-Ala219].Other combination can comprise polypeptide fragment except that aforesaid combination (as the syzygy of [Tyr1-Lys47] and [Gly183-Ala219] and [Val133-Ala182] and [Leu48-Thr75] of SEQ ID NO:30).Other combination also can comprise heterologous polypeptide fragment as herein described and/or other polypeptide of the present invention or polypeptide fragment (as [Leu48-Thr75] and [Ile76-Lys94] and [Gly95-Gln132] and [Val133-Ala182] of SEQ IDNO:30 and the syzygy of Fc receptor marker).The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Neutrokine-α that other embodiment of the present invention contains and/or Neutrokine-α SV polypeptide fragment comprise or are made up of the functional area of polypeptide of the present invention, α district as Garnier-Robaon, the β district, the corner regions and the district of curling, the α district of Chou-Fasman, the β district and the district of curling, Kyte-Doolittle hydrophilic area and hydrophobic region, the α of Eisenberg and β amphiphilic district, the Karplus-Schulz flex region, Emini surface forms district and Jameson-Wolf high antigenic index region, as Fig. 3,6 and table 1 shown in.In an embodiment preferred, polypeptide fragment of the present invention is antigenic.VIII in the table 1, IX, data can be used for the zone that routine determines to be highly potential antigenic Neutrokine-α shown in XIII and the XIV row.High antigenicity zone is by from VIII, IX, shown in XIII and/or the IV row in the data, select representative under certain environment, to be easy to the numerical value in the polypeptide zone that on the polypeptide surface, exposes and determine that described environment is that antigen recognition can occur in the initial process of immunne response therein.The particularly preferred fragment of the present invention is those fragments that comprise the zone of the Neutrokine-α that made up some constructional features such as above-mentioned some (as 1,2,3 or 4) characteristics and/or Neutrokine-α SV.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
In another embodiment, the invention provides and comprise or by the polypeptide that epi-position is partly formed that carries of polypeptide of the present invention.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.The epi-position of polypeptide partly is the immunogenicity or the antigenic epitopes of polypeptide of the present invention." immunogenicity epi-position " is meant a proteinic part that excites antibody response when whole protein is immunogen.On the other hand, " antigenic epitopes " is meant the zone of the combinative protein molecule of antibody.Proteinic immunogenicity epi-position number generally is less than the antigenic epitopes number.For example see Geysen etc., institute of American Academy of Sciences reports 81:3998-4002 (1983).
For selecting to carry the polypeptide (zone of promptly containing the combinative protein molecule of antibody) of antigenic epitopes, the short relatively synthetic peptide of well known simulation part protein sequence can excite the antiserum(antisera) with the partial simulation albumen test usually.For example see Sutcliffe, J.G.Shinnick, T.M.Green, N. and Learrer, R.A (1983) " with the antibody of predetermined site reaction on the protein ", science 219:660-666 is described.Can elicitor protein the peptide of qualitative response serum in proteinic primary sequence, embody usually, can be qualitative, and be defined as neither the immunodominance zone of intact proteins (being the immunity epi-position) by some easy chemical rule, neither amino or C-terminal.Therefore peptide or polypeptide that the present invention carries antigenic epitopes are used to produce antibody, comprise the monoclonal antibody of specific combination polypeptide of the present invention.For example referring to Wilson etc., cell 37:767-778 (1984) is at the p777 page or leaf.
Peptide or polypeptide that the present invention carries antigenic epitopes preferably contain at least 4, and at least 5, at least 6, at least 7, preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, more preferably about 15-30 sequence that is included in the amino acid composition in the amino acid sequence of polypeptide of the present invention.The length that comprises the preferred polypeptide of immunogenicity or antigenic epitopes is at least 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 amino-acid residues.Other preferred antigenic epitopes comprises antigenic epitopes disclosed herein and its part.
The antigenic polypeptide or the peptide that can be used for producing Neutrokine-α and/or Neutrokine-α SV specific antibody for example comprise without limitation: comprise or by the about polypeptide formed of Phe115-Leu147 amino acids residue shown in Figure 1A and the 1B (SEQID NO:2); Comprise or by the about polypeptide formed of Ile150-Tyr163 amino acids residue shown in Figure 1A and the 1B (SEQ ID NO:2); Comprise or by the about polypeptide formed of Ser171-Phe194 amino acids residue shown in Figure 1A and the 1B (SEQ ID NO:2); The polypeptide that comprises or form by Glu223-Tyr246 amino acids residue shown in Figure 1A and the 1B (SEQID NO:2); Comprise or by the about polypeptide formed of Ser271-Phe278 amino acids residue shown in Figure 1A and the 1B (SEQ ID NO:2); " approximately " is meant specific scope in the literary composition, and in this scope the many of aminoterminal or carboxyl terminal or two ends or few, several, 5,4,3,2 or 1 amino acid.These polypeptide fragments are analyzed by the Jameson-Wolf antigenic index, have determined to carry the antigenic epitopes of Neutrokine-α polypeptide, shown in Fig. 3 and table 1.
The antigenic polypeptide or the peptide that can be used for producing Neutrokine-α and/or Neutrokine-α SV specific antibody for example comprise without limitation: comprise or by the about polypeptide formed of Pro32-Leu47 amino acids residue shown in Fig. 5 A and the 5B (SEQID NO:19); Comprise or by the about polypeptide formed of Glu116-Ser143 amino acids residue shown in Fig. 5 A and the 5B (SEQ ID NO:19); Comprise or by the about polypeptide formed of Phe153-Tyr173 amino acids residue shown in Fig. 5 A and the 5B (SEQ ID NO:19); Comprise or by the about polypeptide formed of Pro218-Tyr227 amino acids residue shown in Fig. 5 A and the 5B (SEQID NO:19); Comprise or by the about polypeptide formed of Ala232-Gln241 amino acids residue shown in Fig. 5 A and the 5B (SEQ ID NO:19); Comprise or by the about polypeptide formed of Ile244-Ala249 amino acids residue shown in Fig. 5 A and the 5B (SEQ ID NO:19); Comprise or by the about polypeptide formed of Ser252-Val257 amino acids residue shown in Fig. 5 A and the 5B (SEQID NO:19)." approximately " is meant specific scope in the literary composition, and the inherent aminoterminal of this scope or carboxyl terminal or two ends are many or few, several, 5,4,3,2 or 1 amino-acid residues.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.These polypeptide fragments have determined to carry the antigenic epitopes of Neutrokine-α SV polypeptide by the analysis of Jameson-Wolf antigenic index, as shown in Figure 6 and shown in the data of the sheet form that is produced by the Protean assembly of DNA*STAR computer program.
Peptide and the polypeptide that carries epi-position of the present invention can be by any ordinary method production.For example see Houghten, R.A. (1985) is the general method of a large amount of peptides of solid phase synthesis fast: in the specificity of the horizontal antigen-antibody interaction of individual amino acids, institute of American Academy of Sciences reports 82:5131-5135; (1986) such as method Houghten of " simultaneously multiple peptide synthetic (SMPS) " see that U.S. Patent No. 4631211 is described.
The purposes of peptide that carries epi-position of the present invention and polypeptide comprises but non-being limited to according to well known method induced antibody.For example see Sutcliffe etc., as preceding; Wilson etc. are as preceding; Chow, M. etc., institute of American Academy of Sciences reports 82:910-914; And Bittle, F.J. etc., gene viruses magazine 66:2347-2354 (1985). the present invention carries the peptide of immunogenicity epi-position, promptly when whole protein is immunogen, excites the proteinic part of antibody response, differentiates according to means known in the art.For example see Geysen etc., as described above.In addition, the U.S. Patent No. 5194392 of Geysen has been set forth a kind of the detection or the method for definite monomer (amino acid or other compound) sequence, and this monomer is the homeomorphic thing (being mimotope) of epi-position that is complementary to the special paratope (antigen binding site) of corresponding antibodies.The United States Patent (USP) of Geysen (1989) has been set forth a kind of the detection or the method for definite sequence monomer, and this monomer is the homeomorphic thing of part that is complementary to the ligand-binding site point of corresponding specific receptor.Similarly, Houghten, R.A. etc. (1996) are about crossing the U.S. Patent No. 5480971 of alkylating oligo peptide, disclosed the library of linear C1-C7-alkyl-alkylating oligopeptides of mistake and this peptide, and the method for determining preferentially to cross with corresponding acceptor molecule bonded alkylating oligopeptides sequence with this oligopeptides library.Therefore, the present invention's non-peptide analogs of carrying the peptide of epi-position also can be produced by these methods are conventional.
A peptide species is contained in the present invention, it comprises or is made up of the epi-position of following polypeptide: the polypeptide with aminoacid sequence shown in the SEQ ID NO:2, or by the preservation thing of ATCC preserving number 97768 contained the clone in the polypeptide of the polynucleotide sequence coding that comprises, or by with the sequence of SEQID NO:1 or ATCC preserving number 97768 in the cDNA sequence that comprises complementary sequence hybridization (as described under the hybridization conditions) the polypeptide of polynucleotide encoding.Polynucleotide sequence has also been contained in the present invention, it comprises or by the sequence (sequence shown in SEQ ID NO:1) of code book invention polypeptide epitope, the polynucleotide sequence of the complementary strand of the polynucleotide sequence of code book invention epi-position and form with the polynucleotide sequence of complementary strand hybridization (under described hybridization conditions).
A peptide species has also been contained in the present invention, it comprises or has the epi-position of the polypeptide of aminoacid sequence shown in the SEQ ID NO:19, or the epi-position of the peptide sequence of the polynucleotide sequence coding that is comprised by ATCC preserving number 203518, or by with the complementary sequence of sequence shown in the SEQ ID NO:18 or the cDNA sequence hybridization that ATCC preserving number 203518 comprises (as described under the hybridization conditions) the epi-position of peptide sequence of polynucleotide encoding form.A kind of polynucleotide sequence has also been contained in the present invention, it comprises or by the epi-position of code book invention polypeptide (sequence shown in SEQ IDNO:18), the polynucleotide sequence of the complementary strand of the polynucleotide sequence of code book invention epi-position and with complementary strand hybridization (as described under the hybridization conditions) polynucleotide sequence.
Used term " epi-position " is meant animal in the literary composition, and preferred mammal more preferably has the part of antigenicity or the active polypeptide of immunogenicity in human body.In an embodiment preferred, a kind of polynucleotide that comprise the polypeptide of epi-position and this peptide species of encoding have been contained in the present invention." immunogenicity epi-position " is meant a proteinic part that excites antibody response in the animal body, as determining by any method known in the art, the method by producing antibody as described above (see for example Geysen etc., institute of American Academy of Sciences reports the 81:3998-4002 (1983) described) for example.Term " antigenic epitopes " is meant the antibody capable immunologic opsonin in conjunction with its antigenic proteinic part, as determining by any method well known in the art, for example determines by described immunoassay.The immunologic opsonin combination does not comprise non-specific binding, but is not must not comprise and other antigenic cross reactivity.Antigenic epitopes is nonessential to be immunogenic.
Fragment as epi-position can be by any ordinary method production (see for example Houghten, institute of American Academy of Sciences reports 82:5131-5135 (1985), and it is described to see U.S. Patent No. 4631211 in addition)
In the present invention, antigenic epitopes preferably contains at least 4, and at least 5, painted 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, most preferably about 15-30 amino acid whose sequence.The polypeptide length that preferably comprises immunogenicity or antigenic epitopes is at least 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90 or 100 amino-acid residues.Other preferred antigenic epitopes comprises antigenic epitopes disclosed herein, and 2,3,4,5 or any combination of a plurality of these antigenic epitopes.Antigenic epitopes can be used as target molecule in the immunoassay (Wilson etc. for example, cell 37:767-778 (1984); Sutcliffe etc., science 219:660-666 (1983) is described).
Similarly, the immunogenicity epi-position for example can be used for inducing antibody (to see for example Sutcliffe etc., as preceding according to well known method; Wilson etc. are as preceding; Chow etc., institute of American Academy of Sciences reports 82:910-914; With Bittle etc., gene viruses magazine 66:2347-2354 (1985) is described).Preferred immunogenicity epi-position comprises the immunogenicity epi-position that this paper discloses, and 2,3,4,5 or the combination of a plurality of these immunogenicity epi-positions.The polypeptide that comprises one or more immunogenicity epi-position can exist with carrier proteins such as albumin, and with the antibody response of (as rabbit or the mouse) that excite animal system, if perhaps polypeptide is sufficiently long (about at least 25 amino acid), this polypeptide can be without carrier.Yet, comprise few as 8-10 amino acid whose immunogenicity epi-position illustrated the antibody of effective generation energy in conjunction with the linear epitope in the polypeptide of sex change at least.(as in the Western trace).
The polypeptide that the present invention carries epi-position can be used to induce antibody according to well known method, and described method comprises but non-body interior immunization, external immunization and the phage display method of being limited to.See for example Sutcliffe etc., as preceding; Wilson etc. are as preceding; With Bittle etc., gene viruses magazine 66:2347-2354 (1985).If immunization method in the use body, animal can inoculate with free peptide; Yet anti-peptide antibody titre can be by strengthening peptide and macromolecular carrier coupling, and described carrier is keyhole chirp hemocyanin (KLH) or tetanus toxin in this way.For example, contain cysteine residues peptide can with joint as-the maleimide phenylformic acid-N-hydroxy-succinamide ester (MBS) is coupled to carrier, and other peptide can be with more generally linking agent such as glutaraldehyde are coupled to carrier.Animal such as rabbit, rat and mouse with free peptide or with carrier link coupled peptide immunization, for example inoculate by intraperitoneal and/or a kind of emulsion of intradermal injection, described emulsion contains the adjuvant that about 100mg peptide or carrier proteins and Freud ' s adjuvant or any other known immune stimulatory are replied.Need carry out some booster shots, for example carry out once every about two weeks, so that the anti-peptide antibody of effective titre to be provided, this antibody for example can detect through elisa assay by the free peptide that use is adsorbed in solid surface.The titre of anti-peptide antibody can improve by selecting anti-peptide antibody in the animal serum of immunization, for example the antibody by peptide being adsorbed on the solid support and selecting according to well known method wash-out.
Those skilled in the art recognize and as mentioned above, the polypeptide of the present invention that comprises immunogenicity or antigenic epitopes can be blended in other peptide sequence.For example, polypeptide of the present invention can with immunoglobulin (Ig) (IgA, IgE, IgG, constant region IgM), or its part (CH1, CH2, CH3 or its arbitrary combination and a part thereof) merge to produce chimeric polyeptides.This fusion rotein is easy to purifying and the intravital transformation period can improve.Chimeric protein has been shown to be made up of the various structural domains of the constant region of preceding two structural domains of people CD4 polypeptide and Mammals heavy chain or light chain.For example see Ep394827; Tyaunecker etc., nature, 331:84-86 (1988) is described.Antigen passes the epithelium barrier and is delivered to immunity system and strengthens and antigen (leading element as pancreas) and FcRn binding partners such as IgG or Fc fragment to be shown to put together (seeing that for example PCT announces WO 96/22024 and WO 99/04813).Owing to the desulfurization key of IgG part has the IgG fusion rotein of the dimeric structure that disulfide linkage connects, also found to have the ability of more effective combination and other molecule that neutralizes than monomeric polypeptide or its fragment.See for example Fountoulakis etc., journal of biological chemistry 270:3958-3964 (1995).The nucleic acid of the above-mentioned epi-position of encoding also can be recombinated with the corresponding gene (as hemagglutinin (HA) mark or flag mark) as the epi-position mark, to help detecting and the purifying polypeptide expressed.For example, the system by elaborations such as Janknecht is easy to the unmodified fusion rotein (Janknecht etc., institute of American Academy of Sciences reports 88:89072-897) that purifying is expressed the human cell line.In this system, corresponding gene is gone in the bovine vaccine recombinant plasmid by subclone, and the open reading frame of this gene is blended in the aminoterminal mark of being made up of 6 histidine residues through translation like this.This mark as the matrix of fusion rotein in conjunction with the territory.Will be with the extract application of sample of the cell of recombinant vaccinia virus infection in Ni 2+On the nitrilo acetate agarose chromatography post, and the protein of histidine mark is optionally with the damping fluid selective elution that contains imidazoles.
In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide and the fragment of carrying epi-position thereof are to merge with heterologous antigen (as polypeptide, carbohydrate, phosphatide or nucleic acid).In special embodiment, heterologous antigen is an immunogen.
In more special embodiment, heterologous antigen is gpl20 albumen or its fragment of HIV.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide and carry the fragment of epi-position are that the peptide sequence (or its bioactive fragment or its variant) with another tnf ligand family member merges.In a special embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide merge with the CD40L peptide sequence.In preferred embodiments, the CD40L peptide sequence is soluble.
Gene reorganization, motif reorganization, the method of exon reorganization and/or codon reorganization (being referred to as " DNA reorganization "), can be used for regulating the activity of Neutrokine-α and/or Neutrokine-α SV, thereby produce agonist and the antagonist of Neutrokine-α and/or Neutrokine-α SV effectively.See U.S. Patent No. 5605793,5811238,5830721,5834252 and 5837458, and Patten, P.A. etc., the general suggestion 8:724-33 of biotechnology (1997); Harayama, S. biotechnology trend 16 (2): 76-82 (1998); Hansson, L.O, etc., molecular biology magazine 287:265-76 (1999); And Lorenzo, M.M. and Blasco, R. biotechnology 24 (2): 308-13 (1998) is described, and described document is all incorporated reference into.In one embodiment, to Neutrokine-α and or Neutrokine-α SV polynucleotide and corresponding polypeptide change and can be undertaken by DNA reorganization.DNA reorganization comprises by homology or locus specificity reorganization, two or more DNA section is fitted in required Neutrokine-α and/or the Neutrokine-α SV molecule.In another embodiment, Neutrokine-α and/or Neutrokine-α SV polynucleotide and corresponding polypeptide can by the reorganization before through the error-prone PCR random mutagenesis, insert Nucleotide or other method at random and change.In another embodiment, one or more component of Neutrokine-α and/or Neutrokine-α SV, motif, section, part, structural domain, fragment etc. can with one or more component of one or more heterologous molecule, motif, section, part, structural domain, reorganization such as fragment.In preferred embodiments, heterologous molecule for example is TNF-α, lymphotoxin-α (LT-α, be also referred to as TNF-β), LT-β (in heterotrimer LT-α 2-beta composite, finding), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (international open No.WO 96/14328), AIM-I (international open WO 97/33899), AIM-II (international open WO 97/34911), APRIL (experimental technique magazine 188 (6): 1185-1190), endokine-α (international open WO 98/07880), OPG, OX40, and the Fas of nerve growth factor (NGF) and soluble form, CD 30, CD27, CD40 and 4-IBB, TR2 (international open WO 96/34095), DR3 (international open WO 97/33904), DR4 (international open WO 98/32856), TR5 (international open WO 98/30693), TR6 (international open WO 98/30694), TR7 (international open WO 98/41629), TRANK, the international open WO 98/56892 of TR9), the international open WO 98/54202 of TR10), 312C2 (international open WO 98/06842), TR12, CDA and V-FLIP.In other embodiments, heterologous molecule is any member of TNF family.
In preferred embodiments, Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide (comprising its bioactive fragment or variant) and soluble CD40L polypeptide or its bioactive fragment or variant fusion.
For the feature of improvement or change Neutrokine-α and/or Neutrokine-α SV polypeptide, can carry out protein engineering.Can be used for producing new mutant protein or " mutain " in recombinant DNA method well known by persons skilled in the art, it comprises one or more aminoacid replacement, and disappearance is added or fusion rotein.This modified polypeptides can show that for example enhanced is active or the stability of raising.In addition, they are compared with corresponding natural polypeptide, at least under some purifying and condition of storage, and can the high yield purifying and stability preferably.For example, with regard to numerous protein, comprise the extracellular domain of the secretory protein of mature form, N-terminal known in the art or C-terminal can lack one or more amino acid and not lose biological function substantially.For example, Ron etc., journal of biological chemistry, 268:2984-2988 (1993) have reported the KGF albumen of modifying, even it loses 3,8 or 27 aminoterminal amino acid, but still have heparin binding activity.
Because protein of the present invention is TNF peptide family member, the N-terminal amino acid of Figure 1A and 1B (SEQ IDNO:2) is until the disappearance of 191 Gly (G) residue, still can keep some biological activitys as stimulating lymphocyte (as the B cell) propagation, differentiation and/or activation, and suitable target cell is had the ability of cellular toxicity.Have further N-terminal and comprise that the polypeptide of Gly (G) residue disappearance do not expect retains biological activity, because this residue in the known TNF related polypeptide is the starting point of the required conserved domain of biological activity.Yet even one or more amino acid of protein N terminal deletion and lost proteinic one or more function, other functionally active still can keep.Therefore, the protein induce of shortening and/or in conjunction with the ability of the antibody of identification whole protein or its extracellular domain, when the small part residue of whole protein or its extracellular domain when N-terminal is removed, still can keep.The special peptides of disappearance intact proteins N-terminal residue is to keep this immunocompetence, can determine by ordinary method described herein and other method known in the art.
Therefore, the present invention further provides the polynucleotide of a peptide species and this peptide species of encoding, described polypeptide has disappearance from the aminoacid sequence aminoterminal of Neutrokine-α shown in Figure 1A and the 1B (SEQ ID NO:2) one or more residue until 191 glycine residues (N-terminal Gly191).In particular, polypeptide provided by the invention comprises or by the n of SEQ IDNO:2 1The aminoacid sequence of-285 residues is formed, wherein n 1It is an integer in the 2-190 amino acids position of aminoacid sequence shown in the SEQ ID NO:2.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.More particularly, the polynucleotide of coded polypeptide provided by the invention comprise or are made up of the aminoacid sequence that is selected from next group: the 2-285 of SEQ ID NO:2,3-285,4-285,5-285,6-285,7-285,8-285,9-285,10-285,11-285,12-285,13-285,14-285,15-285,16-285,17-285,18-285,19-285,20-285,21-285,22-285,23-285,24-285,25-285,26-285,27-285,28-285,29-285,30-285,31-285,32-285,33-285,34-285,35-285,36-285,37-285,38-285,39-285,40-285,41-285,42-285,43-285,44-285,45-285,46-285,47-285,48-285,49-285,50-285,51-285,52-285,53-285,54-285,55-285,56-285,57-285,58-285,59-285,60-285,61-285,62-285,63-285,64-285,65-285,66-285,67-285,68-285,69-285,70-285,71-285,72-285,73-285,74-285,75-285,76-285,77-285,78-285,79-285,80-285,81-285,82-285,83-285,84-285,85-285,86-285,87-285,88-285,89-285,90-285,91-285,92-285,93-285,94-285,95-285,96-285,97-285,98-285,99-285,100-285,101-285,102-285,103-285,104-285,105-285,106-285,107-285,108-285,109-285,110-285,111-285,112-285,113-285,114-285,115-285,116-285,117-285,118-285,119-285,120-285,121-285,122-285,123-285,124-285,125-285,126-285,127-285,128-285,129-285,130-285,131-285,132-285,133-285,134-285,135-285,136-285,137-285,138-285,139-285,140-285,141-285,142-285,143-285,144-285,145-285,146-285,147-285,148-285,149-285,150-285,151-285,152-285,153-285,154-285,155-285,156-285,157-285,158-285,159-285,160-285,161-285,162-285,163-285,164-285,165-285,166-285,167-285,168-285,169-285,170-285,171-285,172-285,173-285,174-285,175-285,176-285,177-285,178-285,179-285,180-285,181-285,182-285,183-285,184-285,185-285,186-285,187-285,188-285,189-285, and 190-285.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.The invention still further relates to a kind of nucleic acid molecule, this nucleic acid molecule comprises or has at least 80%, 85% by the polynucleotide sequence with coding Neutrokine-α and/or Neutrokine-α SV polypeptide, 90%, 92%, 95%, the polynucleotide sequence of 96%, 97%, 98% or 99% homogeny is formed.The above-mentioned polynucleotide sequence that merges with the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide encoding also is encompassed in the present invention, these polypeptide be comprise or by with above-mentioned aminoacid sequence and at least 80%, 85%, 90%, 92%, 95%, 96%, the polypeptide that the aminoacid sequence of 97%, 98% or 99% homogeny is formed, the polynucleotide of this peptide species of encoding also are encompassed in the present invention.
In addition, but because the extracellular domain oneself excitation biological activity of inferring of Neutrokine-α polypeptide of the present invention, the extracellular domain that this polypeptide is inferred (crossing over the Gln73-Leu285 amino acids of SEQ ID NO:2) disappearance N-terminal and C-terminal amino-acid residue still can keep some biological activitys, for example part combination, stimulate lymphopoiesis, differentiation and/or activation, and regulate cellular replication or regulate the target cell activity.Yet even the extracellular domain N-terminal that Neutrokine-α polypeptide is inferred lacks one or more biological function forfeiture that one or more amino acid causes this polypeptide, other functionally active still can keep.Therefore, the polypeptid induction of shortening and/or in conjunction with the ability of the antibody of identification complete polypeptide or its extracellular domain, when the small part residue of complete or mature polypeptide or its extracellular domain when N-terminal is removed, still can keep.Whether the special peptides that lacks complete polypeptide N-terminal residue keeps this immunocompetence, can determine by ordinary method described herein and other method known in the art.
Therefore, the present invention also provides the polynucleotide of a peptide species and this peptide species of encoding, and this polypeptide has disappearance from the aminoterminal of Neutrokine-alpha amino acid sequence shown in the SEQ ID NO:2 one or more residue until 280 glycine residues.In particular, polypeptide provided by the invention comprises or by the n of SEQ ID NO:2 2-285 amino acids sequences are formed, wherein n 2Be the integer-bit in the 73-280 amino acids positional number of SEQ ID NO:2,73 is first residue position of Neutrokine-α polypeptide (SEQ ID NO:2) the extracellular domain N-terminal of inferring.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.More particularly, in certain embodiments, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or forms by being selected from next group aminoacid sequence: the Q-73 to L-285 of SEQ ID NO:2; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-283; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-235; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; 1-114 to L-285; F-115 to L-285; EI16 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-235; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-280; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; 1-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-235; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-280; I-195 to L-285; Y196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-203 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-235; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; 1-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285 G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; With G-280 to L-285.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.The invention still further relates to a kind of nucleic acid molecule, this nucleic acid molecule comprises or has at least 80% by the polynucleotide sequence with above-mentioned Neutrokine-α of coding and/or Neutrokine-α SV polypeptide, 85%, 90%, 92%, 95%, 96%, the polynucleotide sequence of 97%, 98% or 99% homogeny is formed.The above-mentioned polynucleotide sequence that is blended in the fusion of heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide encoding also is encompassed in the present invention, these polypeptide are to comprise or by having at least 80% with above aminoacid sequence, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny is formed, and the polynucleotide of this peptide species of encoding also are encompassed in the present invention.
The present invention particularly embodiment preferred relates to a kind of nucleic acid molecule, this nucleic acid molecule comprises or is made up of the polynucleotide with a kind of nucleotide sequence, the polynucleotide sequence that this nucleotide sequence and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 95%, 96%, 97%, 98%, 99% or 100% homogeny.The preferred embodiment of the invention relates to the nucleic acid molecule that comprises or be made up of polynucleotide, these polynucleotide have a kind of nucleotide sequence, and the polynucleotide sequence that this nucleotide sequence sequence and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 90% homogeny.The preferred embodiment of the present invention relates to the nucleic acid molecule that comprises or be made up of polynucleotide, these polynucleotide have a kind of nucleotide sequence, and the polynucleotide sequence that this sequence and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 95% homogeny.The preferred embodiment of the present invention relates to the nucleic acid molecule that comprises or be made up of polynucleotide, these polynucleotide have a kind of nucleotide sequence, and the polynucleotide sequence that this sequence and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 96% homogeny.
In addition, the preferred embodiment of the present invention relates to the nucleic acid molecule that comprises or be made up of polynucleotide, these polynucleotide have a kind of nucleotide sequence, and the polynucleotide sequence that this sequence and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 97% homogeny.In addition, the preferred embodiment of the present invention relates to the nucleic acid molecule that comprises or be made up of polynucleotide, these polynucleotide have a kind of nucleotide sequence, and the polynucleotide sequence that this sequence and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 98% homogeny.In addition, the preferred embodiment of the present invention relates to the nucleic acid molecule that comprises or be made up of polynucleotide, these polynucleotide have a kind of nucleotide sequence, and the polynucleotide sequence that this sequence and coding have 134-285 amino acids sequence of N eutrokine-α polypeptide shown in Figure 1A and the 1B (SEQ ID NO:2) has at least 99% homogeny.
In special embodiment, the polypeptide of being made up of one of the N-terminal of following Neutrokine-α and/or Neutrokine-α SV disappearance polypeptide fragment is preferred: the Ala71-Leu285 amino acids residue of SEQ IDNO:2, Ala81-Leu285 amino acids residue, Leu112-Leu285 amino acids residue, Ala134-Leu285 amino acids residue, Leu147-Leu285 amino acids residue, with Gly161-Leu285 amino acids residue, the polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Similarly, known a lot of biological functionality C-terminal deletion mutantion albumen.For example, IFN-improves 10 times of activity (Dobeli etc. by 8-10 the amino-acid residue that lacks its protein carboxyl terminal, biotechnology magazine 7:199-216 (1988)). because albumen of the present invention is a member of TNF peptide family, C-terminal amino acid is until the disappearance of 284 leucine residues, expectation keeps most of biological activity, and for example part combination stimulates lymphocyte (as the B cell) propagation, differentiation and/or activatory ability, the ability of regulating cellular replication.Disappearance is until the polypeptide of about 10 other C-terminal or residue (promptly until 274 glycine residues), also can keep some activity, as receptor-binding activity, although this peptide species forfeiture part extends to the conservative TNF structural domain of the Leu284 of SEQ ID NO:2.Yet, even lacking one or more amino acid, c-terminal of protein causes losing one or more proteinic biological function, other function still can keep.Therefore, when a small amount of residue of complete or mature protein when C-terminal is removed, the ability of the antibody of the protein induce of shortening and/or or maturation protein complete in conjunction with identification still keeps.Whether the special peptides of disappearance intact proteins C-terminal residue keeps this immunocompetence, can determine by ordinary method described herein and other method known in the art.
Therefore, the present invention further provides the polynucleotide of a peptide species and this peptide species of coding, this polypeptide has disappearance from one or more residue until 274 glycine (Gly274) of aminoacid sequence carboxyl terminal shown in Figure 1A and the 1B (SEQ ID NO:2).Especially, the invention provides and comprise or by the 1-m of SEQ ID NO:2 aminoacid sequence 1The polypeptide that the aminoacid sequence of position residue is formed, wherein m 1It is the arbitrary integer between the 274-284 amino acids residue of SEQ ID NO:2.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.More particularly, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or is made up of the aminoacid sequence that is selected from next group: the 1-274 of SEQ ID NO:2,1-275,1-276,1-277,1-278,1-279,1-280,1-281,1-282, the aminoacid sequence that 1-283 and 1-284 position residue are formed.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.The invention still further relates to a kind of nucleic acid molecule, it comprises or has at least 80%, 85% by the polynucleotide sequence with coding Neutrokine-α and/or Neutrokine-α SV polypeptide, 90%, 92%, 95%, the polynucleotide sequence of 96%, 97%, 98% or 99% homogeny is formed.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, these polypeptide are to comprise or by having at least 80% with above-mentioned aminoacid sequence, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny is formed, and the polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
The present invention also provides a peptide species, and it comprises or is made up of from one or more amino acid of aminoterminal and carboxyl terminal disappearance, can be described as the n with SEQ ID NO:2 1-m 1Residue, wherein n 1And m 1It is integer as the aforementioned.The present invention also comprises the nucleotide sequence of coded polypeptide, this polypeptide comprises or is made up of the part of the complete Neutrokine-alpha amino acid sequence of the cDNA clones coding of the preservation of ATCC preserving number 97768, wherein this part does not comprise the N-terminal 1-190 amino acids of complete amino acid sequence (or arbitrary combination of these N-and C-terminal deletion body), or the 1-11 amino acids of C-terminal.The polynucleotide of all above-mentioned disappearance polypeptide of encoding also are encompassed in the present invention.
Similarly, the C-terminal residue of the extracellular domain of inferring from Neutrokine-α can keep some biological activitys until 79 leucine residue deletants of SEQID NO:2, as the part combination, stimulate lymphocyte (as the B cell) propagation, differentiation and/or activation, and regulate cellular replication or regulate the target cell activity.Have further C-terminal disappearance and comprise that the polypeptide of the Leu79 of SEQ ID NO:2 do not expect retains biological activity.
Yet even one or more amino acid of peptide C terminal deletion causes losing one or more polypeptide biological function, other biological activity still can keep.Therefore, the polypeptid induction of shortening and/or complete in conjunction with identification, the ability of the antibody of mature polypeptide or its extracellular domain, when complete, a small amount of residue of mature polypeptide or its extracellular domain still can keep when C-terminal is removed.Whether the special peptides that disappearance is inferred extracellular domain C-terminal residue keeps this immunocompetence, can determine by ordinary method described herein and other method known in the art.
Therefore, this area also provides the polynucleotide sequence of polypeptide and this peptide species of encoding, and this polypeptide has disappearance and infers the aminoacid sequence carboxyl terminal of extracellular domain from Neutrokine-α polypeptide shown in the SEQ ID NO:2 until 79 leucic one or more residues.In particular, the invention provides and comprise or by the 73-m of aminoacid sequence shown in the SEQ ID NO:2 2The polypeptide that the aminoacid sequence that residue is formed is formed, wherein m 2Be the integer-bit in the 79-285 amino-acid residue position of aminoacid sequence shown in the SEQ ID NO:2,78 residues are first residues that Neutrokine-α polypeptide (SEQ ID NO:2) is inferred the extracellular domain C-terminal.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.More particularly, the invention provides in certain embodiments that coding comprises or by the polynucleotide that are selected from next polypeptide of forming of group aminoacid sequence: the Q-73 to Leu-285 of SEQ ID NO:2; Q-73 to L-284; Q-73 to K-283; Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279; Q-73 to F-278; Q-73 to T-277; Q-73 to V-276; Q-73 to D-275; Q-73 to G-274; Q-73 to D-273; Q-73 to L-272; Q-73 to S-271; Q-73 to I-270; Q-73 to Q-269; Q-73 to A-268; Q-73 to N-267; Q-73 to E-266; Q-73 to R-265; Q-73 to P-264; Q-73 to I-263; Q-73 to A-262; Q-73 to L-261; Q-73 to Q-260; Q-73 to L-259; Q-73 to E-258; Q-73 to D-257; Q-73 to G-256; Q-73 to E-255; Q-73 to E-254; Q-73 to L-253; Q-73 to K-252; Q-73 to A-251; Q-73 to I-250; Q-73 to G-249; Q-73 to A-248; Q-73 to 8-247; Q-73 to Y-246; Q-73 to C-245; Q-73 to S-244; Q-73 to N-243; Q-73 to N-242; Q-73 to P-241; Q-73 to L-240; Q-73 to T-239; Q-73 to E-238; Q-73 to P-237; Q-73 to M-236; Q-73 to N-235; Q-73 to Q-234; Q-73 to I-233; Q-73 to C-232; Q-73 to R-231; Q-73 to F-230; Q-73 to L-229; Q-73 to T-228; Q-73 to V-227; Q-73 to L-226; Q-73 to S-225; Q-73 to L-224; Q-73 to E-223; Q-73 to D-222; Q-73 to G-221; Q-73 to F-220; Q-73 to V-219; Q-73 to H-218; Q-73 to V-217; Q-73 to K-216; Q-73 to K-215; Q-73 to R-214; Q-73 to Q-213; Q-73 to I-212; Q-73 to L-211; Q73 to H-210; Q-73 to G-209; Q-73 to M-208; Q-73 to A-207; Q-73 to Y-206; Q-73 to T-205; Q-73 to K-204; Q-73 to D-203; Q-73 to T-202; Q-73 to Y-201; Q-73 to L-200; Q-73 to V-199; Q-73 to Q-198; Q-73 to G-197; Q-73 to Y-196; Q-73 to I-195; Q-73 to F-194; Q-73 to F-193; Q-73 to Y-192; Q-73 to G-191; Q-73 to T-190; Q-73 to E-189; Q-73 to K-188; Q-73 to V-187; Q-73 to L186; Q-73 to I-185; Q-73 to K-184; Q-73 to N-183; Q-73 to E-182; Q-73 to K-181; Q-73 to E-180; Q-73 to E-179; Q-73 to L-178; Q-73 to A-177; Q-73 to S-176; Q-73 to G-175; Q-73 to R-174; Q-73 to K-173; Q-73 to F-172; Q-73 to S-171; Q-73 to L-170; Q-73 to L-169; Q-73 to W-168; Q-73 to P-167; Q-73 to V-166; Q-73 to F-165; Q-73 to T-164; Q-73 to Y-163; Q-73 to S-162; Q-73 to G-161; Q-73 to K-160; Q-73 to Q-159; Q-73 to I-158; Q-73 to T-157; Q-73 to P-156; Q-73 to T-155; Q-73 to E-154; Q-73 to S-153; Q-73 to D-152; Q-73 to A-151; Q-73 to 1-150; Q-73 to L-149; Q-73 to Q-148; Q-73 to L-147; Q-73 to C-146; Q-73 to D-145; Q-73 to Q-144; Q-73 to T-143; Q-73 to V-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-S7; Q73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Q-73 to L-79.Polypeptide by these polynucleotide encodings also is encompassed in the present invention.The invention still further relates to the nucleic acid molecule that comprises or form by a kind of polynucleotide sequence, this polynucleotide sequence has at least 80% with the polynucleotide sequence of above-mentioned Neutrokine-α of coding and/or Neutrokine-α SV polypeptide, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny, the above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, these polypeptide are to comprise or by having at least 80% with above-mentioned aminoacid sequence, 85%, 90%, 92%, 95%, 96%, the polypeptide that the aminoacid sequence of 97%, 98% or 99% homogeny is formed, the polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
The present invention also provides to have disappearance and infers one or more amino acid whose polypeptide of extracellular domain aminoterminal and carboxyl terminal from Neutrokine-α, and it can be described as the n with SEQ ID NO:2 2-m 2Residue, wherein n 2And m 2Be integer as the aforementioned.
In another embodiment, the one nucleotide sequence coded polypeptide of forming by the part of Neutrokine-alpha amino acid sequence extracellular domain, Neutrokine-alpha amino acid sequence is plasmid-encoded by the cDNA of No.97768 among the ATCC, the part of described extracellular domain does not comprise the N-terminal 1-206 amino acids of Neutrokine-alpha amino acid sequence extracellular domain, or the 1-206 amino acids of carboxyl terminal, or the arbitrary combination of above-mentioned aminoterminal and carboxy terminal deletion.
As mentioned above, even the polypeptide N-terminal lacks one or more functionally active (being biological activity) that one or more amino acid causes losing polypeptide, other function or biological activity still can keep.Therefore, the Neutrokine-alpha muteins of shortening is induced and/or in conjunction with the ability of the antibody of identification total length or mature form polypeptide or its extracellular domain, when a small amount of residue of complete or mature polypeptide or its extracellular domain when N-terminal is removed, still can keep.Whether the special peptides that lacks complete polypeptide N-terminal residue keeps this immunocompetence, can determine by methods described herein and means known in the art.It is not impossible that the Neutrokine-alpha muteins that lacks a large amount of N-terminal amino-acid residues still keeps some functions (as biological or immunity) activity.In fact, also can excite immunne response usually by few peptide of forming as 6 Neutrokine-alpha amino acid residues.
Therefore, the invention provides the polynucleotide of a peptide species and this peptide species of coding, this polypeptide has disappearance from the full length amino acid sequence aminoterminal of inferring of Neutrokine-α shown in the SEQ ID NO:2 one or more residue until 280 glycine residues.Especially the invention provides and comprise sequence n shown in the SEQ ID NO:2 3The polypeptide of the aminoacid sequence that-285 residues are formed, wherein n 3It is the integer in the position of aminoacid sequence 1-280 amino-acid residue shown in the SEQ ID NO:2.
More particularly, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or is made up of the aminoacid sequence that is selected from next group: the D-2 to L-285 of SEQ ID NO:2; D-3 to L-285; S-4 to L-285; T-5 to L-285; E-6 to L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to L-285; R-11 to L-285; L-12 to L-285; T-13 to L-285; S-14 to L-285; C-15 to L-285; L-16 to L-285; K-17 to L-285; K-18 to L-285; R-19 to L-285; E-20 to L-285; E-21 to L-285; M-22 to L-285; K23 to L-285; L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; V-28 to L-285; S-29 to L-285; I-30 to L-285; L-31 to L-285; P-32 to L-285; R-33 to L-285; K-34 to L-285; E-35 to L-285; S-36 to L-285; P-37 to L-285; S-38 to L-285; V-39 to L-285; R-40 to L-285; S-41 to L-285; S-42 to L-285; K43 to L-285; D-44 to L-285; G-45 to L-285; K-46 to L-285; L-47 to L-285; L-48 to L-285; A-49 to L-285; A-50 to L-285; T-51 to L-285; L-52 to L-285; L-53 to L-285; L-54 to L-285; A-55 to L-285; L-56 to L-285; L-57 to L-285; S-58 to L-285; C-59 to L-285; C-60 to L-283; L-61 to L-285; T-62 to L-285; V-63 to L-285; V-64 to L-285; S-65 to L-285; F-66 to L-285; Y-67 to L-285; Q-68 to L-285; V-69 to L-285; A-70 to L-285; A-71 to L-285; L-72 to L-285; Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-283; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; 1-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; B-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; And G-280 to L-285.The invention still further relates to the nucleic acid molecule that comprises or form by a kind of polynucleotide sequence, this polynucleotide sequence has at least 80% with the polynucleotide sequence of above-mentioned Neutrokine-α of coding and/or Neutrokine-α SV polypeptide, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny, the above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, these polypeptide comprise with above-mentioned aminoacid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny, the polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
As mentioned above, cause protein to lose one or more functionally active (as biological activity) even c-terminal of protein lacks one or more amino acid, other functionally active still can keep.Therefore, when a small amount of residue of complete or mature polypeptide or extracellular domain when C-terminal is removed, the Neutrokine-alpha muteins of shortening is induced and/or the ability of the antibody of or mature polypeptide or extracellular domain complete in conjunction with identification still can keep.Lack the terminal special peptides of complete peptide C and whether keep this immunocompetence, can determine by methods described herein and other method known in the art.It is not impossible that the Neutrokine-alpha muteins that lacks a large amount of C-terminal amino-acid residues still keeps some functions (as biological or immunity) activity.In fact, also can excite immunne response usually by few peptide of forming as 6 Neutrokine-alpha amino acid residues.
Therefore, the invention provides have disappearance from the carboxyl terminal of Neutrokine-alpha amino acid sequence shown in the SEQ ID NO:2 until polypeptide at one or more residue of 6 L-glutamic acid, the polynucleotide of this peptide species of encoding also are provided.Especially the invention provides the 1-m that comprises SEQID NO:2 3The polypeptide of the aminoacid sequence that residue is formed, wherein m 3It is the integer-bit in the amino-acid residue of aminoacid sequence 6-284 shown in the SEQ IDNO:2 position.
More particularly, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or forms by being selected from next group aminoacid sequence: the M-1 to L-284 of SEQ ID NO:2; M-1 to K-283; M-1 to L-282; M-1 to A-281; M-1 to G-280; M-1 to F-279; M-1 to F-278; M-1 to T-277; M-1 to V-276; M-1 to D-275; M-1 to G-274; M-1 to D-273; M-1 to L-272; M-1 to S-271; M-1 to I-270; M-1 to Q-269; M-1 to A-268; M-1 to N-267; M-1 to E-266; M-1 to R-265; M-1 to P-264; M-1 to I-263; M-1 to A-262; M-1 to L-261; M-1 to Q-260; M-1 to L-259; M-1 to E-258; M-1 to D-257; M-1 to G-256; M-1 to E-255; M-1 to E-254; M-1 to L-253; M-1 to K-252; M-1 to A-251; M-1 to I-250; M-1 to G-249; M-1 to A-248; M-1 to S-247; M-1 to Y-246; M-1 to C-245; M-1 to S-244; M-1 to N-243; M-1 to N-242; M-1 to P-241; M-1 to L-240; M-1 to T-239; M-1 to E-238; M-1 to P-237; M-1 to M-236; M-1 to N-235; M-1 to Q-234; M-1 to I-233; M-1 to C-232; M-1 to R-231; M-1 to F-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-1 to L-226; M-1 to S-225; M-1 to L-224; M-1 to E-223; M-1 to D-222; M-1 to G-221; M-1 to F-220; M-1 to V-219; M-1 to H-118; M-1 to V-117; M-1 to K-216; M-1 to K-115; M-1 to R-114; M-1 to Q-113; M-1 to I-112; M-1 to L-211; M-1 to H-210; M-1 to G-209; M-1 to M-208; M-1 to A-207; M-1 to Y-206; M-1 to T-205; M-1 to K-204; M-1 to D-203; M-1 to T-202; M-1 to Y-201; M-1 to L-200; M-1 to V-199; M-1 to Q-198; M-1 to G-197; M-1 to Y-196; M-1 to 1-195; M-1 to F-194; M-1 to F-193; M-1 to Y-192; M-1 to G-191; M-1 to T-190; M-1 to E-189; M-1 to K-188; M-1 to V-187; M-1 to L-186; M-1 to I-185; M-1 to K-184; M-1 to N-183; M-1 to E-132; M-1 to K-181; M-1 to E-180; M-1 to E-179; M-1 to L-178; M-1 to A-177; M-1 to S-176; M-1 to G-175; M-1 to R-174; M-1 to K-173; M-1 to F-172; M-1 to S-171; M-1 to L-170; M-1 to L-169; M-1 to W-168; M-1 to P-167; M-1 to V-166; M-1 to F-165; M-1 to T-164; M-1 to Y-163; M-1 to S-162; M-1 to G-161; M-1 to K-160; M-1 to Q-159; M-1 to I-158; M-1 to T-157; M-1 to P-156; M-1 to T-155; M-1 to E-154; M-1 to S-153; M-1 to D-152; M-1 to A-151; M-1 to I-150; M-1 to L-149; M-1 to Q-148; M-1 to L-147; M-1 to C-146; M-1 to D-145; M-1 to Q-144; M-1 to T-143; M-1 to V-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-35; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to 1-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R-7; With M-1 to E-6.The invention still further relates to the nucleic acid molecule that comprises or form by a kind of polynucleotide sequence, this polynucleotide sequence has at least 80% with the polynucleotide sequence of above-mentioned Neutrokine-α of coding and/or Neutrokine-α SV polypeptide, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, this polypeptide comprises with above-mentioned aminoacid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny, the polynucleotide of this peptide species of encoding also are encompassed in the present invention.
The present invention also provides has disappearance one or more amino acid whose polypeptide from Neutrokine-α polypeptide aminoterminal and carboxyl terminal, and it can be described as the n with SEQ ID NO:2 3-m 3Position residue, wherein n 3And m 3Be integer as the aforementioned.
In addition, but since Neutrokine-α SV polypeptide of the present invention infer extracellular domain oneself excitation functionally active (as biological activity), the polypeptide of the Gln73-Leu266 position of SEQ ID NO:19 infer extracellular domain N and the C-terminal aminoacid deletion still can keep some functionally activies, as the part combination, stimulate lymphocyte (as the B cell) propagation, cellular replication is regulated in differentiation and/or activation, regulates target cell activity and/or immunocompetence.Yet, even lacking one or more amino acid, the N-terminal of inferring extracellular domain of Neutrokine-α SV polypeptide causes losing one or more functionally active, other functionally active still can keep.Therefore, the polypeptid induction of shortening and/or in conjunction with the ability of the antibody of identification complete or mature polypeptide or its extracellular domain, when a small amount of residue of ripe or complete polypeptide or extracellular domain when N-terminal is removed, still can keep.Whether the special peptides that lacks complete polypeptide N-terminal residue keeps this immunocompetence, can determine by ordinary method described herein and other method known in the art.
Therefore, the present invention also provides the polynucleotide of a peptide species and this peptide species of coding, and this polypeptide has disappearance from Neutrokine-α SV aminoacid sequence aminoterminal one or more residue until 261 glycine residues.Especially the invention provides the n that comprises SEQ ID NO:19 4The polypeptide of-266 aminoacid sequences, wherein n 4Be the integer-bit in the SEQ ID NO:19 aminoacid sequence 73-261 amino-acid residue position, and 261 is first residue position of inferring the extracellular domain N-terminal of Neutrokine-α SV (shown in SEQ ID NO:19) polypeptide.
More particularly, in certain embodiments, the invention provides the polynucleotide of coding one peptide species, this polypeptide comprises or is made up of the aminoacid sequence that is selected from next group: the Q-73 to L-266 of SEQ IDNO:19; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266, G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-203 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-233 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266; With G-261 to L-266.The invention still further relates to the nucleic acid molecule that comprises or be made up of a kind of polynucleotide sequence, this polynucleotide sequence has at least 80%, 85% with the polynucleotide sequence of the above-mentioned Neutrokine-α SV polypeptide of coding, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, this polypeptide comprises with above-mentioned aminoacid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny, the polynucleotide of this peptide species of encoding also are encompassed in the present invention.
Similarly, the C-terminal amino acid of inferring extracellular domain of Neutrokine-α SV can keep some functionally activies until the 79th leucic disappearance of SEQ ID NO:19, as the part combination, stimulate lymphocyte (as the B cell) propagation, differentiation and/or activation, regulate cellular replication, regulate target cell activity and/or immunogenicity.C-terminal with further SEQ ID NO:19 lacks the polypeptide that comprises the Leu79 disappearance and does not expect the energy retains biological activity.
Yet even one or more amino acid of peptide C terminal deletion causes polypeptide to lose one or more functionally active (as biological activity), other functionally active still can keep.Therefore, the polypeptid induction of shortening and/or the complete polypeptide of combination identification, the ability of the antibody of mature polypeptide or its extracellular domain is worked as maturation, and a small amount of residue of complete polypeptide or extracellular domain still can keep when C-terminal is removed.Whether the special peptides that disappearance is inferred the C-terminal residue of extracellular domain keeps this immunocompetence, can determine by ordinary method described herein and other method known in the art.
Therefore, the present invention further provides the polynucleotide of a peptide species and this peptide species of coding, this polypeptide have disappearance from the aminoacid sequence carboxyl terminal of inferring extracellular domain of Neutrokine-α SV shown in the SEQ ID NO:19 until 79 leucic one or more residues.Especially the invention provides 73-m with aminoacid sequence shown in the SEQ ID NO:19 4The polypeptide of the aminoacid sequence that the position residue is formed, wherein m 4It is an integer-bit in the 79-266 amino acids residue position of aminoacid sequence shown in the SEQ ID NO:19.
More particularly, in certain embodiments, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or is made up of the aminoacid sequence that is selected from next group: the Q-73 to K-264 of SEQ IDNO:19; Q-73 to L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to F-259; Q-73 to T-258; Q-73 to V-257; Q-73 to D-256; Q-73 to G-255; Q-73 to D-254; Q-73 to L-253; Q-73 to S-252; Q-73 to I-251; Q-73 to Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-247; Q-73 to R-246; Q-73 to P-240; Q-73 to I-244; Q-73 to A-243; Q-73 to L-242; Q-73 to Q-241; Q-73 to L-240; Q73 to E-239; Q-73 to D-238; Q-73 to G-237; Q-73 to E-236; Q-73 to E-235; Q-73 to L-234; Q-73 to K-233; Q-73 to A-232; Q-73 to I-231; Q-73 to G-230; Q-73 to A-229; Q-73 to S-228; Q-73 to Y-227; Q-73 to C-226; Q-73 to S-225; Q-73 to N-224; Q-73 to N-223; Q-73 to P-222; Q-73 to L-221; Q-73 to T-220; Q-73 to E-219; Q-73 to P-218; Q-73 to M-217; Q-73 to N-216; Q-73 to Q-215; Q-73 to I-214; Q-73 to C-213; Q-73 to R-212; Q-73 to F-211; Q-73 to L-210; Q-73 to T-209; Q-73 to V-208; Q-73 to L-207; Q-73 to S-206; Q-73 to L-205; Q-73 to E-204; Q-73 to D-203; Q-73 to G-202; Q-73 to F-201; Q-73 to V-200; Q-73 to H-199; Q-73 to V-198; Q-73 to K-197; Q-73 to K-196; Q-73 to R-195; Q-73 to Q-194; Q-73 to I-193; Q-73 to L-192; Q-73 to H-191; Q-73 to G-190; Q-73 to Q-73; Q-73 to A-188; Q-73 to Y-187; Q-73 to T-186; Q-73 to K-185; Q-73 to D-184; Q-73 to T-183; Q-73 to Y-182; Q-73 to L-181; Q-73 to V-180; Q-73 to Q-179; Q-73 to G-178; Q-73 to Y-177; Q-73 to I-176; Q-73 to F-175; Q-73 to F-174; Q-73 to Y-173; Q-73 to G-172; Q-73 to T-171; Q-73 to E-170; Q-73 to K-169; Q-73 to V-168; Q-73 to L-167; Q-73 to I-166; Q-73 to K-165; Q-73 to N-164; Q-73 to E-163; Q-73 to K-162; Q-73 to E-161; Q-73 to E-160; Q-73 to L-159; Q-73 to A-158; Q-73 to S-157; Q-73 to G-156; Q-73 to R-155; Q-73 to K-154; Q-73 to F-153; Q-73 to S-152; Q-73 to L-151; Q-73 to L-150; Q-73 to W-149; Q-73 to P-148; Q-73 to V-147; Q-73 to F-146; Q-73 to T-145; Q-73 to Y-144; Q-73 to S-143; Q-73 to G-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Q-73 to L-79; And Q-73 to S-78.The invention still further relates to the nucleic acid molecule that comprises or be made up of a kind of polynucleotide sequence, this polynucleotide sequence has at least 80%, 85% with the polynucleotide sequence of the above-mentioned Neutrokine-α SV polypeptide of coding, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, this peptide species contains with above-mentioned aminoacid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny, the polynucleotide of this peptide species of encoding also are encompassed in the present invention.
The present invention also provides has disappearance one or more amino acid whose polypeptide of inferring extracellular domain aminoterminal and carboxyl terminal from Neutrokine-α SV, and it can be described as the n with SEQ IDNO:19 4-m 4Position residue, wherein n 4And m 4Be integer as the aforementioned.
Another embodiment this, an one nucleotide sequence coded peptide species, this polypeptide is made up of the part of the extracellular domain of the Neutrokine-α SV aminoacid sequence of the cDNA clones coding of the preservation of ATCC preserving number 203518, the part of described extracellular domain does not comprise the N-terminal 1-260 amino acids of extracellular domain, or the 1-187 amino acids of extracellular domain carboxyl terminal, or the arbitrary combination of above-mentioned aminoterminal of extracellular domain and aminoterminal disappearance.
As mentioned above, cause losing one or more polypeptide functionally active (as biological activity) even the polypeptide N-terminal lacks one or more amino acid, other functionally active still can keep.Therefore, when a small amount of residue of total length or mature polypeptide or its extracellular domain when N-terminal is removed, the Neutrokine-α SV mutain of shortening is induced and/or is still kept in conjunction with the ability of the antibody of identification total length or mature polypeptide or its extracellular domain.Whether the special peptides that lacks complete polypeptide N-terminal residue keeps this immunocompetence, can determine by ordinary method described herein and other method known in the art.The Neutrokine-α SV mutain of a large amount of disappearance N-terminal amino-acid residues still reservation function (as immunogenicity) activity is not impossible.In fact, also can cause immunne response usually by few peptide of forming as 6 Neutrokine-α SV amino-acid residues.
Therefore, the invention provides the polynucleotide of a peptide species and this peptide species of coding, this polypeptide has disappearance from the full length amino acid sequence aminoterminal of inferring of Neutrokine-α SV shown in the SEQ ID NO:19 one or more residue until 261 glycine.Especially the invention provides the n that comprises sequence shown in the SEQ ID NO:19 5-266 aminoacid sequences that residue is formed, wherein m 5It is the integer-bit in the 1-261 amino-acid residue position of aminoacid sequence shown in the SEQ ID NO:19.
More particularly, the invention provides the polynucleotide of coded polypeptide, this polypeptide is selected from the aminoacid sequence with next group: the D-2 to L-266 of SEQ ID NO:19; D-3 to L-266; S-4 to L-266; T-5 to L-266; E-6 to L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to L-266; R-11 to L-266; L-12 to L-266; T-13 to L-266; S-14 to L-266; C-15 to L-266; L-16 to L-266; K-17 to L-266; K-18 to L-266; R-19 to L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to L-266; L-24 to L-266; K-25 to L-266; E-26 to L-266, C-27 to L-266; V-28 to L-266; S-29 to L-266; I-30 to L-266; L-31 to L-266; P-32 to L-266; R-33 to L-266; K-34 to L-266; E-35 to L-266; S-36 to L-266; P-37 to L-266; S-38 to L-266; V-39 to L-266; R-40 to L-266; S-41 to L-266; S-42 to L-266; K-43 to L-266; D-44 to L-266; G-45 to L-266; K-46 to L-266; L-47 to L-266; L-48 to L-266; A-49 to L-266; A-50 to L-266; T-51 to L-266; L-52 to L-266; L-53 to L-266; L-54 to L-266; A-55 to L-266; L-56 to L-266; L-57 to L-266; S-58 to L-266; C-59 to L-266; C-60 to L-266; L-61 to L-266; T-62 to L-266; V-63 to L-266; V-64 to L-266; S-65 to L-266; F-66 to L-266; Y-67 to L-266; Q-68 to L-266; V-69 to L-266; A-70 to L-266; A-71 to L-266; L-72 to L-266; Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-36 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; 1-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-223 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; U-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266; And G-261 to L-266.The invention still further relates to the nucleic acid molecule that comprises or form by polynucleotide sequence, this polynucleotide sequence has at least 80% with the polynucleotide sequence of above-mentioned Neutrokine-α of coding and/or Neutrokine-α SV polypeptide, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, these polypeptide have with above-mentioned aminoacid sequence and have at least 80%, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny, the polynucleotide of these polypeptide of encoding are also in the present invention.
As mentioned above, cause losing one or more protein function activity (as biological activity) even c-terminal of protein lacks one or more amino acid, other functionally active still can keep.Therefore, the Neutrokine-α SV mutain of shortening is induced and/or in conjunction with the ability of the antibody of identification complete or mature polypeptide or its extracellular domain, when a small amount of residue of complete or mature polypeptide or extracellular domain still can keep when C-terminal is removed.Whether the special peptides that lacks complete peptide C terminal residue keeps this immunocompetence, can determine by ordinary method described herein and other method known in the art.It is not impossible that the Neutrokine-α SV mutain of a large amount of disappearance C-terminal amino-acid residues still keeps some functions (as immunogenicity) activity.In fact, also can excite immunne response usually by few peptide of forming as 6 Neutrokine-α SV amino-acid residues.
Therefore, the invention provides the polynucleotide of a peptide species and this peptide species of encoding, this polypeptide have disappearance from the aminoacid sequence of Neutrokine-α SV shown in SEQ ID NO:19 carboxyl terminal until one or more residue at the 6th L-glutamic acid.Especially the invention provides the 1-m that comprises SEQ ID NO:19 5The polypeptide of residue aminoacid sequence, wherein m 5It is the integer-bit in the amino-acid residue of aminoacid sequence 6-265 shown in the SEQ IDNO:19 position.
More particularly, the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or forms by being selected from next group aminoacid sequence: the M-1 to G-265 of SEQ ID NO:19; M-1 to G-264; L-263; M-1 to A-262; M-1 to G-261; M-1 to F-260; M-1 to F-259; M-1 to T-258; M-1 to V-257; M-1 to D-256; M-1 to G-255; M-1 to D-254; M-1 to L-253; M-1 to S-252; M-1 to I-251; M-1 to Q-250; M-1 to A-249; M-1 to N-248; M-1 to E-247; M-1 to R-246; M-1 to R-245; M-1 to I-244; M-1 to A-243; M-1 to L-242; M-1 to Q-241; M-1 to L-240; M-1 to E-239; M-1 to D-238; M-1 to G-237; M-1 to E-236; M-1 to E-235,5 M-1 to L-234; M-1 to K-233; M-1 to A-232; M-1 to I-231; M-1 to G-230; M-1 to A-229; M-1 to S-228; M-1 to Y-227; M-1 to C-226; M-1 to S-225; M-1 to N-224; M-1 to N-223; M-1 to P-222; M-1 to L-221; M-1 to T-220; M-1 to E-219; M-1 to P-218; M-1 to M-217; M-1 to N-216; M-1 to Q-115; M-1 to I-214; M-1 to C-213; M-1 to R-112; M-1 to F-211; M-1 to L-210; M-1 to T-209; M-1 to V-208; M-1 to L-207; M-10 to S-206; M-1 to L-205; M-1 to E-204; M-1 to D-203; M-1 to G-202; M-1 to F-201; M-1 to V-200; M-1 to H-199; M-1 to V-198; M-1 to K-197; M-1 to K-196; M-1 to R-195; M-1 to Q-194; M-1 to I-193; M-1 to L-192; M-1 to H-191; M-1 to G-190; M-1 to M-189; M-1 to A-188; M-1 to Y-187; M-1 to T-186; M-1 to K-185; M-1 to D-184; M-1 to T-183; M-1 to Y-182; M-1 to L-181; M-1 to V-180; M-1 to Q-179; M-1 to G-178; M-1 to Y-177; M-1 to I-176; M-1 to F-175; M-1 to F-174; M-1 to Y-173; M-1 to G-172; M-1 to T-171; M-1 to E-170; M-1 to K-169; M-1 to V-168; M-1 to L-167; M-1 to 1-166; M-1 to K-165; M-1 to N-164; M-1 to E-163; M-1 to K-162; M-1 to E-161; M-1 to E-160; M-1 to L-159; M-1 to A-158; M-1 to S-157; M-1 to G-156; M-1 to R-155; M-1 to K-154; M-1 to F-153; M-1 to S-152; M-1 to L-151; M-1 to L-150; M-1 to W-149; M-1 to P-148; M-1 to V-147; M-1 to F-146; M-1 to T-145; M-1 to Y-144; M-1 to S-143; M-1 to G-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-11 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-110; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R-7; With M-1 to E-6.The invention still further relates to the nucleic acid molecule that comprises or be made up of polynucleotide sequence, this polynucleotide sequence has at least 80%, 85% with the polynucleotide sequence of the above-mentioned Neutrokine-α SV polypeptide of coding, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, these polypeptide comprise with above-mentioned aminoacid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny, the polynucleotide of these polypeptide of encoding are also in the present invention.
The present invention also provides has disappearance one or more amino acid whose polypeptide from Neutrokine-α SV polypeptide aminoterminal and carboxyl terminal, and it can be described as the n with SEQ ID NO:19 5-m 5Residue, wherein n 5And m 5It is above-mentioned integer.In other embodiments, the invention provides the 134-m that comprises SEQ ID NO:2 6The polypeptide of the aminoacid sequence of position residue, wherein m 6It is the integer in the residue of sequence 140-285 amino acids shown in the SEQ ID NO:2.For example the invention provides the polynucleotide of coded polypeptide, this polypeptide comprises or forms by being selected from next group aminoacid sequence: the A-134 to Leu-285 of SEQ ID NO:2; A-134 to L-284; A-134 to K-283; A-134 to L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-134 to F-278; A-134 to T-277; A-134 to V-276; A-134 to D-275; A-134 to G-274; A-134 to D-273; A-134 to L-272; A-134 to S-271; A-134 to I-270; A-134 to Q-269; A-134 to A-268; A-134 to N-267; A-134 to E-266; A-134 to R-265; A-134 to P-264; A-134 to I-263; A-134 to A-262; A-134 to L-261; A-134 to Q-260; A-134 to L-239; A-134 to E-258; A-134 to D-257; A-134 to G-256; A-134 to E-255; A-134 to E-254; A-134 to L-253; A-134 to K-252; A-134 to A-251; A-134 to I-250; A-134 to G-249; A-134 to A-248; A-134 to S-247; A-134 to Y-246; A-134 to C-245; A-134 to S-244; A-134 to N-243; A-134 to N-242; A-134 to P-241; A-134 to L-240; A-134 to T-239; A-134 to E-238; A-134 to P-237; A-134 to M-236; A-134 to N-235; A-134 to Q-234; A-134 to I-233; A-134 to C-232; A-134 to R-231; A-134 to F-230; A-134 to L-229; A-134 to T-228; A-134 to V-227; A-134 to L-226; A-134 to S-225; A-134 to L-224; A-134 to E-223; A-134 to D-222; A-134 to G-221; A-134 to F-220; A-134 to V-219; A-134 to H-218; A-134 to V-217; A-134 to K-216; A-134 to K-215; A-134 to R-214; A-134 to Q-213; A-134 to I-212; A-134 to L-211; A-134 to H-210; A-134 to G-209; A-134 to M-208; A-134 to A-207; A-134 to Y-206; A～134 are to T-205; A-134 to K-204; A-134 to D-203; A-134 to T-202; A-134 to Y-201; A-134 to L-200; A-134 to V-199; A-134 to Q-198; A-134 to G-197; A-134 to Y-196; A-134 to I-195; A-134 to F-194; A-134 to F-193; A-134 to Y-192; A-134 to G-191; A-134 to T-190; A-134 to E-189; A-134 to K-188; A-134 to V-187; A-134 to L-186; A-134 to I-185; A-134 to K-184; A-134 to N-183; A-134 to E-182; A-134 to K-181; A-134 to E-180; A-134 to E-179; A-134 to L-178; A-134 to A-177; A-134 to S-176; A-134 to G-175; A-134 to R-174; A-134 to K-173; A-134 to F-172; A-134 to S-171; A-134 to L-170; A-134 to L-169; A-134 to W-168; A-134 to P-167; A-134 to V-166; A-134 to F-165; A-134 to T-164; A-134 to Y-163; A-134 to S-162; A-134 to G-161; A-134 to K-160; A-134 to Q-159; A-134 to I-158; A-134 to T-157; A-134 to P-156; A-134 to T-155; A-134 to E-154; A-134 to S-153; A-134 to D-152; A-134 to A-151; A-134 to I-150; A-134 to L-149; A-134 to Q-148; A-134 to L-147; A-134 to C-146; A-134 to D-145; A-134 to Q-144; A-134 to T-143; A-134 to V-142; A-134 to T-141; And A-134 to E-140.The invention still further relates to the nucleic acid molecule that comprises or form by a kind of polynucleotide sequence, these polynucleotide have at least 80% with the polynucleotide sequence of above-mentioned Neutrokine-α of coding and/or Neutrokine-α SV polypeptide, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny.The above-mentioned polynucleotide sequence that is blended in the heterologous polynucleotide sequence has also been contained in the present invention.Polypeptide by these nucleic acid and/or polynucleotide sequence coding also is encompassed in the present invention, these polypeptide have with above-mentioned aminoacid sequence and have at least 80%, 85%, 90%, 92%, 95%, 96%, the aminoacid sequence of 97%, 98% or 99% homogeny, the polynucleotide of these polypeptide of encoding are also in the present invention.
Other preferred polypeptide fragment of the present invention comprises or forms by being selected from next group aminoacid sequence: the M-1 to C-15 of SEQ ID NO:2; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; A-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S～58 are to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-83 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-I17; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-103 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-110 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-123 to V-142; S-129 to T-143; R-130 to Q-144; N-131 to D-145; K-132 to C-146; R-133 to L-147; A-134 to Q-148; V-135 to L-149; Q-136 to I-150; G-137 to A-151; P-138 to D-152; E-139 to S-153; E-140 to E-154; T-141 to T-155; V-142 to P-156; T-143 to T-157; Q-144 to I-158; D-145 to Q-159; C-146 to K-160; L-147 to G-161; Q-148 to S-162; L-149 to Y-163; I-150 to T-164; A-151 to F-165; D-152 to V-166; S-153 to P-167; E-154 to W-168; T-155 to L-169; P-156 to L-170; T-157 to S-171; I-158 to F-172; Q-159 to K-173; K-160 to R-174; G-161 to G-175; S-162 to S-176; Y-163 to A-177; T-164 to L-178; F-165 to E-179; V-166 to E-180; P-167 to K-181; W-168 to E-182; L-169 to N-183; L-170 to K-184; S-171 to iI-185; F-172 to L-186; K-173 to V-187; R-174 to K-188; G-175 to E-189; S-176 to T-190; A-177 to G-191; L-178 to Y-192; E-179 to F-193; E-180 to F-194; K-181 to I-195; E-182 to Y-196; N-183 to G-197; K-184 to Q-198; I-185 to V-199; L-186 to L-200; V-187 to Y-201; K-188 to T-202; E-189 to D-203; T-190 to K-204; G-191 to T-205; Y-192 to Y-206; F-193 to A-207; F-194 to M-208; I-195 to G-209; Y-196 to H-210; G-197 to L-211; Q-198 to I-212; V-199 to Q-213; L-200 to R-214; Y-201 to K-215; T-202 to K-216; D-203 to V-217; K-204 to H-218; T-205 to V-219; Y-206 to F-220; A-207 to G-221; M-208 to D-222; G-209 to E-223; H-210 to L-224; L-211 to S-225; I-212 to L-226; Q-213 to V-227; R-214 to T-228; K-215 to L-229; K-216 to F-230; V-217 to R-231; H-218 to C-232; V-219 to I-233; F-220 to Q-234; G-221 to N-235; D-222 to M-236; E-223 to P-237; L-224 to E-238; S-225 to T-239; L-226 to L-240; V-227 to P-241; T-228 to N-242; L-229 to N-243; F-230 to S-244; R-231 to C-245; C-232 to Y-246; I-233 to S-247; Q-234 to A-248; N-235 to G-249; M-236 to 1-250; P-237 to A-251; E-238 to K-252; T-239 to L-253; L-240 to E-254; P-241 to E-255; N-242 to G-256; N-243 to D-257; S-244 to E-258; C-245 to L-259; Y-246 to Q-260; S-247 to L-261; A-248 to A-262; G249 to I-263; I-250 to P-264; A-251 to R-265; K-252 to E-266; L-253 to N-267; E-254 to A-268; E-255 to Q-269; G-256 to I-270; D-257 to S-271; E-258 to L-272; L-259 to D-273; Q-260 to G-274; L-261 to D-275; A-262 to V-276; I-263 to T-277; P-264 to F-278; R-265 to F-279; E-266 to G-280; N-267 to A-281; A-268 to L-282; Q-269 to K-283; I-270 to L-284; With S-271 to L-285.Preferably, these polypeptide fragments have one or more functionally active (as biological activity, antigenicity and immunogenicity) of Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide, and can for example be used for producing or screening antibody, and are as described below.The invention still further relates to and comprise or by having at least 80%, 85% with above-mentioned aminoacid sequence, the aminoacid sequence of 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny is formed.The above-mentioned aminoacid sequence that is blended in the allogeneic amino acid sequence has also been contained in the present invention.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Other preferred polypeptide fragment of the present invention comprises or forms by being selected from next group aminoacid sequence: the M-1 to C-15 of SEQ ID NO:19; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-43 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; A-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-73; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A～93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to G-142; S-129 to S-143; R-130 to Y-144; N-131 to T-145; K-132 to F-146; R-133 to V-147; A-134 to P-148; V-135 to W-149; Q-136 to L-150; G-137 to L-151; P-138 to S-152; E-139 to F-153; E-140 to K-154; T-141 to R-155; G-142 to G-156; S-143 to S-157; Y-144 to A-158; T-145 to L-159; F-146 to E-160; V-147 to E-161; P-148 to K-162; W-149 to E-163; L-150 to N-164; L-151 to K-165; S-152 to I-166; F-153 to L-167; K-154 to V-168; R-155 to K-169; G-156 to E-170; S-157 to T-171; A-158 to G-172; L-159 to Y-173; E-160 to F-174; E-161 to F-175; K-162 to I-176; E-163 to Y-177; N-164 to G-178; K-165 to Q-179; I-166 to V-180; L-167 to L-181; V-168 to Y-182; K-169 to T-183; E-170 to D-184; T-171 to K-185; G-172 to T-186; Y-173 to Y-187; F-174 to A-188; F-175 to M-189; I-176 to G-190; Y-177 to H-191; G-178 to L-192; Q-179 to I-193; V-180 to Q-194; L-181 to R-195; Y-182 to K-196; T-183 to K-197; D-184 to V-198; K-185 to H-199; T-186 to V-200; Y-187 to F-201; A-188 to G-202; M-189 to D-203 G-190 to E-204; H-191 to L-205; L-192 to S-206; I-193 to L-207; Q-194 to V-208; R-195 to T-209; K-196 to L-210; K-197 to F-211; V-198 to R-212; H-199 to C-213; V-200 to I-214; F-201 to Q-213; G-202 to N-216; D-203 to M-217; E-204 to P-218; L-205 to E-219; S-206 to T-220; L-207 to L-221; V-208 to P-222; T-209 to N-223; L-210 to N-224; F-211 to S-225; R-212 to C-226; C-213 to Y-227; I-214 to S-228; Q-215 to A-229; N-216 to G-230; M-217 to I-231; P-218 to A-232; E-219 to K-233; T-220 to L-234; L-221 to E-235; P-222 to E-236; N-223 to G-237; N-224 to D-238; S-225 to E-239; C-226 to L-240; Y-227 to Q-241; S-228 to L-242; A-229 to A-243; G-230 to I-244; I-231 to P-245; A-232 to R-246; K-233 to E-247; L-234 to N-24S; E-235 to A-249; E-236 to Q-250; G-237 to I-251; D-238 to S-252; E-239 to L-253; L-240 to D-254; Q-241 to G-250; L-242 to D-256; A-243 to V-257; I-244 to T-258; P-245 to F-259; R-246 to F-260; E-247 to G-261; N-248 to A-262; A-249 to L-263; Q-250 to K-264; I-251 to L-265; And S-252 to L-266.Preferably, these polypeptide fragments have one or more functionally active (as biological activity, antigenicity and immunogenicity) of Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide, and can for example be used for producing or screening antibody, and are as described below.The invention still further relates to and comprise or by having 80%, 85% the polypeptide that the aminoacid sequence of 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny is formed at least with above-mentioned aminoacid sequence.The above-mentioned aminoacid sequence that is blended in the allogeneic amino acid sequence has also been contained in the present invention.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Other preferred polypeptide fragment of the present invention comprises or forms by being selected from next group aminoacid sequence: the M-1 to F-15 of SEQ ID NO:38; D-2 to C-16; E-3 to S-17; S-4 to E-18; A-5 to K-19; K-6 to G-20; T7 to E-21; L-8 to D-22; P-9 to M-23; P-10 to K-24; P-11 to V-25; C-12 to G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to I-30; S-17 to T-31; E-18 to P-32; K-19 to Q-33; G-20 to K-34; E-21 to E-35; D-22 to E-36; M-23 to G-37; K-24 to A-38; V-25 to W-39; G-26 to F-40; Y-27 to G-41; D-28 to I-42; P-29 to C-43; I-30 to R-44; T-31 to D-45; P-32 to G-46; Q-33 to R-47; K-34 to L-48; E-35 to L-49; E-36 to A-50; G-37 to A-51; A-38 to T-52; W-39 to L-53; F-40 to L-54; G-41 to L-55; I-42 to A-56; C-43 to L-57; R-44 to L-58; D-45 to S-59; G-46 to S-60; R-47 to S-61; L-48 to F-62; L-49 to T-63; A-50 to A-64; A-51 to M-65; T-52 to S-66; L-53 to L-67; L-54 to Y-68; L-55 to Q-69; A-56 to L-70; L-57 to A-71; L-58 to A-72; S-59 to L-73; S-60 to Q-74; S-61 to A-73; F-62 to D-76; T-63 to L-77; A-64 to M-78; M-65 to N-79; S-66 to L-80; L-67 to R-81; Y-68 to M-82; Q-69 to E-83; L-70 to L-84; A-71 to Q-85; A-72 to S-86; L-73 to Y-87; Q-74 to R-88; A-75 to G-89; D-76 to S-90; L-77 to A-91; M-78 to T-92; N-79 to P-93; L-80 to A-94; R-81 to A-95; M-82 to A-96; E-83 to G-97; L-84 to A-98; Q-85 to P-99; S-86 to E-100; Y-87 to L-101; R-88 to T-102; G-89 to A-103; S-90 to G-104; A-91 to V-105; T-92 to K-106; P-93 to L-107; A-94 to L-108; A-93 to T-109; A-96 to P-110; G-97 to A-111; A-98 to A-112; P-99 to P-113; E-100 to R-114; L-101 to P-115; T-102 to H-116; A-103 to N-117; G-104 to S-118; V-105 to S-119; K-106 to R-120; L-107 to G-121; L-108 to H-122; T-109 to R-123; P-110 to N-124; A-111 to 125; A-112 to R-126; P-113 to A-127; R-114 to F-128; P-115 to Q-129; H-116 to G-130; N-117 to P-131; S-118 to E-132; S-119 to E-133; R-120 to T-134; G-121 to E-135; H-122 to Q-136; R-123 to D-137; N-124 to V-138; R-125 to D-139; R-126 to L-140; A-127 to S-141; F-128 to A-142; Q-129 to P-143; G-130 to P-144; P-131 to A-145; E-132 to P-146; E-133 to C-147; T-134 to L-148; E-135 to P-149; Q-136 to G-150; D-137 to C-151; V-138 to R-152; D-139 to H-153; L-140 to S-154; S-141 to Q-105; A-142 to H-156; P-143 to D-157; P-144 to D-158; A-145 to N-159; P-146 to G-160; C-147 to M-161; L-148 to N-162; P-149 to L-163; G-150 to R-164; C-151 to I-165; R-152 to I-166; H-153 to I-167; S-154 to Q-168; Q-155 to D-169; H-156 to C-170; D-157 to L-171; D-158 to Q-172; N-159 to L-173; G-160 to I-174; M-161 to 175; N-162 to D-176; L-163 to S-177; R-164 to D-178; N-165 to T-179; I-166 to P-180; I-167 to A-181; Q-163 to L-182; D-169 to E-183; C-170 to E-184; L-171 to K-185; Q-172 to E-186; L-173 to N-187; I-174 to K-188; A-175 to I-189; D-176 to V-191; S-177 to V-191; D-178 to R-192; T-179 to Q-193; P-180 to T-194; A-181 to G-195; L-182 to Y-196; E-183 to F-197; E-184 to F-198; K-185 to I-199; E-186 to Y-200; N-187 to S-201; K-188 to Q-202; I-189 to V-203; V-190 to L-204; V-191 to Y-205; R-192 to T-206; Q-193 to D-207; T-194 to P-203; G-195 to I-209; Y-196 to F-210; F-197 to A-211; F-198 to M-212; I-199 to G-213; Y-200 to H-214; S-201 to V-215; Q-202 to I-216; V-203 to Q-217; L-204 to R-218; Y-205 to K-219; T-206 to K-220; D-207 to V-221; P-208 to H-222; I-209 to V-223; F-210 to F-224; A-211 to G-225; M-212 to D-226; G-213 to E-227; H-214 to L-228; V-215 to S-229; I-216 to L-230; Q-217 to V-231; R-218 to T-232; K-219 to L-233; K-220 to F-234; V-221 to R-235; H-222 to C-236; V-223 to 1-237; F-224 to Q-238; G-225 to N-239; D-226 to M-240; E-227 to P-241; L-228 to K-242; S-229 to T-243; L-230 to L-244; V-231 to P-245; T-232 to N-246; L-233 to N-247; F-234 to S-248; R-235 to C-249; C-236 to Y-250; I-237 to S-250; Q-238 to A-252; N-239 to G-253; M-240 to I-254; P-241 to A-253; K-242 to R-256; T-243 to L-257; L-244 to E-253; P-245 to E-259; N-246 to G-260; N-247 to D-261; S-248 to E-262; C-249 to I-263; Y-250 to Q-264; S-251 to L-265; A-252 to A-266; G-253 to I-267; I-234 to P-268; A-255 to R-269; R-256 to E-270; L-257 to N-271; E-258 to A-272; E-259 to Q-273; G-260 to I-274; D-261 to S-275; E-262 to R-276; I-263 to N-277; Q-264 to G-278; L-265 to D-279; A-266 to D-280; I-267 to T-281; P-268 to F-282; R-269 to F-283; E-270 to G-284; N-271 to A-285; A-272 to L-286; Q-273 to K-287; I-274 to L-288; And S-275 to L-289.Preferably, these polypeptide fragments have one or more activity of Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide, and can for example be used for producing or screening antibody, and are as described below.The invention still further relates to and comprise or by having 80%, 85% the polypeptide that the aminoacid sequence of 90%, 92%, 95%, 96%, 97%, 98% or 99% homogeny is formed at least with above-mentioned aminoacid sequence.The above-mentioned aminoacid sequence that is blended in the allogeneic amino acid sequence has also been contained in the present invention.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Those skilled in the art recognize that some Neutrokine-α and Neutrokine-α SV amino acid sequence of polypeptide can change and not obvious structure or the function that influences polypeptide.If this sequence difference is expected, should remember to exist the critical area of determining polypeptide active.
Therefore, the present invention also provides Neutrokine-α variant polypeptides, and it presents the functionally active (as biological activity) of Neutrokine-α polypeptide or it comprises the zone of Neutrokine-α polypeptide, polypeptide fragment as described herein.The present invention also comprises Neutrokine-α SV variant polypeptides, and it presents the functionally active (as biological activity) of Neutrokine-α SV polypeptide, or it comprises the zone of Neutrokine-α SV polypeptide, polypeptide fragment as described herein.This mutant comprises the insertion of selecting according to general rule known in the art, disappearance, and conversion repeats and pattern replaces, and is therefore less to activity influence.For example, see Bowie about how producing the guidance that the phenotype silent amino acid replaces, J.U. etc., " information lexical or textual analysis in the protein sequence of tolerance aminoacid replacement ", science 247:1306-1310 (1990), wherein the author points out the tolerance of two kinds of main method research aminoacid sequences to changing.First method depends on evolutionary process, and wherein sudden change is accepted by natural selection or repelled.Second method is to carry out amino acid in the special position of cloned genes with genetically engineered to change, and selects or screen to differentiate to keep functional sequence.
As the author said, these researchs have confirmed that protein tolerates aminoacid replacement astoundingly.The author points out that also the amino acid variation allows in more proteinic positions.For example, most of hidden amino-acid residues require non-polar sidechain, and surface side chains is normally guarded.The reticent replacement of other this phenotype sees Bowie, J.U. etc., and as preceding, and reference is described shown in this paper.Conspicuous conservative replacement is aliphatic amino acid Ala, Val, the displacement between Leu and the Ile; Exchange between hydroxyl residue Ser and the Thr, the exchange between acidic residues Asp and the Glu, the replacement between amide residues Asn and the Gln, the exchange between alkaline residue Lys and the Arg, and aromatic residue Phe, the displacement between the Tyr.
Therefore, the fragment of the polypeptide that polypeptide or preservation cDNA shown in Figure 1A and the 1B (SEQ ID NO:2) are plasmid-encoded, derivative or analogue can be following a kind of: (i) wherein one or more amino-acid residue replaces (preferred conservative amino-acid residue) with conservative or nonconservative amino-acid residue, and the amino-acid residue of this replacement can or not encoded by genetic code; (ii) wherein one or more amino-acid residue comprises a substituting group; Or (iii) wherein the extracellular domain of polypeptide and another compound merge, as the compound (for example polyoxyethylene glycol) of transformation period of improving polypeptide; Or (iv) wherein other amino acid and polypeptide extracellular domain merge, as IgG FC corresponding circle of sensation peptide or leading or secretion sequence or be used for the sequence or the preceding protein sequence of purified polypeptide extracellular domain.This fragment, derivative and analogue are instructed according to this paper by those skilled in the art and are thought within the scope of the present invention.
In addition, shown in Fig. 5 A and the 5B (SEQ ID NO:19) or by the fragment of the plasmid-encoded polypeptide of preservation cDNA, derivative or analogue can be so a kind of: (i) wherein one or more amino-acid residue replaces with conservative or nonconservative amino-acid residue (preferred conservative amino-acid residue), and the amino-acid residue of this replacement can or not encoded by genetic code, or (ii) wherein one or more amino-acid residue comprise a substituting group, or (iii) wherein the extracellular domain of polypeptide and another compound merge, as improve the compound (for example polyoxyethylene glycol) of the transformation period of polypeptide, or the (iv) wherein extracellular domain of other amino acid and polypeptide fusion, solvable bioactive fragment as other tnf ligand family member (as the CD40 part), IgGFC corresponding circle of sensation peptide or leading or secretion sequence, or be used for the sequence or the preceding protein sequence of purified polypeptide extracellular domain.This fragment, derivative and analogue are instructed according to this paper by those skilled in the art and are thought within the scope of the present invention.
Therefore, Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide can comprise one or more aminoacid replacement, disappearance or interpolation, and it is through natural sudden change or artificial the manipulation.As mentioned above, change preferably lessly, replace (seeing Table 2) as not influencing protein folding or active conserved amino acid substantially.
Table 2 conserved amino acid replaces
Aromatic series Phenylalanine tryptophane tyrosine
Hydrophobicity Leucine Isoleucine Xie Ansuan
Polarity The glutamine l-asparagine
Alkalescence Arginine Methionin Histidine
Acid Aspartic acid L-glutamic acid
Small molecules L-Ala serine threonine methionine(Met) glycine
In an embodiment of the present invention, polypeptide comprises or is made up of Neutrokine-α and/or Neutrokine-α SV amino acid sequence of polypeptide, this Neutrokine-α and/or Neutrokine-α SV amino acid sequence of polypeptide contain at least 1 but be no more than 50, preferably be no more than 40, more preferably no more than 30, particularly preferably be no more than 20 conserved amino acids and replace.Certainly most preferably contain at least 1 but be no more than 10,9,8,7,6,5,4,3,2 or 1 conserved amino acids replace.
For example, the site-directed mutagenesis at the amino acid levels of Neutrokine-α can be by carrying out with the special amino acid of conservative substituting group displacement.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.Can carry out conventional screening Neutrokine-α and/or Neutrokine-α SV functionally active and/or physical properties (as stability and/or the solvability that strengthens or reduce) to gained Neutrokine-alpha protein of the present invention.Preferably, gained protein of the present invention has Neutrokine-α and/or the Neutrokine-α SV functionally active that improves and/or reduce.More preferably, gained Neutrokine-α of the present invention and/or Neutrokine-α SV albumen have Neutrokine-α and/or the Neutrokine-α SV functionally active and/or the physical properties of more than one raisings and/or reduction.
In another embodiment, the site-directed mutagenesis at Neutrokine-α SV amino acid levels can be by carrying out with conservative substituting group displacement specific amino acids.The preferred conservative replacement sudden change of the aminoacid sequence of Neutrokine-α SV shown in the SEQ ID NO:19 comprises: use A, G, I, L, S, T or V displacement M1; Replace D2 with E; Replace D3 with E; Use A, G, I, L, T, M or V displacement S4; Use A, G, I, L, S, M or V displacement T5; Replace E6 with D; With H or K displacement R7; Replace E8 with D; Replace Q9 with N; Use A, G, I, L, T, M or V displacement S10; With H or K displacement R11; Use A, G, I, S, T, M or V displacement L12; Use A, G, I, L, S, M or V displacement T13; Use A, G, I, L, T, M or V displacement S14; Use A, G, I, S, T, M or V displacement L16; With H or R displacement K17; With H or R displacement K18; With H or K displacement R19; Replace E20 with D; Replace E21 with D; Use A, G, I, L, S, T or V displacement M22; With H or R displacement K23; Use A, G, I, S, T, M or V displacement L24; With H or R displacement K25; Replace E26 with D; Use A, G, I, L, S, T or M displacement V28; Use A, G, I, L, T, M or V displacement S29; Use A, G, L, S, T, M or V displacement I30; Use A, G, I, S, T, M or V displacement L31; With H or K displacement R33; With H or R displacement K34; Replace E35 with D; Use A, G, I, L, T, M or V displacement S36; Use A, G, I, L, T, M or V displacement S38; Use A, G, I, L, S, T or M displacement V39; With H or K displacement R40; Use A, G, I, L, T, M or V displacement S41; Use A, G, I, L, T, M or V displacement S42; With H or R displacement K43; Replace D44 with E; Use A, I, L, S, T, M or V displacement G45; With H or R displacement K46; Use A, G, I, S, T, M or V displacement L47; Use A, G, I, S, T, M or V displacement L48; Use G, I, L, S, T, M or V displacement A49; Use G, I, L, S, T, M or V displacement A50; Use A, G, I, L, S, M or V displacement T51; Use A, G, I, S, T, M or V displacement L52; Use A, G, I, S, T, M or V displacement L53; Use A, G, I, S, T, M or V displacement L54; Use G, I, L, S, T, M or V displacement A55; Use A, G, I, S, T, M or V displacement L56; Use A, G, I, S, T, M or V displacement L57; Use A, G, I, L, T, M or V displacement S58; Use A, G, I, S, T, M or V displacement L61; Use A, G, I, L, S, M or V displacement T62; Use A, G, I, L, S, T or M displacement V63; Use A, G, I, L, S, T or M displacement V64; Use A, G, I, L, T, M or V displacement S65; With W or Y displacement F66; With F or W displacement Y67; Replace Q68 with N; Use A, G, I, L, S, T or M displacement V69; Use G, I, L, S, T, M or V displacement A70; Use G, I, L, S, T, M or V displacement A71; Use A, G, I, S, T, M or V displacement L72; Replace Q73 with N; Use A, I, L, S, T, M or V displacement G74; Replace D75 with E; Use A, G, I, S, T, M or V displacement L76; Use G, I, L, S, T, M or V displacement A77; Use A, G, I, L, T, M or V displacement S78; Use A, G, I, S, T, M or V displacement L79; With H or K displacement R80; Use G, I, L, S, T, M or V displacement A81; Replace E82 with D; Use A, G, I, S, T, M or V displacement L83; Replace Q84 with N; Use A, I, L, S, T, M or V displacement G85; With K or R displacement H86; With K or R displacement H87; Use G, I, L, S, T, M or V displacement A88; Replace E89 with D; With H or R displacement K90; Use A, G, I, S, T, M or V displacement L91; Use G, I, L, S, T, M or V displacement A93; Use A, I, L, S, T, M or V displacement G94; Use G, I, L, S, T, M or V displacement A95; Use A, I, L, S, T, M or V displacement G96; Use G, I, L, S, T, M or V displacement A97; With H or R displacement K99; Use G, I, L, S, T, M or V displacement A100; Use A, I, L, S, T, M or V displacement G101; Use A, G, I, S, T, M or V displacement L102; Replace E103 with D; Replace E104 with D; Use G, I, L, S, T, M or V displacement A105; Use G, I, L, S, T, M or V displacement A107; Use A, G, I, L, S, T or M displacement V108; Use A, G, I, L, S, M or V displacement T109; Use G, I, L, S, T, M or V displacement A110; Use A, I, L, S, T, M or V displacement G111; Use A, G, I, S, T, M or V displacement L112; With H or R displacement K113; Use A, G, L; S, T, M or V displacement I114; With W or Y displacement F115; Replace E116 with D; Use G, I, L, S, T, M or V displacement A119; Use A, I, L, S, T, M or V displacement G121; Replace E122 with D; Use A, I, L, S, T, M or V displacement G123; Replace N124 with Q; Use A, G, I, L, T, M or V displacement S125; Use A, G, I, L, T, M or V displacement S126; Replace Q127 with N; Replace N128 with Q; Use A, G, I, L, T, M or V displacement S129; With H or K displacement R130; Replace N131 with Q; With H or R displacement K132; With H or K displacement R133; Use G, I, L, S, T, M or V displacement A134; Use A, G, I, L, S, T or M displacement V135; Replace Q136 with N; Use A, I, L, S, T, M or V displacement G137; Replace E139 with D; Replace E140 with D; Use A, G, I, L, S, M or V displacement T141; Use A, I, L, S, T, M or V displacement G142; Use A, G, I, L, T, M or V displacement S143; With F or W displacement Y144; Use A, G, I, L, S, M or V displacement T145; With W or Y displacement F146; Use A, G, I, L, S, T or M displacement V147; With F or Y displacement W149; Use A, G, I, S, T, M or V displacement L150; Use A, G, I, S, T, M or V displacement L151; Use A, G, I, L, T, M or V displacement S152; With W or Y displacement F153; With H or R displacement K154; With H or K displacement R155; Use A, I, L, S, T, M or V displacement G156; Use A, G, I, L, T, M or V displacement S157; Use G, I, L, S, T, M or V displacement A158; Use A, G, I, S, T, M or V displacement L159; Replace E160 with D; Replace E161 with D; With H or R displacement K162; Replace E163 with D; Replace N164 with Q; With H or R displacement K165; Use A, G, L; S, T, M or V displacement I166; Use A, G, I, S, T, M or V displacement L167; Use A, G, I, L, S, T or M displacement V168; With H or R displacement K169; Replace E170 with D; Use A, G, I, L, S, M or V displacement T171; Use A, I, L, S, T, M or V displacement G172; With F or W displacement Y173; With W or Y displacement F174; With W or Y displacement F175; Use A, G, L; S, T, M or V displacement I176; With F or W displacement Y177; Use A, I, L, S, T, M or V displacement G178; Replace Q179 with N; Use A, G, I, L, S, T or M displacement V180; Use A, G, I, S, T, M or V displacement L181; With F or W displacement Y182; Use A, G, I, L, S, M or V displacement T183; Replace D184 with E; With H or R displacement K185; Use A, G, I, L, S, M or V displacement T186; With F or W displacement Y187; Use G, I, L, S, T, M or V displacement A188; Use A, G, I, L, S, T or V displacement M1 89; Use A, I, L, S, T, M or V displacement G190; With K or R displacement H191; Use A, G, I, S, T, M or V displacement L192; Use A, G, L; S, T, M or V displacement I193; Replace Q194 with N; With H or K displacement R195; With H or R displacement K196; With H or R displacement K197; Use A, G, I, L, S, T or M displacement V198; With K or R displacement H199; Use A, G, I, L, S, T or M displacement V200; With W or Y displacement F201; Use A, I, L, S, T, M or V displacement G202; Replace D203 with E; Replace E204 with D; Use A, G, I, S, T, M or V displacement L205; Use A, G, I, L, T, M or V displacement S206; Use A, G, I, S, T, M or V displacement L207; Use A, G, I, L, S, T or M displacement V208; Use A, G, I, L, S, M or V displacement T209; Use A, G, I, S, T, M or V displacement L210; With W or Y displacement F211; With H or K displacement R212; Use A, G, L; S, T, M or V displacement I214; Replace Q215 with N; Replace N216 with Q; Use A, G, I, L, S, T or V displacement M217; Replace E219 with D; Use A, G, I, L, S, M or V displacement T220; Use A, G, I, S, T, M or V displacement L221; Replace N223 with Q; Replace N224 with Q; Use A, G, I, L, T, M or V displacement S225; With F or W displacement Y227; Use A, G, I, L, T, M or V displacement S228; Use G, I, L, S, T, M or V displacement A229; Use A, I, L, S, T, M or V displacement G230; Use A, G, L; S, T, M or V displacement I231; Use G, I, L, S, T, M or V displacement A232; With H or R displacement K233; Use A, G, I, S, T, M or V displacement L234; Replace E235 with D; Replace E236 with D; Use A, I, L, S, T, M or V displacement G237; Replace D238 with E; Replace E239 with D; Use A, G, I, S, T, M or V displacement L240; Replace Q241 with N; Use A, G, I, S, T, M or V displacement L242; Use G, I, L, S, T, M or V displacement A243; Use A, G, L; S, T, M or V displacement I244; With H or K displacement R246; Replace E247 with D; Replace N248 with Q; Use G, I, L, S, T, M or V displacement A249; Replace Q250 with N; Use A, G, L; S, T, M or V displacement I251; Use A, G, I, L, T, M or V displacement S252; Use A, G, I, S, T, M or V displacement L253; Replace D254 with E; Use A, I, L, S, T, M or V displacement G255; Replace D256 with E; Use A, G, I, L, S, T or M displacement V257; Use A, G, I, L, S, M or V displacement T258; With W or Y displacement F259; With W or Y displacement F260; Use A, I, L, S, T, M or V displacement G261; Use G, I, L, S, T, M or V displacement A262; Use A, G, I, S, T, M or V displacement L263; With H or R displacement K264; Use A, G, I, S, T, M or V displacement L265; And/or use A, G, I, S, T, M or V displacement L266.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.Can carry out conventional screening Neutrokine-α and/or Neutrokine-α SV functionally active and/or physical properties (as stability and/or the solubility that strengthens or reduce) to gained Neutrokine-alpha protein of the present invention.Preferably, gained protein of the present invention has Neutrokine-α and/or the Neutrokine-α SV functionally active that improves and/or reduce.More preferably, gained Neutrokine-α of the present invention and/or Neutrokine-α SV protein have Neutrokine-α and/or the Neutrokine-α SV functionally active and/or the physical properties of more than one raisings and/or reduction.
In another embodiment, can be in the direct mutagenesis in the site of Neutrokine-alpha amino acid level by carrying out with the special amino acid of conservative substituting group displacement.The preferred conservative replacement sudden change of Neutrokine-alpha amino acid sequence shown in the SEQ ID NO:23 comprises: with H or K displacement R1; Use A, G, I, L, S, T or M displacement V2; Use A, G, I, L, S, T or M displacement V3; Replace D4 with E; Use A, G, I, S, T, M or V displacement L5; Use A, G, I, L, T, M or V displacement S6; Use G, I, L, S, T, M or V displacement A7; Use G, I, L, S, T, M or V displacement A10; Use A, G, I, S, T, M or V displacement L13; Use A, I, L, S, T, M or V displacement G15; With H or K displacement R17; With K or R displacement H18; Use A, G, I, L, T, M or V displacement S19; Replace Q20 with N; With K or R displacement H21; Replace D22 with E; Replace D23 with E; Replace N24 with Q; Use A, I, L, S, T, M or V displacement G25; Use A, G, I, L, S, T or V displacement M26; Replace N27 with Q; Use A, G, I, S, T, M or V displacement L28; With H or K displacement R29; Replace N30 with Q; With H or K displacement R31; Use A, G, I, L, S, M or V displacement T32; With F or W displacement Y33; Use A, G, I, L, S, M or V displacement T34; With W or Y displacement F35; Use A, G, I, L, S, T or M displacement V36; With F or Y displacement W38; Use A, G, I, S, T, M or V displacement L39; Use A, G, I, S, T, M or V displacement L40; Use A, G, I, L, T, M or V displacement S41; With W or Y displacement F42; With H or R displacement K43; With H or K displacement R44; Use A, I, L, S, T, M or V displacement G45; Replace N46 with Q; Use G, I, L, S, T, M or V displacement A47; Use A, G, I, S, T, M or V displacement L48; Replace E49 with D; Replace E50 with D; With H or R displacement K51; Replace E52 with D; Replace N53 with Q; With H or R displacement K54; Use A, G, L; S, T, M or V displacement I55; Use A, G, I, L, S, T or M displacement V56; Use A, G, I, L, S, T or M displacement V57; With H or K displacement R58; Replace Q59 with N; Use A, G, I, L, S, M or V displacement T60; Use A, I, L, S, T, M or V displacement G61; With F or W displacement Y62; With W or Y displacement F63; With W or Y displacement F64; Use A, G, L; S, T, M or V displacement I65; With F or W displacement Y66; Use A, G, I, L, T, M or V displacement S67; Replace Q68 with N; Use A, G, I, L, S, T or M displacement V69; Use A, G, I, S, T, M or V displacement L70; With F or W displacement Y71; Use A, G, I, L, S, M or V displacement T72; Replace D73 with E; Use A, G, L; S, T, M or V displacement I75; With W or Y displacement F76; Use G, I, L, S, T, M or V displacement A77; Use A, G, I, L, S, T or V displacement M78; Use A, I, L, S, T, M or V displacement G79; With K or R displacement H80; Use A, G, I, L, S, T or M displacement V81; Use A, G, L; S, T, M or V displacement I82; Replace Q83 with N; With H or K displacement R84; With H or R displacement K85; With H or R displacement K86; Use A, G, I, L, S, T or M displacement V87; With K or R displacement H88; Use A, G, I, L, S, T or M displacement V89; With W or Y displacement F90; Use A, I, L, S, T, M or V displacement G91; Replace D92 with E; Replace E93 with D; Use A, G, I, S, T, M or V displacement L94; Use A, G, I, L, T, M or V displacement S95; Use A, G, I, S, T, M or V displacement L96; Use A, G, I, L, S, T or M displacement V97; Use A, G, I, L, S, M or V displacement T98; Use A, G, I, S, T, M or V displacement L99; With W or Y displacement F100; With H or K displacement R101; Use A, G, L; S, T, M or V displacement I103; Replace Q104 with N; Replace N105 with Q; Use A, G, I, L, S, T or V displacement M106; With H or R displacement K108; Use A, G, I, L, S, M or V displacement T109; Use A, G, I, S, T, M or V displacement L110; Replace N112 with Q; Replace N113 with Q; Use A, G, I, L, T, M or V displacement S114; With F or W displacement Y116; Use A, G, I, L, T, M or V displacement S117; Use G, I, L, S, T, M or V displacement A118; Use A, I, L, S, T, M or V displacement G119; Use A, G, L; S, T, M or V displacement I120; Use G, I, L, S, T, M or V displacement A121; With H or K displacement R122; Use A, G, I, S, T, M or V displacement L123; Replace E124 with D; Replace E125 with D; Use A, I, L, S, T, M or V displacement G126; Replace D127 with E; Replace E128 with D; Use A, G, L; S, T, M or V displacement I129; Replace Q130 with N; Use A, G, I, S, T, M or V displacement L131; Use G, I, L, S, T, M or V displacement A132; Use A, G, L; S, T, M or V displacement I133; With H or K displacement R135; Replace E136 with D; Replace N137 with Q; Use G, I, L, S, T, M or V displacement A138; Replace Q139 with N; Use A, G, L; S, T, M or V displacement I140; Use A, G, I, L, T, M or V displacement S141; With H or K displacement R142; Replace N143 with Q; Use A, I, L, S, T, M or V displacement G144; Replace D145 with E; Replace D146 with E; Use A, G, I, L, S, M or V displacement T147; With W or Y displacement F148; With W or Y displacement F149; Use A, I, L, S, T, M or V displacement G150; Use G, I, L, S, T, M or V displacement A151; Use A, G, I, S, T, M or V displacement L152; With H or R displacement K153; Use A, G, I, S, T, M or V displacement L154; And/or use A, G, I, S, T, M or V displacement L155.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.Can carry out conventional screening Neutrokine-α and/or Neutrokine-α SV functionally active and/or physical properties (as stability and/or the solubility that strengthens or reduce) to gained Neutrokine-α of the present invention albumen.Preferably, gained protein of the present invention has Neutrokine-α and/or the Neutrokine-α SV functionally active that improves and/or reduce.More preferably, gained Neutrokine-α of the present invention and/or Neutrokine-α SV protein have Neutrokine-α and/or the Neutrokine-α SV functionally active and/or the physical properties of more than one raisings and/or reduction.
In another embodiment, can be in the direct mutagenesis in the site of Neutrokine-alpha amino acid level by carrying out with the special amino acid of conservative substituting group displacement.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.Can carry out conventional screening Neutrokine-α and/or Neutrokine-α SV functionally active and/or physical properties (as stability and/or the solubility that strengthens or reduce) to gained Neutrokine-α of the present invention albumen.Preferably, gained protein of the present invention has Neutrokine-α and/or the Neutrokine-α SV functionally active that improves and/or reduce.More preferably, gained Neutrokine-α of the present invention and/or Neutrokine-α SV protein have Neutrokine-α and/or the Neutrokine-α SV functionally active and/or the physical properties of more than one raisings and/or reduction.
For the amino acid in necessary Neutrokine-α of the present invention of function and/or the Neutrokine-α SV polypeptide can be differentiated by means known in the art, as direct mutagenesis in site or alanine scanning mutagenesis (Cunnirgham and Wells, science 244:1081-1085 (1989)).Back one method is that each residue carries out single alanine mutation in molecule.Test the functionally active of gained mutant then, as part combination and stimulation lymphocyte (as the B cell) propagation, differentiation and/or activatory ability.
Interested especially is that charged or neutral amino acids replaces charged amino acid with other, can produce to have the protein of required improved characteristics as low aggregation.Aggregation not only reduces activity, and is a problem when preparing formula of medicine, because aggregation can be immunogenic (Pinckard etc., clinical experiment immunology 2:331-340 (1967); Robbins etc., diabetes 36:838-845 (1987); Cleland etc., Crit.Rev.Therepeutic DrugCarrien Systems 10:307-377 (1993)).
In another embodiment, the invention provides and have the polypeptide that contains the aminoacid sequence of the non-conservative replacement of aminoacid sequence shown in the SEQ ID NO:2.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.Can carry out conventional screening Neutrokine-α described herein and known in the art and/or Neutrokine-α SV functionally active and/or physical properties (as stability and/or the solubility that strengthens or reduce) to gained Neutrokine-α of the present invention albumen.Preferably, gained protein of the present invention has Neutrokine-α and/or the Neutrokine-α SV functionally active that strengthens and/or reduce.More preferably, gained Neutrokine-α of the present invention and/or Neutrokine-α SV protein have Neutrokine-α and/or the Neutrokine-α SV functionally active and/or the physical properties of more than one raisings and/or reduction.
In another embodiment, Neutrokine-α polypeptide of the present invention comprises more than one (as 2,3,4,5,6,7,8,9,10,15,20,30 and 50) amino acid (conservative or non-conservation) with above-mentioned amino-acid substitution.
The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.Can carry out conventional screening Neutrokine-α described herein and known in the art and/or Neutrokine-α SV functionally active and/or physical properties (as stability and/or the solubility that strengthens or reduce) to gained Neutrokine-alpha protein of the present invention.Preferably, gained protein of the present invention has Neutrokine-α and/or the Neutrokine-α SV functionally active that strengthens and/or reduce.More preferably, gained Neutrokine-α of the present invention and/or Neutrokine-α SV protein have Neutrokine-α and/or the Neutrokine-α SV functionally active and/or the physical properties of more than one enhancings and/or reduction.
In another embodiment, Neutrokine-α polypeptide of the present invention comprises with above-mentioned substituted amino acid (conservative or nonconservative) metathetical more than (as 2,3,4,5,6,7,8,9,10,15,20,30 and 50) amino acid.
The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.Can carry out routine screening Neutrokine-α described herein and known in the art and/or Neutrokine-α SV functionally active and/or solubility to gained Neutrokine-alpha protein of the present invention.Preferably, gained protein of the present invention has Neutrokine-α and/or the Neutrokine-α SV functionally active that strengthens or reduce.More preferably, gained Neutrokine-α of the present invention and/or Neutrokine-α SV protein have Neutrokine-α and/or the Neutrokine-α SV functionally active and/or the physical properties of more than one enhancings or reduction.
In another embodiment, Neutrokine-α polypeptide of the present invention comprises with above-mentioned substituted amino acid (conservative or nonconservative) metathetical more than (as 2,3,4,5,6,7,8,9,10,15,20,30 and 50) amino acid.
The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.Can carry out conventional screening Neutrokine-α described herein and known in the art and/or Neutrokine-α SV functionally active and/or physical properties (as stability and/or the solubility that strengthens or reduce) to gained Neutrokine-alpha protein of the present invention.Preferably, gained protein of the present invention has Neutrokine-α and/or the Neutrokine-α SV functionally active that strengthens and/or reduce.More preferably, gained Neutrokine-α of the present invention and/or Neutrokine-α SV protein have Neutrokine-α and/or the Neutrokine-α SV functionally active and/or the physical properties of more than one enhancings and/or reduction.
In another embodiment, Neutrokine-α polypeptide of the present invention comprises with above-mentioned substituted amino acid (conservative or nonconservative) metathetical more than (as 2,3,4,5,6,7,8,9,10,15,20,30 and 50) amino acid.
Amino acid whose displacement also can change the selective binding of part and cell surface receptor.Ostade etc. for example, natural 361:266-268 (1993) has set forth some sudden changes and has caused TNF-alpha selective ground only to combine with one of two kinds of known TNF acceptors.Because Neutrokine-α and Neutrokine-α SV are TNF peptide family members, those sudden changes that are similar among the TNF-α have similar action in Neutrokine-α and/or Neutrokine-α SV.
Ligand-receptor bonded critical sites also can be definite by structural analysis, as crystallization, and nucleus magnetic resonance, or radiation affinity marker (Smith etc., molecular biology magazine 224:899-904 (1992) and Uos etc., science 255:306-312 (1992)).
Because Neutrokine-α is the member of TNF associated protein family, for the functionally active (as biological activity) of regulating rather than eliminate fully Neutrokine-α can suddenly change to the amino acid whose sequence in the conservative structural domain of coding TNF, described amino acid is the amino acid in the Gly191-Leu284 position shown in Figure 1A and the 1B (SEQ ID NO:2), preferably this zone is all inherent, great majority or some TNF family members are (as TNF-α, TNF-β, LT-3 and Fas part) in the amino-acid residue (seeing Fig. 2 A-B) that cannot not be conservative.Carry out special sudden change among the Neutrokine-α by the position of typically in relevant TNFs, finding at this conservative amino acid, the Neutrokine-alpha muteins will work as antagonist, thereby have inhibition lymphocyte (as the B cell) propagation, break up and/or activation.Therefore, polypeptide of the present invention comprises the Neutrokine-alpha-mutant.This Neutrokine-alpha-mutant comprises or by the total length of Neutrokine-alpha amino acid sequence shown in Figure 1A and the 1B (SEQ ID NO:2) or the fragment of preferred extracellular domain, variant or derivative are formed.The polynucleotide of above-mentioned Neutrokine-alpha-mutant of encoding also are encompassed in the present invention.
Because Neutrokine-α SV is the member of TNF associated protein family, for regulating rather than eliminate fully the functionally active of Neutrokine-α SV, can suddenly change to amino acid whose sequence in the coding TNF conserved domain, described amino acid is the amino acid in the Gly172-Leu265 position among Fig. 5 A and the 5B (SEQ IDNO:19), preferably this zone is all inherent, great majority or some TNF family members are (as TNF-α, TNF-β, LT-β and Fas part) in be non-conservative amino-acid residue (seeing Fig. 2 A-B).By carrying out special sudden change among the Neutrokine-α SV in this seed amino acid position that the typical case finds in relevant TNFs, Neutrokine-α SV mutain will work as antagonist, thereby have and for example suppress lymphocyte (as the B cell) propagation, differentiation and/or activation.Therefore, polypeptide of the present invention comprises Neutrokine-α SV mutant.This Neutrokine-α SV mutant comprises or by the total length of Neutrokine-α SV aminoacid sequence shown in Fig. 5 A and the 5B (SEQ ID NO:19) or the fragment of preferred extracellular domain, variant or derivative are formed.The polynucleotide of above-mentioned Neutrokine-α SV mutant of encoding also are encompassed in the present invention.
In addition, those skilled in the art recognize that the sudden change that is oriented to some zones of Neutrokine-α polypeptide of the present invention can influence the functionally active (as biological activity) that Neutrokine-α polypeptide is observed, and described zone is encompassed in undiscovered 19 amino-acid residue insertion sequences in the Neutrokine-α SV peptide sequence (being the Val142-Lys160 amino acids residue of sequence shown in Figure 1A and 1B and the SEQ ID NO:2).More particularly, this residue of the Neutrokine-α peptide sequence of orientable sudden change for example comprises the amino-acid residue of Neutrokine-α peptide sequence shown in the following SEQ ID NO:2: V142 without limitation; T143; Q144; D145; C146; L147; Q148; L149; I150; A151; D152; S153; E154; T155; P156; T157; I158; Q159; And K160.
Recombinant DNA method known in the art (see for example DNA reorganization, as described above) can be used for producing new mutant protein or mutain, comprise single or multiple aminoacid replacement, add or fusion rotein by disappearance.This modified polypeptides can present the stability that enhanced for example is active or improve.In addition, they under certain purifying and condition of storage, are compared with corresponding natural polypeptides at least, but high yield purifying and present solubility preferably.
Therefore, derivative and the analogue of Neutrokine-α and/or Neutrokine-α SV also contained in the present invention, they have one or more amino-acid residue disappearance, add or replacement, be more suitable in the host cell of selecting, expressing the Neutrokine-α of amplification and/or Neutrokine-α SV polypeptide with generation.For example, cysteine residues can lack or replace to eliminate disulfide linkage with other amino-acid residue; The glycosylation site that N connects can change or eliminate for example to reach the expression of homology product, and this homology product is easier to recovery and purifying from yeast host, and yeast host is known to have super glycosylation site.Therefore, various aminoacid replacement in Neutrokine-α and/or Neutrokine-α SV polypeptide on first or the 3rd amino acid position of one or more glycosylation recognition sequence or these two, and/or the aminoacid deletion on second amino acid position of one or more this recognition sequence, with stop Neutrokine-α and/or Neutrokine-α SV the tripeptide sequence glycosylation of modifying (referring to Miyajimo etc., EMBO magazine 5 (6): 1193-1197).
In addition, one or more amino-acid residue of polypeptide of the present invention (as arginine and lysine residue) can lack or replace with other residue, to eliminate undesirable processing by proteolytic enzyme such as furin or Kexins.A possible outcome of this sudden change is that Neutrokine-α polypeptide of the present invention is not cut and discharges from cell surface.
In a special embodiment, the Lys132 of Neutrokine-α sequence and/or Arg133 shown in the SEQ ID NO:2 sport other amino-acid residue or disappearance together, discharge from the cell of expressing Neutrokine-α with the Neutrokine-α that stops or reduce soluble form.In more special embodiment, the Lys132 of Neutrokine-α sequence sports Ala132 shown in the SEQ ID NO:2.In another embodiment, the Arg133 of the sequence of Neutrokine-α shown in the SEQ ID NO:2 sports Ala133.The protein of these sudden changes and/or these proteinic polynucleotide of encoding have the effect of external treatment for example or gene therapy, express the cell of the Neutrokine-α polypeptide that is retained in the through engineering approaches cell surface with through engineering approaches.
In a special embodiment, the Cys146 of Neutrokine-α sequence sports other amino-acid residue or disappearance shown in the SEQ ID NO:2, with the oligomerize that for example helps to stop or reduce Neutrokine-α polypeptide mutant when expressing in expression system (substantially as embodiment 1 as described in).In a special embodiment, Cys146 replaces with serine residue.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
In another special embodiment, the Cys232 of Neutrokine-α sequence sports other amino-acid residue or disappearance shown in the SEQ ID NO:2, with the oligomerize that helps to stop or reduce Neutrokine-α polypeptide mutant when expressing in expression system (substantially as embodiment 1 as described in).In a special embodiment, Cys232 replaces with serine residue.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
In another special embodiment, the Cys245 of Neutrokine-α sequence sports other amino-acid residue or disappearance shown in the SEQ ID NO:2, with the oligomerize that for example helps to stop or reduce Neutrokine-α polypeptide mutant when expressing in expression system (substantially as embodiment 1 as described in).In a special embodiment, Cys245 replaces with serine residue.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Polypeptide of the present invention is unpack format preferably, and basic purifying preferably.The Neutrokine-α of recombinant production and/or Neutrokine-α SV polypeptide can be by Smith and Johnson in the single stage method described in the gene 67:31-40 (1988) and basic purifying.
Polypeptide of the present invention comprises the complete polypeptide by the cDNA of preservation (ATCC preserving number 97768) coding, comprise born of the same parents' internal area by the cDNA encoded polypeptides of preservation, membrane-spanning domain and extracellular domain, ripe soluble polypeptide by the cDNA of preservation coding, proteinic extracellular domain deducts born of the same parents' internal area and membrane-spanning domain, the complete polypeptide of Figure 1A and 1B (the 1-285 amino acids residue of SEQ ID NO:2), the ripe solvable polypeptide of Figure 1A and 1B (the 134-285 amino acids residue of SEQ ID NO:2), the extracellular domain (the 73-285 amino acids residue of SEQ ID NO:2) of figure IA and 1B deducts born of the same parents' internal area and membrane-spanning domain, and have at least 80% with aforementioned polypeptides, 85%, 90%, preferably at least 95%, more preferably at least 96%, the polypeptide of 97%, 98% or 99% similarity.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Polypeptide of the present invention comprises the complete polypeptide by the cDNA of preservation (ATCC preserving number 203518) coding, comprise born of the same parents' internal area by the cDNA encoded polypeptides of preservation, membrane-spanning domain and extracellular domain, ripe soluble polypeptide by the cDNA of preservation coding, proteinic extracellular domain deducts born of the same parents' internal area and membrane-spanning domain, the complete polypeptide of Fig. 5 A and 5B (the 1-266 amino acids residue of SEQ ID NO:19), the extracellular domain of Fig. 5 A and 5B (the 73-266 amino acids residue of SEQ ID NO:2) deducts born of the same parents' internal area and membrane-spanning domain, and have at least 80%, 85%, 90% with aforementioned polypeptides, preferably at least 95%, more preferably at least 96%, 97%, 98% or the polypeptide of 99% similarity.The polynucleotide of these polypeptide of encoding also are encompassed in the present invention.
Other polypeptide of the present invention comprise with the cDNA (ATCC No.97768) of preservation coding or Figure 1A and 1B (SEQ ID NO:2) shown in polypeptide have at least 80% or 85%, preferably at least 90% or 95%, more preferably at least 96%, 97%, the polypeptide of 98% or 99% homogeny, have at least 30 that also comprise this peptide species, preferably at least 50 amino acid whose parts.The polynucleotide of this peptide species of encoding also are encompassed in the present invention.
Other polypeptide of the present invention comprise with the cDNA (ATCC No.203518) of preservation coding or Fig. 5 A and 5B (SEQ ID NO:19) shown in polypeptide have at least 80% or 85%, preferably at least 90% or 95%, more preferably at least 96%, 97%, the polypeptide of 98% or 99% homogeny, have at least 30 that also comprise this peptide species, preferably at least 50 amino acid whose parts.The polynucleotide of this peptide species of encoding also are encompassed in the present invention.
" similarity percentage " between two polypeptide is meant with Bestfit program (Wisconsin sequence analysis software bag, the 8 version genetics computer groups that are used for Unix, the university research garden, 575 Science Drive, Madison, WI53711) and set up two amino acid sequence of polypeptide of default parameters contrast, the similarity scope that produces to determine similarity.Bestit uses the local homologous sequence contrast (Advances in AppliedMathematics 2:482-498,1981) of Smith and Waterman, to find best similar section between two sequences.
The Neutrokine-α and/or the Neutrokine-α SV amino acid sequence of polypeptide that have with reference have for example polypeptide of the aminoacid sequence of at least 95% " homogeny ", be meant that this amino acid sequence of polypeptide and canonical sequence are identical, 5 changes at the most arranged except this peptide sequence can comprise per 100 amino acid with reference to Neutrokine-α and/or Neutrokine-α SV amino acid sequence of polypeptide.In other words, for obtaining to contain and the polypeptide that the aminoacid sequence of at least 95% homogeny is arranged with reference to aminoacid sequence, 5% amino-acid residue can lack or use other aminoacid replacement in the canonical sequence, or the amino acid that accounts for canonical sequence total amino acid residue several 5% can be inserted in the canonical sequence.These variations of canonical sequence can occur in the aminoterminal or the carboxyl terminal of canonical sequence, or occur in any position between these ends, are dispersed between each residue of canonical sequence or at one or more of canonical sequence continuously in the group.
In fact, whether any special polypeptide has at least 80% with following sequence, 85%, 90%, 95%96%, 97%, 98% or 99% homogeny, available known computer program such as Bestfit program (as preceding) are determined, described sequence is an aminoacid sequence shown in Figure 1A and the 1B (SEQ IDNO:2) for example, by cDNA clone HNEDU15 (ATCC NO.97768) amino acid sequence coded of preservation, or their fragment, or for example be aminoacid sequence shown in Fig. 5 A and the 5B (SEQID NO:19), by cDNA clone's HDPMC52 (ATCCNO.203518) amino acid sequence coded or their fragment of preservation.When with Bestfit or any other sequence contrast program when determining that whether a special sequence for example has at least 95% homogeny with canonical sequence of the present invention, certainly to set up parameter, on the total length canonical sequence, calculate the homogeny percentage like this, and allow the homology breach that accounts for reference sequences total amino acid residue several 5%.
In special embodiment, with reference to the homogeny between (reference) sequence (sequence of the present invention) and the target sequence, being also referred to as the contrast of whole sequence, is to use the correlated FASTDB computer program of sequence based on Brutlag etc. (Comp.App.Biosei.6:237-245 (1990) determines.Being used for the correlated preferred parameter of FASTDB aminoacid sequence is: Matrix=PAM 0, K-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or target sequence length are got shorter.According to this embodiment, if target sequence is owing to N or C-terminal disappearance but not because inherent disappearance is shorter than reference sequence, to manually adjust the result, because the FASTDB program reckons without the brachymemma of target sequence N and C-terminal when calculating whole homogeny per-cent.Just with respect to reference sequence, target sequence is in N and C-terminal brachymemma, the homogeny percentage by calculate not with the respective objects sequences match be that the residue number of the reference sequence of target sequence N and C-terminal is adjusted, as the per-cent of reference sequence total alkali radix.It is definite to determine whether a residue mates by FASTDB sequence comparing result.Then this percentage is deducted from the homogeny percentage that calculates with special parameter by the FASTDB program, obtain final homogeny percentage scope.This final homogeny percentage just can be used for the present invention.Have only the N and the C-terminal residue of target sequence not to mate, just need the artificial homogeny percentage of adjusting with reference sequence.Promptly has only reference sequence residue position outside the N farthest and C-terminal residue of target sequence.For example, the homogeny percentage is determined in the contrast of the reference sequence of the target sequence of 90 amino-acid residues and 100 residues.Disappearance occurs in the N-terminal of target sequence, thereby the FASTDB contrast does not demonstrate the coupling at preceding 10 residues of N-terminal.These 10 not paired residues are represented 10% (total residue number in residue number/reference sequence that N and C-terminal do not mate) of sequence, therefore this 10% are deducted from the homogeny per-cent that calculates through the FASTDB program.If remaining 90 residues fully mate, then final homogeny per-cent is 90%.In another embodiment, the reference sequence of the target sequence of 90 residues and 100 residues contrast.Current disappearance is inherent disappearance, therefore not have not the residue that mates with reference sequence at the N of target sequence and C-terminal.In the case, the homogeny percentage different people wage adjustment of calculating through FASTDB is put in order.Moreover, only be positioned at the outer residue of target sequence N and C-terminal and do not mate with reference sequence, shown in the contrast of FASTDB sequence, need artificial adjustment this moment.The present invention does not need to carry out other artificial adjustment.
The purposes of polypeptide of the present invention comprises but non-being limited on the SDS-PAGE gel or on molecular sieve gel filtration column, uses well known method as molecular weight marker.In addition, as detailed below, the purposes of polypeptide of the present invention comprises but non-being limited to produces polyclone and monoclonal antibody, be used to detect the expression of Neutrokine-α and/or Neutrokine-α SV, or as the agonist and the antagonist that can strengthen or suppress Neutrokine-α and/or Neutrokine-α SV function.Polypeptide of the present invention also has therapeutic action described as follows.In addition, to be used for yeast two-hybrid system also be that the Neutrokine-α and/or the Neutrokine-α SV of candidate's agonist of the present invention and antagonist is conjugated protein with " seizure " to this peptide species.This yeast two-hybrid system sees Fields and Song, and natural 340:245-246 (1989) is described.Transgenosis and " rejecting "
Polypeptide of the present invention also can be expressed in transgenic animal.Any species animal comprises but non-ly is limited to rat, mouse, and rabbit, hamster, cavy, pig, piggy, goat, sheep, milk cow and inhuman primate such as baboon, monkey and chimpanzee can be used for producing transgenic animal.In a special embodiment, methods described herein and other method known in the art are used for expressing polypeptide of the present invention in human body, as the part of gene therapy.
Any currently known methods in this area can be used for transgenosis (being polynucleotide of the present invention) is imported in the animal body, to produce the person's of foundation strain of transgenic animal.This method comprises but non-pronucleus microinjection (Paterson etc., the applied microbiology biotechnology 40:691-698 (1994) of being limited to; Carver etc., biotechnology (NY) 11:1263-1270 (1993); Wright etc., biotechnology (NY) 9:830-834 (1991); With Hoppe etc., U.S. Patent No. 4873191 (1989)); The transgenosis of retrovirus-mediated method to germ cell line (Vander putten etc., institute of American Academy of Sciences reports 82:6148-6152 (1985), among blastocyst or the embryo; Gene orientation in the embryonic stem cell (Thompson etc., cell 56:313-321 (1989)); Cell or embryo's electroporation (Lo, 1983, molecular cytobiology 3:1803-1814 (1983)); Import polynucleotide of the present invention with particle gun and (see Ulmer etc., science 259:1745 (1993); Import nucleic acid construct in embryo's multipotent stem cells and retract this stem cell in the blastocyst again; And transgenosis (Lavitrano etc., the cell 57:717-723 (1989) of sperm mediation; Deng.Summary to these methods is seen Gordon, " transgenic animal ", Int ' l Rev Cytol.115:171-229 (1989) is described, and it incorporates reference in full into.Also see U.S. Patent No. 5464764 (Capecchi etc., male/female system of selection and carrier); U.S. Patent No. 5631153 (Capecchi etc. contain cell and non-human being's body of predetermined genomic modification and produce their male/female system of selection and carrier); U.S. Patent No. 4736866 (Leder etc., transgenic nonhuman animal); With U.S. Patent No. 4873191 (Wagner etc., the genetic transformation of zygote); Above document is all incorporated reference in full into.
Any method known in the art all can be used for producing the transgene clone that contains polynucleotide of the present invention, for example, nucleus is moved to the embryo who induces to the cultivation of stationary state, fetus or become (Campell etc., natural 380:64-66 (1996) in the somatic enucleation oocyte; Wilmut etc., natural 385:810-813 (1997)).
The invention provides and in its all cells, all carry genetically modified transgenic animal, and at it some rather than all carry genetically modified animal in the cell, promptly inlay or chimaeric animals.This transgenosis can be used as single transgenosis or a plurality of copy such as concatermer for example head's series connection or the integration of series connection form end to end.Transgenosis also optionally imports in the particular cell types and activation therein, for example by instructions (Lasko etc., institute of American Academy of Sciences report 89:6232-6236 (1992)) such as Lasko.Decide for the required adjusting sequence of this cell type specificity activation depends on corresponding special cells type, and be that those skilled in the art institute is apparent.In the time the polynucleotide transgenosis need being integrated into the chromosome position of native gene, the preferred gene orientation.In brief, when making in this way, design contains with coming from the carrier of some nucleotide sequences of native gene, is integrated in the nucleotide sequence of native gene and destroys its function, and described carrier is by using the chromosome sequence homologous recombination.Transgenosis also optionally imports in the particular cell types, so the deactivation native gene in this cell type only, for example by instructions (Gu etc., science 265:103-106 (1994)) such as Gu.For the required adjusting sequence of this cell type specificity deactivation will depend on corresponding special cells type, and be that those skilled in the art institute is apparent.Except in transgenic animal, expressing the polypeptide of the present invention with omnipresence or tissue specificity mode, producing the construct (for example expression of developmental character or chemical adjusting) of regulating expression of polypeptides by various alternate manners, also is easy to do to those skilled in the art.
In case produced transgenic animal, can utilize standard method to analyze the expression of recombination.Just begin by Southern engram analysis or PCR screening, integrate with analyze animal tissue verification transgenosis and whether take place.Transgenosis determines that in the also available following method of the in-house mRNA expression level of transgenic animal described method comprises but the non-tissue sample that is limited to deriving from animal carries out the Northern engram analysis, hybridization analysis in the body, reversed transcriptive enzyme-PCR (rt-PCR); With TagMan PCR.The also available antibody that is specific to transgene product of the tissue sample of express transgenic carries out immunocytochemistry or immunohistochemistry evaluation.
In case produce the person of foundation animal, they can be bred, endogamy, out breeding or outbreeding are to produce particular animals group.This propagation method for example comprises but non-ly is limited to out breeding and has the person of foundation animal of an above integration site to set up isolating strain; The isolating strain of endogamy is to be created in the genetically modified transgenosis compound of high level expression, and high level expression is because due to the influence that each genetically modified expression adds up; The breeding heterozygote transgenic animal of intersecting produce the animal homozygote of given integration site to increase expression and the elimination need by DNA analysis screening animal; Intersect the isolating homozygote strain of breeding to produce heterozygote or homozygote product based compound; Transgenosis is bred in being suitable for the different background of trial model; And with transgenic animal breedings for carrying different transgenosiss or rejecting other animal of sudden change.
The purposes of transgenosis of the present invention and " rejecting " animal comprises but non-being limited to is used for setting forth Neutrokine-α and/or Neutrokine-α SV polypeptide biological function, research is expressed relevant disease with unusual Neutrokine-α and/or Neutrokine-α SV, and screens in the active compound that improves this disease as animal model system.
In other embodiments of the present invention, with genetically engineered expression or the cell of not expressing (as reject) polypeptide of the present invention be applied to the patient in vivo.This cell can derive from patient's (be animal, comprise the people) or the compatible donor of MHC, and can comprise but non-inoblast, medullary cell, hemocyte (as lymphocyte), adipocyte, myocyte, the endotheliocyte etc. of being limited to.Cell is genetically engineered in the external use recombinant DNA method, encoding sequence transfered cell with polypeptide of the present invention, perhaps destroy the encoding sequence of polypeptide of the present invention and/or associated endogenous and regulate sequence, as passing through transduction (use virus vector, preferably transgenosis is integrated into the carrier of cellular genome), or infection protocol comprises but non-being limited to used plasmid, clay, YACs, naked DNA, electroporation, liposome etc.The encoding sequence of polypeptide of the present invention can place under the control of strong composing type or inducible promoter or promotor/enhanser, to express preferred secretion polypeptide of the present invention.But the through engineering approaches cell general ground of expressing and preferably secreting polypeptide of the present invention imports in patient's body, as in circulation or intraperitoneal import.
Perhaps, but in this cell doped matrix and implant, can be used as the part of skin graft and implant as genetically engineered inoblast; Genetically engineered endotheliocyte can be used as the part of lymphatic vessel or blood vessel graft and implants and (see for example Anderson etc., U.S. Patent No. 5399349; With Mulligan and Wilson, U.S. Patent No. 5460959 is described, all incorporates reference into).
When the cell cell right and wrong of using self or non-MHC consistency, the host that organizes that they are known by use can be used the method that the cell that imports produces immunne response.For example, cell can capsule form import, and component is exchanged with born of the same parents' external environment immediately, and the cell of importing is discerned by host immune system.
Antibody
Other polypeptide of the present invention relates to antibody and T cell antigen receptor (TCR), its immunologic opsonin ground is in conjunction with the polypeptide of SEQ ID No:2 of the present invention and/or SEQ ID No:19, polypeptide fragment or variant, and/or epi-position (determining) by analysis specific antibody well known in the art-antigen bonded immunoassay.Antibody of the present invention comprises but the non-polyclonal antibody that is limited to, monoclonal antibody, multi-specificity antibody, people's antibody, humanized antibodies or chimeric antibody, single-chain antibody, the Fab fragment, F (ab ') fragment, by the fragment that the Fab expression library produces, the epi-position binding fragment of antiidiotype (anti-Id) antibody (antiidiotypic antibody that comprises anti-antibody of the present invention) and above any antibody.Term " antibody " is meant immunoglobulin molecules and immunocompetence part thereof, promptly contains the molecule of the antigen binding site of immunologic opsonin conjugated antigen.Immunoglobulin molecules of the present invention can be any kind (as IgG, IgE, IgM, IgD, IgA and IgY), classification (as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass immunoglobulin molecules.Immunoglobulin (Ig) can be heavy chain or light chain.A collection of IgG, IgE, IgM, IgD, IgA and IgY heavy chain can match with the light chain of κ or λ form.
Most preferred antibody is the antibody fragment in conjunction with the human antigen of the present invention, comprises but the non-Fab of being limited to Fab ', and F (ab ') 2, Fd, strand Fvs (scFv), the Fvs (sdFv) that single-chain antibody, disulfide linkage connect and comprise VL or the fragment of VH structural domain.The antibody fragment of conjugated antigen comprises single-chain antibody, can only comprise the variable region or comprises and the combination of all or part with lower area: hinge region, CH1, CH2 and CH3 structural domain.The present invention also comprises and also comprises variable region and hinge region, CH1, the fragment of the conjugated antigen of CH2 and CH3 structural domain arbitrary combination.Antibody of the present invention can comprise bird and Mammals from any animal.Preferably, antibody is the people, mouse (as rat and mouse), donkey, rabbit, goat, cavy, camel, horse or chicken.Used in the literary composition " people " antibody comprises the antibody of the aminoacid sequence with human normal immunoglobulin, and comprise separation from the human normal immunoglobulin library or to separate personal one or more human normal immunoglobulin genetically modified and do not express the antibody of the animal of endogenous immunoglobulin, it is described to reach U.S. Patent No. 5939598 such as Kucherlapati as previously mentioned.
Antibody of the present invention can be monospecific, dual specific, triple specificitys or multiple specific more.Multi-specificity antibody can be the different epi-positions that are specific to polypeptide of the present invention, maybe can be specific to polypeptide of the present invention and allos epi-position such as heterologous polypeptide or solid support.For example see PCT publication WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt etc., Journal of Immunology 147:60-69 (1991); U.S. Patent No. 4474893; 4714681; 5573920; 5601819; Kostelng etc., Journal of Immunology 148:1547-1553 (1992).
Antibody of the present invention can be set forth with terms such as the epi-position of their identification or the polypeptide of the present invention of specific combination or parts.The part of described epi-position or polypeptide can be for example by N-terminal and C-terminal position, the size of continuous amino acid residue, or list among table and the figure and set forth explanation.The antibody of any epi-position of specific combination the present invention or polypeptide also can be excluded.Therefore, the present invention includes the antibody of specific combination polypeptide of the present invention, and allow to get rid of identical antibody.
In special embodiment, antibody of the present invention combines with the polypeptide that comprises following aminoacid sequence: the Phe115-Leu147 of SEQ ID No:2, Ile150-Tyr163, Ser171-Phe194, the aminoacid sequence of Glu223-Tyr246 and Ser271-Phe278.In another special embodiment, antibody of the present invention combines with the polypeptide that comprises following aminoacid sequence: the Phe115-Leu147 of SEQ ID No:2, Ile150-Tyr163, Ser171-Phe194, the aminoacid sequence of Glu223-Tyr246 and Ser271-Phe278.In an embodiment preferred, antibody of the present invention combines with the polypeptide of the Glu223-Tyr246 that comprises SEQ ID No:2.In another embodiment preferred, antibody of the present invention combines with the polypeptide that Glu223-Tyr246 by SEQ ID No:2 forms.In a preferred embodiment, antibody of the present invention combines with the polypeptide that Phe230-Asn242 by SEQ ID No:2 forms.In another embodiment preferred, antibody of the present invention is by specificity one or more biological activity in conjunction with inhibition Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide.In a more preferred embodiment, antibody of the present invention suppresses the B cell proliferation of Neutrokine-α and/or Neutrokine-α SV mediation.
Antibody of the present invention can also its cross reactivity be set forth or is illustrated.The present invention includes any other analogue of debond polypeptide of the present invention, directly to homologue, or the antibody of homologue.The present invention also comprises in conjunction with having 95%, 90% with polypeptide of the present invention, the antibody of the polypeptide of 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50% homogeny (calculating with means known in the art and methods described herein) at least.In special embodiment, the rat of antibody of the present invention and human body protein, mouse and/or rabbit homologue and corresponding epi-position cross reaction thereof.The present invention comprises that also the homogeny of debond and polypeptide of the present invention is less than 95%, 90%, the antibody of the polypeptide of 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% (calculating with means known in the art and methods described herein).In a special embodiment, above-mentioned cross reactivity is about arbitrary specific antigenic or immunogenic polypeptide, or 2,3,4,5 or the combination of a plurality of specific antigenicity and/or immunogenic polypeptide.The present invention also comprises the antibody in conjunction with a peptide species, and this polypeptide is by the polynucleotide encoding of (as described herein) and multi-nucleotide hybrid of the present invention under hybridization conditions.Antibody of the present invention can also itself and polypeptide of the present invention binding affinity set forth or illustrated.Preferred binding affinity comprises that those have dissociation constant or Kd is less than 5 * 10 -5M, 10 -5M, 5 * 10 -6M, 10 -6M, 5 * 10 -7M, 10 -7M, 5 * 10 -8M, 10 -8M, 5 * 10 -9M, 10 -9M, 5 * 10 -10M, 10 -10M, 5 * 10 -11M, 10 -11M, 5 * 10 -12M, 10 -12M, 5 * 10 -13M, 10 -13M, 5 * 10 -14M, 10 -14M, 5 * 10 -15M, or 10 -15The antibody of M.
The present invention also provides competitive inhibition antibody and epi-position bonded antibody of the present invention, as determine immunoassay for example as herein described by any method of definite competitive bonded known in the art.In preferred embodiments, antibody competition inhibition and epi-position is combined with at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, or 50%.
Antibody of the present invention can be used as the agonist or the antagonist of polypeptide of the present invention.For example, the present invention includes part or all of destruction receptor/ligand and the interactional antibody of polypeptide of the present invention.Preferably, antibodies of the present invention antigenic epitopes as herein described or its part.The present invention includes receptor specific antibody and ligand specificity's antibody.The present invention also comprises the receptor specific antibody that does not stop the part combination but stop receptor activation.Receptor activation (being signal) can be determined by other method described herein or known in the art.For example, receptor activation can be determined that detection is carried out Western engram analysis (as described above) subsequently by immunoprecipitation and carried out by the phosphorylation (as tyrosine or serine/threonine) that detects acceptor or its substrate.In the special embodiment, the antibody that suppresses ligand activity or receptor active is provided, the specific activity that is suppressed does not have antibody and exists under the situation and suppress 95%, 90%, 85%, 80%, 75%, 70%, 60% at least, or 50%.
The present invention also comprises the receptor specific antibody that stops part combination and receptor activation, and identification receptor-ligand complex, the antibody of preferred not unconjugated acceptor of specific recognition or unconjugated part.The present invention also comprises binding partner and stops part and the neutralisation antibody of receptors bind, and binding partner, thereby stops receptor activation but do not stop the antibody of part and receptors bind.The present invention comprises the antibody of activated receptor in addition.These antibody can be used as receptor stimulant, promptly strengthen or activate the biological activity of all or part of ligand-mediated receptor activation, for example by inducing receptor dimerization to carry out.Antibody can be used as agonist, and antagonist or inverse agonist are illustrated its biological activity, comprises the special biological activity of peptide of the present invention.Above-mentioned antibody agonist can be produced with means known in the art.See that for example PCT announces WO92/40281; U.S. Patent No. 581109; Deng etc., blood 92 (6): 1981-1988 (1998); Cher etc., cancer research 58 (16): 3668-3678 (1998); Harrop etc., Journal of Immunology 161 (4): 1786-1794 (1998); Zhu etc., cancer research 58 (15): 3209-3214 (1998); Yoon etc., Journal of Immunology 160 (7): 3170-3179 (1998): Prat etc., cell science magazine 111 (Pt2): 237-247 (1998); Pitard etc., immunological method magazine 205 (2): 177-190 (1997); Liautard etc., cytokine 9 (4): 233-241 (1997); Carlson etc., journal of biological chemistry 272 (17): 11295-11301 (1997); Taryman etc., neurone 14 (4): 755-762 (1995); Muller etc., structure 6 (9): 1153-1167 (1998); , Bartunek etc., cytokine 8 (1): 14-20 (1996) (all documents are all incorporated reference in full into).
Antibody of the present invention can for example be used for but the non-purifying that is limited to, and detects and directed polypeptide of the present invention, comprises being used for external and in-vivo diagnostic and methods of treatment.For example, antibody can be used in the immunoassay quantitatively to reach the level of polypeptide of the present invention in the qualitative test biological sample.For example see Harlow etc., antibody laboratory manual (cold spring harbor laboratory publishes, second volume, 1988) (incorporate in full with reference to).
Following more detailed elaboration, antibody of the present invention can use separately or with other combination of compositions.Antibody can be blended in heterologous polypeptide at N or C-terminal, or chemically conjugated in (comprising covalency and non-covalent puting together) polypeptide or other composition.For example, antibody of the present invention can be recombinated and be merged or sew in molecule and effector molecule such as heterologous polypeptide, medicine, radionuclide or toxin as mark in the check and analysis.See that for example PCT announces WO 92/08495; WO91/14438; WO 89/12624; U.S. Patent No. 5314995; With EP 396387.
Antibody of the present invention comprises the derivative of modification, promptly is covalently attached to antibody by any kind molecule, and covalent attachment does not stop antibody generation antiidiotype to be replied like this.Antibody derivatives for example but non-being limited to comprise in the following manner the antibody of modifying: for example by glycosylation, acetylize, PEGization; phosphorylation, amidation is by the derivatize of known protection/blocking group; proteolytic cleavage is connected etc. with cell ligand or other protein.Can carry out any chemically modified by currently known methods, comprise but non-ly be limited to special chemical cracking, acetylize, formylation, the metabolism of tunicamycin is synthetic etc.In addition, derivative can contain one or more non-classical amino acid.
Antibody of the present invention can produce by any means known in the art.The polyclonal antibody of corresponding antigens can produce by the whole bag of tricks well known in the art.For example, uniqueness of the present invention can be applied to that various host animals comprise but non-ly be limited to rabbit, rat, mouse etc. contain the serum that is specific to antigenic polyclonal antibody and produce to induce.Can use various adjuvants to improve the rabbit immunne response according to host species, adjuvant comprises but the non-Freund of being limited to ' s (fully or not exclusively) adjuvant, inorganics gel such as aluminum oxide, tensio-active agent such as lysolecithin, how pure, polyanion, peptide, oil emulsion, keyhole chirp hemocyanin, dinitrophenol(DNP) and potential effective people's adjuvant such as BCG (bacille Calmette-Guerin vaccine) and short and small excellent bacillus.These adjuvants are well-known in the art.
Monoclonal antibody can comprise the use hybridoma with various means known in the art preparations, recombinates and the phage display method, or they are used in combination.For example, monoclonal antibody can produce with hybridoma method, this method comprise those methods known in the art and as Harlow etc., antibody laboratory manual (cold spring harbor laboratory publishes, and second rolls up 1988); Hammerling etc., (Elsevier N.Y.1981) instructs (described document is incorporated reference in full into) for monoclonal antibody and T quadroma 563-681.The non-antibody that produces by hybridoma method that is limited to of term " monoclonal antibody ".Term " monoclonal antibody " refers to comprise any eucaryon derived from monospecific polyclonal, the antibody of protokaryon or phage clone, rather than the method that refers to produce it.
" monoclonal antibody " can comprise or be made up of two kinds of protein, i.e. heavy chain and light chain.
Method with hybridoma method production and screening specific antibody is known in the art, and sees for details in (as embodiment 9) described in the embodiment.In a non-restrictive example, available uniqueness of the present invention or the cell inoculation of expressing this uniqueness.In case detect immunne response, be specific to antigenic antibody as in mouse serum, detecting, the spleen of mouse is taken and separating Morr. cell.Then splenocyte is blended in any suitable myeloma cell by well-known process, for example derives from the cell of the SP20 clone of ATCC.Select hybridoma also by restricted dilution clone.Analyzing hybridoma clone's secretion by means known in the art then can be in conjunction with the cell of the antibody of uniqueness of the present invention.Usually the ascites that contains high-level antibody can be by producing with positive hybridoma clone inoculation mouse.
Therefore, the invention provides the antibody that produces monoclonal antibody method and produce by this method, this method comprises the hybridoma of cultivating secretion antibody of the present invention, wherein preferably this hybridoma produces by merging splenocyte and myeloma cell, splenocyte separates the mouse of antigen immune inoculation personal of the present invention, screening derives from the hybridoma of syzygy then, selects the secretion can be in conjunction with the hybridoma clone of the antibody of uniqueness of the present invention.
The antibody fragment of identification specificity epi-position can produce by currently known methods.For example, Fab of the present invention and F (ab ') 2 fragments can produce by the proteolysis immunoglobulin molecules, and the enzyme of use is papoid (to produce the Fab fragment) in this way, or trypsin produces F (ab ') 2 fragments).F (ab ') 2 fragments contain the variable region, the CH1 structural domain of constant region of light chain and heavy chain.
For example, the also available various phage display methods known in the art of antibody of the present invention produce.In the phage display method, the functional antibodies structural domain is illustrated in the phage particle surface of carrying the polynucleotides encoding them sequence.In special embodiment, this phage can be used for the antigen binding domains of presenting and expressing from repertoire or combinatorial antibody library.Expression can be with antigen selection or discriminating in conjunction with the phage of the antigen binding domains of corresponding antigens, as with the antigen of mark in conjunction with or catch in the antigen of solid surface or pearl.The phage of using in these methods is typical filobactivirus, comprise fd and the M13 binding domains of expression from phage, this phage has reorganization and is blended in phage gene III or the proteinic Fab of gene VIII, the Fv antibody structure territory that Fv or disulfide linkage are stable.The phage display rule that can be used for production antibody of the present invention is as comprising below with reference to described those methods of document: as Brinkman etc., immunization method magazine 182:41-50 (1995); Ames etc., immunization method magazine 184:177-186 (1995); Kettleborough etc., European Journal of Immunology 24:952-958 (1994); Persic etc., gene 187:9-18 (1997); Burton etc., immunology progress 57:191-280 (1994); PCT applies for No.PCT/GB91/01134; PCT announces WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/20401; With U.S. Patent No. 5698426,5223409,5403484,5580717,5427908,5750753,5821047,5571698,5427908,5516637,5780225,5658727,5733743 and 5969108 is described, and above document is all incorporated reference in full into.
As described in above reference, after phage is selected, antibody coding region in the phage is separable and be used to produce whole antibody, comprise people's antibody, or any other required Fab, and can comprise mammalian cell any required host, insect cell, vegetable cell is expressed in yeast and the bacterium, as detailed below.For example, reorganization produces Fab, and Fab ' and F (ab ') 2 segmental methods also can be used by means known in the art, announces WO 92/22324 as PCT; Mullinax etc., biotechnology 12 (6): 864-869 (1992); With Sawai etc., AJRI 34:26-34 (1995); With Better etc., science 240:1041-1043 (1988) is described, and described document is all incorporated reference in full into.
The method that can be used for producing strand Fvs and antibody for example comprises United States Patent (USP) 4946778 and 5258498; Huston etc., Enzymology method 203:46-88 (1991); Shu etc., PNAS90:7995-7999 (1993); With SKerra etc., described those methods of science 240:1038-1040 (1988).For some application, be included in and use antibody and vitro detection analysis in the human body, preferably use chimeric, peopleization or people's antibody.Chimeric antibody is a kind of molecule, and wherein the different piece of antibody is derived from the animal of different plant species, has variable region and human normal immunoglobulin constant region derived from mouse monoclonal antibody as antibody.The method that produces chimeric antibody is known in the art.See for example Morrison, science 229:1202 (1985); Oi etc., biotechnology 4:214 (1986); Gillies etc. (1989), immunization method magazine 125:191-202; U.S. Patent No. 5807715,4816567 and 4816397 is described.The humanized antibodies is the antibody molecule from inhuman species antibody, and it is in conjunction with the required antigen of the framework region of one or more complementary determining region (CDR) with inhuman species and human normal immunoglobulin molecule.Usually, the framework residue in people's frame area will replace with the corresponding residue of CDR donor antibody, to change the combination of preferred improvement antigen.These frameworks replace by well known method to be differentiated, as by setting up the interaction model of CDR and framework residue, to differentiate antigen in conjunction with important framework residue, and carry out sequence contrast and (for example see Queen etc., U.S. Patent No. 5585089 to differentiate framework residue in the uniqueness of specific position; Riechmann etc., natural 332:323 (1988) incorporates reference in full into).Antibody can for example comprise CDR displacement (EP239400 with several different methods humanization known in the art; The PCT publication; / 09967 U.S. Patent No. 5225539,5530101 and 5585089), the Veneering or the (EP592106 that comes to the surface again; EP 519596; Padlan, molecular immunology 28 (4/5): 489-498 (1991); Studnicka etc., protein engineering 7 (6): 805-814 (1994); Roguska etc., PNAS 91:969-973 (1994)) and chain reorganization (U.S. Patent No. 5565332).
Fully human antibodies is that the treatment patient special procures.People's antibody can comprise the phage display method of the antibody library of above-mentioned use derived from human immunoglobulin sequences by produced in several ways known in the art.See U.S. Patent No. 4444887 and 4716111; And PCT publication WO 98/46645, WO 98/50433, and WO 98/24893, WO 9,8/1 6654, WO96/34096, WO 96/33735 and WO 91/10741 are described, and each document is all incorporated reference in full into.
The also available transgenic mouse of people's antibody produces, and this transgenic mouse can not the expressive function endogenous immunoglobulin, but can the expressing human immunoglobulin gene.For example, people's heavy chain and light chain immunoglobulin gene mixture can import at random or homologous recombination is gone in the mouse embryonic stem cell.Perhaps, except people's heavy chain and light chain gene, the people variable region, constant region and diversity region can import in the mouse embryonic stem cell.Mouse heavy chain and light chain immunoglobulin gene can non-functional separate or import human immunoglobulin gene's seat simultaneously by homologous recombination.In particular, the homozygous deletion in JH district stops endogenous antibody to produce.With the embryonic stem cell expansion of modifying and microinjection in the blastocyst to produce chimeric mouse.Breed the homozygote generation that chimeric mouse produces expressing human antibody then.With transgenic mouse with normal way with the antigen of selecting such as all or part of immunization of polypeptide of the present invention.Can the use by oneself transgenic mouse of conventional hybridization knurl method immunization of directly anti-antigenic monoclonal antibody.The human normal immunoglobulin transgenosis is reset during the B cytodifferentiation by transgenic mouse, carries out classification conversion and somatic mutation subsequently.Therefore, make the IgG that may produce therepic use in this way, IgA, IgM and IgE antibody.See Lonberg and Huszar about this general Study that produces the method for people's antibody, immunology research Int.13:65-93 (1995) is described.The scheme that produces the method for people's antibody and human monoclonal antibodies and produce this antibody sees for example PCT publication WO 98/24893 for details, and WO 92/01047, WO96/34096, and WO 96/33735; European patent No.0598877; U.S. Patent No. 5413923,5625126,5633425,5569825,5661016,5545806,5814318,5885793,5916771 and 5939598 is described, and above document is all incorporated reference in full into.In addition, Abgenix company (Freemont, CA) and Genpharm company (San Jose CA) can provide directly anti-antigenic people's antibody of selecting, uses to be similar to above-mentioned those methods and to produce.
The fully human antibodies of the epi-position of identification selection can produce with the method that is called " instruct and select ".In this method, the non-human monoclonal antibodies such as the murine antibody of selection are used to instruct the fully human antibodies (Jespers etc., biotechnology 12:899-903 (1988)) of selecting the identical epi-position of identification.
In addition, the antibody of polypeptide of the present invention can be used for producing the antiidiotypic antibody of " simulation " polypeptide of the present invention, use method well known in the art (to see for example Greenspan and Bona, FASEB magazine 7 (5): 437-444, (1989) and Nissinoff, Journal of Immunology 147 (8): 2429-2438 (1991)).For example, in conjunction with and competitive inhibition polypeptide polymerization and/or polypeptide of the present invention and part bonded antibody, can be used for producing " simulation " polypeptide polymerization and/or binding domains, and the therefore combination antiidiotype of neutralizationization polypeptide and/or its part also.In this and the Fab fragment of antiidiotypic antibody or this antiidiotypic antibody can be used in the treatment plan with in and polypeptide ligand.For example, this antiidiotypic antibody can be used in conjunction with polypeptide of the present invention and/or in conjunction with its ligand/receptor, thereby blocks its biological activity.
The polynucleotide of encoding antibody
The present invention also provides nucleotide sequence and the segmental polynucleotide thereof that comprise code book invention antibody.The present invention has also been contained under strict or low stringent hybridization condition (as previously mentioned), polynucleotide with the multi-nucleotide hybrid of encoding antibody, described antibody is preferably specifically in conjunction with polypeptide of the present invention, more preferably in conjunction with the polypeptide with aminoacid sequence shown in the SEQ ID NO:2.In another embodiment preferred, antibody is specifically in conjunction with the polypeptide with SEQ ID NO:19 aminoacid sequence.In another embodiment preferred, antibody is specifically in conjunction with the polypeptide with SEQ ID NO:23 aminoacid sequence.In another embodiment preferred, antibody is specifically in conjunction with the polypeptide with SEQ ID NO:28 aminoacid sequence.In another embodiment preferred, antibody is specifically in conjunction with the polypeptide with SEQ ID NO:30 aminoacid sequence.
Polynucleotide can obtain by any method known in the art, and the nucleotide sequence of polynucleotide can be determined by any method known in the art.For example, if the nucleotide sequence of known antibodies, the polynucleotide of encoding antibody can assemble oligonucleotide from chemosynthesis (as Kutmeier etc., biotechnology 17:242 (1994) is described), in brief, comprise the synthetic overlapping oligonucleotide that contains the sequence part of encoding antibody, anneal and connect these oligonucleotide, the oligonucleotide that connects through pcr amplification then.
Perhaps, the polynucleotide of encoding antibody can produce the nucleic acid from appropriate sources.If containing the clone of the nucleic acid of the specific antibodies of encoding can not obtain, but the sequence of known antibodies molecule, but the nucleic acid chemosynthesis of the immunoglobulin (Ig) of then encoding, or use can with specific gene sequence to be identified, for example from the cDNA clone's in the cDNA library of encoding antibody 3 ' and the synthetic primer of 5 ' terminal hybridization derive from appropriate sources or derive from appropriate sources (antibody cDNA library for example through the clone through pcr amplification with the oligonucleotide probe that is specific to this sequence, or produce from any tissue of expressing antibodies or cell as selecting to be used to express the cDNA library of the hybridoma of antibody of the present invention, or separate from any tissue of expressing antibodies or the nucleic acid of cell, preferred poly-A+RNA).Any well-known process in the available then this area of nucleic acid of the amplification that produces by PCR is cloned in the reproducible cloning vector.
In case determine the nucleotide sequence and the amino acid sequence corresponding of antibody, available well known method is handled the nucleotide sequence of antibody, as recombinant DNA method, site-directed mutagenesis, PCR etc. (see for example Sambrook etc., 1990, the molecular cloning laboratory manual, second edition, cold spring harbor laboratory, cold spring port, editor such as NY and Ausubel, 1998, molecular biology universal method, John Wiley and Sons, the described document of NY is incorporated reference in full into), antibody so that generation has the different aminoacids sequence for example produces aminoacid replacement, disappearance and/or insertion.
In a special embodiment, aminoacid sequence to heavy chain and/or light chain variable structural domain is checked, to differentiate the sequence of complementary determining region (CDR), by well known method inspection, as contrast the known amino acid sequence of other heavy chain and variable region of light chain, to determine the zone of sequence highly variable.Use conventional recombinant DNA method, one or more CDR can be inserted in the framework region, as inserting in people's framework region with the humanization non-human antibody, as previously mentioned.Framework region can be a framework region natural generation or total, and people's framework region (seeing for example Chothia etc., people's framework region that molecular biology magazine 278:457-479 (1998) is listed) preferably.Preferably, the antibody of the polynucleotide encoding specific combination polypeptide of the present invention that produces by group frame district and CDR.Preferably, as previously mentioned, in framework region, can carry out one or more aminoacid replacement, and preferably, this aminoacid replacement improvement antibody and its antigenic combination.In addition, this method can be used for producing the aminoacid replacement or the disappearance of one or more variable region cysteine residue that participates in intrachain disulfide bond, to produce the antibody molecule of one or more intrachain disulfide bond of disappearance.Other variation of polynucleotide also is encompassed in the present invention.
In addition, (Morrison etc., institute of American Academy of Sciences report 81:851855 (1984) can to use the method for production " chimeric antibody "; Neuberger etc., natural 312:604-608 (1984); Takeda etc., natural 314:452-454 (1985)), by the gene of montage, and from the gene of suitable bioactive human antibody molecules and carry out from the mouse antibody molecule of suitable antigen-specific.As previously mentioned, chimeric antibody is a kind of molecule, and wherein different piece has derived from the variable region of mouse mAb and the chimeric antibody of human normal immunoglobulin constant region, for example humanized antibodies as those derived from the different plant species animal.
Perhaps, can use the method (U.S. Patent No. 4946778 that produces single-chain antibody; Bird, science 242:423-42 (1988); Huston etc., institute of American Academy of Sciences report 85:5879-5883 (1988); With Ward etc., natural 334:544-54 (1989) produces single-chain antibody.Single-chain antibody is heavy chain and the light chain segments that meets the Fv district by the amino acid bridging, produces single-chain antibody.Also can use the segmental method of assembling function Fv in intestinal bacteria (Skerra etc., science 242:1038-1041 (1988)).
Produce the method for antibody
Antibody of the present invention can pass through any synthetic antibody known in the art, especially by chemosynthesis or preferably produce by recombinant expression method.Antibody of the present invention or its fragment, derivative or analogue (as the heavy chain or the light chain of antibody of the present invention, or single-chain antibody of the present invention) recombinant expressed requires to make up an expression vector that contains the polynucleotide of encoding antibody.In case obtained the polynucleotide of code book invention antibody molecule or its heavy chain or light chain or its part (part that preferably contains heavy chain or light chain variable structural domain), the carrier of producing antibody molecule can produce by recombinant DNA method with method well known in the art.Therefore, this paper has set forth the polynucleotide that contain antibody coding sequence by expression and has prepared method of protein.Available method well known in the art makes up the expression vector that contains antibody coding sequence and suitably transcribe and translate control signal.These methods for example comprise the extracorporeal recombinant DNA method, synthesis method, and genetic recombination in the body.Therefore the invention provides a kind of replicable vector, it comprises and operably is connected in promotor, code book invention antibody molecule or its heavy chain or light chain, or the nucleotide sequence of heavy chain or variable region of light chain.This carrier can comprise that the nucleotide sequence of encoding antibody molecule constant region (sees for example PCT publication WO 86/05807; PCT publication WO 89/01036; With U.S. Patent No. 5122464), and the variable domains of antibody can be cloned in this carrier to express whole heavy chains or light chain.
Expression vector is moved in the host cell by ordinary method, then cells transfected is cultivated to produce antibody of the present invention by ordinary method.Therefore, the present invention includes to contain and operably be connected in allogeneic promoter, code book invention antibody, its heavy chain or light chain, or the host cell of the polynucleotide of single-chain antibody of the present invention.In expressing the embodiment preferred of double-stranded antibody, the carrier of encoding heavy chain and light chain can be in host cell co expression, to express all immunoglobulins molecule, as described below.
Available various host expresses carrier system is expressed antibody molecule of the present invention.This host expression system is represented a kind of carrier, can produce corresponding coding sequence and subsequent purificn by it, but also represents a kind of cell, when with suitable nucleotide coding sequence conversion or transfection, but this cell expressed in situ antibody molecule of the present invention.These host expression systems comprise but non-ly are limited to microorganism, as with the recombinant bacteria phage DNA that contains antibody coding sequence, and the bacterium that plasmid DNA or cosmid DNA expression vector transform (for example intestinal bacteria, Bacillus subtilus); Yeast (as sugar yeast, pichia) with the recombinant yeast expression vector conversion that contains antibody coding sequence; Insect cell system with the recombinant virus expression vector that contains antibody coding sequence (as baculovirus) infection; With the recombinant virus expression vector that contains antibody coding sequence (as cauliflower mosaic virus, CaMV; Tobacco mosaic virus (TMV) TMV) infect, or with the recombinant plasmid expression vector that contains antibody coding sequence (as Ti-plasmids) plant transformed cell system; Have contain derived from the genomic promotor of mammalian cell (as metallothionein promoter) or derived from the promotor of mammalian virus (as gland virus stage starting; The mammal cell line system (as COS, CHO, BHK, 293,3T3 cell) of recombinant expression construct body vaccinia virus 7.5k promotor).Preferably, bacterial cell such as intestinal bacteria, more preferably, the eukaryotic cell of especially expressing whole recombinant antibody molecule is used for the expressing recombinant antibody molecule.For example, mammalian cell such as Chinese hamster ovary cell (CHO), uniting with carrier is the effective expression system of antibody, this carrier is the main middle sub-factor of early gene promoter (Foecking etc., the gene 45:101 (1986) of human cytomegalic inclusion disease virus in this way; Biology/technology such as Cockett 8: 2 (1990).
In bacterial system, used according to the antibody molecule of being expressed, can select many favourable expression vectors.For example, when producing a large amount of this protein, be the pharmaceutical composition that produces antibody molecule, need direct high level expression to be easy to the carrier of the fusion protein product of purifying.This carrier comprises but the non-coli expression carrier pUR278 (Ruther etc., EMBO magazine 2:1791 (1983)) that is limited to, and wherein in the independent carrier that connects in the framework with LacZ coding region of antibody coding sequence, produces fusion rotein; PIN carrier (Inouye and InouYe, nucleic acids research 13:3101-3109 (1985); Van Heeke and Schuster, journal of biological chemistry 24:5503-5509 (1989)) etc.The pGEX carrier also can be used for expressing as with the allogenic polypeptide of the fusion rotein of glutathione-S-transferase (GST).Normally, this albumen is soluble, and is easy to purifying from the cracked cell, by absorption and be incorporated into matrix gsh-sepharose 4B, the purifying there being wash-out under the situation of free glutathione subsequently.The pGEX carrier comprises zymoplasm or factor Xo proteolysis site, so that clone's target gene product can disengage from the GST component.
In the insect system, autographa california multiple nuclear polyhedrosis virus (AcNPV) is as the carrier of expression alien gene.This virus is grown in the fall army worm cell.Antibody coding sequence can be cloned into the nonessential region (for example polyhedron gene) of virus separately, and places under the control of AcNPV promotor (for example polyhedrin promotor).
In mammalian host cell, can use many expression systems based on virus.When adenovirus as under the situation of expression vector, corresponding antibody coding curve can be connected in adenovirus transcribes/translate and control mixture, as late promoter and tripartite leader[.This mosaic gene can insert in the adenoviral gene group by reorganization in external or the body then.Produce recombinant virus in the insertion of virus genomic nonessential region (as E1 district or E3 district), it survive, and can be in the host of infection expressed antibody molecule (for example see Logan and Shenk, institute of American Academy of Sciences reports 81:355-359 (1984)).Also can need special start signal to translate antibody coding sequence effectively.These signals comprise ATG initiator codon and adjacent sequence.In addition, initiator codon must have the frame of required encoding sequence, to guarantee that all inserting body all translates.These external source translation control signals and initiator codon can be various origins, as natural and synthetic.Expressing effectiveness can be by comprising the suitable transcriptional enhancer factor, and transcription terminator etc. are strengthened (seeing Bitther etc., Enzymology method 153:51-544 (1987)).
In addition, can select to regulate the expression of insertion sequence or modify and the host cell bacterial strain of processed gene product in special required mode.This modification (as glycosylation) of protein and processing (as cutting) are important to proteinic function.Different host cells has the special and special translation post-treatment and the mechanism of modifying protein and gene product.Can select suitable clone or host system to guarantee the correct foreign protein that to express of modifying and process.For this reason, can use to have correct processing primary transcript, make the eukaryotic host cell of the cell mechanism of gene product glycosylation and phosphorylation.This mammalian host cell comprises but the non-CHO of being limited to, VERY, BHK, Hela, COS, MDCK, 293,3T3, WI38, especially breast cancer cell line and BT 483, Hs 578T, HTB2, HT20, and T47D, and MCF-10 such as CRL 7030 and Hs 578Bst.
With regard to the extended high rate amount is produced recombinant protein, preferred stably express.For example, the clone of stably express antibody molecule can be genetically engineered.Unless use the expression vector that contains the virus replication origin, host cell can use the DNA by suitable expression controlling elements (as promotor, enhanser, sequence, transcription terminator, polyadenylation site etc.) and selectable marking of control to transform.After importing foreign DNA, the cell of through engineering approaches was grown in enriched medium 1-2 days, was converted to selective medium then.But the selective marker in the recombinant plasmid is given the selection resistance, and make cytotostatic ground with plasmid integration to its chromatin, and growth forms colony, can be cloned and extend to clone thereupon.This method can be used for the clone of genetically engineered expressed antibody molecule.The clone of this through engineering approaches can be used for screening especially and estimate direct or indirect and the interactional compound of antibody molecule.
Many selective systems can be used, and comprise but non-herpes simplex virus thymidine kinase (Wigler etc., cell 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (the Szybalska ﹠amp of being limited to; Szybalski, institute of American Academy of Sciences report 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy etc., cell 22:817 (1980) gene can be used for tk-respectively, hgprt-or aprt-cell.Equally, the metabolic antagonist resistance can be used as the basis of selecting following gene: dhfr, and it grants the methotrexate resistance, and (Wigler etc., institute of American Academy of Sciences report 77:357 (1980); O ' Hare etc., institute of American Academy of Sciences report 78:1527 (1981)); Gpt, it grants mycophenolic acid resistance (Mulligan ﹠amp; Berg, institute of American Academy of Sciences report 78:2072 (1981)); Neo, it grants aminoglycoside G418 resistance (clinical pharmacology 12:488-505; Wu and Wu, biotherapy 3:87-95 (1991); Tolstoshev, drug toxicity analysis and research 32:573-596 (1993); Mulligan, science 260:926-932 (1993); And Morgan and Anderson, biochemical analysis research 62:191-217 (1993); May, 1993, TIB, TECH 11 (5): 155-215); And hygro, it grants hygromycin resistance (Santerre etc., gene 30:147 (1984).The known recombinant DNA method of the present invention can conventional be used to select required recombinant clone, and this method is for example by Ausubel etc., (editor), molecular biology universal method, John Wiley ﹠amp; Sons, NY (1993); Kriegler, transgenosis and expression laboratory manual, Stockton press, NY (1990); Reach (editors) such as Dracopoli, Human genome is learned common method, the 12nd and 13 chapters, John Wiley ﹠amp; Sons, NY (1994); Colbene-Garapin etc., molecular biology magazine 150:1 (1981) is described, all incorporates reference in full at this.
The expression level of antibody molecule can improve by carrier amplification and (referring to Bebbington, with Hentschel, uses the gene based on carrier cloning by expression in mammalian cell of gene amplification, dna clone, the 3rd volume (Science Press, New York, 1987)).When the mark in the carrier system of expressing antibodies be can increase the time, inhibitor level in the host cell culture improves the copy number that will increase marker gene.Because the zone of amplification is relevant with antibody gene, so antibody production also increases (Crouse etc., molecular cytobiology 3:257 (1983)).
Host cell can be with two kinds of common transfections of expression vector of the present invention, first kind of vector encoded heavy chain polypeptides derived, the polypeptide of second kind of vector encoded derived light chain.These two kinds of carriers can contain the mark of identical selection, and this mark can identical expression heavy chain and light chain polypeptide.Perhaps can use one he, this vector encoded and can express heavy chain and light chain polypeptide.In this case, light chain should place before the heavy chain, to avoid the free heavy chain of toxicity too much (Proudfoot, natural 322:52 (1986); Kohler, institute of American Academy of Sciences report 77:2197 (1980).The encoding sequence of heavy chain and light chain can comprise cDNA or genomic dna.
In case antibody molecule of the present invention has passed through animal, chemosynthesis, or recombinant expressed and produce, its can be by any method of purifying immunoglobulin molecules known in the art purifying, for example, by chromatography (ion exchange chromatography for example, affinity chromatography is especially by the specific antigen affinity chromatography after albumin A, and big or small column chromatography), centrifugal, different solvability, or the standard method by any other protein purification.In addition, antibody of the present invention or its fragment can be blended in other heterologous sequence described herein or known in the art, to promote purifying.
The present invention has been contained that reorganization is merged or is chemically conjugated in the antibody of (comprising covalency and non-covalent conversion) polypeptide of the present invention (or its part, preferably at least 10,20,30,40,50,60,70,80,90 or 100 amino acid whose polypeptide), to produce fusion rotein.Fusion needs not to be directly, also can take place by joint sequence.Antibody can be specific to the antigen that is different from polypeptide of the present invention (or its part, preferably at least 10,20,30,40,50,60,70,80,90 or 100 amino acid whose polypeptide).For example, antibody be used in external or body in polypeptide of the present invention is oriented to the specific type cell, by polypeptide of the present invention being merged or puting together in the antibody that is specific to the special cells surface receptor and carry out.Merge or put together and also can be used for external immunoassay and purification process in the antibody of polypeptide of the present invention.See for example Harbor etc., as preceding and PCT publication WO 93/21232; EP 439095; Naramura etc., immunology communication, 39:91-99 (1994); United States Patent (USP) 5474981; Gillies etc., PNAS89:1428-1432 (1992); Fecl etc., Journal of Immunology 146:2446-2452 (1991); All incorporate reference in full at this.
The present invention also comprises a kind of composition, and said composition comprises fusion or puts together in the polypeptide of the present invention in other antibody structure territory except that the variable region.For example, polypeptide of the present invention can be blended in or put together the Fc zone in antibody, or its part.The antibody moiety that is blended in polypeptide of the present invention can comprise constant region, hinge region, CH1 structural domain, CH2 structural domain and CH3 structural domain, or the arbitrary combination of all structural domains or its part.Polypeptide also can merge or put together in an above-mentioned antibody part to form polymer.For example, the Fc part that is blended in polypeptide of the present invention can form dimer by the disulfide linkage between the Fc part.Higher polymer form can produce by the part fusion of polypeptide and IgA and IgM.Known in the artly polypeptide of the present invention is merged or put together method in antibody.See for example U.S. Patent No. 5336603; 5622929; 5359046; 5349053; 5447851; 5112946; EP 307434; EP367166; PCT publication WO 96/04388; WO 91/06570; Ashkenazi etc., institute of American Academy of Sciences report 88:10535-10539 (1991); Zheng etc., Journal of Immunology 154:5590-5600 (1995); With Vil etc., institute of American Academy of Sciences reports 89:11337-11341 (1992) described, all incorporates reference in full at this.
As previously mentioned, corresponding to SEQ ID NO:2 polypeptide, the polypeptide of polypeptide fragment or its variant can merge or put together in above-mentioned antibody moiety, with the transformation period in the body that improves polypeptide, or is used for immunoassay.In addition, be equivalent to the polypeptide of SEQ ID NO:19, can merge or put together in above-mentioned antibody moiety to promote purifying.For example one piece of report has been set forth the chimeric protein of being made up of the various structural domains of the constant region of the light chain of preceding two structural domains of people CD4-polypeptide and mammalian immune sphaeroprotein or heavy chain.(EP 394827; Traunecker etc., natural 331:84-86 (1988)).Merge or put together in the polypeptide of the present invention of antibody with dimeric structure (because due to IgG) that disulfide linkage connects, also comparable monomeric secretory protein or its fragment be combination and other molecule that neutralizes (Fountoulakis etc., journal of biological chemistry 270:3958-3964 (1995) more effectively.In many cases, the Fc part in the fusion rotein is favourable to treatment and diagnosis, therefore can for example improve pharmacokinetic property (EP A232262).Perhaps, can express disappearance Fc part behind detection and the purifying at fusion rotein.For example, if fusion rotein is used as the antigen of immunization, the Fc part can hinder treatment and diagnosis.In drug development, for example human body protein such as hIL-5 are to differentiate the antagonist of hIL-5 with the Fc meromixis to carry out the high throughput screening analysis.(see Bennett etc., molecular recognition magazine 8:52-58 (1995); Johanson etc., journal of biological chemistry 270:9459-9471 (1995).
In addition, antibody of the present invention or its fragment can be blended in flag sequence, as merging to promote purifying with peptide.In preferred such scheme, marker amino acid sequence is 6 Histidine peptides, as the mark (QIAGEN, Inc.9259 Eton Avenue, Chatsworth, CA, 91311) that provides in the pQE carrier, wherein many can being purchased.As Gentz etc., institute of American Academy of Sciences reports 86:821-824 (1989), and is described, and 6 Histidines can conventional purified fusion protein.Be used for other of purifying peptide-labeled comprise but non-being limited to " HA " mark, it is equivalent to epi-position (Wilson etc., cell 37:767 (1984)) and " flag " mark derived from influenza hemagglutinin protein.
Antibody or its fragment of puting together in diagnosis or therapeutical agent also contained in the present invention.This antibody for example is used to diagnosticability monitor the growth or the progress of tumour, as the part of clinical trial, for example to determine the effect of treatment plan.Antibody coupling can be convenient to detect in detectable material.Detectable material for example comprises various enzymes, prothetic group, and fluorescence, shiner, the noclilucence thing, radioactive substance uses the positron emitting metal and the on-radiation paramagnetic metal ion of various positron emission emission computer tomography developings.Detectable material can use the direct coupling of means known in the art or put together in antibody (or its fragment), or indirectly by intermediate (joint as known in the art) coupling or put together in antibody (or its fragment).See that for example U.S. Patent No. 4741900 has been set forth the metal ion that can put together in antibody, it can be used as diagnostic reagent according to the present invention.Suitable enzyme for example comprises horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes, or acetylcholinesterase; Suitable prothetic group mixture for example comprises Streptavidin/vitamin H and avidin/biotin; Suitable fluorescence comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine amine, dansyl chloride or phycoerythrin; Shiner for example comprises luminol,3-aminophthalic acid cyclic hydrazide; The noclilucence thing for example comprises luciferase, fluorescein and aequorin; Suitable radioactive substance comprises 125I, 131I, 111In, or 99Tc.
In addition, antibody or its fragment can be puted together in therapeutic component such as cytotoxin for example cytostatics or cytocide, and for example alpha emitter is for example for therapeutical agent or radioactive metal ion 213Bi.Cytotoxin or cytotoxic agent comprise the deleterious any preparation of pair cell.For example comprise paclitaxol, cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, mitomycin, Etoposide, teniposide, vincristine(VCR), vincaleucoblastine, colchicine, Zorubicin, daunorubicin, dihydroxy anthracin, mitoxantrone, mithramycin, dactinomycin, 1-dehydrogenation testosterone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Propranololum and tetracycline and their analogue or homologue.Therapeutical agent comprises but the non-metabolic antagonist (methotrexate for example that is limited to, Ismipur, 6-thioguanine, cytosine arabinoside, 5 FU 5 fluorouracil decarbazine), alkylating agent (dichloromethyldiethylamine for example, the thioepa Chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, the dibromo mannitol, streptozotocin, ametycin, with dichloro diamines cis-platinum (II) (DDP), anthracycline antibiotics (daunorubicin for example, (daunomycin) and Zorubicin) in the past, microbiotic (as daunomycin (actinomycin) in the past, bleomycin, mithramycin, ammonia lucerne mycin (AMC)), and antimitotic agent (as vincristine(VCR) and vincaleucoblastine).
Conjugate of the present invention can be used for modifying given biological response, and therapeutical agent or drug component are not necessarily limited to traditional chemotherapeutics.For example, drug component can be to have required bioactive protein or polypeptide.This protein can for example comprise toxin such as toxalbumin, ricin, false pseudomonas bacillus extracellular toxin, or diphtheria toxin; Protein such as tumour necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived somatomedin, tissue plasminogen activator, apoptosis agent such as α-TNF, β-TNF, AIMI (seeing international publication No.WO 97/33899), AIMII (seeing international publication No.WO 97/34911), Fas part (Takahashi etc., immunology research 6:1567-1574 (1994)), VEGI (seeing international publication No.WO 99/23105), CD 40 parts, thrombosis (thrombotic) agent or anti-angiogenic agent be angiostatin or endostatin for example; Or biological response modifier such as lymphokine, interleukin 1 (" IL-1 "), interleukin II (IL-2), interleukin-6 (IL-6), rHuGM-CSF (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or other somatomedin.
Antibody also can be attached to solid support, and it is used in particular for the immunoassay or the purifying of target antigen.This solid support comprises but the non-glass that is limited to, Mierocrystalline cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
The method that this treatment component and antibody are puted together is known, seen for example Arnon etc., " monoclonal antibody of cancer therapy Chinese traditional medicine immune disorders ", monoclonal antibody and cancer therapy, Reisfeld etc. (editor).P243-56(Alan R.Liss，Inc。1985); Hellstrom etc., " antibody of drug conveying ", the drug conveying of control (second edition), Robinson etc. (editor), 623-53 (Marcel Dekker, Inc.1987); Thorpe, " antibody carrier of cytotoxic agents in the cancer therapy ", monoclonal antibody 84: biology and clinical application, Pinchera etc. (editor), P475-506 (1985); " therapeutic is used the analysis of radiolabeled antibody in the cancer therapy; result and expected results ", monoclonal antibody in cancer detection and the treatment, Balolwin etc., P303-16 (Science Press 1985), with Thorpe etc., " preparation and the cellular toxicity of antibody-toxin conjugate ", immunology research 62:119-58 (1982).
Perhaps, antibody can be puted together formation antibody allos conjugate with another kind of antibody, described in the U.S. Patent No. 4676980, in full incorporates reference at this as Segal.
Have or do not have the therapeutic component and put together antibody, use separately or with born of the same parents' poison factor and/or combination of cytokines and can be used for treatment in it.
Immunophenotyping
Antibody of the present invention can be used for the immunophenotyping of clone and biological sample.The translation product of gene of the present invention can be used as cell specific marker, or more particularly as each differentiation and/or the different cell markings of expressing of stage of maturity at the specific type cell.The directly anti-special epi-position or the monoclonal antibody of epi-position combination can be screened the cell mass of presentation markup.Can utilize various methods of screening the cell mass of presentation markup with monoclonal antibody, comprise that the magnetic bead with antibody sandwich carries out magnetic resolution, with the antibody elutriation that is attached to solid substrate (promptly dull and stereotyped), and flow cytometry (is seen for example United States Patent (USP) 5985660; With Morrison etc., cell 96:737-49 (1999)).
These methods are used to screen the special group of cell, as " non-self " cell that can find leukemia (being the MRD of acute leukemia philtrum) and transplant, with prevention graft/host disease (GVHD).Perhaps, these methods can be used for screening hemopoietic stem cell and the progenitor cell that can breed and/or break up, and these cells are found in the human cord blood.
Antibodies is analyzed
Antibody of the present invention can carry out the immunologic opsonin binding analysis by any method known in the art.Spendable immunoassay comprises but non-ly is limited to competitiveness or noncompetitive analytical system, as uses Western engram analysis, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoprecipitation analysis, precipitin reaction, GDP reaction, immunodiffusion(ID) analysis, aggegation is analyzed, the complement binding analysis, immunoradiometric assay(IRMA), fluoroimmunoassay, the albumin A immunoassay, or the like.This analysis for this area be use always and know (see for example editor such as Ausubel, 1994, molecular biology universal method, the first roll, John Wiley ﹠amp; Sons, Inc. New York; Incorporate reference in full at this).Some immunoassays (but unrestricted meaning) have below been set forth briefly for example.
Immunoprecipitation method generally comprises is adding protein phosphatase and/or proteinase inhibitor (as EDTA, PMSF, aprotinin, vanadic acid sodium) RIPA damping fluid (1%NP-40 or Triton X-100,1% Sodium desoxycholate, 0.01%SDS, 0.15M Nacl, 0.01M sodium phosphate, pH 7.2, lysing cell group 1%Trasylol) adds corresponding antibody in the cell lysate, 4 ℃ of insulation for some time (as 1-4 hour), albumin A and/or Protein G sepharose pearl are added in the cell lysate, 4 ℃ the insulation about 1 hour or the longer time, in lysate the flushing this pearl, and with its resuspending in the SDS/ sample buffer.The special antigenic ability of corresponding antibodies immunoprecipitation can be determined by for example Western engram analysis.Those skilled in the art will know that modifiable parameter combines and reduces background (as clearing up cell lysate in advance with the sepharose pearl) with antigenic to improve antibody.See for example editor such as Ausubel, 1994, molecular biology universal method, the first roll, John Wiley ﹠amp about the further elaboration of immunoprecipitation method; Sons, set forth at 10.16.1 in Inc. New York.
The Westeyn engram analysis generally comprises the preparation protein example, electrophoresis protein example in polyacrylamide gel (as selecting 8%-20%SDS-PAGE for use) according to antigenic molecular weight, from polyacrylamide gel, move to protein example on the film, as Nitrocellulose, on PVDF or the nylon membrane, this film of sealing in confining liquid (as have 3%BSA PBS or skimming milk), flushing this film (as in PBS-Tween 20) in dcq buffer liquid, be used in one anti-(corresponding antibodies) that dilutes in the confining liquid and seal this film, this film of flushing in dcq buffer liquid, anti-(its identification one is anti-with two, as anti-people's antibody) seal this film, this two resists in confining liquid and dilutes, put together in enzymatic substrate (as horseradish peroxidase or alkaline phosphatase) or Geigers (as 32P or 125I), this film of flushing in washing fluid, and detect the antigenic situation that exists.Those skilled in the art will know that modifiable parameter is to improve the signal that detects and to reduce background interference.See for example editor such as Ausubel, 1994, molecular biology universal method, the first roll, John Wiley﹠amp about the further elaboration of Western trace method; Sons, set forth at 10.8.1 in Inc. New York.
ELISAs comprises preparation antigen, and with antigen coated 96 hole microtitre flat boards, but Xiang Kongzhong adds the corresponding antibodies of puting together with detection compound such as enzyme substrates (as horseradish peroxidase or alkaline phosphatase), insulation for some time, detects the antigenic situation that exists.In ELISAs, but corresponding antibodies needn't necessarily be connected in detection compound; On the contrary, but put together in two anti-(its identification one is anti-) of detection compound and can add in the hand-hole.In addition, can antigen coated hole and replace and use the antibody sandwich hole.In this case, but put together in detection compound two anti-can adding after in the hole of bag quilt, adding corresponding antigen.Those skilled in the art will know that and to make amendment to improve detection signal to parameter, also know other variation of ELISAs known in the art.See for example editor such as Ausubel, 1994, molecular biology universal method, the first roll, John Wiley ﹠amp about the further elaboration of ELISA; Sons, set forth in 11.2.1 in Inc. New York.
The disengaging rate of antibody and antigenic binding affinity and antibody-AI can be determined by competitive binding analysis.Competitive binding analysis for example is a radioimmunoassay, comprises that with corresponding antibody the antigen of incubation mark (for example under the situation of the unlabelled antigen that has increasing amount 3H or 125And the antigen bonded antibody of detection and mark I).Corresponding antibodies is with special antigenic affinity and combine the disengaging rate, can determine from Scatchard engram analysis gained data.Determine with the also available radioimmunoassay of the competition of second kind of antibody.In this case, antigen with put together compound in mark (as 3H or 125I) corresponding antibodies, incubation under the situation of the unlabelled another kind of antibody that has increasing amount.
Therepic use
The invention still further relates to a kind of methods of treatment, comprise antibody of the present invention is applied to animal based on antibody, preferred mammal, more preferably people, patient is to treat one or more described disease, functional disorder or pathological condition.Treatment compound of the present invention comprises but non-antibody of the present invention (comprising its fragment, analogue and derivative) and the code book invention antibody (comprising its fragment, analogue and derivative and described antiidiotypic antibody) of being limited to.Antibody of the present invention can be used for treatment, inhibition or prevention and polypeptide unconventionality expression of the present invention and/or active relevant disease.Functional disorder or pathological condition, comprise but non-ly be limited to arbitrary or multiple disease as herein described, functional disorder or pathological condition (autoimmune hemolytic anemia for example, autoimmunity newborn infant thrombopenia, spontaneous thrombopenic purpura, the autoimmunity cytopenia; Hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, the recurrent polychondritis, rheumatic heart disease, glomerulonephritis (as IgA nephropathy), multiple sclerosis, neuritis, uveitis, multiple endocrinopathy, purpura (as the Henloch-Scoenlein purpura), Reiter ' s disease, Stiff-Man syndrome, autoimmune pulmonary inflammation, Guillain-Pane syndrome, pancreas is led plain dependent diabetes and autoimmunity ophthalmia, autoimmune thyroiditis, hypothyroidism (being Hashimoto ' s thyroiditis), systemic lupus erythematous, Goodpasture ' s syndrome, pemphigus, the receptor autophosphorylation immunity is (a) Graves disease for example, (b) Myasthenia Gravis and (c) pancreas lead plain resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatic arthritis has the schleroderma of anticol original antibody, blended connective tissue disease (CTD), polymyositis/dermatomyositis, pernicious anemia, spontaneous Addison ' s disease, Infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, BP, SJogren ' s syndrome, diabetes, with adrenin drug resistance (comprising treatment asthma or the Fibrotic adrenin drug resistance of bladder), chronic active hepatitis, primary biliary cirrhosis, other incretory gland pathology, vitiligo, vasculitis, back MI, myocardium syndrome, urticaria, hereditary allergic dermatitis, asthma, inflammatory myositis, with other inflammation, granulamatous degenerates, and the atrophic functional disorder).
In a special embodiment, antibody of the present invention is used for the treatment of, and suppresses prognosis, diagnosis or prevention rheumatic arthritis.
In another special embodiment, antibody of the present invention is used for the treatment of, and suppresses prognosis, diagnosis or prevention system lupus erythematosus.
With polypeptide unconventionality expression of the present invention and/or active relevant disease, the treating and/or preventing of imbalance or pathological condition comprises but non-being limited to alleviates and these diseases, the symptom that imbalance or pathological condition are correlated with.Antibody of the present invention also can be used for orientation and the cell that kills at its surface expression Neutrokine-α, or on its surface Neutrokine-α bonded cell is arranged.Antibody of the present invention can medicine appropriate combination thing form known in the art or as herein described provide.
The method that antibody treatability of the present invention is used comprises the part or systematically in vivo in conjunction with polynucleotide of the present invention or polypeptide, or the direct cellular toxicity by antibody, as by complement (CDC) or effector cell (ADCC) mediation.Some such methods have below been set forth in more detail.By this paper instruction, those skilled in the art will know that how to use antibody of the present invention, diagnosing, monitoring or treatment and without undue experimentation.
Antibody of the present invention and other mono-clonal or chimeric antibody, or to be used in combination with lymphokine or hemopoieticgrowth factor (as IL-2, IL-3 and IL-7) be favourable, for example improves number or activity with the interactional effector cell of antibody.
Antibody of the present invention can be separately or with other type treatment plan combined administration (as radiotherapy, chemotherapy, hormonotherapy, immunotherapy, antineoplastic agent, microbiotic, and immunoglobulin (Ig)).Normally, preferably use source of species or species reactivity (under the situation of antibody) product with patient's same species.Therefore, in an embodiment preferred, people's antibody, fragment derivative, analogue or nucleic acid are applied to human patients to treat or to prevent.
Preferred high-affinity and/or the strong antibody that suppresses in vivo and/or neutralize anti-polypeptide of the present invention or polynucleotide of using is with to comprising that with polynucleotide of the present invention or polypeptide the relevant disease of its fragment carries out immunoassay and treatment.This antibody, fragment or zone preferably comprise that with polynucleotide of the present invention or polypeptide its fragment has affinity.Preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10 -5M, 10 -5M, 5 * 10 -6M, 10 -6M, 5 * 10 -7M, 10 -7M, 5 * 10 -8M, 10 -8M, 5 * 10 -9M, 10 -9M, 5 * 10 -10M, 10 -10M, 5 * 10 -11M, 10 -11M, 5 * 10 -12M, 10 -12M, 5 * 10 -13M, 10 -13M, 5 * 10 -14M, 10 -14M, 5 * 10 -15M and 10 -15M.
Gene therapy
In a special embodiment, use the nucleic acid of the sequence that comprises encoding antibody or its functional deriv, with by the gene therapy treatment, inhibition or prevention and polypeptide unconventionality expression of the present invention and/or relevant disease or the functional disorder of activity.Gene therapy refers to by use the treatment that nucleic acid expression or effable carries out for treatment target.In this embodiment of the present invention, nucleic acid produces its encoded protein matter of mediation treatment effect.
Any suitable gene therapy methods can be used according to the present invention in this area.The method of the following stated for example.
General Study about gene therapy is seen Goldspiel etc., clinical pharmacy 12:488-505 (1993); Wu and Wu, biotherapy 3:87-95 (1991); Tolstoshev, research 32:573-596 (1993) is analysed in the drug toxicity credit; Mulligan, science 260:926-932 (1993); With Morgan and Anderson, biochemical analysis research 62:191-217 (1993); May, TIB TECH 11 (5): 155-215 (1993) is described.Spendable recombinant DNA technology well known in the art is seen (editors) such as Ausubel, molecular biology universal method, John Wiley ﹠amp; Sons, NY (1993); And Kriegler, transgenosis and expression laboratory manual, Stockton press, NY (1990) is described.
In an embodiment preferred, compound comprises the nucleotide sequence of encoding antibody, and described nucleotide sequence is the part of the expression vector of expressing antibodies or its fragment or chimeric protein or heavy or light chain in suitable host.In particular, this nucleotide sequence has the promotor that operably is connected in antibody coding region, and described promotor is derivable or composing type, and randomly is tissue-specific.In another special embodiment, the nucleic acid molecule that uses wherein antibody coding sequence and any other required sequence is to be located at the regional both sides that start homologous recombination in the required site of genome, therefore (Koller and Smithies, institute of American Academy of Sciences report 86:8932-8935 (1989) at intrachromosomal expression to provide antibody encoding nucleic acid; Zijlstra etc., natural 342:435-438 (1989).In special embodiment, the antibody molecule of expression is a single-chain antibody; Perhaps, nucleotide sequence comprises encoding antibody heavy chain and light chain or its fragments sequence.
It can be direct or indirect that nucleic acid is delivered to the patient, and direct mode is that the patient directly is exposed to nucleic acid or carries the carrier of nucleic acid, and indirect mode is at first to use nucleic acid at the vitro conversion cell, is implanted into then in patient's body.These two kinds of methods are known as in the body respectively or come from intravital gene therapy.
In a special embodiment, nucleotide sequence is directly to use in vivo, and it expresses the product that produces coding in vivo.This can be undertaken by the whole bag of tricks known in the art, for example, they are configured to the part of suitable nucleic acid expression vector, and use so that they become intracellular matter, for example by with defective or the reduction retrovirus or other viral vector infection (seeing U.S. Patent No. 4980286), or by the exposed DNA of direct injection, or use the micropartical bombardment (as particle gun; Biolistic; Dupont); or with lipid or cell surface receptor or transfection agents bag quilt; use liposome; particulate or micro-capsule are encapsulated; or use by being connected with the known peptide that enters nuclear, carry out receptor-mediated endocytosis (see for example Wu and Wu, journal of biological chemistry 262:4429-4432 (1987) is described) (it can be used for the cell type of directed specifically expressing acceptor) by being connected with part to use.In another embodiment, can form nucleic acid-ligand complex, wherein part comprises the fusion viral peptide to destroy endosome, makes nucleic acid avoid being degraded by lysosome.In another embodiment, the nucleic acid cell that can be oriented to special absorption and expression by orientation-special acceptor in vivo (is seen for example PCT publication WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188; WO 923/20221).Perhaps, nucleic acid can be by in the homologous recombination transfered cell and mix in the host cell DNA that (Koller and Smithies, institute of American Academy of Sciences report 86:8932-8935 (1989) to express; Zijlstra etc., natural 342:435-438 (1989)).
In a special embodiment, use the virus vector of the nucleotide sequence that contains code book invention antibody.For example, can use retroviral vector (to see Miller etc., Enzymology method 217:581-599 (1993).These reverse transcription carriers contain correct packaging virus genome and integrate the necessary component of λ host cell DNA.The nucleotide sequence of the antibody of use in the encoding gene treatment is cloned in one or more carrier of λ, and described carrier promotes that gene is delivered in patient's body.See Boesen etc. about retroviral more elaborating, biotherapy 6:291-302 (1994), it has been set forth use reverse transcription carrier mdr 1 gene has been delivered to hemopoietic stem cell, to produce the stem cell that chemotherapy is more had resistance.Use other illustration of retroviral vector to see Clowes etc., Journal of Clinical Investigation 93:644-651 (1994) in the gene therapy; Kiem etc., blood 83:1467-1473 (1994); Salmons and Gunzberg, human body gene treatment 4:129-141 (1993); And Grossman and Wilson, hereditary and developmental general viewpoint 3:110-114 (1993).
Adenovirus is other virus vector that can be used for gene therapy.Adenovirus is the launch vehicle that gene is delivered to the particularly suitable of airway epithelial.Adenovirus infects airway epithelial natively, causes minor ailment.Other target based on the delivery system of adenovirus is a liver, central nervous system, endotheliocyte and flesh.Adenovirus has the advantage that can infect undifferentiated cell.Kozansky and Wilson, heredity and the general viewpoint 3:499-503 of growth (1993) have proposed a kind of gene therapy method based on adenovirus.Bout etc., human body gene treatment 5:3-10 (1994) have set forth and have used adenovirus carrier gene to be moved to the adenovirus carrier of asthma monkey airway epithelial.Other that adenovirus is used in the gene therapy is with reference to seeing Rosenfeld etc., science 252:431-434 (1991); Rosenfeld etc., cell 68:143-155 (1992); Mastrangeli etc., Journal of Clinical Investigation 91:225-234 (1993); PCT publication WO 94/12649; With Wang etc., gene therapy 2:775-783 (1995).In an embodiment preferred, use adenovirus carrier.
Adeno associated virus (AAV) has been used for gene therapy for this reason, and (Walsh etc., institute of American Academy of Sciences report 204:289-300 (1993); U.S. Patent No. 5436146).
Another kind of gene therapy method comprises by as electroporation, the fat transfection, and the transfection that calcium phosphate mediates, or method such as virus infection move to gene in the cell in the tissue culture.Normally, transfer method comprises a detectable mark is moved to cell.Separate cell that handled and that express metastatic gene then.Then these cells are applied to patient.
In this embodiment, use in vivo before the gained reconstitution cell, in the nucleic acid transfered cell.This importing can be undertaken by any method known in the art, comprises but non-transfection, the electroporation of being limited to, microinjection infects cytogamy with virus that contains nucleotide sequence or phage vector, the transgenosis of karyomit(e) mediation, the transgenosis of minicell mediation, spheroplast fusion etc.Many methods with the foreign gene transfered cell known in the art (are seen for example Loeffler and Beht, Enzymology method 217:599-618 (1993); Cohen etc., Enzymology method 217:618-644 (1993); Clinical drug therapy 29:69-92m (1985) can use these methods according to the present invention, and does not destroy the basic growth and the physiological function of recipient cell.Method therefor should stably move to cell with nucleic acid, so that nucleic acid can be by cell expressing and preferably can and be expressed by its cell filial generation heredity.
The gained reconstitution cell can be applied to the patient by the whole bag of tricks known in the art.The preferred intravenously of reorganization hemocyte (as hemopoietic stem cell or progenitor cell) is used.Used cell quantity is according to required effect, patient's states etc. and deciding, and can determine by those skilled in the art.
For carrying out gene therapy the cell that nucleic acid imports is wherein contained any required suitable cell type, comprised but the non-epithelial cell that is limited to endotheliocyte, keratinocyte, inoblast, myocyte, liver cell, hemocyte such as T lymphocyte, bone-marrow-derived lymphocyte, monocyte, scavenger cell, neutrophil leucocyte, eosinophil, megalokaryocyte, granulocyte; Various doing or progenitor cells, especially hemopoietic stem cell or progenitor cell, as derive from marrow.Cord blood, peripheral blood, the cell of fetus liver etc.
In preferred embodiments, the cell that is used for gene therapy is patient self.
Be used for the embodiment of gene therapy at reconstitution cell, with the nucleotide sequence transfered cell of encoding antibody, they can be expressed by cell or cell filial generation thus, then reconstitution cell are used in vivo and are treated.In a special embodiment, be used in or progenitor cell.Any separable and external doing and/or progenitor cell of keeping, this embodiment according to the present invention all can be used and (see for example PCT publication WO 94/08598; Stemple and Anderson, cell 71:973-985 (1992); Rheinwald, cell biology method 21A:229 (1980); With Pittelkow and Scott, Mayo Clinic Proc.61：771(1986)。
In special embodiment, comprise the inducible promoter that operably is connected in the coding region for carrying out the nucleic acid that gene therapy imported, expression of nucleic acids can have or not have the suitable elicitor of transcribing and exists and control by control thus.
The proof of treatment or prophylactic activity
Compound of the present invention or pharmaceutical composition are preferred tests required treatment or prophylactic activity then in vivo earlier at vitro test, is used further to human body afterwards.For example, analyzed in vitro confirms the treatment of compound or pharmaceutical composition or the effect that prophylactic activity comprises compound pair cell system or tissue of patient sample.The effect of compound or composition pair cell system or tissue of patient sample can utilize methods known in the art to determine, comprises but non-ly is limited to bow structure and forms and analyze and the lysis analysis.According to the present invention, can be used for determining whether the special compound of using is indicated analyzed in vitro, comprises the cell in vitro culture assays, wherein the tissue of patient sample is grown in substratum, be exposed to or administered compound, and observe of the effect of this compound tissue sample.
Treating and/or preventing property application process and composition
The invention provides by using the compound of the present invention or the pharmaceutical composition of significant quantity for treatment target, preferred antibody of the present invention, and reach treatment suppresses and the method for prevention purpose.In an embodiment preferred, compound is basic purifying (as its effect substantially without limits or produce the material of non-required side effect).Treatment target is animal preferably, comprises but non-being limited to is milk cow, and pig, horse, chicken, cat, dog etc., Mammals preferably, and most preferably be the people.
When compound comprises nucleic acid or immunoglobulin (Ig), spendable use the prescription and method as mentioned above; Other proper formulation and route of administration can be selected from the following stated content.
Known various delivery system also can be used for using compound of the present invention, for example encapsulated in liposome, particulate, micro-capsule, can express the reconstitution cell of compound, receptor-mediated endocytosis (seeing for example Wu and Wu, journal of biological chemistry 262:4429-4432 (1987)) is part of retrovirus or other carrier etc. with nucleic acid construct.Introduction method comprises but the non-intracutaneous that is limited to, muscle, and intraperitoneal, intravenously, the hypogloeeis, in the nose, epidermis or oral.Compound or composition can be used by conventional route, for example by merging or the bolus injection, by epithelium or mucous membrane and skin line of delimitation place's absorption (as oral mucosa, rectum and mucous membrane of small intestine etc.), and can use with other biologically active agent.But whole body or topical application.In addition, can medical compounds of the present invention or composition be imported central nervous system, comprise intravenously and intra-arterial injection by any suitable approach; Intravenous injection is simplified by intravenous catheter, for example is adsorbed in liquid bath such as Ommaya liquid bath.Also can use, as using sucker or atomizer, and have the prescription of smoke substance through lung.
In a special embodiment, need be at the position topical application medical compounds of the present invention or the composition of needs treatment; This can pass through for example in intra-operative regional perfusion, and topical application as using with wound dressings, by injection, is passed through conduit after operation, by suppository, or use by implantation, described implant is porose, atresia, or jelly, comprise film, as the sialastic film, or fiber.Preferably, when using protein of the present invention and comprise antibody, must be noted that and to use the non-absorbent material of protein.
In another embodiment, compound or composition can be carried in vesicle especially liposome and (see Langer, science 249:1527-1533 (1990); Treat etc., the liposome in infectious diseases and the cancer therapy, Lopez-Berestein and Fidler (editor), Liss, New York, p353-365 (1989); Lopez-Berestein, as above, p317-327).
In another embodiment, compound or composition can be carried in controlled release system.In one embodiment, can use pump (to see Langer, as preceding; Sefton, CRC Crit.Ref.Biomed.Eng.14:201 (1987); Buchwald etc., surgery 88:507 (1980); Saudek etc., N.Engl.J.Med.321:574 (1989)).In another embodiment, can use polymkeric substance (to see the medical use of sustained release, Langer and Wise (editor), CRC press, Boca Raton, Florida (1974); The drug bioavailability of control, medicine design and production, Smolen and Ball (editor), Wiley, New York (1984); Ranger and Peppas, J.Macromol, Sci.Rev.Macromol.Chem.23:61 (1983); Also see Levy etc., science 228:190 (1985); During etc., neuroscience research 25:351 (1989); Howanol etc., Neurosurg magazine 71:105 (1989)).In another embodiment, Controlled Release System can place therapeutic goal be brain near, so only need body dose a part (for example see, Goodson, the medical use of sustained release, as preceding, second the volume, p115-138 (1984).
Other Controlled Release System is seen Langer described (science 249:1527-1533 (1990)).
In the one special embodiment of the nucleic acid that compound of the present invention therein is a coded protein, nucleic acid can be used in vivo to start the expression of its encoded protein matter, by it being configured to the part of suitable nucleic acid expression vector and using, make it become an intracellular part, for example by using retroviral vector (seeing U.S. Patent No. 4980286), or by direct injection, or by using microparticle bombardment (as particle gun; Biolistic Dupont), or with lipid or cell surface receptor or transfection agents bag quilt, or uses (for example see Joliot etc., institute of American Academy of Sciences reports 88:1864-1868 (1991)) etc. by being connected with the known class homology frame peptide that can enter in the nuclear.Perhaps, nucleic acid can be by in the homologous recombination transfered cell and mix in the host cell DNA and express.
The present invention also provides pharmaceutical composition.This composition comprises the compound and the medicine suitable carriers for the treatment of significant quantity.In a special embodiment, that term " medicine is suitable " refers to allow through federation or government's structure or the united states drug management structure is listed, or other be it is generally acknowledged and can be used for especially people's article of animal.Term " carrier " refers to thinner, adjuvant, vehicle or the vehicle used with therapeutical agent.This pharmaceutical carrier can be sterile liquid Ru Shui and oil, comprises oil, animal oil, and vegetables oil or synthetic oil, as peanut oil, soybean oil, mineral oil, sesame wet goods.When pharmaceutical composition is when intravenously is used, water is preferred carrier.Salt solution and Glucose Liquid and glycerin liquid also can be used as liquid vehicle.Suitable drug excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, paddy rice, flour, lime, silica dioxide gel, sodium stearate, glycerol monostearate, talc, sodium-chlor, skimming milk, glycerine, propylene, glycol, water, ethanol etc.If desired, composition also can contain a spot of wetting agent or emulsifying agent, or the pH buffer reagent.These compositions can be solution, suspension, and milk sap, tablet, pill, capsule, powder continues the prescription of release etc.Composition can be formulated as suppository with traditional wedding agent and carrier such as triglyceride level.Formula of oral can comprise the mannitol of standard vector such as pharmaceutically grade, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate etc.Suitable pharmaceutical carrier for example by E.W.Martin described in " Remington ' s pharmaceutical science ".This composition contains the compound for the treatment of significant quantity, and the compound of preferred purified form and an amount of carrier are correctly used the formulation of giving the patient to provide.
In an embodiment preferred, composition is to be suitable for the pharmaceutical methods preparation that intravenously is applied to human body according to routine preparation.Typically, the composition used of intravenously is a sterile isotonic solution.If desired, composition also can comprise stablizer and local anesthetic such as lignocaine, to alleviate the pain of injection site.Normally, batching can be separately or is mixed with unitary dose and to provide, for example in the container of sealing with anhydrous lyophilized powder or do not have aqueous concentrate and exist, described container such as ampoule or show the bag of promoting agent quantity.When composition is used by perfusion, its available water or brinish perfusion bottle separatory that contains aseptic pharmaceutically grade.When composition was used by injection, available sterile water for injection or brinish ampoule were with mix before using.
Compound of the present invention can be formulated as neutral or salt form.The salt that medicine is suitable comprise with positively charged ion as originating from hydrochloric acid, phosphoric acid, acetate, oxalic acid, the positively charged ion of tartrate etc., or with negatively charged ion as originating from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ironic hydroxide, isopropylamine, triethylamine, 2-ethylaminoethyl alcohol, histamine, the salt that the negatively charged ion of PROCAINE HCL, PHARMA GRADE etc. forms.
Treatment effectively, the quantity of the The compounds of this invention of the unconventionality expression of inhibition and prevention and polypeptide of the present invention and/or active relevant disease or functional disorder can be definite by standard clinical techniques.In addition, can randomly use analyzed in vitro to help differentiating the optimal dose scope.The actual dose of using in the prescription also depends on route of administration, the severity of disease and deciding, and should be according to the doctor's diagnosis with patient's situation and decide.Effective dose can be by determining in the dose response curve that produces from external or animal model test system.
With regard to antibody, the dosage that is applied to patient is between the 0.1-100mg/kg body weight, more preferably, and between the 1-10mg/kg body weight.Normally, at the intravital long half time of people, this is because to due to the immunne response of allogenic polypeptide to people's antibody than the antibody of other species.Therefore, can use people's antibody of less dosage, and frequency of administration also can be longer.In addition, the application dosage of antibody of the present invention and frequency, thus absorption and the tissue permeability (as going into brain) modified as fatization enhancing antibody by antagonist reduce.
The present invention also provides the drug packages or the test kit of the container that comprises one or more one or more batching that pharmaceutical composition of the present invention is housed.Randomly relevant with this container is government organs' adjusting productions, uses or sell the bulletin that medicine or institute of Biological Products are done, and this bulletin has reflected the production of permission, and use or sale are applied to people's biological products or medicine.
Diagnosis and imaging
With the antibody of the mark of corresponding peptide specific combination, and derivative and analogue, can be used for diagnosing the dried rhizome of rehmannia to detect, diagnosis or monitoring variable and polypeptide unconventionality expression of the present invention and/or relevant disease and/or the functional disorder of activity.The invention provides the method that detects the corresponding polypeptide unconventionality expression, comprise that (a) uses one or more antibody that is specific to corresponding polypeptide, in the cell of individuality or body fluid, analyze the expression of corresponding polypeptide, (b) this gene expression dose and standard gene expression level are compared, thereby compare with standard gene expression level, the polypeptide gene expression level of analysis improves or reduction shows unconventionality expression.
The invention provides the diagnositc analysis of diagnostic functions imbalance, comprise that (a) uses one or more antibody that is specific to corresponding polypeptide, in the cell or body fluid of individuality, analyze the expression of corresponding polypeptide, (b) this gene expression dose and standard gene expression level are compared, thereby compare with the standard expression level, the raising or the reduction of the polypeptide gene expression level of analyzing show specific functional disorder, with regard to cancer, exist the transcript of relative a large amount can represent the susceptibility that disease takes place in the individual biopsy, or can before occurring, true clinical symptom provide the method that detects disease, one more definite diagnosis can make healthy practitioner use preventive measures or courageously treatment earlier, thereby prevents the generation of cancer or further develop.
Antibody of the present invention can be used for (seeing for example Jalkanen etc., cytobiology magazine 101:976-985 (1985) in biological sample analysing protein level with traditional immunohistology method well known by persons skilled in the art; Jalkanen etc., cytobiology magazine 105:3087-3096 (1987)).Be used to detect other method that protein gene expresses and comprise immunoassay, as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) based on antibody.Suitable antibody analysis mark known in the art comprises enzyme labelling such as notatin; Radio isotope such as iodine ( 131I, 125I, 121I), carbon ( 14C), sulphur ( 35S) tritium ( 3H), indium ( 115MIn, 113MIn, 112In, 111In), and technetium ( 99Tc, 99MTc), thallium ( 201Ti), gallium ( 68G A, 67Ga), palladium ( 103Pd), molybdenum ( 99Mo), xenon ( 138Xe), fluorine ( 18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru; Luminescent marking such as Luminol; With fluorescent mark such as fluorescein, and rhodamine, and vitamin H.
Available methods known in the art mark antibody of the present invention.This method comprise but non-be limited to use difunctionally put together agent and (see for example U.S. Patent No. 5756065; 5714631; 5996239; 5652361; 5505931; 5489425; 5435990; 5428139; 5342604; 5274119; 4994560; With 5808003 described, above document all incorporate in full with reference to).
An embodiment of the present invention be in the animal preferred mammal more preferably in human body, disease or functional disorder that detection is relevant with the corresponding polypeptide unconventionality expression with diagnosis.In one embodiment, diagnostic method comprises that (a) uses the molecule of mark of the significant quantity of specific combination corresponding polypeptide for diagnosis object, and as by non-enteron aisle, subcutaneous or intraperitoneal is used; (b) after using, wait for a period of time, make the molecule of mark preferentially concentrate (and removing unlabelled molecule) at the position of expression of polypeptides; (c) determine background level; (d) molecule of mark in the detection diagnosis object, the tagged molecule of Jian Ceing is higher than background level and shows that diagnosis object has special disease or the functional disorder relevant with the corresponding polypeptide unconventionality expression like this.Background level can be determined by the whole bag of tricks, comprises that tagged molecule quantity that will detect and the standard value of determining in particular system in advance compare.As described herein, the special embodiment of the present invention is to use the cell or the monocytic cell of the quantitative or qualitative concentrated B clone of antibody of the present invention.
Also as described herein, antibody of the present invention can be used for treatment, the individuality that diagnosis or prevention have immune deficiency, and in a special embodiment, antibody of the present invention is used for the treatment of, the individuality that diagnosis and/or prevention have CVID or its hypotype.In another embodiment, antibody of the present invention is used for diagnosis, and prevention, treatment are characterised in that serum immune globulin produces defective, recurrent infection, and/or the disease of function of immune system imbalance.
As described herein equally, antibody of the present invention can be used for treatment, the individuality that diagnosis or prediction have autoimmune disease or functional disorder.In a special embodiment, antibody of the present invention is used for the treatment of, the individuality that diagnosis and/or prediction have systemic lupus erythematous or this disease subtypes.In another special embodiment, antibody of the present invention is used for the treatment of, the individuality that diagnosis and/or prediction have rheumatic arthritis or its hypotype.
The size of treatment target and the quantity that used visualization system will determine to need to produce the visual developing component of diagnosis should be known in this area.Under the situation of radio isotope component, ratione personae, the radioactivity quantity of injection is normally in about 5-20 millicurie 99Between the mTc.The antibody of mark or antibody fragment be preferred accumulation in containing the cell of specific protein then.The in-vivo tumour developing sees S.W.Burchiel etc., (the 13rd chapter in the tumour developing: the radiological chemistry of cancer detects " radiolabeled antibody and segmental immune pharmacokinetics thereof ", S.W.Burchiel and B.A.Rhocles edit, Masson Publishing Inc. (1982)) described.
According to some variablees, comprise used labeling pattern and mode of administration, using the back interlude is 6-48 hour, or 6-24 hour or 6-12 hour, certain hour is that the molecule of mark is preferentially concentrated in target bit at interval, and unconjugated tagged molecule is removed in background level.In another embodiment, the timed interval after using is 5-20 days or 5-10 days.
In one embodiment, disease or functional disorder are monitored, for example after initial diagnosis one month, after 6 months, after 1 year etc. by the method that repeats to diagnose the illness.
Whether available body interscan method known in the art detects in the existence of patient body's internal labeling molecule.These methods depend on the type of used mark.Those of skill in the art can determine to detect the proper method of special marking.The method and the instrument that can be used in the diagnostic method of the present invention comprise but the non-computerized tomography (CT) that is limited to body scan such as positron emission computerized tomography (PET), nucleus magnetic resonance (MRI) and ultrasonic.
In a special embodiment, molecule detects (Thurston etc., U.S. Patent No. 5441050) with labelled with radioisotope and in the patient body with radiation responsiveness surgery instrument.In another embodiment, molecule fluorescent chemicals mark, and in the patient body, reply scanner with fluorescence and detect, in another embodiment, the molecule metal mark of emission positron, and in the patient body, detect with positron emission computerized tomography.In another embodiment, molecule paramagnetism marker mark, and in the patient body, detect with nucleus magnetic resonance (MRI).
Test kit
The invention provides the test kit that can be used for aforesaid method.In one embodiment, test kit comprises one or more container, wherein contains antibody of the present invention, preferred antibody purified, in a special embodiment, test kit of the present invention contains basic isolated polypeptide, this polypeptide comprise with test kit in the immunoreactive epi-position of antibodies specific.Preferably, test kit of the present invention also comprises not the control antibodies with corresponding polypeptide reaction.In another special embodiment, test kit of the present invention comprises identical and/or different sequence or regional two or the multiple antibody (mono-clonal and/or polyclone) of discerning polypeptide of the present invention.In another special embodiment, test kit of the present invention comprises and detects antibody and corresponding polypeptide bonded facility (for example antibody can be puted together with detectable substrate, as fluorescent chemicals, the enzymatic substrate, radioactive compound, or luminophor, perhaps discern first kind of antibody second kind of antibody can with can detect substrate and put together).
In another special embodiment of the present invention, test kit is the diagnostic test kit that is used to screen the serum of the antibody that contains special anti proliferative and/or cancer polynucleotide and polypeptide.This test kit can comprise not the control antibodies with the corresponding polypeptide reaction.This test kit can comprise basic isolated polypeptide antigen, and this antigen comprises and the immunoreactive epi-position of the antibodies specific of at least a anti-polypeptide antigen.In addition, this test kit comprises described antibody and the antigen bonded means (for example antibody can be puted together in fluorescent chemicals as passing through the fluorescein or the rhodamine of Flow cytometry) of detecting.In special embodiment, test kit can comprise reorganization polypeptide antigen that produce or chemosynthesis.The polypeptide antigen of test kit also can be attached to solid support.
In a more special embodiment, the detection means of mentioned reagent box comprises the solid support that described polypeptide antigen adheres to, and this test kit also can comprise anti-people's antibody of the receptor marker of not adhering to.In this embodiment, the combination that combines antibody that can be by described receptor marker of antibody and polypeptide antigen detects.
In another embodiment, the present invention includes the diagnostic kit that is used to screen the serum that contains polypeptide antigen of the present invention.This diagnostic kit comprises the basic isolated antibody with polynucleotide or polypeptide antigen specific immune response, and detects the means of polynucleotide or polypeptide antigen and antibodies.In one embodiment, antibody adheres to solid support, and in a special embodiment, antibody can be monoclonal antibody.Detection means in the test kit can comprise the monoclonal antibody of second kind of mark.Perhaps, detection is the competition antigen that can comprise mark.
In a diagnosis configuration, test sera and solid-phase reagent reaction, this solid-phase reagent has the surface bonding antigen that obtains by method of the present invention.After the antibody of specific antigen combined with reagent and removes the bonded serum component by flushing, anti-people's antibody response of reagent and receptor marker, acceptor combine ratio with reagent be to resist antigenic antibody quantity on the solid support.Wash reagent is removed the antibody of unconjugated mark again, and determines the quantity of the acceptor relevant with reagent.Especially, acceptor is a kind of enzyme, and it is being by existing suitable fluorescence, incubation solid phase under the situation of luminous or colorimetric substrates and detect (Sigma, St.Louis, Mo).
Solid surface reagent in the above-mentioned analysis is by known in the art the method for protein attachment in solid support to be prepared, as polymeric beads, and dipping bar, 96 hole flat board or filter membranes.These attachment meanss generally comprise protein non-specific adsorption upholder, or protein is covalently attached to chemical reaction gene on the solid support by free amine group, as active carboxyl, and hydroxyl or aldehyde radical.Perhaps, the flat board of Streptavidin bag quilt can be used for biotinylated antigenic puting together in.
Therefore, the invention provides analytical system or the test kit that carries out this diagnostic method.This test kit generally comprises upholder with surface bonding recombinant antigen and anti-people's antibody of receptor marker, to detect the anti-antigenic antibody of surface bonding.
The invention still further relates to as the agonist of polypeptide of the present invention or the antibody of antagonist.For example, the present invention includes part or all of destruction receptor/ligand and the interactional antibody of polypeptide of the present invention, this comprises two kinds of receptor specific antibody and ligand specificity's antibody.Wherein receptor specific antibody does not stop the part combination but stops receptor activation.Receptor activation (being signalling) can be determined by as herein described or methods known in the art.Promptly stop in part is also included within conjunction with the receptor specific antibody that stops receptor activation again.In addition, also comprise binding partner and stop the neutral antibody of part and receptors bind, thereby and binding partner prevention receptor activation, but do not stop the antibody of part and receptors bind, comprise the antibody of activated receptor in addition.These antibody can be used as agonist, influence all or part of biological activity by ligand-mediated receptor activation.Antibody can be specifically as the bioactive agonist or the antagonist that comprise specific activity described herein.Comprise in addition be no matter Neutrokine-α or Neutrokine-α SV whether with Neutrokine-α receptors bind, and in conjunction with the antibody of Neutrokine-α and/or Neutrokine-α SV.These antibody are as Neutrokine-α and/or Neutrokine-α SV agonist, under the situation that has these antibody, improve the cell proliferation that Neutrokine-α and/or Neutrokine-α SV and Neutrokine-α receptors bind are replied.Above-mentioned antibody agonist can produce with means known in the art.See for example WO 96/40281; United States Patent (USP) 5811097; Deng, blood such as B. 92 (6): 1981-1988 (1998); Chen, Z. etc., cancer research 58 (16): 3668-3678 (1998); Harrop, J.A. etc., Journal of Immunology 161 (4): 1786-1794 (1998); Zhu, Z. etc., cancer research 58 (15): 3209-3214 (1998); Yoon, D.Y. etc., Journal of Immunology 160 (7): 3170-3179 (1998); Prat, M. etc., cell science magazine 111 (Pt2): 237-247 (1998); Ptard, V. etc., immunological method magazine 205 (2): 177-190 (1997); Lautard, J. etc., cytokine 9 (4): 233-241 (1997); Carlson, N.G. etc., journal of biological chemistry 272 (17): 11295-11301 (1997); Taryman, R.E. etc., neurone 14 (4): 755-762 (1995); Muller, Y.A. etc., structure 6 (9): 1153-1167 (1998); Bartunek, P. etc., cytokine 8 (1): 14-20 (1996) described (full text of described document is incorporated reference into).
Now produced at least 14 monoclonal antibodies of anti-Neutrokine-α, these monoclonal antibodies are called 12D6,2E5,9B6,1B8,5F4,9A5,10G12,11G12,16B4,3D4,16C9,13D5,15C10, and 12C5.Initial analysis to these antibody shows in the Western engram analysis and when the Neutrokine-alpha protein combines with the ELISA flat board, all combines with Neutrokine-α.Yet to 12D6,2E5,9B6,1B8,5F4,9A5,10G12, the further analysis revealed of 11G12 and 16B4 antibody has only 12D6,9B6,2E5,10G12,9A5 and 11G12 antibody combine with the Neutrokine-α of film combining form.Therefore, the hypotype of the monoclonal antibody of definite anti-Neutrokine-α that produces, only combine (being that this hypotype does not combine with the Neutrokine-α of the soluble form of the 134-285 amino acids that is equivalent to SEQ ID NO:2) with the Neutrokine-α of film combining form, as described herein, it is limited on monocyte and dendritic cell expresses.
Have now found that the Neutrokine-α of 9B6 antibody binding film combining form specifically, but do not combine with the Neutrokine-α of soluble form.
The epi-position figure of antibody 9B6 shows that this antibody is specifically in conjunction with the aminoacid sequence in the Ser171-Phe194 amino-acid residue that is contained in SEQ ID NO:2.More particularly, epi-position figure shows that antibody 9B6 is specifically in conjunction with the peptide that comprises the Lys 173-Lys188 amino-acid residue of SEQ ID NO:2.
On the contrary, found antibody 16C9 and 15C10 Neutrokine-α (being the 134-285 amino acids of SEQ ID NO:2), and suppressed the alpha mediated B cell proliferation of Neutrokine-in conjunction with soluble form.See for example embodiment 10, found that 15C10 antibody suppresses combining of Neutrokine-α and its acceptor.The epi-position figure of antibody 15C10 has shown that this antibody is specifically in conjunction with the aminoacid sequence in the Glu223-Tyr246 amino acids residue that is contained in SEQ ID NO:2.More particularly, epi-position figure shows that antibody 15C10 is specifically in conjunction with the peptide that comprises the Val227-Asn242 amino acids residue of SEQ ID NO:2.Antibody 15C10 is also specifically in conjunction with the peptide that comprises the Phe 230-Cys245 amino acids residue of SEQ ID NO:2.
As mentioned above, the monoclonal antibody that has now prepared anti-Neutrokine-α.The hybridoma that produces 9B6 and 15C10 antibody is deposited among the ATCC, and preserving number is respectively PTA-1158 and PTA-1159.In one embodiment, antibody of the present invention has one or more and the identical biological nature of hybridoma cell line excretory antibody by preserving number PTA-1158 or PTA-1159." biological nature " is meant the external or activity in vivo or the character of antibody, for example in conjunction with the ability of Neutrokine-α (as the polypeptide of SEQ ID NO:2, the Neutrokine-α of mature form, the Neutrokine-α of film combining form, the Neutrokine-α of soluble form (the 134-285 amino acids of SEQ ID NO:2), antigenic region and/or epi-position district with Neutrokine-α), the ability of basic blocking-up Neutrokine-α and its receptors bind, or the alpha mediated bioactive ability (producing) of blocking-up Neutrokine-as stimulating B cell proliferation and immune protein.Randomly, antibodies of the present invention and the identical epi-position of at least a antibody of being refered in particular to.This epi-position is determined in conjunction with available means known in the art are conventional.
Therefore, in one embodiment, the invention provides the combining form of specific combination film and the antibody of debond soluble form Neutrokine-α.The purposes of these antibody comprises but non-being limited to as diagnostic probe, differentiates and/or separate the monocytic series of expression film combining form Neutrokine-α.For example, on the activatory monocyte, the Neutrokine-alpha expression of film combining form improves, and therefore, the antibody that the present invention is contained can be used for detecting and/or the monocytic level of definite activatory.In addition, the antibody of a binding film combining form Neutrokine-α can be used for toxin is oriented to tumorigenicity, pre-tumorigenicity, and/or express other cell (as monocyte and dendritic cell) of film combining form Neutrokine-α.
In another embodiment, the Neutrokine-α of an antibody of the present invention specific combination soluble form (the 134-285 amino acids of SEQ ID NO:2).The purposes of these antibody comprises but non-being limited to as the Neutrokine-α of diagnostic probe with soluble form in the analysis of biological samples, and as therapeutical agent, toxin guiding is expressed the cell (as the B cell) of Neutrokine-α acceptor, and/or reduce or block the alpha mediated biological activity (producing) of Neutrokine-in external or the body as stimulating B cell proliferation and/or immunoglobulin (Ig).
The present invention also provides the antibody of binding film combination specifically and soluble form Neutrokine-α.
As mentioned above, the present invention has been contained and has been suppressed or reduce Neutrokine-α and/or Neutrokine-α SV in vivo and/or the antibody of external ability in conjunction with its acceptor, in a special embodiment, antibody of the present invention suppresses or reduces Neutrokine-α and/or the Neutrokine-α SV antibody in external ability in conjunction with its acceptor, in another special embodiment, antibody of the present invention suppresses or reduces Neutrokine-α and/or Neutrokine-α SV in vivo in conjunction with the antibody of the ability of its acceptor.The available as herein described or methods known in the art analysis of this inhibition.
Specific combination Neutrokine-α and/or Neutrokine-α SV have also been contained in the present invention, but do not suppress Neutrokine-α and/or the Neutrokine-α SV antibody in conjunction with its acceptor ability in external and/or body.In a special embodiment, antibody of the present invention does not suppress or reduces Neutrokine-α and/or Neutrokine-α SV in external ability in conjunction with its acceptor.In another special embodiment, antibody of the present invention does not suppress or reduces Neutrokine-α and/or Neutrokine-α SV in vivo in conjunction with the ability of its acceptor.
As mentioned above, the present invention has been contained inhibition or has been reduced in bioactive antibody external and/or interior Neutrokine-α of body and/or Neutrokine-α SV mediation.In a special embodiment, antibody of the present invention suppresses or is reduced in the B cell proliferation of external Neutrokine-α and/or Neutrokine-α SV mediation.This inhibition can be analyzed by conventional method of modifying B cell proliferation described herein or known in the art.In another special embodiment, the B cell proliferation that antibody inhibition of the present invention or reduction Neutrokine-α and/or Neutrokine-α SV mediate in vivo.In a special embodiment, antibody of the present invention is 15C10, or its peopleization form.In another preferred special embodiment, antibody is 16C9, or its peopleization form.Therefore, in the special embodiment of the present invention, 16C9 and/or 15C10 antibody or its peopleization form are used for Neutrokine-α and/or Neutrokine-α SV in conjunction with soluble form, and/or its agonist and/or antagonist, thereby suppress (partially or completely) B cell proliferation.
Perhaps, specific combination Neutrokine-α and/or Neutrokine-α SV have also been contained in the present invention, but do not suppress or reduce the antibody of the interior biological activity of external and/or body (as stimulating B cell proliferation) of Neutrokine-α and/or Neutrokine-α SV mediation.In a special embodiment, antibody of the present invention does not suppress or reduces the external biological activity of Neutrokine-α and/or Neutrokine-α SV mediation.In another special embodiment, antibody of the present invention does not suppress or reduces the interior biological activity of body of Neutrokine-α and/or Neutrokine-α SV mediation.In a special embodiment, antibody of the present invention is 9B6, or its peopleization form.
As mentioned above, the antibody of the epi-position that specific combination is identical with at least a antibody that is refered in particular in external and/or body has been contained in the present invention.
In a special embodiment, antibody of the present invention is contained in aminoacid sequence in the Ser171-Phe194 amino acids residue of SEQ ID NO:2 in external specific combination.In another special embodiment, antibody of the present invention specific combination in vivo is contained in aminoacid sequence in the Ser171-Phe194 amino acids residue of SEQID NO:2.In another special embodiment, antibody of the present invention is contained in aminoacid sequence in the Lys173-Lys188 amino acids residue of SEQ IDNO:2 in external specific combination.In another special embodiment, antibody of the present invention specific combination in vivo is contained in aminoacid sequence in the Lys173-Lys188 amino acids residue of SEQ IDNO:2.
In another special embodiment, antibody of the present invention is contained in aminoacid sequence in the Glu223-Tyr 246 amino acids residues of SEQ ID NO:2 in external specific combination.In another special embodiment, antibody of the present invention specific combination in vivo is contained in aminoacid sequence in the Glu 223-Tyr 246 amino acids residues of SEQID NO:2.In another special embodiment, antibody of the present invention is contained in aminoacid sequence in the Val227-Asn242 amino acids residue of SEQ IDNO:2 in external specific combination.In another special embodiment, antibody of the present invention specific combination in vivo is contained in aminoacid sequence in the Val227-Asn242 amino acids residue of SEQ ID NO:2.In another special embodiment, antibody of the present invention is contained in aminoacid sequence in the Phe 230-Cys245 bit amino acidic group of SEQ ID NO:2 in external specific combination.In another special embodiment, antibody of the present invention specific combination in vivo is contained in aminoacid sequence in the Phe230-Cys245 amino acids residue of SEQ IDNO:2.
The present invention also provides 9B6 monoclonal antibody and of the present invention polypeptide of competitive inhibition by the PTA-1159 generation of preservation, preferred SEQ ID NO:2 polypeptide more preferably has the polypeptide bonded antibody of aminoacid sequence of the Ser171-Phe194 position residue of SEQID NO:2.Competitive inhibition can be determined by any method known in the art, for example use competitive binding analysis method as herein described.In preferred embodiments, this antibody competition inhibition 9B6 monoclonal antibody and SEQ ID NO:2 polypeptide, preferably have the Ser171-Phe194 amino acids polypeptide of sequence bonded at least 95%, 90% of SEQ ID NO:2,85%, 80%, 75%, 70%, 60%, 50%.
The present invention also provides the 15C10 monoclonal antibody of competitive inhibition by the PTA-1158 hybridoma generation of preservation, with polypeptide of the present invention, preferred SEQ ID NO:2 polypeptide, the bonded antibody that more preferably has the Glu223-Try246 amino acids polypeptide of sequence of SEQ ID NO:2.In preferred embodiments, this antibody competition presses down property 15C10 monoclonal antibody and SEQ ID NO:2 polypeptide, preferably has the Glu223-Tyr246 amino acids polypeptide of sequence bonded at least 95%, 90% of SEQ ID NO:2,85%, 80%, 75%, 70%, 60%, 50%.
Other embodiments of the present invention relate to 9B6 antibody and express the hybridoma cell line of this antibody.The hybridoma of expressing 9B6 antibody lies in and was deposited among the ATCC on January 7th, 2000, and preserving number is PTA-1159.In an embodiment preferred, antibody 9B6 is the peopleization.
Other embodiments of the present invention relate to 15C10 antibody and express the hybridoma cell line of this antibody.The hybridoma of expressing antibodies 15C10 lies in and was deposited among the ATCC on January 7th, 2000, and preserving number is PTA-1158.In an embodiment preferred, antibody 15C10 is the peopleization.
In a special embodiment, above-mentioned special antibody is with as herein described or methods known in the art peopleization, then as therapeutical agent.
In another special embodiment, above-mentioned arbitrary antibody uses with soluble form.
In another special embodiment, above-mentioned arbitrary antibody is puted together with toxin or mark (as described above).This antibody of puting together is used to kill special cell mass, or quantitative special cell mass.In an embodiment preferred, this antibody of puting together is used to kill the B cell at its surface expression Neutrokine-α acceptor.In another embodiment preferred, this antibody of puting together is used for quantitative B cell at its surface expression Neutrokine-α acceptor.
In another special embodiment, above-mentioned arbitrary antibody is puted together with toxin or mark (as described above).This antibody of puting together is used to kill special cell mass or quantitative special cell mass.In an embodiment preferred, this sew and antibody be used to kill the monocyte of the Neutrokine-α that expresses film combining form.In another embodiment preferred, this antibody of puting together is used for the monocyte of the Neutrokine-α of quantitative expression film combining form.
Antibody of the present invention also has the purposes for the treatment of and/or preventing, comprise but non-ly be limited to activated monocyte or block monocyte activation and/or kill monocytic series, this monocyte is in the Neutrokine-α of its surface expression film combining form (for example treatment, prevention and/or diagnosis myeloid leukemia, leukemia on the monocyte basis and lymphoma, mononucleosis, monopenia, rheumatic arthritis, illness or the pathological state relevant with the activatory monocyte with other).In a special embodiment, antibody complement-fixing of the present invention.In other special embodiment, antibody of the present invention (or its fragment) combine with heterologous polypeptide or nucleic acid (toxin for example, as in conjunction with and the compound of activation of endogenous cellular toxicity effector system, and radio isotope; And cellular toxicity medicine).
In another embodiment, produced one or more monoclonal antibody, their identification or in conjunction with Neutrokine-α and/or its mutain, but nonrecognition or in conjunction with Neutrokine-α SV and/or its mutain.In a relevant embodiment, produced one or more monoclonal antibody, wherein their identification or, but nonrecognition or in conjunction with Neutrokine-α and/or its mutain in conjunction with Neutrokine-α and/or its mutain.
As mentioned above, the antibody of Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide, can be used for producing the antiidiotypic antibody of " simulation " Neutrokine-α, use be that method well known to those skilled in the art (is seen for example Greenspan ﹠amp; Bona, FASEB magazine 7 (5): 437-444 (1989), and Nissionff, Journal of Immunology 147 (8): 2429-2438 (1991).For example, in conjunction with Neutrokine-α and/or Neutrokine-α SV and competitive inhibition Neutrokine-α and/or Neutrokine-α SV polymer and/or with part bonded antibody, can be used for producing the antiidiotypic antibody of " simulation " Neutrokine-α TNF polymerization and/or integrated structure, thus in conjunction with and in Neutrokine-α or Neutrokine-α SV and/or its part.In this and antiidiotypic antibody or its Fab fragment, can be used in the treatment plan with in and the Neutrokine-alpha ligands.For example, this antiidiotypic antibody can be used in conjunction with Neutrokine-α and/or Neutrokine-α SV, or be combined in the Neutrokine-α and/or the Neutrokine-α SV acceptor of B cell surface, thereby the B cell activation of blocking-up Neutrokine-α and/or Neutrokine-α SV mediation, propagation and/or differentiation.
The diagnosis of immunity system relative disease
Neutrokine-α is in kidney, lung, peripheral blood leucocyte, marrow, and t cell lymphoma, B cell lymphoma, the activated T cell, cancer of the stomach, unstriated muscle is expressed in scavenger cell and bleeding of the umbilicus tissue and the monocytic special cells.In addition, Neutrokine-α SV expresses in initial dendritic cell.In addition, Neutrokine-α is at the cell surface expression of following non-hematopoiesis tumor cell line: colorectal carcinoma HCT 116 (ATCC preserving number NO.CCL-247) and HT-29 (ATCC preserving number NO.HTB-38); Adenocarcinoma of colon Caco-2 (ATCC preserving number NO.HTB-37), COLO 201 (ATCC preserving number NO.CCL-224) and WiDr (ATCC preserving number NO.CCL-218); Mammary cancer MDA-MB-231 (ATCC preserving number NO.HTB-26); Bladder squama cancer SCaBER (ATCC preserving number NO.HTB-3); Bladder cancer HT-1197 (ATCC preserving number NO.CRL-1473); Kidney A-498 (ATCC preserving number NO.HTB-44); Caki-1 (ATCC preserving number NO.HTB-46), and Caki-2 (ATCC preserving number NO.HTG-47); Kidney Wilms tumour SK-NEP-1 (ATCC preserving number NO.HTB-48); With carcinoma of the pancreas Hs766T (ATCC preserving number NO.HTB-134); MIA PaCa-2 (ATCC preserving number NO.CRL-1420) and SU.86.86 (ATCC preserving number NO.CRL-1837).With regard to the disease that many immunity systems are correlated with, the variation that Neutrokine-α and/or Neutrokine-α SV gene expression dose are compared with standard level (improve or reduce), can be at the immunity system tissue of taking from individuality or other cell or body fluid (as serum with this disease, blood plasma, urine, synovia or cerebrospinal fluid) the middle detection, " standard " Neutrokine-α and/or Neutrokine-α SV gene expression dose are at the immunity system tissue of the individuality of taking from no disease of immune system or the expression level in the body fluid.Therefore; the invention provides a kind of diagnostic method that during the diagnosis disease of immune system, uses; be included in and take from individual the immunity system tissue or other cell or body fluid; measure the expression of gene level of coding Neutrokine-α and/or Neutrokine-α SV polypeptide; and compare with standard Neutrokine-α and/or Neutrokine-α SV gene expression dose; this gene expression dose improves or reduction shows that disease of immune system or normal activation are arranged; propagation, differentiation and/or dead.
Especially, it is believed that at some and suffer from the mammiferous tissue of cancer, compare with " standard " level, the expression level of Neutrokine-α and/or Neutrokine-α SV polypeptide and coding Neutrokine-α and/or Neutrokine-α SV and mRNA obviously improves or reduces.In addition, it is believed that this and suffer from more mammiferous body fluid of cancer (as serum, blood plasma, urine, and cerebrospinal fluid) or in the cell or tissue, raising or reduction that the serum of the same species of Neutrokine-α and/or Neutrokine-α SV polypeptide expression level and no cancer compares and showed can detect.
For example, as described herein, Neutrokine-α expresses at monocytic cell camber.Therefore, the antibody of polynucleotide of the present invention (as be complementary to all or part of Neutrokine-α mRNA and/or Neutrokine-α SV mRNA polynucleotide sequence) and anti-polypeptide of the present invention (and antibody fragment) can be used for the concentration of quantitative or qualitative monocytic cell at its surface expression Neutrokine-α (as the monocytic leukemia cell).These antibody are in addition in that to detect Neutrokine-α gene expression dose unusual, or structure and/or the sequential of Neutrokine-α and/or Neutrokine-α SV, in the tissue, unusual in cell or the Subcellular Localization diagnostic uses arranged.These diagnositc analysiss can be in vivo or external carrying out, and for example at blood sample, carries out in biopsy or the autopsy tissue.
In addition, as described herein, Neutrokine-α acceptor is initially on the cell of B clone and expresses.Therefore, Neutrokine-α polypeptide of the present invention (the Neutrokine-α polypeptide and the Neutrokine-alpha fusion protein that comprise mark), with the anti-Neutrokine-Alpha antibodies (fragment that comprises anti-Neutrokine-Alpha antibodies) of anti-polypeptide of the present invention, can be used for the concentration of quantitative or qualitative B cell line cell (leukemia or the lymphoma cell relevant) as the B cell at its surface expression Neutrokine-α acceptor.These Neutrokine-α polypeptide and antibody are unusual at detection Neutrokine-α acceptor gene expression level in addition, or the structure and/or the sequential of Neutrokine-α acceptor, tissue, unusual in cell or the Subcellular Localization, and/or in the activity/defective in the diagnosis signal pathway relevant, diagnostic uses is arranged with Neutrokine-α.These diagnositc analysiss can be in vivo or external carrying out, and for example carries out with as herein described or methods known in the art at blood sample or biopsy.
In one embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, or the agonist of Neutrokine-α and/or Neutrokine-α SV or antagonist (as the antibody of anti-Neutrokine-α and/or anti-Neutrokine-α SV), be used for the treatment of, prevention, diagnosis or prediction suffer from the individuality of immune deficiency.
Can be with Neutrokine-α of the present invention and/or Neutrokine-α polynucleotide or polypeptide, or Neutrokine-α and/or Neutrokine-α SV agonist or antagonist (as the antibody of anti-Neutrokine-α and/or Neutrokine-α SV) treatment, prevention, the immune deficiency of diagnosis and/or prediction comprises but non-ly is limited to one or more and is selected from the chain severe combined immunodeficiency of following immune deficiency: X (SCID), euchromosome SCID; Adenosine deaminase defective (ADA defective); the agammaglobulinaemia that X is chain; (XLA); Bruton ' s disease; congenital agammaglobulinemia's disease; third kind of hypoproteinosis of baby that X is chain; acquired agammaglobulinaemia; the agammaglobulinaemia of adult onset; the agammaglobulinaemia of late onset; dysgammaglobulinemia disease; Hypogammaglobulinemia; agammaglobulinaemia; common mutability immune deficiency (CVID) (acquired); Wiskott-Aldrich syndrome (WAS); the high IgM immune deficiency that X is chain; the high IgM immune deficiency that non-X is chain; selective IgA deficiency; IgG subclass defective (have or do not have IgA defective), the antibody defective of the normal or IgS that improves, thymoma immune deficiency; the Ig heavy chain deletion; the k chain is damaged, B cell lymphocytic hyperplasia disease (BLPD), selective IgM immune deficiency; recessive agammaglobulinaemia (Swiss type); reticular dysgenesis, newborn infant's neutropenia, the similar oligoleukocythemia of severe; immunodefiiciency thymic alymphoplasia-underdevelopment or heteroplasia; ataxia-telangiectasis, short-limb dwarfism disease, the lymphocytic hyperplasia syndrome (XLP) that X is chain; the Nezelof syndrome of associating IgS immune deficiency; purine nucleotides Phosphoric acid esterase defective (PNP), MHC II class defective (Bare lymphocyte syndrome) and severe severe combined immunodeficiency.
According to this embodiment, to suffer from the individuality of immune deficiency and compare with the individuality of no immune deficiency, the horizontal abnormality ground of Neutrokine-α and/or Neutrokine-α SV is low.Any method as herein described or known in the art all can be used to detect Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide (as facs analysis or ELISA detection and hybridization or PCR detection), and is used for determining Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or the polypeptide expression pattern at biological sample.
The biological sample feature of suffering from the people of immune deficiency be with no immune deficiency individuality in observed result compare, the expression level of its Neutrokine-α and/or Neutrokine-α SV is low.Therefore, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, and/or its agonist or antagonist, but the method according to this invention is used for diagnosis and/or prediction immune deficiency.For example, can analyze the Neutrokine-α of the present invention that derives from the biological sample (" target ") of suspecting the people suffer from immune deficiency and/or the correlated expression level of Neutrokine-α SV polynucleotide and/or polypeptide.Expression level and same molecular with one or more these molecules of the present invention compares at the intravital expression level of the people of known no immune deficiency then.Deriving between target and the control sample, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide, and/or the expression level of its agonist and/or antagonist is obviously different, the prompting target suffers from immune deficiency.
In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or Neutrokine-α SV), be used for the treatment of, diagnosis and/or prediction suffer from common mutability immune deficiency disorder, the individuality of (CVID is also referred to as acquired agammaglobulinaemia, and acquired hypogammaglobulinemia) or its subclass disease.According to this embodiment, the individuality of patient CVID or its subclass shows as when the individuality with no CVID compares, and Neutrokine-α and/or the expression level of Neutrokine-α SV acceptor on its B cell and/or monocyte are unusual.Any method as herein described and known in the art can be used for detecting Neutrokine-α polynucleotide of the present invention or polypeptide and/or its receptor polypeptides and (for example detects the facs analysis or the ELISA of polypeptide, and the hybridization or the PCR method of detection polynucleotide), and be used for determining Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and receptor polypeptides thereof, in the sample that contains monocyte at least or its some components (as RNA), with in the sample that contains B cell at least or its component (as RNA), different expression patterns.Determining containing at least in the sample of monocyte or its some components (as RNA), Neutrokine-α and/or Neutrokine-α SV polynucleotide or expression of polypeptides strengthen, and determine in the sample that contains B cell or its some components (as RNA) at least, under Neutrokine-α and/or Neutrokine-α SV polynucleotide or the subnormal situation of expression of polypeptides level, (be acquired agammaglobulinaemia, or acquired hypogammaglobulinemia) can be pointed out and suffered from CVID to this sample.
People's feature of suffering from CVID be when with human body at no CVID in the result that observes when comparing, Neutrokine-α and acceptor thereof (NAR) expression level in peripheral blood or circulation blood B cell is all high.On the contrary, the feature of not suffering from the people of CVID is that Neutrokine-alpha expression level is low in periphery or circulation blood B cell, and NAR expression level height.Therefore, Neutrokine-α of the present invention, Neutrokine-α SV polypeptide and/or NAR polypeptide, polynucleotide, and/or its agonist or antagonist, but the method according to this invention is used in the middle of the difference diagnosis of this CVID hypotype, for example, analyze Neutrokine-α of the present invention, the correlated expression level of Neutrokine-α SV and/or NAR polynucleotide and/or polypeptide to deriving from the peripheral blood B cell sample of doubting the people's (target) who suffers from CVID.The expression level and the expression level of same molecular in the human body (contrast) of known no CVID of one or more these molecules of the present invention are compared.Between target and control sample, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, and/or the obvious difference of NAR expression of polypeptides level, the prompting target suffers from CVID hypotype disease.
In a special embodiment, Neutrokine-α and/or Neutrokine-α SV polypeptide, or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or anti-Neutrokine-α SV), be used for diagnosis, prediction, treatment or prevention are characterised in that serum immune globulin produces defective, repeatedly the disease of infection of Fa Shenging and/or function of immune system imbalance.In addition, Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or Neutrokine-α SV), can be used for diagnosis, prediction, treatment or prevention joint, bone, the infection of the skin and/or the parotid gland, blood infection (as septicemia, meningitis, septicemia sacroiliitis, and/or osteomyelitis), autoimmunization systemic disease (as described herein), inflammation, and malignant tumour and/or with these diseases, infect, any disease or functional disorder or pathological state that functional disorder and/or malignant tumour are relevant comprise but non-limit CVID other immune deficiency of former, the HIV disease, CLL, the bronchitis of Fa Shenging repeatedly, fistula, otitis media, inflammation of connective tissue, pneumonia, hepatitis, meningitis, zoster (as the severe zoster) and/or pneumocystiscapnii.
In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or Neutrokine-α SV), be used for the treatment of, diagnosis or prediction suffer from the individuality of autoimmunization systemic disease.
Available Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or Neutrokine-α SV) treatment, the autoimmune disease of diagnosis or prediction comprises but non-one or more following disease that is limited to: autoimmune hemolytic anemia, the autoimmunity neonatal thrombocytopenia, idiopathic thrombocytopenic purpure, the autoimmunity cytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, the recurrent polychondritis, rheumatic heart disease, glomerulonephritis (as IgA nephropathy), multiple sclerosis, neuritis, uveitis, multiple endocrinopathy, purpura (as the Henloch-Scoenlein purpura), Reiter ' s disease, Stiff-Man syndrome, autoimmune pulmonary inflammation, Guillain-Pane syndrome, pancreas is led plain dependent diabetes, with the autoimmunity ophthalmia, autoimmune thyroiditis, hypothyroidism (being Hashimoto ' s thyroiditis), systemic lupus erythematous, Goodpasture ' s syndrome, pemphigus, receptor autophosphorylation immunity be (a) Graves disease for example, (b) Myasthenia Gravis, (c) pancreas is led plain resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatic arthritis, schleroderma with anticol original antibody, the blended connective tissue disease (CTD), polymyositis/dermatomyositis, pernicious anemia, spontaneous Addison ' s disease, Infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, BP, Sjogren ' s syndrome, diabetes and adrenin drug resistance (comprising treatment asthma or the Fibrotic adrenin drug resistance of bladder), chronic active hepatitis, primary biliary cirrhosis, other incretory gland pathology, vitiligo, vasculitis, back MI, cardiac muscle syndrome, urticaria, hereditary allergic dermatitis, asthma, inflammatory myositis and other inflammation, granulamatous, degenerate, and the atrophic functional disorder).
According to this embodiment, the individuality of suffering from autoimmune disease or functional disorder shows as with the individuality that does not have autoimmune disease or functional disorder and compares, its Neutrokine-α, the expression level of Neutrokine-α SV and/or NAR is high unusually, any method as herein described and known in the art all can be used for detecting Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, and/or the NAR polypeptide (for example detects the facs analysis or the ELISA of polypeptide, and the hybridization or the PCR of detection polynucleotide), and can be used for determining Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/polypeptide, and/or the expression pattern of NAR polypeptide in biological sample.
The feature of suffering from the human-body biological sample of autoimmune disease is when comparing with the result that observes in the individuality of no autoimmune disease or functional disorder, Neutrokine-α, the expression level height of Neutrokine-α SV and/or NAR.Therefore, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide, and/or its agonist or antagonist, but the method according to this invention is used for diagnosis and/or prediction autoimmune disease or functional disorder.For example, can analyze and derive from people's (target) biological sample that is suspected to have autoimmune disease or functional disorder the correlated expression level of Neutrokine-α and/or Neutrokine-α SV polynucleotide and/or polypeptide and/or NAR polypeptide.Expression level and the expression level of same molecular in the human body of known nothing self systemic disease with one or more these molecules of the present invention compares then.Between the sample that derives from target and contrast body, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide, and/or its agonist and/or antagonist, and/or the obvious difference of NAR expression of polypeptides level, the prompting objective body suffers from autoimmune disease or functional disorder.
In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or anti-NEUTROKINE-ASV), be used for the treatment of, diagnosis or prediction suffer from the individuality of systemic lupus erythematous or its hypotype disease.According to this embodiment, suffer from the individuality of systemic lupus erythematous or its hypotype, when comparing with the individuality of unsystematic lupus erythematosus, the expression level of Neutrokine-α and/or Neutrokine-α SV is high unusually.Any method as herein described and known in the art all can be used for detecting Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and (for example detects the facs analysis or the ELISA of polypeptide, and the hybridization or the PCR method of detection polynucleotide), and be used for determining Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide expression pattern at biological sample.
People's biological sample feature of suffering from systemic lupus erythematous is when comparing with the result that observes in the individuality of unsystematic lupus erythematosus, the expression level height of Neutrokine-α and/or Neutrokine-α SV.Therefore, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide, and/or its agonist or anti-agent, recurrence according to the present invention can be used for diagnosis and/or prognoses system lupus erythematosus or its hypotype.For example, analyze the correlated expression level of Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide to deriving from the biological sample of doubting the patient's (target) who suffers from systemic lupus erythematous.Expression level and the expression level of same molecular in the human body of known unsystematic lupus erythematosus with one or more these molecules of the present invention compares then.Between the sample that derives from target and contrast body, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide, and/or the obvious difference of expression level in its agonist and/or the antagonist, the prompting objective body suffers from systemic lupus erythematous or its hypotype.
In addition, between the severity extent of systemic lupus erythematous or its hypotype and Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide (RNA) and/or the polypeptide direct relation is arranged.Therefore, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide (RNA), polypeptide and/or agonist or antagonist, the method according to this invention can be used for prognoses system lupus erythematosus or its hypotype disease degree.For example, analysis derives to doubt and suffers from the biological sample of systemic lupus erythematous patient (target) relative expression's level of Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide.Expression level and the expression level of same molecular in this disease of known representative one group of disease in various degree with one or more these molecules of the present invention compares then.According to this method, expression level meets which member among this group patient, then shows the degree with this member.
In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or Neutrokine-α SV), be used for the treatment of, diagnosis or prediction suffer from the individuality of rheumatic arthritis or its hypotype.According to this embodiment, to suffer from the individuality of rheumatic arthritis or its hypotype and the individuality of no rheumatic arthritis or its hypotype and compare, the expression level of Neutrokine-α and/or Neutrokine-α SV is high unusually.Any method as herein described and known in the art all can be used for detecting Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and (for example detects the facs analysis or the ELISA of polypeptide, and the hybridization or the PCR method of detection polynucleotide), and can be used for determining Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or the expression pattern of polypeptide in biological sample.
The biological sample feature of suffering from the patient of rheumatic arthritis be when with individuality in no rheumatic arthritis in the result that observes when comparing, the expression level of Neutrokine-α and/or Neutrokine-α SV improves.Therefore, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide, and/or its agonist or antagonist, but the method according to this invention is used for diagnosis and/or prediction rheumatic arthritis or its hypotype.For example, analysis derives from the biological sample of doubting the patient's (target) who suffers from rheumatic arthritis, relative expression's level of Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide.Expression level and the expression level of same molecular in the human body of known no rheumatic arthritis with one or more these molecules of the present invention compares then.Between the sample that derives from target and contrast body, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide and/or polypeptide, and/or the expression level of its agonist and/or antagonist is obviously different, and the prompting objective body suffers from rheumatic arthritis or its hypotype.
Therefore, the invention provides a kind of the imbalance and comprise this system's cancer at the diagnosis function of immune system, and the efficient diagnosis method in immune deficiency and/or the autoimmune disease, comprise the gene of measuring coding Neutrokine-α and/or Neutrokine-α SV polypeptide, at the immunity system tissue of individuality or the expression level in other cell or the body fluid, and this expression level and standard Neutrokine-α and/or Neutrokine-α SV gene expression dose compared, thereby comparing to improve or reduce with standard level then shows to suffer from disease of immune system.
The diagnosis disease of immune system comprises but the non-diagnosing tumour that is limited to, immune deficiency, and/or autoimmune disease, carry out according to conventional methods, the present invention is as the prediction indicator, thereby patient presents Neutrokine-α and/or Neutrokine-α SV gene expression dose to strengthen or reduces, and with respect to the be near the mark patient of level of gene expression dose, the clinical effectiveness that is showed is more serious.
Analyze or determine the expression of gene level of coding Neutrokine-α and/or Neutrokine-α SV polypeptide, be meant the expression level of the mRNA of quantitative directly or indirectly or qualitative test or estimation Neutrokine-α and/or Neutrokine-α SV polypeptide or coding Neutrokine-α and/or Neutrokine-α SV polypeptide at first biological sample, directly measuring is to determine or estimating absolute protein matter level or mRNA level, indirect measurement be with Neutrokine-α in second biological sample and/or Neutrokine-α SV polypeptide level or mRNA level relatively.Preferably, measure or estimate Neutrokine-α and/or Neutrokine-α SV polypeptide level or mRNA level in first biological sample, and compare with standard Neutrokine-α and/or Neutrokine-α SV polypeptide level or mRNA level, standard level derives from second biological sample of normal human, or gets by the mean level (ML) of determining the normal human.In case one skilled in the art will appreciate that known standard Neutrokine-α and/or Neutrokine-α SV polypeptide level or mRNA level, it can repeat as reference standard.
" biological sample " is meant and derives from the individuality that contains Neutrokine-α and/or Neutrokine-α SV polypeptide or mRNA, body fluid, clone, any biological sample in tissue culture or other source.As mentioned above, biological sample comprises that body fluid is (as serum, blood plasma, urine, synovia and cerebrospinal fluid), it contains the free ectodomain of Neutrokine-α and/or Neutrokine-α SV polypeptide, and immunity system tissue and other are found the tissue of the ectodomain of The expressed or free Neutrokine-α and/or Neutrokine-α SV or its acceptor.The well known method that obtains biopsy and body fluid from Mammals.When biological sample comprised mRNA, biopsy was preferred source.
Compound of the present invention is used for diagnosis, prediction or the relevant disease of the intravital various immunity systems of the treatment preferred people of Mammals.This disease comprises but non-tumour (as B cell and monocytic leukemia and lymphoma) and the metastases of being limited to, bacterium, virus and other parasitic infection, immune deficiency, inflammation, lymphadenopathy, and autoimmune disease (as rheumatic arthritis, systemic lupus erythematous, Sjogren syndrome, blended connective tissue disease, and inflammatory myositis), and graft/host disease.
Can from biological sample, isolate total cell RNA with any appropriate means, as Chomczynski and Sacchi in biochemical analysis 162:156-159 (1987)) described in a step guanidine thiocyanate-benzene phenol-chloroform method.Use the mRNA level of any appropriate means analysis of encoding Neutrokine-α and/or Neutrokine-α SV polypeptide then.These methods comprise the Northern engram analysis, S1 nuclease mapping, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), and reverse transcription-ligase chain reaction (RT-LCR).
Available based on Neutrokine-α in the methods analyst biological sample of antibody and/or Neutrokine-α SV polypeptide level.For example, Neutrokine-α and/or Neutrokine-α SV polypeptide expression can be with traditional immunohistology method research (Jalkanen, M. etc., cytobiology magazine 101:976-985 (1985) in the tissue; Jalkanen, M. etc., cytobiology magazine 105:3087-3096 (1987)).Other method that detects Neutrokine-α and/or the expression of Neutrokine-α SV polypeptide gene based on antibody comprises immunoassay, as enzyme-linked immunosorbent assay (ELISA) radioimmunoassay (RIA).Suitable antibody analysis mark known in the art, comprise enzyme labelling such as notatin and radio isotope such as iodine ( 131I, 125I, 123I, 121I), carbon ( 14C), sulphur ( 35S), tritium ( 3H), indium ( 115mIn, 113mIn, 112MIn, 111MIn), and technetium ( 99Tc, 99MTc), titanium ( 201Ti), gallium ( 68Ga, 67Ga), palladium ( 103Pd), molybdenum ( 99Mo) xenon ( 133Xe), fluorine ( 18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 188Re, 142Pr, 105Rh, 97Ru; Luminescent marking such as luminol,3-aminophthalic acid cyclic hydrazide and fluorescent mark such as fluorescein and rhodamine, and vitamin H.
Methods known in the art can be used for mark antibody of the present invention.This method comprise but non-be limited to use difunctionally put together agent and (see for example U.S. Patent No. 5756065; 5714631; 5696239; 5652361; 55055931; 5489425; 5435990; 5428139; 5342604; 5274119; 4994560; With 5808003; Above document is incorporated reference in full into).
Tissue of being analyzed or cell type generally comprise known or suspect the cell or tissue (as the monocytic series cell) of expressing Neutrokine-α gene, or cell or tissue (as B clone and splenocyte) known or suspection expression Neutrokine-α acceptor gene.The used method for protein isolation of the present invention is for example by Harlow and the described method of Lane (Harlow, E and Lane, D., 1988, " antibody laboratory manual ", press of cold spring harbor laboratory, cold spring port, New York), incorporate reference in full into it.Isolated cells can be derived from cell culture or patient.The cell that culture is taken from analysis is to determine that this cell can be used as the part based on the gene therapy method of cell, or the necessary step of effect of test compounds Neutrokine-α gene or the expression of Neutrokine-α acceptor gene.
For example, antibody as herein described or its fragment can be used for the situation that exists of quantitative or qualitative detection Neutrokine-α gene product or its conservative variant or peptide fragment.This can for example be undertaken by immunofluorescence method, uses opticmicroscope, and flow cytometry or fluorometry detect fluorescently-labeled antibody.
Antibody of the present invention (or its fragment) or Neutrokine-α polypeptide can be used for histologic analysis such as immunofluorescence analysis in addition, in immunoelectron microscope or the non-immune analysis, with in situ detection Neutrokine-α gene product or its conservative variant or peptide fragment, or in conjunction with the Neutrokine-α of Neutrokine-α acceptor.In situ detection can be by getting the histology sample in the patient body, or it is used the antibody or the Neutrokine-α polypeptide of mark of the present invention.Antibody (or fragment) or Neutrokine-α polypeptide preferably cover on the biological sample by the antibody (or fragment) with mark and use.By making in this way, not only can determine Neutrokine-α gene product, or conservative variant or peptide fragment, or the combination of Neutrokine-α polypeptide, also can determine its distribution in test organization.Use the present invention, those skilled in the art can be to many Histological methods (as dyeing process) correct, to carry out in situ detection with easy to understand.
Immunity and non-immunoassay that the conservative variant of Neutrokine-α gene product or its or peptide fragment are carried out, be included under the situation of antibody of detectable label of variant that existence can differentiate that Neutrokine-α gene product or its are conservative or peptide fragment, incubation sample such as biological fluid, tissue extract, the new cell of collecting, or in cell culture the cell lysate of incubation, and detect the bonded antibody by many methods well known in the art.
Immunity and non-immunoassay that the conservative variant of Neutrokine-α acceptor gene product or its or peptide fragment are carried out, be included in variant that existence can differentiate that Neutrokine-α acceptor gene product or its are conservative or peptide fragment detecting or situation that the Neutrokine-α polypeptide of mark exists under, incubation sample such as biological fluid, tissue extract, the new cell of collecting or in cell culture the cell lysate of incubation, and by many methods detection bonded Neutrokine-α polypeptide well known in the art.
Biological sample can contact and be fixed thereon with solid support or carrier, as is fixed in Nitrocellulose or energy fixed cell, other solid support of cell granulations or soluble protein.Then this upholder is washed with suitable buffer reagent, handle with the anti-Neutrokine-Alpha antibodies or the detectable Neutrokine-α polypeptide of detectable label subsequently.Then this solid support is washed once with damping fluid again, to remove unconjugated antibody or polypeptide.Randomly this antibody is mark subsequently.Bonded mark quantity can detect by ordinary method then on the solid support.
" solid support or carrier " is meant any upholder of energy conjugated antigen or antibody.Upholder of knowing or carrier comprise glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, the natural and Mierocrystalline cellulose modified, polyacrylamide, Hui Kayan, and magnetite.The character of carrier can be soluble in some cases, or for the object of the invention insoluble.In fact solid support can have any possible configuration, as long as link coupled molecular energy conjugated antigen or antibody.Therefore, the upholder configuration can be spheric such as pearl, or right cylinder is as the internal surface at test tube, or the outside surface of rod.Perhaps, this surface can be smooth as thin slice, test paper etc.Preferred upholder comprises polystyrene bead.The known many binding antibodies of those skilled in the art or antigenic other suitable carrier maybe can use routine test to determine.
Except analyze Neutrokine-α and/or Neutrokine-α SV polypeptide level or polynucleotide level in deriving from individual biological sample, Neutrokine-α and/or Neutrokine-α SV polypeptide or polynucleotide also can detect by developing in vivo.For example, in one embodiment of the invention, Neutrokine-α and/or Neutrokine-α SV polypeptide and/or anti-Neutrokine-Alpha antibodies are used for the developing B cell lymphoma.In another embodiment, the antibody of Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide and/or anti-Neutrokine-α and/or Neutrokine-α polynucleotide (as be complementary to all or part of Neutrokine-α and/or Neutrokine-α SV mRNA polynucleotide) are used for developing lymphoma (as monocyte and B cell lymphoma).
Being used for the antibody labeling of developing Neutrokine-α in the body and/or Neutrokine-α SV polypeptide or mark comprises and can pass through the X line, NMR, MRI, cat scan, or the mark of ESR detection.With regard to the X line, suitable mark comprises radio isotope such as barium or caesium, and it launches detectable ray, but tested object is not had obvious injury.The suitable mark that carries out NMR and ESR comprises mark such as the tritium with detected characteristics spin, and it can mix in the antibody by the nutrient substance of the relevant hybridoma of mark.When developing is used to detect Neutrokine-α and/or Neutrokine-α SV polypeptide level and strengthens in vivo so that human body is diagnosed, preferred end user's antibody or " peopleization " chimeric mAb.This antibody can be used for as herein described or means known in the art produce.For example, the method for generation chimeric antibody known in the art.For example see Morrison, science 229:1202 (1985); Oi etc., biotechnology 4:214 (1986); Cabilly etc., United States Patent (USP) 4816567; Taniguchi etc., EP171496; Morrison etc., EP173494; Neuberger etc., WO 8601533; Robinson etc., WO 8702671; Boulianne etc., natural 312:643 (1984); Neuberger etc., natural 314:268 (1985).
Perhaps, can use any Neutrokine-α polypeptide that its existence can be detected.For example, can use polypeptide through the Neutrokine-α of radiopaque or other suitable compound mark, and the antibody of evaluation mark in vivo as mentioned above.This in addition Neutrokine-α polypeptide can be used for the in-vitro diagnosis method.
With with Neutrokine-α and/or the Neutrokine-α SV polypeptid specificity antibody or the antibody fragment of suitable detected developing component mark, import in the Mammals (as through non-enteron aisle, subcutaneous or intraperitoneal is used) detecting disease of immune system, described detectable developing component for example be radio isotope (for example 131I, 112In, 99MTc, ( 131I, 125I, 123I, 121I), carbon ( 14C), sulphur ( 35S), tritium ( 3H), indium ( 115MIn, 113MIn, 112In, 111In), and technetium ( 99Tc, 99MTc), titanium ( 201Ti), gallium ( 68Ga, 67Ga), palladium ( 103Pd), molybdenum ( 99Mo) xenon ( 133Xe), fluorine ( 18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru), radiopaque material, or by the detectable material of nucleus magnetic resonance.Those skilled in the art should know the size of tested object and used visualization system will determine to produce diagnosis imaging the developing component.Using under the radioisotopic situation, ratione personae, the radioactivity quantity of injection is generally about 5-20 millicurie 99MTc.The antibody of mark and antibody fragment are preferentially containing the accumulation of the proteic cell of Neutrokine-α place then.The in-vivo tumour imaging sees S.W.Burchiel etc., " radiolabeled antibody and segmental immune pharmacokinetics thereof " (the 13rd chapter, the tumour imaging: the radiological chemistry of cancer detects, and S.W.Burchiel and B.A.Rhodes edit, Masson Publishing Inc. (1982)).
With regard to antibody, it is that it is connected with enzyme that the antibody of wherein anti-Neutrokine-α can detect one of mode of ground mark, and (EIA) uses the product (Voller that connects in enzyme immunoassay, A. " enzyme-linked immunosorbent assay (ELISA) ", 1978, diagnostic level 2:1-7, the microbiology four fens publications of being correlated with, Walkersville, MD); Voller etc., clinical pathology magazine 31:507-520 (1978); Bulter, J.E. Enzymology method 73:482-523 (1981); Maggio, E. etc. (editor) 1980, enzyme immunoassay, CRC press, Boca Raton, FL; Ishikawa, E. etc. (editor), 1981, enzyme immunoassay, Kgaku Shoin, Tokyo).With the enzyme of antibody structure will with suitable substrate reactions, preferably with the chromogenic substrate reaction, producing in this way can be by for example spectrophotometry, the chemical composition that fluorometry or appearance method detect.The enzyme that can be used for detecting ground mark antibody comprises but the non-malate dehydrogenase (malic acid dehydrogenase) that is limited to staphylococcal nuclease, δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate, desaturase, triose-phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, notatin, beta-galactosidase enzymes, rnase, urase, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.In addition, can detect by the colorimetry of use with the chromogenic substrate of enzyme reaction.Also can substrate be compared to the standard of similar preparation to the response situation of enzyme and detect by range estimation.
Also available any other immunoassay detects.For example, by radiolabelled antibody or antibody fragment, can detect Neutrokine-α through radioimmunoassay (RIA) and (see for example Weintraub, B. radioimmunoassay principle, to the 7th training Course of radioligand analytical procedure, The Endocrine Society, March, 1986, incorporate reference in full into it).Radio isotope can be by comprising but the non-γ of being limited to calculating instrument, and methods such as scintillation counter or radioautograph detect.
Also available fluorescent chemicals traget antibody.When fluorescently-labeled antibody is exposed under the light of proper wavelength, can detects it owing to fluorescein and have situation.Fluorescent mark compound commonly used is a fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, ophthaldehyde and fluorescamine.
Antibody also available transmission fluorescence metal as 152Eu, or other lanthanide series metal can detect ground mark.These metals can be with the chelation group of this metal such as diethylenetriamine pentaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA)s (EDTA) and are attached to antibody.
Antibody also can be by with itself and chemiluminescence compound coupling and can detect ground mark.Then by detecting the luminescence phenomenon in chemical reaction process, occur, and determine the antibody of chemoluminescence substance markers.Particularly effective chemical luminescent marking compound for example is a luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, theromatic acridinum ester, imidazoles, acridinium salt and oxalate.
In addition, bioluminescent compound can be used for mark antibody of the present invention.The noclilucence thing is a kind of chemiluminescent substance of finding in biosystem, and wherein catalytic protein improves the effectiveness of chemiluminescent substance reaction.The situation that exists of bioluminescent protein can detect by the situation that exists that detects shiner.The important biomolecule luminophor that carries out mark comprises but the non-fluorescein that is limited to luciferase, and aequorin.
The treatment of immunity system relative disease
As mentioned above, the antibody of Neutrokine-α and/or Neutrokine-α SV polypeptide and polynucleotide and anti-Neutrokine-α is used to diagnose the disease of Neutrokine-α and/or the active unusual high or low expression of Neutrokine-α SV.Provide that Neutrokine-α wherein and/or Neutrokine-α SV expressed with and active cell and the tissue of regulating by Neutrokine-α and/or Neutrokine-α SV, the expression level of comparing Neutrokine-α in the individuality and/or Neutrokine-α SV with standard or " normally " level changes (improve or reduce), produce with wherein Neutrokine-α and/or Neutrokine-α SV be expression and/or the relevant pathological change of active airframe systems.
Those skilled in the art also know, because Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide are the members of TNF family, representative proteinic ectodomain can soluble form from the cell of expressing Neutrokine-α and/or Neutrokine-α SV, discharge by proteolysis, and therefore work as Neutrokine-α and/or Neutrokine-α SV polypeptide (the especially representative ectodomain of soluble form) and add individual tissue from external source, when cell or body, this polypeptide presents the activity of any its target cell through regulating to individuality.Equally, the cell of expressing this II type transmembrane protein can add individual cell, in tissue or the body, thereby the cell that adds combines with the cell of expressing Neutrokine-α and/or Neutrokine-α SV acceptor, thus, the cell of expression Neutrokine-α and/or Neutrokine-α SV can be to the target cell generation effect (as propagation or cellular toxicity) of carrying acceptor.
In one embodiment, the invention provides the composition that contains polypeptide of the present invention for the cell delivery of orientation (as contains and heterologous polypeptide, heterologous nucleic acids, the Neutrokine-α that toxin or prodrug are relevant and/or the composition of Neutrokine-α SV polypeptide or anti-Neutrokine-α and/or anti-Neutrokine-α SV antibody) method, the cell of described orientation is expressed the B cell of Neutrokine-α and/or Neutrokine-α SV acceptor in this way, or the monocyte of the Neutrokine-α of express cell surface bonding form and/or Neutrokine-α SV.Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or anti-Neutrokine-α and/or Neutrokine-α SV antibody, by wetting ability, hydrophobicity, ion and/or covalent interaction and and heterologous polypeptide, heterologous nucleic acids, toxin or prodrug combine.
In one embodiment, the invention provides and a kind of composition of the present invention is delivered to the method for cell specifically, undertaken by using the polypeptide of the present invention relevant (as the antibody of Neutrokine-α and/or Neutrokine-α SV polypeptide or anti-Neutrokine-α and/or anti-Neutrokine-α) with heterologous polypeptide or nucleic acid.In one embodiment, the invention provides a kind of method that therapeutic protein is delivered to directed cell.In another embodiment, the invention provides a kind of nucleic acid (as antisense or ribozyme molecule) with strand, or double-stranded nucleic acid (DNA that if can be integrated into the genome of cell or additional copy and can transcribe).
In another embodiment, the invention provides a kind of special destruction cell method of (as destroying tumour cell), undertaken by using with the polypeptide of the present invention that combines with toxin or cellular toxicity prodrug (as Neutrokine-α and/or Neutrokine-α SV polypeptide or anti-Neutrokine-α and/or anti-Neutrokine-α SV antibody).
In a special embodiment, the invention provides the method for a kind of special destruction B cell line cell (leukemia or the lymphoma relevant), undertaken by using the Neutrokine-α and/or the Neutrokine-α SV polypeptide that combine with toxin or cellular toxicity prodrug as the B cell.
In another special embodiment, the invention provides the method for a kind of special destruction monocytic series cell (as monocytic leukemia or lymphoma), by using the anti-Neutrokine-α relevant and/or anti-Neutrokine-α SV antibody carries out with toxin or cellular toxicity prodrug.
" toxin " is meant combination and activates the compound of endogenous cellular toxicity effector system, radio isotope, holotoxin, the toxin of modifying, the catalytic subunit of toxin, born of the same parents' toxin (cytotoxic agents), or under the condition that causes necrocytosis that limits, be present in or at any molecule or the enzyme of cell surface improperly.The spendable toxin of the method according to this invention comprises but non-ly is limited to radio isotope known in the art that compound is as the antibody (or containing its a part of complement fixation(CF) district) in conjunction with inherent or inductive endogenous cellular toxicity effector system, thymidine kinase, endonuclease, RNase, alpha-toxin, ricin, abrin, pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, α-sarcin and Toxins,exo-, cholera." toxin " also comprises cytostatics or cytocide, therapeutical agent or radioactive metal ion, as alpha emitter for example 213Bi, or other radio isotope is for example 133Xe, 131I, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 90Y, 153Sm, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, 90Yttrium, 117Tin, 186Rhenium, 166Holmium and 188Rhenium, luminescent marking such as luminol,3-aminophthalic acid cyclic hydrazide; With fluorescent mark such as fluorescein and rhodamine, and vitamin H.
Methods known in the art can be used for mark antibody of the present invention.This method comprise but non-be limited to use difunctionally put together agent and (see for example United States Patent (USP) 5756065; 5714631; 5696239; 5652361; 5505931; 5489425; 5435990; 5428139; 5342604; 5274119; 4994560; With 5808003; Described document is all incorporated reference in full into).Born of the same parents' toxin or cytotoxic agents comprise the disadvantageous any preparation of pair cell.For example comprise paclitaxol, cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, mitomycin, etoposide, teniposide, vincristine(VCR), vincaleucoblastine, colchicine, Zorubicin, daunorubicin, dihydroxy anthracin dione mitoxantrone, mithramycin, dactinomycin, 1-dehydrogenation testosterone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Propranololum and tetracycline and analogue thereof or homologue.Therapeutical agent comprise but the non-metabolic antagonist that is limited to (as methotrexate, Ismipur, 6-thioguanine, cytosine arabinoside, 5 FU 5 fluorouracil decarbazine), alkylating agent (dichloromethyldiethylamine for example, the thioepa Chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, the dibromo mannitol, streptozotocin, ametycin, with dichloro diamines cis-platinum (II) (DDP), anthracycline antibiotics (daunorubicin for example, (daunomycin) and Zorubicin) in the past, microbiotic (as daunomycin (actinomycin) in the past, bleomycin, mithramycin, anthramycin (AMC)), and antimitotic agent (as vincristine(VCR) and vincaleucoblastine).
" cellular toxicity prodrug " is meant that the enzyme by normal presence in the cell is converted to the non-toxic chemical of cellular toxicity compound.The spendable cellular toxicity prodrug of the method according to this invention comprises but the non-glutamyl derivative that is limited to M-nitro benzoic acid mustard seed alkylating agent, the phosphate derivative of etioposide or ametycin, cytosine arabinoside, the phenoxy-acetamide derivative of daunorubicin and Zorubicin.
Should know by Neutrokine-α in the individuality and/or Neutrokine-α SV activity level and be lower than disease due to standard or the normal level, especially disease of immune system can be handled by using Neutrokine-α and/or Neutrokine-α SV polypeptide (with soluble ectodomain or the proteic cells form of The expressed) or agonist.Therefore, the present invention also provides a kind of method for the treatment of the individuality that needs raising Neutrokine-α and/or Neutrokine-α SV activity level, comprise to this body and use a kind of pharmaceutical composition, said composition comprises Neutrokine-α isolating of the present invention and/or Neutrokine-α SV polypeptide or its agonist of some amount, improves Neutrokine-α and/or Neutrokine-α SV activity level in this individuality effectively.
It is also understood that by individual Neutrokine-α and/or Neutrokine-α SV activity level and be higher than disease due to standard or the normal level, especially disease of immune system can be treated by using Neutrokine-α and/or Neutrokine-α SV polypeptide (the proteic cells form of soluble ectodomain or The expressed) or its antagonist (as the antibody of anti-Neutrokine-α).Therefore the present invention also provides a kind of method that needs to reduce Neutrokine-α and/or the active individuality of Neutrokine-α SV for the treatment of, be included as this individuality and use a kind of pharmaceutical composition, said composition comprises isolating Neutrokine-α of a certain amount of the present invention and/or Neutrokine-α SV polypeptide, or its antagonist, to reduce Neutrokine-α and/or the Neutrokine-α SV activity level in this individuality effectively.
Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide or its antagonist can be used for treating infectious disease.For example, especially improve B cell proliferation and differentiation, can treat infectious disease by improving immunne response.Immunne response can be passed through to strengthen existing immunne response, or is improved by starting new immunne response.Perhaps, Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide or its antagonist also can directly suppress infectious factors, and need not excite immunne response.
Virus for example is a kind of infectant that can produce disease or clinical symptom, and this disease or clinical symptom can be by Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide or its agonists and treated.Virus for example comprises but non-following DNA and RNA viruses and the Viraceae of being limited to: arboviruses, adenovirus, arenavirus, arteritis virus, birnavirus, bunyavirus, Calicivirus, Circoviridae, coronavirus, dengue virus, EBV, HIV, flavivirus, hepadnavirus (hepatitis virus), simplexvirus is (as cytomegalovirus, hsv, varicella zoster virus), Mononegavirus is (as paramyxovirus, Measles virus, rhabdovirus), orthomyxovirus is (as influenza A virus, Influenza B virus and parainfluenza virus), papilloma virus, papovavirus, parvovirus, picornavirus, poxvirus (as smallpox or vaccinia virus), reovirus (as rotavirus), retrovirus (HTLV-I, HTLV-II, lentivirus), and togavirus (as rubella virus).Virus in these families can cause various diseases or clinical symptom, comprises but the non-sacroiliitis that is limited to bronchitis, respiratory syncytial virus, encephalitis, ocular infection (as conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (first, second, the third, penta type, the chronic active type, the δ type), Japanese Type B encephalitis, Junin, Chikungunya, Rift Valley heat, yellow jack, meningitis, opportunistic infection (as AIDS), pneumonia, Burkitt ' s lymphoma, varicella, hemorrhagic fever, measles, mumps, parainfluenza, rabies, common cold poliomyelitis, leukemia, rubella, venereal disease, tetter (as Kaposi ' wart) and viremia.Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide or its agonist or antagonist can be used for treatment, and any these symptoms or disease is diagnosed and/or detected in prevention.In special embodiment, Neutrokine-α-polynucleotide, polypeptide or agonist are used for the treatment of, prevention and/or diagnosis: meningitis, singapore hemorrhagic fever, EBV, and/or hepatitis (as B-mode).In another special embodiment, Neutrokine-α-polynucleotide, polypeptide or agonist are used for the treatment of and are purchased the unresponsive patient of hepatitis vaccine to one or more other.In another special embodiment, Neutrokine-α polynucleotide, polypeptide or agonist are used for the treatment of, prevention and/or diagnosis AIDS.In another special embodiment, Neutrokine-α and/or Neutrokine-α SV and/or its acceptor polynucleotide, polypeptide, agonist and/or antagonist are used for the treatment of, prevention and/or diagnosis cryptosporidiosis.
Similarly, can cause disease or clinical symptom and available Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or the bacterium of its agonist or antagonist for treating or mycosis are because of comprising but non-ly be limited to following Gram-negative and gram positive bacterium and bacterium section and fungi: the unwrapping wire order is (as excellent bacillus, mycobacterium, Nocardia bacteria), novel Cryptococcus, aspergillus, Bacillaceae is (as anthrax-bacilus, Clostridium), class Cordycepps, blastomycete, Bordetella, burgdorferi (as Borrelia Burgdorferi), brucella, Candida, Campylobacter, coccidioides immitis, cryptococcus, Dermatocycoses, intestinal bacteria (as enterotoxigenic E.Coli and enterohemorrhagic Escherichia coli), enterobacter (Klebsiella, salmonella (as Corynebacterium diphtheriae, paratyphosum Bacterium), Serratiia, yersinia's genus), Erysipelothrix, convolution bacillus, legionella, leptospira, listeria (as Listeriamonocytogenes), mycoplasma, leprosy bacillus, vibrio cholerae, and neisseria (as actinobacillus, Gomorrhea, meningococcus), Meisseriameningitidis, and the Pasteurella infection (as the unwrapping wire genus bacillus, influenzae (as the Type B hemophilus influenzae), Pasteurella), Rhodopseudomonas, Rickettsiae, chlamydozoan, syphilis, Shigella, Staphylococcus, meningococcus, streptococcus pneumoniae, and suis (as streptococcus pneumoniae and B group B streptococcus B).These bacteriums or fungi section can produce following disease or symptom, comprise but the non-bacterium mass formed by blood stasis that is limited to endocarditis, ocular infection (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infection (infection relevant) as AIDS, paronychia, the infection that prosthese is relevant, Reiter ' s disease, respiratory tract infection, as Whooping cough or empyema, septicemia, Lyme disease, Cat-Seratch disease, dysentery, paratyphoid, food poisoning, typhoid fever, pneumonia, gonorrhoea, meningitis (as first type, epidemic cerebrospinal meningitis B), chlamydiosis, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, sausage poisoning, gangrene, tetanus, pustulosis, rheumatic fever, scarlet fever, venereal disease, tetter is (as cellulitis, dermatocycoses), toxicaemia, urinary tract infections, wound infection.Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist can be used for handling, and any these sick disease or symptom are diagnosed and/or detected in prevention.In special embodiment, Neutrokine-α polynucleotide, polypeptide or its agonist are used for the treatment of, prevention and/or diagnosis: tetanus, Diptheria, sausage poisoning and/or epidemic cerebrospinal meningitis B.
In addition, cause disease or symptom and can use Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or the parasitosis of its agonist treatment because of, comprise but non-following family or the type of being limited to: loeschiasis, babesiosis, the ball spore, cryptosporidiosis, Dientarnoebiasis, covering disease, Ectoparasitic, giardiasis, verminosis, leishmaniasis, Taylor's that piroplasmosis, toxoplasmosis, a trypanosomiasis and hair genus and sporozoite are (as Plasmodium virax, plasmodium falciparum, malaria plasmodium and Plasmodium ovale).These parasites can cause various diseases or symptom, comprise but the non-scabies that is limited to autumn tsutsugamushi fever, ocular infection, intestinal tract disease (as dysentery, giardiasis), hepatic diseases, pulmonary disorder, opportunistic infection (infection relevant) as AIDS, malaria, pregnancy complication, and toxoplasmosis.Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist can be used for treatment, prevention, diagnosis and/or detection any of these symptom or disease.In special embodiment, Neutrokine-α-polynucleotide, polypeptide or its agonist are used for the treatment of, prevention and/or diagnosis malaria.
In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist and/or antagonist, be used for the treatment of, prevention and/or diagnosis inner ear infections (as otitis media), and further feature is to infect due to streptococcus pneumoniae and other pathogenic micro-organism.
In a special embodiment, Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or Neutrokine-α SV), be used for the treatment of or prevent to be characterised in that serum immune globulin produces defective, the disease of recurrent infection and/or function of immune system imbalance.Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist (as the antibody of anti-Neutrokine-α and/or Neutrokine-α SV) in addition can be used for treatment or prevention joint, bone, skin and/or the parotid gland infect, and infect (as septicemia due to the blood, meningitis, septicemia sacroiliitis, and/or osteomyelitis), autoimmunization systemic disease (as described herein), inflammation, and malignant tumour, and/or with these infection, disease, function are closed accent or relevant any disease or functional disorder or the pathological change of malignant tumour, comprise but the non-CVID of being limited to, other former immune deficiency, HIV, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, zoster (as the severe zoster), and/or Pneumocystis carinii.
Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist, can be used for diagnosis, prediction, treat or prevent one or more following disease or functional disorder, or pathological state: former immune deficiency, immune-mediated thrombocyte anaemia, Kawasaki syndrome, bone marrow transplantation (as adult or children's bone marrow transplantation), chronic B cell lymphoid leukemia, HIV infects (infecting as adult or children HIV), chronic inflammation demyelination polyneuropathy, and post-transfusion purpura.
In addition, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist or antagonist, can be used for diagnosis, prediction, treat or prevent one or more following disease, functional disorder or pathological state: Guillian-Barre syndrome, anaemia is (as the anaemia relevant with assays for parvovirus B 19, patient with stable a plurality of myelomatosis, the danger of its infection very high (as recurrent infection), autoimmune hemolytic anemia (as the heat-type autoimmune hemolytic anemia), thrombocyte anaemia (as blood of neonate platelet anaemia), with immune-mediated nervosa anaemia), transplant (as the negative acceptor of the CMV of the positive organ of CMV), hypogammag lobulinemia (as have infect or newborn infant's hypogammag lobulinemia of initiation potential factor), epilepsy (as the epilepsy of refractory), the general vascular syndrome, myasthenia (as the cardiac decompensation in the myasthenia), dermatomyositis, and polymyositis.
The other preferred embodiment of the present invention comprises but non-being limited to Neutrokine-α and/or Neutrokine-α SV polypeptide that polynucleotide and function agonist thereof are used for following application:
(as mouse, rat is exempted from, hamster, cavy to be applied to animal, pig, piggy, chicken, camel, goat, horse, ox, sheep, dog, cat, inhuman Primates, and people, preferred people), produce one or more antibody (as IgG, IgA, IgM and IgE) of increasing amount with the skeptophylaxis system, induce than high-affinity antibody and produce (as IgG, IgA, IgM and IgE), and/or improve immunne response.In a special embodiment, use Neutrokine-α polypeptide of the present invention and/or its agonist, produce the IgG of increasing amount with the skeptophylaxis system.In another special embodiment, use Neutrokine-α polypeptide of the present invention and/or its agonist, produce the IgA of increasing amount with the skeptophylaxis system.In another special embodiment, use Neutrokine-α polypeptide of the present invention and/or its agonist, produce the IgM of increasing amount with the skeptophylaxis system.
Be applied to animal (comprise but non-be limited to above listed and also comprise transgenic animal), described animal can not produce functional endogenous antibody molecule, or has an endogenous immunity system of other composition, but can not be by from other animal, rebuilding or part is rebuild immunity system and produced the human normal immunoglobulin molecule and (see for example PCT publication No.WO 98/24893, WO 96/34096, and WO 96/33735 and WO 91/10741 are described).
A kind of vaccine adjuvant strengthens the immunne response to specific antigen.In a special embodiment, vaccine adjuvant is Neutrokine-α as herein described and/or Neutrokine-α SV polypeptide.In another special embodiment, vaccine adjuvant is Neutrokine-α described herein and/or α SV polynucleotide (being that Neutrokine-α and/or Neutrokine-α SV polynucleotide are vaccine adjuvants of a kind of heredity).As described herein, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide can use methods known in the art to use, and comprise but non-liposome conveying, recombinant vectors conveying, the injection naked DNA, and particle gun conveying of being limited to.
A kind of adjuvant strengthens tumour-specific immune response.
A kind of adjuvant strengthens antiviral immunity and replys.Antiviral immunity is replied available composition of the present invention and is made adjuvant and strengthen, and antiviral immunity is replied and comprised but non-virus of the present invention or known in the art disease or the symptom relevant with virus of being limited to.In special embodiment, composition of the present invention is as adjuvant, to strengthen being selected from following virus disease or the immunne response of symptom: AIDS, meningitis, singapore hemorrhagic fever, EBV, and hepatitis (as hepatitis B).In another special embodiment, composition of the present invention is as adjuvant, to strengthen being selected from following virus, the immunne response of disease or symptom: HIV/AIDS, respiratory syncytial virus, singapore hemorrhagic fever, rotavirus, Japanese Type B encephalitis, first type and second type influenza virus, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley heat, herpes simplex, and yellow jack.In another special embodiment, composition of the present invention strengthens the antigenic immunne response of HIV gp120 as adjuvant.
A kind of adjuvant strengthens antibiotic or antifungal immunity is replied.Available composition of the present invention is made the antibiotic or antifungal immunity of adjuvant enhanced and is replied, and comprises bacterium described herein and known in the art or fungi and disease or the symptom relevant with bacterium or fungi.In special embodiment, composition of the present invention is used as adjuvant to strengthen being selected from following bacterium or fungi, the immunne response of disease or symptom: tetanus, diphtheria, sausage poisoning, and epidemic cerebrospinal meningitis B.In another special embodiment, composition of the present invention is as adjuvant, to strengthen being selected from following bacterium or fungi, the immunne response of disease or symptom: vibrio cholerae, leprosy bacillus, Corynebacterium diphtheriae, paratyphosum Bacterium, Meisseria meningitidis, streptococcus pneumoniae, the B group B streptococcus B, shigella, enterotoxigenic E.Coli, enterohemorrhagic Escherichia coli, Borreliaburgdorferi, plasmodium (malaria).
A kind of adjuvant strengthens antiparasitic immunity and replys.Available composition of the present invention is made adjuvant enhanced antiparasitic immunity and is replied, and comprises parasite described herein or known in the art and disease or the symptom relevant with parasite.In special embodiment, composition of the present invention strengthens parasitic immunne response as adjuvant.In another special embodiment, composition of the present invention is used as adjuvant to strengthen the immunne response to plasmodium (malaria).
The stimulant of pathogenic agent being replied as the B cell.
As before accepting immunosuppressant therapy, estimate the factor of individual immunity state.
As the factor of inducing high-affinity antibody.
As the factor that improves serum immune globulin concentration.
Comprise the individual factor that reclaims as quickening immunity.
The factor as auxiliary the elderly's immunne response.
As before in bone marrow transplantation and/or other transplanting (as of the same race or xenotransplant), during or afterwards, immune enhanser.Consider transplanting, composition of the present invention can simultaneously, and/or be used before transplanting afterwards.In a special embodiment, composition of the present invention is used before beginning to reclaim the T cell after transplanting.In another special embodiment, composition of the present invention at first begins to recover after the T cell mass after transplanting, but uses before the B cell recovering fully.
As the factor of the immunne response of auxiliary B cellular immunity deficiency individuality, splenectomy postoperative patient partially or completely for example.Can be by using Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide; or its agonist and be improved or the B cellular immunity deficiency disease for the treatment of comprises but the chain severe combined immunodeficiency (SCID) of the non-X of being limited to; euchromosome SCID; adenosine deaminase damaged (ADA is damaged); the agammaglobulinaemia (XLA) that X is chain; Bruton ' s disease; congenital agammaglobulinemia's disease; baby's agammaglobulinaemia that X is chain; acquired agammaglobulinaemia; the adult onset agammaglobulinaemia; the late onset agammaglobulinaemia; dysgammaglobulinemia; hypogammag lobulinemia; transient hypogammaglobulinemia of infancy; non-specific hypogammag lobulinemia; agammaglobulinaemia; the immune deficiency of common variation (CVID) (acquired); Wiskott-Aldrich syndrome (WAS); the high IgM immune deficiency that X one is chain; the high IgM immune deficiency that non-X is chain; selective IgA deficiency; IgG subclass defective (have or do not have IgA defective); normal or high IgS antibody defective; the thymoma immune deficiency; the Ig heavy chain deletion; the k chain is damaged; B cell lymphocytic hyperplasia disease (BLPD); the selective IgM immunodeficiency; recessive agammaglobulinaemia (Swiss type); reticular dysgenesis; newborn infant's neutropenia; the severe congenital leukemi; the immunodefiiciency thymic alymphoplasia; ataxia telangiectasia, short-limb dwarfism, the lymphocytic hyperplasia syndrome (XLP) that X is chain; the Nozelof syndrome of associating Igs immunodeficiency; purine nucleoside phosphorylase (PNP) defective, mhc class ii defective (Bare lymphocyte syndrome), and severe combined immunodeficiency.
Reply preparation as booster immunization with B cell function loss of atachment individuality, can be by using Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist and be improved or the disease of acquired B cell function forfeiture of causing for the treatment of comprises but the non-HIV of being limited to infects, AIDS, bone marrow transplantation and B cell lymphocytic leukemia (CLL).
As the factor of assisting temporary immunodeficiency individual immunity to reply.Can be by using Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist and be improved or the disease that causes temporary transient immunodeficiency for the treatment of comprises but the non-virus infection (as influenza) that is limited to, the disease relevant with malnutrition, mononucleosis infects, or with disease that stress be relevant, measles, blood transfusion, surgical operation.
As passing through monocyte, the conditioning agent that dendritic cell and/or B cell carry out antigen presentation.In one embodiment, Neutrokine-α and/or Neutrokine-α SV polypeptide (solvable, film in conjunction with or stride form membrane) or polynucleotide, enhancement antigen is presented or the antagonism antigen presentation in external or body.In addition, in relevant embodiment, this enhancing of antigen presentation or antagonistic action can be used for antineoplaston or regulate immunity system.
Mediation agent as mucosal immune response.Monocytes Neutrokine-α and B cell are replied prompting to other, its can be contained between B cell and the monocyte or the filial generation of their differentiation between in the handshaking.This activity in many aspects with B cell and T cell between the CD40-CD154 signal identical.Therefore, Neutrokine-α can be the important conditioning agent to the T cell dependent/non-dependent immunne response of environment cause of disease.Especially relevant with the mucous membrane place and can reply many natural immunitys in the human body unconventional B cell mass (CD5+) can be replied Neutrokine-α, therefore strengthens individual protective immune state.
As instructing the individual immunity system that the factor of the humoral response (be TH2) opposite with the TH1 cell response takes place.
As the factor of induced tumor propagation, and therefore make it be more suitable for antineoplastic agent.For example, multiple myeloma is a kind of disease of slow differentiation, and therefore in fact all inoperative to all anti-tumor methods.If make the faster propagation of these cells, its ability to accept to anti-tumor method will be improved.
Conjugated protein as the B cell-specific, the specificity activator or the inhibitor of cell growth can be attached to it.The result is that the activity with this activator or inhibitor concentrates on normally, the disease attitude or tumprigenicity B cell mass.
As the factor that detects the B cell line cell by its specificity.This application can require with vitamin H or other preparation (as described herein) labelled protein, to detect.
As the stimulant of the generation of B cell in some diseases, as AIDS, chronic lymphocytic functional disorder and/or common mutability immunodeficiency.
As the part of the method for selecting the B cell, its function is to separate the B cell from the heterogeneous mixture of various types of cells.Neutrokine-α can with solid support coupling, B cell and its specific combination then.Rinse out unconjugated cell, subsequently the cell of elution of bound.A kind of non-limiting application of this selection is to make before transplanting, with tumour cell from for example marrow or remove the blood on every side.
As in operation, generate and/or the adenoid therapy of regenerating after wound or the genetic deficiency.
As the gene therapy that causes immunoincompetent genetic diseases as in patient SCID, observing.
As generating the antigen of replying that suppresses or strengthen Neutrokine-α medium.
Material as activated mononuclear cell/scavenger cell acts on monocytic parasitic disease to resist, as leishmaniasis.
Before transplanting as the agent of marrow The pretreatment.This processing improves the B cell presents again, and therefore quickens to reclaim.
As the material of regulating the excretory cytokine, the secretion of this cytokine is excited by Neutrokine-α.
Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist is used in external or the interior IgE concentration of regulating of body.
In addition, Neutrokine-α of the present invention or Neutrokine-α SV polypeptide or Nucleotide, or its agonist can be used for treatment, the transformation reactions of prevention and/or diagnosis IgE mediation.This transformation reactions comprises but the non-asthma that is limited to, rhinitis and eczema.
In a special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevention is diagnosed and/or is improved selective IgA deficiency.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevention is diagnosed and/or is improved ataxia telangiectasia.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevent, diagnose and/or improve the immunodeficiency of common variation.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevention is diagnosed and/or is improved the chain agammaglobulinaemia of X.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevention is diagnosed and/or is improved severe combined immunodeficiency (SCID).
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevention is diagnosed and/or is improved Wiskott-Aldrich syndrome.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevention is diagnosed and/or to improve the Ig of the chain high IgM of X damaged.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist or antagonist (as anti-Neutrokine-Alpha antibodies), with treatment, prevention, diagnosis and/or diagnosing chronic myelomatosis, acute myelogenous leukemia, leukemia, the hystiocytic leukemia, monocytic leukemia (as acute monocytic leukemia), reticulosis leukemia, Shilling type monocytic leukemia, and/or derived from other leukemia of the cell and/or the tissue of monocyte and/or monokaryon.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevention is diagnosed and/or is improved the monocytic leukemia reaction, for example sees tuberculosis.
In another special embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its agonist, with treatment, prevention, diagnosis and/or improvement, monocarpotic cellularity leukocytosis, the monocarpotic cellularity leukopenia, monocarpotic cellularity anaemia and/or mononucleosis.
In a special embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, and/or the antibody of anti-Neutrokine-α and/or its agonist or antagonist, be used for the treatment of, prevention detects and/or diagnoses former bone-marrow-derived lymphocyte functional disorder and/or associated disease and/or pathological state.In one embodiment, this primary bone-marrow-derived lymphocyte functional disorder, disease and/or pathological state, feature are to lose humoral immunization wholly or in part.Be characterised in that and lose humoral immunization wholly or in part, and available composition prevention of the present invention, treatment, detect and or the primary bone-marrow-derived lymphocyte functional disorder of diagnosis, disease and/or pathological state, comprise but the chain agammaglobulinemia (XLA) of the non-X of being limited to that severe combined immunodeficiency (SCID) and selectivity IgA are damaged.
In an embodiment preferred, Neutrokine-α and/or Neutrokine-α SV polynucleotide, polypeptide and/or its agonist or antagonist are used for the treatment of, the disease that prevention and/or diagnosis are relevant with one or more various mucous membranes of body.This disease comprises but the non-mucositis that is limited to, mucoclasis, mucous colitis, mucous membrane and cutaneous leishmaniosis (for example U.S. state leishmaniasis, nasopharyngeal leishmaniasis and New World leishmaniasis), mucous membrane and skin lymph method syndrome (for example Kawasaki disease), mucoenteritis, mucus epidermoid carcinoma, mucus epiderm-like tumour, the mucous epithelium dysplasia, adenocarcinoma,mucoid, mucinous degeneration, mucinous degeneration; Myxomatosis; Multiple myxoma, floor height in the mucoid (as the capsule Medionecrosis), sticking fat disease (comprises that the I type glues the fat disease, the II type glues the fat disease, the III type glues the fat disease, with the sticking fat disease of IV type), mucoenteritis, mucopolysaccharidosis is (as I type mucopolysacchariduria (being Hurler ' s syndrome), IS type mucopolysaccharidosis (being Scheie ' s syndrome or V-type mucopolysaccharidosis), II type mucopolysaccharidosis (being Hunter ' s syndrome), III type mucopolysaccharidosis (being Sanfilippo ' s syndrome), IV type mucopolysaccharidosis (being Morquio ' s syndrome), VI type mucopolysaccharidosis (being Naroteaux-Lamy syndrome), VII type mucopolysaccharidosis (promptly because beta-Glucuronidase defective due to mucopolysaccharidosis), (mucus vitriol disease) mucopolysacchariduria, MPC, mycopus, mucormycosis, mucosal disease (being bovine viral diarrhoea), mucous colitis (as mucus and mucous colitis), and fibrocystic disease of pancreas is (as pancreatic fibrosis, Carke-Hadfield syndrome, the pancreas Fibrocystic disease becomes, fibrocystic disease of pancreas).In a particularly preferred embodiment, Neutrokine-α and/or Neutrokine-α SV polynucleotide, polypeptide and/or its agonist and/or antagonist are used for the treatment of, prevention and/or diagnosis mucositis, especially relevant with chemotherapy mucositis.
In an embodiment preferred, Neutrokine-α and/or Neutrokine-α SV polynucleotide, polypeptide and/or its agonist and/or antagonist are used for the treatment of, the disease that prevention and/or diagnosis are relevant with sinusitis.
Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist can treat, and the another kind of disease or the symptom of prevention and/or diagnosis are osteomyelitis.
Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its agonist can treat, and the another kind of disease or the symptom of prevention and/or diagnosis are endocarditises.
All above-mentioned application all can be used for animal doctor's pharmacy.
The antagonist of Neutrokine-α comprises combination and/or inhibiting antibody, antisense nucleic acid, ribozyme and Neutrokine-α polypeptide of the present invention.These can expect to reverse some activity of above-mentioned part, and find some clinical or practical applications, as:
Be used to block each side immunne response to external source or self material.For example comprise autoimmune disease such as lupus, and sacroiliitis, and to allergic, inflammation, intestinal tract disease, the immunne response of damage and pathogenic agent.Although available data has directly illustrated the latent effect of Neutrokine-α in the B cell pathology relevant with monocyte, Neutrokine-α may be expressed or reply to other cell type also.Therefore, Neutrokine-α is as CD40 and part thereof, can be by immune situation and the residing microenvironment of cell and regulate.
Be used for preventing the treatment of B cell proliferation relevant and Ig excretory with autoimmune disease, as idiopathic thrombocytopenic purpure, systemic lupus hung classes and MS.
The disease of resisting mutually as graft and host or the inhibitor of transplant rejection.
Treatment B cell malignancies such as ALL, Hodgkins disease, non-Hodgkins lymphoma, lymphocytic leukemia, plasmoma, multiple myeloma, the disease that Burkitt ' s lymphoma and EBV-transform.
Treat chronic hypergammaglobulinemia, as in the single celled gammopathy (MGUS) of not determining importance, Waldenstrom ' s disease is in spontaneous relatively unicellular gammopathy and the plasmoma.
The cell proliferation of treatment large B cell lymphoid tumor reduces.
Reduce B cell relevant and Ig connection thing with chronic lymphocytic leukemia.
As immunosuppressor.
Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its antagonist is used in external or the interior IgE concentration of regulating of body.
In another embodiment, use Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide or polynucleotide, or its antagonist, can be used for treatment, the transformation reactions of prevention and/or diagnosis IgE mediation comprises but the non-asthma that is limited to, rhinitis and eczema.
As comprising ERK1, the inhibitor of the signal pathway of COX2 and Cyclin D2, this approach is relevant with Neutrokine-α inductive B cell activation.
Above-mentioned being applied among numerous hosts all can be used.This host comprises but the non-people of being limited to, mouse, rabbit, goat, cavy, camel, horse, mouse, rat, hamster, pig, piggy, chicken, ox, sheep, dog, cat, inhuman primate, and people.In special embodiment, the host is a mouse, rabbit, goat, cavy, chicken, rat, hamster, pig, sheep, dog or cat.In preferred embodiments, the host is a Mammals.In a more preferred embodiment, the host is the people.
Agonist and antagonist can with medicine suitable carriers applied in any combination.
Antagonist for example can be used for suppressing Neutrokine-chemotactic and activated macrophage and precursor thereof alpha mediated and/or Neutrokine-α SV mediation, and neutrophil, basophilic granulocyte, bone-marrow-derived lymphocyte, with some T cell subclass such as activatory and CD8 cellular toxicity T cell, and natural killer cell, in some autoimmune diseases and chronic inflammatory diseases and infectious diseases.Autoimmune disease for example comprises that multiple sclerosis and insulin-dependent diabetes mellitus, antagonist also can be used for treatment, prevention and/or diagnose infections disease, comprise silicosis, sarcoidosis, the primary pulmonary fibrosis can be undertaken by stoping mononuclear phagocyte collection and activation.They also can be used for treatment, and prevention and/or diagnosing primary eosinophil syndrome are by stoping eosinophil to produce and moving and carry out.Interior toxicogenic shock is antagonist for treating thus also, by stoping macrophage migration and its to produce Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide carries out.This antagonist also can be used for treating arteriosclerosis, is undertaken by stoping the monocyte infiltration arterial wall.This antagonist also can be used for treatment, the transformation reactions and the immune dysfunction of prevention and/or diagnosis histamine-mediated comprise the transformation reactions in late period, chronic urticaria, and atopic dermatitis, undertaken by chemokine inhibiting inductive mastocyte and basophil degranulation and histamine release.Also can treat transformation reactions such as allergic asthma, rhinitis and the eczema of IgE mediation.Antagonist also can be used for treatment, and prevention and/or diagnosing chronic and acute inflammation are undertaken by stoping monocyte to invade damage field.They also can be used for regulating the normal scavenger cell group of lung, because chronic and acute pneumonia is relevant with compiling of the interior mononuclear macrophage of lung.Antagonist also can be used for treatment, and prevention and/or diagnosticating rheumatic sacroiliitis are undertaken by stoping monocyte to invade the IA synovia of patient.Monocyte influx and activation play an important role in degenerative and struvite arthropathy.Antagonist can be used for disturbing the insalubrious connection reaction of threading by due to IL-1 and the TNF, is undertaken by the biosynthesizing that stops other struvite cytokine.In this mode, antagonist can be used for preventing inflammation.Antagonist also is used for suppressing by Neutrokine-α and/or the heating of Neutrokine-α SV inductive prostaglandin(PG) dependent/non-dependent.Antagonist also can be used for treatment, prevention and/or diagnosis bone marrow lesion, for example aplastic anemia and myelodysplastic syndrome.Antagonist also can be used for treatment, and prevention and/or diagnosis asthma and transformation reactions are undertaken by eosinophil accumulation in the prevention lung.Antagonist also can be used for treatment, prevents and/or diagnose inferior basement membrane of epithelium fibrosis, and this is an obvious characteristic of asthma lung.Antagonist also can be used for treatment, prevention and/or diagnosis lymphoma (as one or more lymphoma cited herein, but non-be limited to this).
All above-mentioned application all can be used for animal doctor's pharmacy.
Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist and/or antagonist can be used for treatment, prevention, and/or the various immunity system relative diseases of the preferred human body of diagnosis Mammals.Many autoimmune diseases be by immunocyte to self thing as due to the non-suitable identification of xenobiotics.This non-suitable identification causes destroying the immunne response of host tissue.Therefore, use Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist and/or antagonist, can suppress immunne response, the especially generation of the propagation of B cell and/or immunoglobulin (Ig) can treat and/or prevent autoimmune disease effectively.Therefore, in preferred embodiments, Neutrokine-α of the present invention and/or Neutrokine-α SV antagonist (as polypeptide fragment and the anti-Neutrokine-Alpha antibodies of Neutrokine-α and/or Neutrokine-α SV) are used for the treatment of, prevention and/or diagnosis autoimmune disease.
Available Neutrokine-α polynucleotide of the present invention, polypeptide and/or antagonist (as anti-Neutrokine-Alpha antibodies) treatment, the autoimmune disease of prevention and/or diagnosis, comprise but the non-autoimmune hemolytic anemia that is limited to, autoimmunity newborn infant thrombopenia, primary thrombocytopenic purpura, the autoimmunization cell reduces, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (being IgA nephropathy), multiple sclerosis, neuritis, uveitis, multiple incretopathy, purpura (as the Henloch-Scoenlein purpura), Reiter ' s disease, Stiff-Man syndrome, autoimmune pulmonary inflammation, Guillain-Barre syndrome, insulin-dependent diabetes mellitus and autoimmune ocular inflammation.
Available combination treatment of the present invention, the other autoimmune disorder of prevention and/or diagnosis, comprise but the non-autoimmune thyroiditis that is limited to, methylene shape adenositis (being Hashimoto ' s thyroiditis), (usually feature is by cell-mediated and body fluid Tiroidina cellular toxicity), systematicness lupus hung classes (feature is blood circulation and the local immunocomplex that produces usually), Goodpasture ' s syndrome (feature is an ABMA usually), the receptor autophosphorylation immunity is as (a) Grave ' s disease (feature is a tsh receptor antibody usually), (b) Myasthenia Gravis (being characterized as acetylcholine receptor antibodies usually) and (c) insulin resistance (being characterized as insulin receptor antibody usually), autoimmune hemolytic anemia (being characterized as the phagolysis of immune body sensitized RBCs usually), autoimmune thrombocytopenic purpura (being characterized as immune body sensitized hematoblastic phagolysis usually).
Available combination treatment of the present invention, the other autoimmune disease of prevention and/or diagnosis, comprise but the non-rheumatic arthritis (immunocomplex that is characterized as usually in the joint is qualitative) that is limited to, schleroderma (being characterised in that kernel antibody and other antinuclear antibodies usually) with anticol original antibody, blended connective tissue disease (being characterised in that the antibody (as ribonucleoprotein) that can extract nuclear antigen usually), polymyositis/epidermis myositis (being characterised in that non-group of ANA usually), pernicious anemia (is characterized as anti-parietal cell usually, microsome, with intrinsic factor antibody) primary Addison ' s disease (being characterized as body fluid and cell-mediated suprarenal gland toxicity usually), infertility (being characterized as antisperm antibody usually), glomerulonephritis (being characterized as glomerular basement membrane antibody or immunocomplex usually) is as primary glomerulonephritis and IgA nephropathy, BP (being characterized as IgG and compensation in the basement membrane usually), Sjogren ' s syndrome (be characterized as usually and organize antibody and/or non-group of ANA (SS-B) more), diabetes (be characterized as usually cell-mediated with body fluid ICA) and suprarenin medicine resistance (comprising asthma or cystic fibrosis suprarenin medicine resistance) (being characterized as Beta-3 adrenergic receptor antibody usually).
Available combination treatment of the present invention, the other autoimmune disease of prevention and/or diagnosis, comprise but the non-chronic active hepatitis (being characterized as smooth muscle antibody (SMA) usually) that is limited to, primary biliary cirrhosis (being characterized as mitochondrial antibody usually), other incretory gland pathology (being characterized as the specific tissue's antibody in the certain situation usually), vitiligo (being characterized as melanocyte antibody usually), vascular lesion (being characterized as Ig and complement and/or low serum complement in the vessel wall usually), MI sequela (being characterised in that heart antibody), postcardiotomy syndrome (being characterized as heart antibody usually), urticaria (being characterized as IgG and the IgM antibody of IgE usually), atopic dermatitis (being characterized as IgG and the IgM antibody of IgE usually), asthma (being characterized as IgG and the IgM antibody of IgE usually), inflammatory myositis and many other inflammation, granulamatous, degenerative and other atrophic disease.
In an embodiment preferred, above-mentioned autoimmune disorder and functional disorder and/or pathological state, with anti-Neutrokine-Alpha antibodies and/or anti-Neutrokine-α SV Antybody therapy, prevention and/or diagnosis.
In a special preferred embodiment, rheumatic arthritis is with anti-Neutrokine-Alpha antibodies of the present invention and/or anti-Neutrokine-α SV antibody and/or other antagonist for treating, prevention and/or diagnosis.
In a special preferred embodiment, lupus is with anti-Neutrokine-Alpha antibodies of the present invention and/or Neutrokine-α SV antibody and/or other antagonist for treating, prevention and/or diagnosis.
In a special preferred embodiment, the ephritis relevant with lupus is with anti-Neutrokine-Alpha antibodies of the present invention and/or Neutrokine-α SV antibody and/or other antagonist for treating, prevention and/or diagnosis.
In a special embodiment, Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its antagonist (as anti-Neutrokine-Alpha antibodies and/or Neutrokine-α SV antibody), be used for the treatment of or prevention system lupus erythematosus and/or associated disease.Available Neutrokine-Alpha antibodies of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, or the disease relevant of its antagonist for treating with lupus, comprise but the non-hematologic disease that is limited to (as hemolytic anemia, leukemia, lymphocytic anemia and thrombocyte anaemia), immunological disease is (as anti-DNA antibody, and anti-Sm antibody), fash, photosensitivity, stomatocace, sacroiliitis, heating, fatigue, body weight reduces, serosity disease (as pleuritis), kidney disease (as ephritis), DPN is (as ictal peripheral neuropathy, the disease that CNS is relevant), gastrointestinal tract disease, the Raynaud phenomenon is as pericarditis.In an embodiment preferred, Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its antagonist (as anti-Neutrokine-α and/or Neutrokine-α SV antibody), be used for the treatment of or kidney disease that prevention is relevant with systemic lupus erythematous.In a preferred embodiment, Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, or its antagonist (as anti-Neutrokine-α and/or Neutrokine-α SV antibody), be used for the treatment of or ephritis that prevention is relevant with systemic lupus erythematous.
Similarly, allergic disease such as asthma (especially allergic asthma) or other respiratory tract disease, also available Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist and/or antagonist for treating.In addition, these molecules can be used for treatment, and prevention and/or diagnosis are to the supersensitivity of antigen molecule, hypersensitivity, or the uncompatibility of blood group.
Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, and/or its agonist and/or antagonist, also can be used for treatment, prevention and/or diagnosis organ rejection or transplanting host repel disease (GVHD) and/or associated disease.The tissue of transplanting destroys the host immune cell by immunne response the organ rejection is taken place.Similarly, in GVHD, also comprise immunne response, but in this case, the immune cell destruction host tissue that external source is transplanted.Use the inhibition immunne response, especially suppressor T cell is bred, differentiation or chemotactic Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its excitement/or antagonist, can prevent organ rejection or GVHD effectively.
Similarly, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist and/or antagonist also can be used for regulating inflammation.For example, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist and/or antagonist, but inflammation-inhibiting is replied the propagation and the differentiation of middle cell.These molecules can be used for treatment, prevention and/or diagnosing acute and chronic inflammatory diseases, comprise chronic prostatitis, the granuloma prostatitis, and malakoplakia, the inflammation relevant with infection (as septic shock, or systemic inflammatory reaction syndromes (SIRS)), the ischemia reperfusion damage, the intracellular toxin fatal disease, sacroiliitis, the acute cellular rejection excessively of complement-mediated, ephritis, cytokine or chemokine inductive pulmonary lesion, inflammatory bowel disease, Crohn ' s disease, or because the excessive generation associated diseases of cytokine (as TNF or IL-1).
In a special embodiment, anti-Neutrokine-α of the present invention and/or Neutrokine-α SV antibody are used for the treatment of, and prevention is regulated, and detect and/or the diagnosis inflammation.
In a special embodiment, anti-Neutrokine-α of the present invention and/or Neutrokine-α SV antibody are used for the treatment of, and prevention is regulated, and detect and/or the diagnosis diseases associated with inflammation.
In another special embodiment, anti-Neutrokine-α of the present invention and/or Neutrokine-α SV antibody are used for the treatment of, and detection and/or diagnosis of allergies and/or hypersensitivity disease are regulated in prevention.
Anti-Neutrokine-α and/or Neutrokine-α SV antibody can be used for combination and suppress Neutrokine-α and/or Neutrokine-α SV activity, with by stoping neutrophil infiltration lung to be treated after damage, prevent and/or diagnosis ARDS.Agonist of the present invention and antagonist can with medicine suitable carriers applied in any combination, as described later.
Neutrokine-α of the present invention and/or Neutrokine-α SV and/or Neutrokine-α acceptor polynucleotide or polypeptide, and/or its agonist and/or antagonist, be used for the treatment of, prevention and/or diagnosis lung pattern disease are (as disease of bronchus, for example sinopulmonary and bronchial infection reach associated disease and other respiratory tract disease).In special embodiment, this disease comprises but the non-bronchial adenoma that is limited to, bronchial asthma, pneumonia is (as bronchopneumonia, and tuberculous bronchopneumonia), chronic obstructive disease of lung (COPD), bronchial polyp, bronchiectasis is (as dry bronchiectasis, tubbiness bronchiectasis, and cystic bronchiectasis), bronchial adenocarcinoma, bronchogenic carcinoma, bronchiolitis (as bronchiolitis exudativa, fibroma bronchiolitis obliterans and proliferative bronchitis), the Bronchio-alveolar cell carcinoma, bronchial asthma, bronchitis (as asthmatic bronchitis, Castellani ' s bronchitis, chronic bronchitis, croupous bronchitis, fibrinous bronchitis, hemorrhagic bronchitis, infectious fowl bronchitis, bronchitis obliterans, the plasticity-bronchitis, membranous bronchitis, putrid bronchitis, and hoose), BG, segmental bronchus oedema, segmental bronchus esophagopathy, segmental bronchus originality cancer, bronchogenic cyst, broncholithiasis, bronchomalacia, bronchomycosis (as bronchopneumonic aspergillosis), bronchopulmonary spirochetosis, hemorrhagic bronchitis, bronchorrhea, bronchospasm, bronchorrhagia, bronchostenosis, Biot ' s respiratory murmur, bronchial breath sounds, the Kussmaul respiratory murmur, Kussmaul-kien respiratory murmur, respiratory acidosis, respiratory alkalosis, hyaline membrane disease of newborn, respiratory insufficiency, scleroma respiratorium disease, respiratory syncytial virus, etc.
In a special embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, and/or its agonist and/or antagonist are used for the treatment of, prevention, and/or diagnosing chronic obstructive disease of lung (COPD).
In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist and/or antagonist, be used for the treatment of, the pathology that prevention and/or diagnosis are relevant with fibrosis, for example but non-ly be limited to cystic fibrosis (comprise cystic fibrosis of the pancreas, Clarke-Hadfield comprehensively demonstrate,proves, pancreas fibering tumour, with the mucus pathology), myocardial fibrosis, primary retroperitoneal fibrosis, pia-arachnoid fibrosis, fibrosis of mediastinum, fibrosis around the brief summary fibrosis under the epidermis, maincenter, all fibrosiss of flesh, the vocal cords fibrosis, fibrosis and Symmer ' s clay fiberization under the adventitia.
Known TNF family part is multi-purpose cytokine, induce a large amount of cell responses, comprise cellular toxicity, antiviral activity, immunoregulatory activity and some gene transcription are regulated (D.V.Goeddet etc., " tumour necrosis factor: gene structure and biological activity ", Symp.Quant.Biol.51:597-609 (1986), the cold spring port; B.Beutler and A.Cerami, biochemical analysis research 57:505-5181 (1988); L.J.Old.Sci.Am.258:59-75 (1988); W.Fiers, the FEBs 284:199-224 (1991) that communicates by letter).TNF family part comprises Neutrokine-α of the present invention and/or Neutrokine-α SV, induces so various cell responses by combining with the TNF family receptors.Neutrokine-α and/or Neutrokine-α SV polypeptide be sure of to excite effective cell response, comprise cell, clone, tissue.Tissue culture or patient's genotype, phenotype and/or form change.As mentioned above, this cell response not only comprises normal physiologic response to TNF family part, also comprises and apoptosis increase or the relevant disease of inhibition.Apoptosis is a kind of physiological mechanism, be contained in the lymphocytic disappearance of immunity system periphery B and/or T, and its functional disorder can cause many different pathological changes (J.C.Ameisen, AIDS8:1197-1213, (1994); P.H.Krammer etc., the general viewpoint 6:279-289 of immunology (1994)).
Available Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide, and the diagnosis of agonist and antagonist, treatment or prevention suppress relevant disease with cell survival raising or apoptosis, comprise cancer (as the cystic lymphangioma knurl, the cancer due to the p53 sudden change, and hormone-dependent tumor, comprise but the non-colorectal carcinoma that is limited to myocardium tumour, pancreatic ductal carcinoma, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, carcinoma of testis, osteocarcinoma, neuroblastoma, myxoma, myomata, lymphoma, endothelioma, skeletonization C knurl, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, mammary cancer, prostate cancer, Kaposi ' s sarcoma and ovarian cancer); Autoimmunization systemic disease (as systemic lupus erythematous and Ia glomerulonephritis, rheumatic arthritis); Virus infection (as simplexvirus, poxvirus, and adenovirus); Inflammation; Graft versus host; Acute grafing repels and chronic transplanting rejection.Therefore, in preferred embodiments, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist or antagonist, be used for the treatment of prevention and/or diagnosis autoimmunization systemic disease and/or inhibition, the growth of cancer, progress and/or transfer, comprise but non-ly be limited to those cancers as herein described that for example Lymphocytic leukemia (comprises for example MLL, and lymphocytic leukemia (CLL) and lymph follicle knurl.In another embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide are used to activate cancer cells or the tissue (cancer (as CLL and MLL) relevant as B clone, Lymphocytic leukemia, or lymphoma) differentiation or propagation, cell is easier accepts cancer therapy (as chemotherapy or radiotherapy) thereby make.
In addition, in other embodiments, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide or its agonist or antagonist, be used to suppress the growth of malignant tumour and relative disease, progress and/or transfer, these diseases such as leukemia (comprise that acute leukemia is (as acute lymphoblastic leukemia, acute cellulous leukemia of bone marrow (comprises former grain C leukemia, promyelocytic leukemia, Myelomonocyte leukemia, monocytic leukemia, and erythroleukemia)) and chronic leukemia (as chronic myeloid (granulocyte) leukemia and lymphocytic leukemia)), polycythemia vera, and lymphoma (Hodgkin ' sick and non-Hdgkin ' the s disease of s), multiple myeloma, Waldenstrom ' s macroglobulinemia, heavy chain disease and solid-state tumour comprise but non-sarcoma and cancer such as fibrosarcoma, the myxosarcoma of being limited to, liposarcoma, chondrosarcoma, osteogenic sarcoma, spinal cord knurl, angiosarcoma, endotheliosarcoma, lymph vessels sarcoma, lymph vessels endotheliosarcoma, synovioma, mesothelioma, Ewing ' s knurl, leiomyosarcoma, rhabdosarcoma, colorectal carcinoma, carcinoma of the pancreas, mammary cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, marrow cancer, bronchiogenic cancer, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilm ' s knurl, cervical cancer, carcinoma of testis, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, neurospongioma, stellate cell cancer, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic tumor, oligodendroglioma, melanoma, neuroblastoma and retinoblastoma.
The diagnosis of available Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and agonist thereof and antagonist, the disease that increases with apoptosis of treatment or prevention comprises AIDS; Neurodegenerative disease (as Alzheimer ' s disease, Parkinson ' s disease, amyotrophic lateral sclerosis, the retinitis, cerebellar degeneration); Myelodysplastic syndromes (as aplastic anemia), local asphyxia damage (after one's own heart obstructing apoplexy and reperfusion injury), the hepatic diseases that toxin causes (as by due to the ethanol), septic shock, emaciation, and apocleisis.Therefore, in preferred embodiments, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist or antagonist are used for the treatment of, and prevent and/or diagnose above-mentioned disease.
In preferred embodiments, Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide and/or its agonist or antagonist (as anti-Neutrokine-Alpha antibodies) suppress the growth of the lymphoma U-937 of human tissue cell cell in the dose-dependently mode.In another embodiment preferred, Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide and/or its agonist or antagonist (as anti-Neutrokine-Alpha antibodies) suppress the PC-3 cell, the HT-29 cell, the Hela cell, the growth of MCF-7 cell and A293 cell.In another preferred embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV polynucleotide or polypeptide and/or its agonist (as anti-Neutrokine-Alpha antibodies) are used to suppress prostate cancer, colorectal carcinoma, the growth of cervical cancer and mammary cancer, progress and/or transfer.
Therefore, in another embodiment preferred, the present invention relates to increase method by TNF family part inductive apoptosis, be included as the cell of expressing Neutrokine-α and/or Neutrokine-α SV acceptor and use the Neutrokine-α and/or the Neutrokine-α SV of significant quantity, or its agonist or antagonist, can improve or reduce the signal that Neutrokine-α and/or Neutrokine-α SV mediate.Preferably, the signalling of Neutrokine-α and/or Neutrokine-α SV mediation improves or reduces and can treat, and prevents and/or diagnose wherein apoptosis reduction or cytokine and adsorbed molecules to express the disease that reduces.Agonist or antagonist can comprise the monoclonal antibody of Neutrokine-α and/or Neutrokine-α SV and the direct anti-Neutrokine-α and/or the Neutrokine-α SV polypeptide of soluble form.
On the other hand, the present invention relates to the method for a kind of inhibition by TNF family part inductive apoptosis, it is included as agonist or antagonist that the cell of expressing Neutrokine-α and/or Neutrokine-α SV acceptor is used the signalling that can improve or reduce Neutrokine-α and/or Neutrokine-α SV mediation of significant quantity.Preferably, the signalling of Neutrokine-α and/or Neutrokine-α SV mediation improves or reduces and can treat, and prevents and/or diagnose wherein apoptosis or NF-KB to express the disease that improves.Agonist or antagonist can comprise the monoclonal antibody of Neutrokine-α and/or Neutrokine-α SV and the direct anti-Neutrokine-α and/or the Neutrokine-α SV polypeptide of soluble form.
Because Neutrokine-α and/or Neutrokine-α SV belong to the TNF superfamily, this polypeptide also should be regulated vasculogenesis.In addition, because Neutrokine-α and/or Neutrokine-α SV suppress immune cell function, this polypeptide has anti-inflammatory activity widely.Neutrokine-α and/or Neutrokine-α SV can be used as anti-neovascularity and generate agent, with treatment, prevention and/or diagnose solid-state tumour is infected and activate by stimulation of host defense cell such as cellular toxicity T cell and scavenger cell, and undertaken by the vasculogenesis of inhibition tumour.Those skilled in the art can discern wherein, and vascular proliferation is other non-required non-cancer indication.They also can be used for strengthening the resistance that the host resists chronic and acute infection, for example anti-flesh bacterial infection by luring and activate microbicidal white corpuscle.Neutrokine-α and/or Neutrokine-α SV also can suppressor T cell breed by suppressing the IL-2 biosynthesizing, with cell-mediated autoimmune disease and the Lymphocytic leukemia (comprising for example lymphocytic leukemia (CLL)) of treatment T.Neutrokine-α and/or Neutrokine-α SV also can be used for stimulating wound healing, and fragment is removed and the reticular tissue of startup inflammatory cell carries out by raising.In an identical manner, Neutrokine-α and/or Neutrokine-α SV also can be used for treatment, prevent and/or diagnose other fibrotic disease, comprise liver cirrhosis, osteoarthritis and pulmonary fibrosis.Neutrokine-α and/or Neutrokine-α SV also increase the content of eosinophil, and this cell has and kills and wounds a large amount of parasitic unique function that infects tissue, as schistosomicide, trichinella and roundworm.It also can be used for regulating hemopoietic, is undertaken by activation and the differentiation of regulating various hematopoietic cell progenitor cells, for example discharges eukocyte the marrow after chemotherapy, promptly in stem cell shifts.Neutrokine-α and/or Neutrokine-α SV also can be used for treatment, prevention and/or diagnosis of sepsis disease.
Polynucleotide of the present invention or/or polypeptide and/or its agonist and/or antagonist can be used for diagnosis and treatment or prevention numerous disease and/or pathological change.This disease comprises with pathological change but the non-cancer (cancer relevant as immunocyte that be limited to, mammary cancer, prostate cancer, ovarian cancer, the lymph follicle knurl, with p53 sudden change or the relevant cancer of change, brain tumor, bladder cancer, cervical cancer, colorectal carcinoma, colorectal carcinoma, nonsmall-cell lung cancer, small cell lung cancer, cancer of the stomach etc.), lymphocytic hyperplasia disease (as the lymph gland pathology), microorganism is (as virus, bacteriums etc.) infect, (infect as HIV-1, HIV-2 infects, herpesvirus infection (comprises but the non-HSV-1 of being limited to, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human papilloma virus infection, and hepatites virus infections (as HAV, HBV, HCV etc.), helicobacter pylori infection, staphylococcus etc.), parasitic infection, ephritis, osteopathy (as osteoporosis), atherosclerosis, pain, cardiovascular disorder (generates as neovascularity, vasculogenesis reduces, or circulation volume reduces (as ischemic disease) as myocardial infarction, apoplexy etc.)), AIDS, transformation reactions, inflammation, neurodegenerative disease is (as Alzheimer ' s disease, Parkinson ' s disease, amyotrophic lateral sclerosis, the pigmentation retinitis, cerebellar degeneration etc.), transplant rejection (acute and chronic), graft versus host disease (GVH disease), the osteomyelodysplasia associated diseases (as aplastic anemia etc.), joint tissue destroys in the rheumatosis, hepatic diseases is (as acute and chronic hepatitis, liver injury and liver cirrhosis), and autoimmune disorder (as multiple sclerosis, rheumatic arthritis, systemic lupus erythematous, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave ' s disease, Hashimoto ' s thyroiditis etc.), myocardosis (as DCM (dilated cardiomyopathy)), diabetes, diabetic complication is (as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, septic shock and ulcerative colitis.
Polynucleotide of the present invention and/or polypeptide and/or its agonist and/or antagonist are used to promote vasculogenesis, wound healing (as wound, burn and fracture).Polynucleotide of the present invention and/or polypeptide and/or its agonist and/or antagonist also are used as adjuvant to strengthen the immunne response to specific antigen, and antiviral immunity is replied.
Normally, polynucleotide of the present invention and/or polypeptide and/or its agonist and/or antagonist are used for regulating (promptly excite or reduce) immunne response.For example, polynucleotide of the present invention and/or polypeptide can be used for preparing or from surgical operation, wound, radiotherapy, chemotherapy and transplant in recover, or be used among senior people and the immune impairer skeptophylaxis and reply and/or recover.Perhaps, polynucleotide of the present invention and/or polypeptide and/or its agonist and/or antagonist are as immunosuppressor, for example in treatment or prevention autoimmune disease.In special embodiment, polynucleotide of the present invention and/or polypeptide are used for the treatment of or prophylaxis of chronic, transformation reactions or autoimmune disease, as described herein and other disease known in the art.
Preferably, with Neutrokine-α and/or Neutrokine-α SV polynucleotide or polypeptide, and/or its agonist or antagonist (as anti-Neutrokine-Alpha antibodies), can be by Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide with significant quantity, or its agonist or antagonist, be applied to patient; Or, support this cell with Neutrokine-α and/or Neutrokine-α SV polynucleotide from patient's emigrated cells, the cell with this through engineering approaches is applied to patient's (ex vivo treatment) more then.In addition, Neutrokine-α and/or Neutrokine-α SV polypeptide or polynucleotide can be used as the adjuvant in the vaccine as further described herein, to produce the immunne response of anti-infectious disease.
Prescription and application process
Consider each patient's clinical condition (especially only using the side effect of Neutrokine-α and/or Neutrokine-α SV polypeptide), the transfer position of Neutrokine-α and/or peptide composition, application process, use program, and other known facts, for obtaining good medical applications, preparation Neutrokine-α and/or Neutrokine-α SV peptide composition (preferably containing is the polypeptide of soluble form Neutrokine-α and/or Neutrokine-α SV extracellular domain) and definite dosage.Therefore the Neutrokine-α of " significant quantity " and/or Neutrokine-α SV polypeptide are determined by this condition.
Generally speaking, total the medicine effective quantity scope of every dose of Neutrokine-α that non-enteron aisle is used and/or Neutrokine-α SV polypeptide at about 1 μ g/kg days to the 10mg/kg body weight/day, although will carry out the therapeutic decision as mentioned above.Preferably this dosage is 0.01mg/kg body weight/day at least, more preferably in the 0.01-1mg/kg body weight/day.
In another embodiment, the dosage that is applied to people's Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide is between 0.0001～0.045mg/kg body weight/day, preferably between 0.0045～0.045mg/kg body weight/day, 45 μ g/kg body weight/day more preferably; The dosage that is applied to mouse is about 3mg/kg body weight/day.
If continuous application, Neutrokine-α and/or Neutrokine-α SV polypeptide application dosage be about 1 μ g/kg body weight/hour～50 μ g/kg body weight/hour, inject 1-4 time every day or continuous subcutaneous perfusion, for example use Micropump.Also can use intravenous fluids.
Treatment time is according to being determined according to effect.
In a special embodiment, total medicine effective quantity scope that non-enteron aisle is used every dose of Neutrokine-α and/or Neutrokine-α SV polypeptide is about 0.1 μ g/kg body weight/day～45 μ g/kg body weight/day, although will carry out the therapeutic decision as mentioned above.Preferably, this dosage is at least 0.1 μ g/kg body weight/day, and the dosage that more preferably is applied to the people is between 0.01～50 μ g/kg body weight/day.But Neutrokine-α and/or Neutrokine-α SV continous pouring, every day, multiple injection was (as every day three times or more times, or twice of every day, injection every day once, or intermittent infusion (as secondary every day, once a day, the next day once, secondary weekly, weekly, once January once two weeks, February, once March once).If continuous application, Neutrokine-α and/or Neutrokine-α SV polypeptide application dosage be about 0.001～10 μ g/kg body weight/hour～about 50 μ g/kg body weight/hour, inject 1-4 time every day or continuous subcutaneous perfusion, for example use Micropump.
The effective dose of the present composition of using can be determined by means known in the art, indicate parameter such as biological half-life, biopotency, and toxicity.This mensuration is well known to those skilled in the art.
Body is exposed in determinacy and/or the medicine effective quantity the biology of Neutrokinne-α and/or Neutrokine-α SV polypeptide and also plays an important role during treating.For some time that the Neutrokine-α of the variation of dosage such as the relative low dosage of repetitive administration and/or Neutrokine-α SV polypeptide are long relatively, Neutrokine-α and/or short relatively for some time of Neutrokine-α SV polypeptide with the relative high dosage of repetitive administration, have different treatments and/or pharmacology effect.See for example test of the serum immunoglobulin level shown in the embodiment 6.
Use Freireich, E.J. etc. (cancer chemotherapy reports 50 (4): the surface-area dosage conversion factor of equal value that 219-44 (1966)) provides, those skilled in the art can conventional will derive from and use Neutrokine-α and/or the data of Neutrokine-α SV in given test, are converted in another test system the accurate assessment value of the medicine effective quantity of every dose of Neutrokine-α that uses and/or Neutrokine-α SV polypeptide.By the testing data (seeing for example embodiment 6) of in mouse, using Neutrokine-α gained, the conversion factor that can provide by Freireich etc., assessment Neutrokine-α is rat exactly, monkey, the medicine effective quantity of dog and philtrum.Following conversion table (table 3) has been summarized the data that Freireich etc. provides.Table 3 provides the dosage that will represent with the mg/kg body weight in species to be converted to the approximation factor of the surface-area dosage of representing with the mg/kg body weight of equal value in another special type.
Table 3 surface-area dose conversion factor of equal value
Since Mouse (20g) Rat (150g) Monkey (3.5kg) Dog (8kg) People (60kg)
Mouse rat monkey dog people 1 2 4 6 12 1/2 1 2 4 7 1/4 1/2 1 5/3 3 1/6 1/4 3/5 1 2 1/12 1/7 1/3 1/2 1
Therefore, the conversion factor that is provided in the use table 3 for example, the dosage of 50mg/kg is converted to and is the suitable dosage of 12.5mg/kg in monkey in mouse, because (50mg/kg) * 1/4=12.5mg/kg.In addition, in mouse, use 0.02,0.08,0.8,2 and the dosage of 8mg/kg equal in the people, to use 1.667 μ g/kg, 6.67 μ g/kg, 66.7 μ g/kg, the dosage role of 166.7 μ g/kg and 0.667mg/kg.
The pharmaceutical composition that contains Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide can be by oral, rectum, non-enteron aisle, subcutaneous, intracistemally, intravaginal, intraperitoneal, powder (is passed through in the part, ointment, drops or emplastrum form), the cheek, through the oral cavity or nasal spray (sucking) etc. as steam or powder use.In one embodiment, " medicine suitable carriers " is meant nontoxic solid, semi-solid or liquid-filled dose, and thinner, the prescription auxiliary of capsule compound or any kind.In a special embodiment, " medicine is suitable " is meant that the permission of government authorities U.S. pencil department is used for especially people's article of animal.For example provided in " Remington ' s pharmaceutical science " by E.W.Martin without limitation according to the suitable pharmaceutical carrier of this embodiment, and comprise sterile liquid, Ru Shui and oil comprise being derived from oil, animal, plant or synthetic are as peanut oil, soya-bean oil, mineral oil, sesame wet goods.When pharmaceutical composition was intravenous injection, water was preferred carrier.Salt brine solution and glucose solution and glycerine solution can be used as liquid vehicle, and especially as injectable solution, if desired, this composition also can contain the wetting agent or the emulsifying agent of trace, or the PH buffer reagent.These compositions can be solution, suspension, milk sap, tablet, pill, capsule, powder, forms such as slowly-releasing prescription.
Term " non-enteron aisle " is meant and comprises intravenously, intraperitoneal, subcutaneous and intra-arterial and gluteal muscle and dabbling mode of administration.
In an embodiment preferred, Neutrokine-α of the present invention and/or Neutrokine-α SV composition (comprising polypeptide, polynucleotide and antibody thereof, agonist and/or antagonist) are through subcutaneous administration.
In another embodiment preferred, Neutrokine-α of the present invention and/or Neutrokine-α SV composition (comprising polypeptide, polynucleotide and antibody thereof, agonist and/or antagonist) are used through intravenously.
Neutrokine-α of the present invention and/or Neutrokine-α SV composition also can suitably be used by slow-released system.Slow releasing composition for example comprises suitable polymkeric substance (as the semipermeability polymeric matrices, as film or micro-capsule), suitable hydrophobic materials (as suitable fat liquor), or ion exchange resin and a small amount of soluble derivative (as a small amount of soluble salt).
Sustained-release matrix comprises poly(lactic acid) (U.S. Patent No. 3773919, EP58481), multipolymer (the Sidman of L-L-glutamic acid and γ-ethyl-L-glutamate, V. etc., biological polymer 22:547-556 (1983)), poly-(second-hydroxyethyl meth acrylate) (R.Langer etc., J.Biomed.Mater.Res.15:167-277 (1981)), with R.Langer. chemical technology 12:98-105 (1982), the ethylethylene acetic ester (R.Langer etc., Id.) or poly--D-3-hydroxybutyric acid (EP 133988).
Slow releasing composition comprises that also liposome carries the present composition (seeing Langer, science 249:1527-1533 (1990)) that stays; Treat etc., the liposome in infectious diseases and the cancer therapy, Lopez-Berestein and Fidler (editor), Liss, New York, p317-327 and 353-365 (1989).The liposome that contains Neutrokine-α and/or Neutrokine-α SV polypeptide can prepare by currently known methods, sees DE 3218121; Epstein etc., institute of American Academy of Sciences report 82:3688-3692 (1985); Hwang etc., institute of American Academy of Sciences report 77:4030-4034 (1980); EP 52322; EP 36676; EP 88046; EP 143949; EP 142641; Japanese patent application 83-118008; U.S. Patent No. 4485045 and 4544545; And EP 102324.Normally, liposome is small single sheet shape (approximately 200-800Angstroms), and wherein lipid content is regulated selection percentage and carried out Neutrokine-α and/or the treatment of Neutrokine-α SV polypeptide with the best greater than the 30mol cholesterol.
In another embodiment, slow releasing composition of the present invention comprises crystal prescription known in the art.
In another embodiment, composition of the present invention (is seen Langer, as preceding by pump delivery; Sefton, CRC Crit Ref.Biomed.Eng.14:201 (1987); Buchwald etc., surgery 88:507 (1980); Saudek etc., N.Engl.J.Med 321:574 (1989)).
Other Controlled Release System sees Langer described (science 249:1527-1533 (1990)).
With regard to non-enteron aisle is used, in one embodiment, Neutrokine-α and/or Neutrokine-α SV polypeptide are generally with required purity, with unitary dose injectable forms (solution, suspension or milk sap), with the medicine suitable carrier, promptly application dose and concentration to the acceptor nontoxicity and with prescription in the compatible carrier of other batching.For example, formula optimization does not comprise oxygenant and known to disadvantageous other compound of polypeptide.
Usually, by with Neutrokine-α and/or Neutrokine-α SV polypeptide and liquid vehicle or the solid carrier that fully separates or the two all even close contact and preparation formula.Then, if desired, product is made desired shape.Preferably, this carrier is non-enteron aisle carrier, is more preferably and the isoosmotic solution of acceptor blood.This carrier for example comprises water, salt solution, ringer's solution, and glucose solution.Nonaqueous phase carrier such as fixed lipid and ethyl oleic acid and liposome also can use at this.
Carrier suitably contains a small amount of additive as strengthening isotonicity and chemical stability.This material is nontoxic at application dose and concentration to acceptor, comprises buffer reagent such as phosphoric acid, citric acid, succsinic acid, acetate and other organic acid or its salt; Antioxidant such as xitix; Lower molecular weight (less than 10 residues) polypeptide such as poly arginine or tripeptides; Protein such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer such as polyvinylpyrrolidone; Amino acid such as glycine, L-glutamic acid, aspartic acid or arginine; Monose, disaccharides or other carbohydrate comprise the Mierocrystalline cellulose or derivatives thereof, glucose, seminose, sucrose or dextrin; Sequestrant such as EDTA; Sugar alcohol such as mannitol or Sorbitol Powder; Gegenion such as sodium ion; Sanitas such as cresols, phenol, butylene-chlorohydrin, benzyl ethanol and parabens and/or nonionic surfactant such as polysorbate, poloxamers or PEG.
The concentration of Neutrokine-α for preparing in this carrier and/or Neutrokine-α SV polypeptide is about 0.001mg/ml～100mg/ml, or 0.1mg/ml～100mg/ml, preferred 1-10mg/ml, or 1-10mg/ml, PH is about 3-10, or 2-8, preferred 5-8, more preferably 6-7.Should know and use some aforementioned acceptors, carrier or stablizer will cause Neutrokine-α and/or Neutrokine-α SV to form salt.
Neutrokine-α that being used for the treatment of property is used and/or Neutrokine-α SV polypeptide must be aseptic.Can filter by aseptic filter membrane (as the film of 0.2micron) and reach aseptic.Therapeutic Neutrokine-α and/or Neutrokine-α SV polypeptide peptide composition generally place in the container with sterile channel, for example intravenous injection solution bag or have in the bottle of the bottle stopper that can be penetrated by hypodermic needle.
Neutrokine-α and/or Neutrokine-α SV polypeptide are stored in unitary dose or multi-dose container usually, for example sealed ampoule or bottle, the lyophilized form that maybe can rebuild with aqueous solution form.The freeze-drying Formulation Example is equipped with 1% (w/v) the Neutrokine-α and/or the Neutrokine-α SV polypeptid solution of 5ml sterile filtration in this way in the 10ml bottle, with the freeze-drying of gained mixture.Perfusion liquid is by rebuilding freeze dried Neutrokine-α and/or Neutrokine-α SV polypeptide prepares with sterile water for injection.
Perhaps, Neutrokine-α and/or Neutrokine-α SV polypeptide are stored in the single dose container with lyophilized form.This perfusion liquid aseptic injection carrier reprovision.
The present invention also provides drug packages or test kit.The container that comprises one or more pharmaceutical composition batching of the present invention of one or more filling.This container also has by responsible production, and the permission that the government organs that use or sale medicine or biological products are examined provide is used for the notice of human body.In addition, polypeptide of the present invention can with other therapeutic compound combined utilization.
Composition of the present invention can be separately or with other adjuvant combined administration.The adjuvant that can use with combination of compositions of the present invention comprises but non-limit alum that alum adds deoxycholate salt (Immuno Ag), and MTP-PE (Biocine Corp.) QS21 (Genentech, Inc.), BCG and MPI.In a special embodiment, composition of the present invention and QS-21 combined administration.Can comprise but the single phosphoric acid lipid of non-limit immunomodulator AdjuVax 100a, QS-21, QS-18, CRL1005, aluminium salt, MF-59 and viral adjuvant with other adjuvant that the present composition is used in combination.Can comprise but the non-MMR of being limited to (measles, mumps, rubella) vaccine Poliomyelitis Vaccine with the vaccine that the present composition uses, chickenpox vaccine, tetanus vaccine, diphtheria vaccine, hepatitis A vaccine, Hepatitis B virus vaccine, influenzae second type influenza virus vaccine, pertussis vaccine, pneumovax, influenza vaccines, Lyme ' s disease vaccine, cholera vaccine, Yellow Fever Vaccine, vaccine of epidemic menigitis, Rabies Vaccine hinders the hot vaccine of plug, pertussis vaccine and/or PNEUMOVAX-23 TMThe property followed combined administration is as mixture, discretely but use simultaneously; Or but successive combination uses, and this comprises presents, and wherein composition is used together as the treatment mixture, comprises that also composition is respectively but side by side as through dividing other venous access to inject in the same individuality." combination " used and also comprised and use a kind of compound respectively earlier, and then uses another kind.
In another special embodiment, composition of the present invention and PNEVMOVAX-23 TMBe used in combination treatment, prevention and/or diagnose infections disease and/or associated any disease.In one embodiment, composition of the present invention and PNEVMOVAX-23 TMCombined administration, treatment prevents and/or diagnoses any gram positive bacteria infection and/or associated any disease.In another embodiment, composition of the present invention and PNEVMOVAX-23 TMCombined administration, treatment prevention with/diagnosis and enterobacter and/or more than one relevant infection of member of streptomyces.In another embodiment, composition of the present invention and PNEOMOVAX-23 TMBe used in combination, treatment, prevention and/or diagnosis belong to the relevant disease of member with one or more beta streptococcus.In another embodiment, composition of the present invention and PNEOMOVAX-23 TMBe used in combination treatment, the disease that prevention and/or diagnosis are relevant with streptococcus pneumoniae.
Composition of the present invention can be used separately, or with other therapeutical agent combined administration, comprise but the non-chemotherapeutics that is limited to microbiotic, antiviral agent, steroid and on-steroidal anti-inflammatory agent, routine immunization therapeutical agent and cytokine.But the combined administration concomitant administration is as mixture, respectively but use simultaneously; Or sequential application.Comprise that composition uses together as the therapeutic mixture, comprise that also composition respectively but use simultaneously, as by dividing other venous access to inject same individual simultaneously, " combination " used and also comprised and use a kind of compound respectively earlier, and then uses another kind.
In one embodiment, other member's combined administration of composition of the present invention and TNF family.Can with the TNF of present composition combined administration, TNF is correlated with or class TNF molecule comprises but non-TNF-α and/or the α-lymphotoxin (α-LT that is limited to soluble form, be also referred to as TNF-β), LT-β (in heterotrimer LT-α 2-beta composite, finding), OPGL, FasL, CD27L, CD30L, CD40L, 40-1BBL, DcR3, OX40L, γ-TNF (international open WO 96/14328), AIM-I (international open WO 97/33899), AIM-II (international open WO 97/34911), APRIL (J.Exp.Med.188 (6): 1185-1190), α-intrinsic factor (international open WO98/07880), TR6 (international open WO 98/30694), OPG and Neutrokine-α (international open WO 98/18921), the Fas of OX40 and nerve growth factor (NGF) and soluble form, CD30, CD27, CD40 and 4-IBB, TR2 (international open WO98/34095), DR3 (international open WO 97/33904), DR4 (international open WO98/32856), TR5 (international open WO 98/30693), TR6 (international open WO98/30694) TR7 (international open WO 98/41629), TRANK, TR9 (international open WO 98/56892), TR10 (international open WO 98/54202), 312C2 (international open WO 98/06842) and TR12.
In an embodiment preferred, composition of the present invention and CD40 part (CD40L), the CD40L of soluble form is (as AVREND TM), the bioactive fragment of CD40L, variant or derivative, anti-CD40L antibodies (as excitability or antagonistic antibodies), and/or anti-cd40 antibody (as excitability or antagonistic antibodies).
In some embodiments, composition of the present invention and antiretroviral preparation, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and/or proteinase inhibitor combined administration.Can comprise with the nucleoside reverse transcriptase of present composition combined administration but the non-RETROVIR of being limited to TM(zidovudine/AZT), VIDEX TM(didanosine/ddI), HIVID TM(zalcitabine/ddC), ZERIT TM(stavudine/d4T), EPIVIR TM(lamivudine/3 TC), and COMBIVIR TM(zidovudine/lamivudine).Can comprise with the non-nucleoside reverse transcriptase of present composition combined administration but the non-VIRAMUNE of being limited to TM(nevirapine), RESCRIPTOR TMAnd SUSTIVA (delavirdine), TM(efavirenz).Can comprise with the proteinase inhibitor of present composition combined administration but the non-CRIXIVAN of being limited to TM(indinavir), NORVIR TM(ritonavir), INVIRASE TMAnd VIRACEPT (saquinavir), TM(nelfinavir).In a special embodiment, antiviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and/or proteinase inhibitor can with present composition arbitrary combination, the treatment, prevention and/or diagnosis AIDS and/or treatment, prevention and/or diagnosis HIV infect.
In other embodiments, composition of the present invention can be used in combination with anti-opportunistic infection agent.Can comprise but the non-TRIMETHOPRIM-SULFAMETHOXAZOLE of being limited to the anti-opportunistic infection agent of present composition combined administration TM, DAPSONE TM, PENTAMIDINE TM, ATOVAQUONE TM, ISONIAZID TM, RIFAMPIN TM, PYRAZINAMIDE TM, ETHAMBUTOL TM, RIFABUTIN TM, CLARITHROMYCIN TM, AZITHROMYCIN TM, GANCICLOVIR TM, FOSCARNET TM, CIDOFOVIR TM, FLUCONAZOLE TM, ITRACONAZOLE TM, KETOCONAZOLE TM, ACYCLOVIR TM, FAMCICOLVIR TM, PYRIMETHAMINE TM, LEUCOVORIN TM, NEVPOGEN TMAnd LEUKINE (filgrastim/G-CSF), TM(sargramostim/GM-CSF).In a special embodiment, composition of the present invention can with TRIMETHOPRIM-SULFAMETHOXAZOLE TM, DAPSONE TM, PENTAMIDINE TMAnd/or ATOVAQUONE TMArbitrary combination, with prophylactic treatment, prevention and/or diagnosis opportunistic Pneumocystis carinii infect.In another special embodiment, composition of the present invention and ISONIAZID TM, RIFAMPIN TM, PYRAZINAMIDE TMAnd/or ETHAMBUTOL TMArbitrary combination, with prophylactic treatment, prevention and/or diagnosis opportunistic Mycobacterium avium mixture infect.In another special embodiment, composition of the present invention and RIFABUTIN TM, CLARITHROMYCIN TMAnd AZITHROMYCIN TMArbitrary combination, with prophylactic treatment, prevention and/or diagnosis opportunistic mycobacterium tuberculosis infection.In another special embodiment, composition of the present invention and GANCICLOVIR TM, FOSCARNET TMAnd/or CIDOFOVIR TMArbitrary combination, with prophylactic treatment, prevention and/or the cytomegalovirus infection of diagnosis opportunistic.In another special embodiment, composition of the present invention and FLUCONAZOLE TM, ITRACONAZOLE TM, and/or KETOCONAZOLE TMArbitrary combination, with prophylactic treatment, prevention and/or diagnosis opportunistic fungal infection.In another special embodiment, composition of the present invention and ACYCOLOVIR TMAnd/or FAMCICOLVIR TMArbitrary combination, with prophylactic treatment, prevention and/or diagnosis opportunistic I type or II herpes simplex virus type infect.In another special embodiment, composition of the present invention and PYRIMETHAMINE TMAnd/or LEUCOVORIN TMArbitrary combination, with prophylactic treatment, prevention and/or diagnosis opportunistic Toxoplasma gondii infect.In another special embodiment, composition of the present invention and LEUCOVORIN TMAnd/or NEUPOGEN TMArbitrary combination, with prophylactic treatment, prevention and/or diagnosis opportunistic infectation of bacteria.
In another embodiment, composition of the present invention and antiviral agent combined administration.Can comprise but the non-acycloguanosine that is limited to virazole, Symmetrel and remantidine with the antiviral agent of present composition combined administration.
In another embodiment, composition of the present invention and microbiotic combined administration.Can comprise but the non-amoxycilline Trihydrate bp that is limited to aminoglycoside antibiotics, beta-lactam (glycopeptide), β-Nei Xiananmei with the microbiotic of present composition combined administration, clindamycin, paraxin, cynnematin, Ogostal, erythromycin, fluorescence quinomycin, macrolide, metronidazole, penicillin, quinomycin, Rifampin, Streptomycin sulphate, sulphonamide, tsiklomitsin, Trimethoprim BP, Trimethoprim BP-thiamines imidazoles, and vancomycin.
Can with the conventional nonspecific immunosuppressive agent of present composition combined administration, comprise but the non-steroid that is limited to, S-Neoral, the cyclosporin analog endoxan, endoxan IV, methyl meticortelone, Ultracortene-H, azathioprine, FK-506, the immunosuppressor of T cell function is replied in 15-deoxidation spergualin and other inhibition.
In special embodiment, composition of the present invention and immunosuppressor combined administration.Can comprise with the immunosuppressor prepared product of present composition combined administration but the non-ORTHOCLONE of being limited to TM(OKT3), SANDIMMUNE TM/ NEORAL TM/ SANGDYA TM(S-Neoral), PROGRAF TM(tacrolimus), CELLCEPT TM(mycophenolate salt), azathioprine, glucorticosteroids, and RAPAMUNE TM(sirolimus).In a special embodiment, immunosuppressor can be used for preventing the rejection of organ or bone marrow transplantation.
In an embodiment preferred, composition of the present invention and steroid therapy combined administration.Can comprise with the steroid that the present invention is used in combination but non-oral corticoid, prednisone and the methyl prednisone (as IV methyl prednisone) of being limited to.In a special embodiment, composition of the present invention and prednisone combined administration.In another special embodiment, composition of the present invention and prednisone and immunosuppressor combined administration.Can with as described herein those of the immunosuppressor of the present composition and prednisone combined administration, comprise but the non-azathioprine that is limited to endoxan and endoxan IV.In another special embodiment, composition of the present invention and methyl prednisone combined administration.In another special embodiment, composition of the present invention and methyl prednisone and immunosuppressor are used in combination.Can with as described herein those of the immunosuppressor of the present composition and methyl prednisone combined administration, comprise but the non-azathioprine that is limited to endoxan and endoxan IV.
In an embodiment preferred, composition of the present invention and antimalarial agent combined administration.Can comprise but the non-Plaquenil that is limited to chloroquine, and/or quinacrine with the antimalarial agent of present composition combined administration.
In an embodiment preferred, composition of the present invention and NSAID combined administration.
In one embodiment, composition of the present invention and one, two, three, four, five, ten or multiple following drug regimen use: NRD-101 (Hoechst Marion Roussel), Ciclofenaziae (Dimethaid), oxaprozine potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522 (JapanTobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelonemesylate (Warner-Lambert), soluble TNF acceptor 1 (Synergen; Amgen), IPR-6001 (Institute for Pharmaceutical Research), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals), BIIL-284 (BoehringerIngelheim), BIIF-1149 (Boehringer Ingelheim), Leuko Vax (Inflammatics), MK-663 (Merch), ST-1482 (Sigma-Tau) and butixocort propionate (Warner Lam bent).
In an embodiment preferred, composition of the present invention with next, two, three, four, five or multiple drug regimen use: methotrexate, sulphasalazine, sodium aurothiomalate, AF, ciclosporin, Trolovol, imuran, antimalarial drug (as described herein), endoxan, Chlorambucil, gold, ENBREL TM(Etanercept), anti-TNF antibodies, and Ultracortene-H.
In a preferred embodiment, composition of the present invention and antimalarial drug, methotrexate, anti-TNF antibodies, ENBREL TMAnd/or sulphasalazine combined administration.In one embodiment, composition of the present invention and methotrexate combined administration.In another embodiment, composition of the present invention and anti-TNF antibodies combined administration.In another embodiment, composition of the present invention and methotrexate and anti-TNF antibodies combined administration.In another embodiment, composition of the present invention and sulphasalazine combined administration.In another special embodiment, composition of the present invention and ENBREL TMCombined administration.In another embodiment, composition of the present invention and ENBREL TM, methotrexate and sulphasalazine combined administration.In other embodiments, one of one or more antimalarial drug and aforesaid combination combined administration.In a special embodiment, composition of the present invention and antimalarial drug (as Plaquenil), sulphasalazine, anti-TNF antibodies and methotrexate combined administration.
In another embodiment, composition of the present invention use separately or with the immunoglobulin (Ig) combined administration of one or more intravenous injection.Can comprise with the intravenously immunoglobulin (Ig) of present composition combined administration but the non-GAMMAR of being limited to TM, IVEEGAM TM, SANDOGLOBULIN TM, GAMMAGARD S/D TM, and GAMIMUNE TMIn a special embodiment, composition of the present invention and intravenously immunoglobulin (Ig) combined administration (as bone marrow transplantation) in transplantation therapy.
CD40 part (CD40L), a kind of CD40L of soluble form is (as AUREND TM), the bioactive fragment of CD40L, variant or derivative, anti-CD40L antibodies (as excitability or antagonistic antibodies), and/or anti-cd40 antibody (as excitability or antagonistic antibodies).
In another embodiment, composition of the present invention use separately or with the anti-inflammatory agent combined administration.Can comprise with the anti-inflammatory agent of present composition combined administration but non-glucocorticosteroid and non-hormone anti-inflammatory agent, ammonia arylcarboxylic acid derivative, the Arylacetic acids derivative of being limited to, arylbutyric acid derivatives, arylbutyric acid derivatives, arylcarboxylic acid derivative, aryl propionic acid derivatives, pyrazoles, pyrazoline, salicyclic acid derivatives, carboxamide thiazine, e-kharophen caproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, benzene Yin acid, 6-benzyladenine, BCP, Difenax, Abbott-36683, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxine, proquazone, proxazole, and tenidap.
In another embodiment, compound of the present invention and chemotherapeutics combined administration.Can comprise with the chemotherapeutics that combination of compositions of the present invention is used but the non-antibiotic derivatives (as Zorubicin, bleomycin, daunorubicin and daunomycin) that is limited to; Estrogen antagonist (as tamoxifen); Metabolic antagonist (as Fluracil, 5-FU, methotrexate, 5 FU 5 fluorouracil deoxynucleoside, alpha-interferon 2b, L-glutamic acid, mithramycin, purinethol and b-thioguanine); Cytotoxic agents (as carmustine, BCNU, 1 lomustine, CCNU, cytosine arabinoside, endoxan, estramustine phosphate, hydroxyl urea, procarbazine, mitomycin, busulfan, cis-platinum, and leucocristine sulfate); Hormone (as Zytron, estramustine phosphate sodium, orgabolin, estradiol, megestrol acetate, methyltestosterone, fosfestrol, chlorotrianisene and testis lactone); Nitrogen mustard derivatives (as mephalen, chorambucil, mechlorethamine (mustargen) and tespamin); Steroid and composition (as the bethamethasone sodium phosphate); And other material (as dicarbazine, asparaginase, mitotane, vincristine sulphate, Vinblastine Sulfate and etioposide).
In a special embodiment, composition of the present invention and CHOP (endoxan, Zorubicin, vincristine(VCR) and prednisone) or with the arbitrary composition combined administration of CHOP component.In another embodiment, composition of the present invention and Rituximab combined administration.In another embodiment, composition of the present invention and Rituximab and CHOP, or the arbitrary composition combined administration of Rituxamab and CHOP component.
In another embodiment, composition of the present invention and combination of cytokines are used.Can comprise but the non-GM-CSF of being limited to G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-α, IFN-β, IFN-γ, TNF-α, TNF-β with the cytokine of present composition combined administration.In another embodiment, composition of the present invention can with any interleukin-combined administration, comprise but the non-IL-1 of being limited to α IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 and Il-22.In preferred embodiments, composition of the present invention and IL4 and IL10 combined administration.The inventor observes IL4 and IL10 all strengthens the alpha mediated B cell proliferation of Neutrokine-.
In another embodiment, composition of the present invention and chemokine combined administration.In another embodiment, composition of the present invention and chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory protein 4 combined administrations.In an embodiment preferred, composition of the present invention and chemokine beta-8 combined administrations.
In another embodiment, composition of the present invention and IL-4 antagonist combined administration.Can comprise but non-ly be limited to soluble IL-4 receptor polypeptides that poly closes the solvable IL-4 receptor polypeptides of form with the IL-4 antagonist of present composition combined administration; In conjunction with the IL-4 acceptor but the anti-IL-4 receptor antibody of the bio signal that excites by IL-4 of not transduceing; The anti-IL-4 antibody of blocking-up IL-4 and one or more IL4 receptors bind and in conjunction with the IL4 acceptor but the IL4 mutain of the bio signal that excites by IL4 of not transduceing.Preferably, the antibody that uses according to this method is monoclonal antibody (comprising antibody fragment, as described herein).
In another embodiment, composition of the present invention and hemopoieticgrowth factor combined administration.Can comprise with the hemopoieticgrowth factor of present composition combined administration but the non-LEUKINE of being limited to TM(SARGRAMOSTIM TM) and NEUPOGEN TM(FILGRASTIM TM).
In another embodiment, composition of the present invention and fibroblast growth factor combined administration.Can comprise but the non-FGF-1 of being limited to FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15 with the fibroblast growth factor of present composition combined administration.
In addition, composition of the present invention can use separately or with other methods of treatment combined administration, comprise but non-ly be limited to radiotherapy.This combination treatment can be in succession and/or concomitant administration.
Agonist and antagonist-analysis and molecule
The present invention also provides a kind of method of SCREENED COMPOUND, differentiate strengthening or the effect of blocking-up Neutrokine-α and/or Neutrokine-α SV polypeptide pair cell, as itself and Neutrokine-α and/or Neutrokine-α SV binding molecule such as the interactional compound of acceptor molecule.Agonist is a kind of enhancing Neutrokine-α and/or Neutrokine-α SV natural biological function, or with the compound of the function of Neutrokine-α and/or Neutrokine-α SV similar manner, and antagonist is the compound that reduces or eliminate this function.
In another embodiment, the invention provides the receptor protein or the protein-bonded method of other part of a kind of specific combination Neutrokine-α and/or Neutrokine-α SV polypeptide.For example, cellular compartment such as cytolemma or its prepared product can prepare from the cell of expressing in conjunction with the molecule of Neutrokine-α and/or Neutrokine-α SV.With Neutrokine-α and/or the Neutrokine-α SV incubation of this prepared product with mark, the Neutrokine-α of separation and combination acceptor and/or Neutrokine-α SV or other protein-bonded mixture, and qualitative according to ordinary method known in the art.Perhaps, Neutrokine-α and/or Neutrokine-α SV can combine with solid support, so that combine with chromatography column from cytolytic binding molecule, and wash-out and qualitative according to conventional methods then.
In agonist of the present invention or antagonist analysis, cellular compartment such as cytolemma or its prepared product, can prepare from the cell of expressing in conjunction with the molecule of Neutrokine-α and/or Neutrokine-α SV, this molecule is in this way by Neutrokine-α and/or synthetic signal of Neutrokine-α SV or adjusting approach molecule.This prepared product is having or is not having incubation under the situation of candidate molecules with the Neutrokine-α of mark and/or Neutrokine-α SV, and this candidate molecules can be Neutrokine-α and/or Neutrokine-α SV agonist or antagonist.Candidate molecules combines in the reduction with the part that the bonded ability of binding molecule is reflected in mark.One-sided bonded molecule is not promptly induced the molecule of Neutrokine-α to Neutrokine-α and/or the effect of Neutrokine-α SV binding molecule bonded, is good antagonist.Good combination and to excite the molecule of similar or closely related effect in Neutrokine-α and/or Neutrokine-α SV be agonist.
The class Neutrokine-α of potential agonist and antagonist and/or Neutrokine-α SV effect, can be for example by after determining that candidate molecules and cell or suitable cellular preparations interact, the activity of second messenger system, and with Neutrokine-α and/or Neutrokine-α SV or excite the effect with the molecule of Neutrokine-α and/or Neutrokine-α SV same function to compare, and determined.The second messenger system that can be used for this comprises but the non-AMP of being limited to guanylate cyclase, ionic channel or phosphoric acid hydrolysis second messenger system.
The another kind analysis of Neutrokine-α and/or α SV antagonist for example is competition analysis, it is to make up Neutrokine-α and/or Neutrokine-α SV and potential antagonist under suitable condition, with membrane-bound acceptor molecule or reorganization Neutrokine-α and/or Neutrokine-α SV acceptor molecule, being at war with property inhibition analysis.Neutrokine-α and/or Neutrokine-α SV can for example pass through radio-labeling, and the number of the Neutrokine-α of bind receptor molecule and/or Neutrokine-α SV molecule can accurately be measured like this, to confirm the effectiveness of potential antagonist.
The potential antagonist comprises the little organic molecule in conjunction with polypeptide of the present invention, peptide, and polypeptide (as IL-13) and antibody, thus suppress or its activity of becoming extinct.The potential antagonism can also be the little organic molecule that is combined in same loci on binding molecule such as the acceptor molecule, peptide, polypeptide such as closely-related protein or antibody, and do not induce Neutrokine-α and/or Neutrokine-α SV inductive activity, thereby stop the effect of Neutrokine-α and/or Neutrokine-α SV by exclusion Neutrokine-α and/or Neutrokine-α SV combination.
Other potential antagonist comprises antisense molecule.The antisense method can be used for by antisense DNA or RNA or the controlling gene expression by triple helical formation.The antisense method is for example by Okara, neurochemistry magazine 56:560 (1991); " as the oligodeoxynucleotide of the antisense inhibitor of genetic expression ", CRC press, Boca Raton, F1 (1988) is described.Triple helical for example forms by Lee etc., nucleic acids research 6:3073 (1979); Cooney etc., science 241:456 (1988); With Dervan etc., science 251:1360 (1991).This method combines based on polynucleotide and complementary DNA or RNA's.For example, 5 ' encoding part of the polynucleotide of code book invention polypeptide extracellular domain can be used for the antisense rna oligonucleotide that design length is approximately the 10-40 base pair.Design a DNA oligonucleotide that is complementary to the zone of the gene that comprises in transcribing, thereby prevent transcribing and producing of Neutrokine-α and/or Neutrokine-α SV.This antisense rna oligonucleotide is hybridized with mRNA in vivo, and blocking-up mRNA is translated as Neutrokine-α and/or Neutrokine-α SV polypeptide.Above-mentioned oligonucleotide also can be delivered to cell, and sense-rna or DNA can express in vivo like this, to suppress the generation of Neutrokine-α and/or Neutrokine-α SV.
In one embodiment, Neutrokine-α of the present invention and/or Neutrokine-α SV antisense nucleic acid produce by transcribing from exogenous array in cell.For example, transcribe a carrier or its part that produces antisense nucleic acid of the present invention (RNA).This carrier contains the sequence of coding Neutrokine-α and/or Neutrokine-α SV antisense nucleic acid.This carrier can keep additive type or become the carrier of chromosomal integration, can be transcribed until it to produce required antisense DNA.This carrier can make up by the recombinant DNA method of this area standard.Carrier can be a plasmid, and virus, or other material known in the art are used for duplicating and expressing at vertebrate cells.Coding Neutrokine-α and/or Neutrokine-α SV, or its fragments sequence can express in vertebrates especially human body cell by any promotor known in the art.This promotor can be derivable or composing type.This promotor comprises but the non-SV40 of being limited to early promoter district (Bernoist and Chambon, nature 29:304-310 (1981), be contained in Rous sarcoma viral 3 ' long promotor (Yamamoto etc. in terminal repetition, cell 22:787-797 (1980)), (Wagner etc., institute of American Academy of Sciences report 78:1441-1445 (1981) to herpes thymidine promoter, the adjusting sequence (Brinster etc. of metallothionein gene, nature 296:39-42 (1982)), etc.
Antisense nucleic acid of the present invention comprises the sequence of the rna transcription thing at least a portion that is complementary to Neutrokine-α and/or Neutrokine-α SV gene.Yet,, be not required although absolute complementarity is preferred.The sequence that " is complementary to RNA at least a portion " is meant to have good complementarity, can form the sequence of stable duplex with RNA hybridization; In the situation of double-stranded Neutrokine-α and/or Neutrokine-α SV antisense nucleic acid, can test the duplex DNA of strand, or the analysis triple helical forms, the hybridization ability depends on the complementary degree of antisense nucleic acid and the length of antisense nucleic acid.Normally, bigger hybrid nucleic acid, many more with the base mispairing of Neutrokine-α and/or Neutrokine-α SV RNA, it can contain and still can form stable duplex (or triple helical).Those skilled in the art use standard method can find out the tolerance level of mispairing, to determine the fusing point of hybridization complex.
Be complementary to courier 5 ' terminal as near and comprise and the oligonucleotide of 5 ' the untranslated sequence of AUG initiator codon should more effectively suppress translation.Yet the sequence that is complementary to 3 ' the untranslated sequence of mRNA has illustrated the translation that suppresses mRNA effectively.See Wagner, R.1994, natural 372:333-335 is described.Therefore, the oligonucleotide of 5 ' or 3 ' the untranslated non-coding region that is complementary to Neutrokine-α and/or Neutrokine-α SV respectively shown in Figure 1A-B and the 5A-B can be used in the antisense method to suppress the translation of endogenous Neutrokine-α and/or Neutrokine-α SV mRNA.The oligonucleotide that is complementary to 5 ' the untranslated district of mRNA should comprise the complementary sequence of AUG initiator codon.The antisense oligonucleotide that is complementary to the mRNA coding region is little effect translational inhibitor, but can use according to the present invention.Whether no matter hybridize with 5 ', 3 ' or the coding region of Neutrokine-α and/or Neutrokine-α SVmRNA, antisense nucleic acid length should be 6 Nucleotide, and preferred length is 6～50 Nucleotide.Especially, this oligonucleotide length is at least 10,17,25 or at least 50 Nucleotide.
Polynucleotide of the present invention can be DNA or RNA or its chimeric mixture or the form of deriving and closing or modifying, and can be strand or two strands.Can be in the base component, sugar component or phosphate backbone are modified oligonucleotide, for example to improve the stability of molecule, hybridization etc.This oligonucleotide can comprise other appended group such as peptide (as directed host cell receptor in vivo), or promote transmembrane transport preparation (for example see Letsinger etc., 1989, institute of American Academy of Sciences reports 86:6553-6556; Lemaitre etc., institute of American Academy of Sciences report 84:648-652 (1987); PCT publication No.WO 88/09810, published on December 15th, 1988) or the preparation that passes hemato encephalic barrier (see for example open WO 89/10134 of PCT, on April 25th, 1988 is open), the cracking agent that hybridization causes (is for example seen Krol etc., biotechnology 6:958-976 (1988)) or intercalator (seeing for example Zon, drug research 5:539-549 (1988)).Therefore, oligonucleotide can be puted together with another molecule, as peptide, and the linking agent that hybridization causes, transport agents, the cracking agent that hybridization causes etc.
Antisense oligonucleotide can comprise a kind of base component of modification, be selected from and comprise but the non-5 FU 5 fluorouracil that is limited to, 5-bromouracil, 5-chlorouracil, 5-iodouracil, xanthoglobulin, xanthine, 4-acetylcytosine, 5-carboxylic methylol uridylic, 5-carboxymethyl aminomethyl-2-sulphur urine purine, 5-carboxylic aminomethyl uridylic, dihydrouracil, β-D-semi-lactosi queosine, inosine, the N6-isopentennyladenine, 1-methyl guanine, 1-methylinosine, 2, the 2-dimethylguanine, 2-methyladenine, 2-methyl guanine, the 3-methylcystein, 5-methylcytosine, N6-VITAMIN B4,7-methyl guanine, 5-methylamino-6-Methyl Uracil, 5-methoxy aminomethyl-2-thiouracil, β-D-mannosylqueosine, 5-methoxy carboxymethyl uracil, 5-methoxy uridylic, 2-first sulphur-N6-isopentennyladenine, uridylic-5-fluoroacetic acid (v), Wybutoxiosine, pseudouracil, queosine, 2-sulphur cytosine(Cyt), 5-methyl-2-thiouracil, the 2-thiouracil, the 4-thiouracil, methyl uracil, uridylic-5-fluoroacetic acid methyl esters, uridylic-the 5-fluoroacetic acid (v), 5-methyl-2-thiouracil, 3-(3-ammonia-3-N-carboxylic propyl group) uridylic, (acp3) w, with 2,6-diamino purine.
Antisense oligonucleotide also can comprise the sugar component of at least one modification, is selected to comprise but the non-pectinose that is limited to 2-fluorine pectinose, xylulose and hexose.
In another embodiment, antisense oligonucleotide comprises the phosphate backbone of at least one modification, is selected to comprise but the non-phosphorodithioate that is limited to thiophosphatephosphorothioate, the sulfo-phosphonic ester, phosphoramidite, inferior phosphorus diamide, methyl phosphorodithioate, alkyl phosphotriester, formacetal or its analogue.
In another embodiment, antisense oligonucleotide is α-end group isomery oligonucleotide.α end group oligonucleotide and complementary RNA form special double-stranded crossbred, and be wherein opposite with general β unit, chain parallel each other (Gautier etc., nucleic acids research 15:6625-6641 (1987)).Oligonucleotide is 2-O-methyl ribonucleotides (Inoue etc., nucleic acids research 15:6131-6148 (1987)), or chimeric RNA-DNA analogue (Inoue etc., FEBS communicate by letter 215:327-330 (1997)).
Polynucleotide of the present invention can be synthetic by standard method known in the art, for example uses automatic dna synthesizer) as can be available from Biosearch, Applied Biosysterms etc.).For example, thiophosphatephosphorothioate can be by the method synthetic (nucleic acids research 16:3209 (1988)) of Stein etc., the methyl phosphorodithioate oligonucleotide can pass through control punch glass, polymer support preparation (Sarin etc., institute of American Academy of Sciences report 85:7448-7451 (1988)) etc.
Can use the antisense nucleotide that is complementary to Neutrokine-α and/or Neutrokine-α SV coding region sequence, preferably be complementary to the antisense nucleotide in the untranslated district that transcribes.
Potential antagonist of the present invention also comprises catalytic RNA or ribozyme, and (see for example international open WO 90/11364 of PCT, October 4 nineteen ninety is open; Sarver etc., science 247:1222-1225 (1990).Can use ribozyme,, preferably use hammerhead ribozyme to destroy Neutrokine-α and/or Neutrokine-α SV mRNA at site specific recognition sequence cracking mRNA.The hammerhead ribozyme cracking is positioned at the mRNAS that forms the right regional both sides of complementary base with said target mrna.Unique demand is that said target mrna has 5 '-UG-3 ' sequence.The structure of well known hammerhead ribozyme and generation, and see for details in Haseloff and Gerlach, natural 334:585-591 (1988) is described.Many potential hammerhead ribozyme cleavage sites (seeing Figure 1A-B and 5A-B respectively) are arranged in the nucleotide sequence of Neutrokine-α and/or Neutrokine-α SV.Preferably, this ribozyme is carried out through engineering approaches,, promptly improve effectiveness and the intracellular accumulation of non-functional mRNA transcript is minimized so that the cutting recognition site is positioned near the 5 ' end of Neutrokine-α and/or Neutrokine-α SVmRNA.
In the antisense method, ribozyme of the present invention can be formed (as improved stability, orientation etc.) by the oligonucleotide of modifying, and should carry in the cell of expressing Neutrokine-α and/or Neutrokine-α SV in vivo.The DNA construct of encoding ribozyme can be as in the above-mentioned same way as transfered cell, to import antisense code dna.Preferred carrying method comprises that use is at strong constitutive promoter such as polIII, or under the control of polII promotor, the DNA construct of " coding " ribozyme, cells transfected will produce the ribozyme of capacity like this, with destruction endogenous Neutrokine-α and/or Neutrokine-α SV courier, and suppress translation.Because ribozyme is a catalytic unlike antisense molecule, need lower intracellular concentration to render a service to increase.
Endogenous gene expression also can be by using directed homologous recombination, deactivation or " rejecting " Neutrokine-α and/or Neutrokine-α SV gene and/or its promotor and reduce (as Smithies etc., natural 317:230-24 (1985); Thomas and Capeahi, cell 51:503-512 (1987); Thompson etc., cell 5:313:321 (1989); Above document is all incorporated reference in full into).For example, can use the non-functional polynucleotide (or the incoherent dna sequence dna of complementary) that suddenly change in the present invention with DNA (coding region of gene or the regulatory region) both sides that come from the endogenous polynucleotide sequence, it has or does not have selectable mark and/or negative selectable mark, expresses the cell of polypeptide of the present invention in vivo with transfection.In another embodiment, use means known in the art to produce rejecting in cell, this cell contains but does not express corresponding gene.Homologous recombination by orientation is inserted DNA construct, causes directed inactivation of gene.This method is particularly suited for research and agriculture field, wherein the modification of embryonic stem cell be can be used for producing the animal filial generation (as table Thomas and Capechi1987 and Thompson1989 such as preceding) of the directed gene with deactivation.Yet this method can routine be applicable to human body, and the recombinant DNA construction body that provides uses suitable virus vector, directly is applied to or is oriented to required site in the body, and this it will be apparent to those skilled in the art that.The document of being quoted is all incorporated reference in full into.
In other embodiment, antagonist of the present invention comprises the Neutrokine-α and/or the Neutrokine-α SV (fragment of the Neutrokine-α shown in Figure 1A-B of soluble form, comprise ligand binding domains, the structural domain that TNF is conservative, and/or the ectodomain of Neutrokine-α and/or Neutrokine-α SV, and the fragment of Neutrokine-α SV shown in Fig. 5 A-B, comprise ligand binding domains, the structural domain that TNF is conservative, and/or the ectodomain of Neutrokine-α and/or Neutrokine-α SV).The Neutrokine-α of this soluble form and/or Neutrokine-α SV, its can be natural generation or synthetic, by combining Neutrokine-α and/or Neutrokine-α SV acceptor with natural Neutrokine-α and/or Neutrokine-α SV competition (as DR5 (seeing international open WO 98/41629), TR10 (seeing international open WO 98/54202), 312C2 (seeing international open WO 98/06842) and TR11, TR11SV1 and TR11SV2 (seeing U.S. Patent No. 09/176200)), by form can in conjunction with or the debond acceptor, but can not inducement signal the polymer of transduction, and the signal of antagonism Neutrokine-α and/or Neutrokine-α SV mediation.Preferably, these antagonists suppress stimulation lymphocyte (as the B cell) propagation, differentiation and/or the activation of Neutrokine-α and/or Neutrokine-α SV mediation.Antagonist of the present invention also comprises the antibody that is specific to TNF family part (as CD30) and Neutrokine-α-Fc and/or Neutrokine-α SV-Fc fusion rotein.
" TNF family part " is meant natural generation, reorganization with the synthetic part, it can be in conjunction with TNF receptor family member, and induces and/or block ligand/receptor signal approach.The tnf ligand family member comprises but the non-TNF-of being limited to α, lymphotoxin-α (LT-α is also referred to as TNF-β), LT-β (in heterotrimer LT-α-2-beta composite, finding), FasL, CD40L, (TNF-γ (international open WO 96/14328), ATIM-I (international open WO 97/33899), AIM-II (international open WO 97/34911), APRIL (J.Exp.Med.188 (6): 1185-1190), α-intrinsic factor (international open WO 98/07880), α-neural factor (international open WO 97/18921), CD27L, CD30L, 4-1BBL, OX40L, CD27, CD30,4-1BB, OX40 and nerve growth factor (NGF).In preferred embodiments, Neutrokine-α of the present invention and/or Neutrokine-α SV TNF family part are DD5 (seeing international open WO 98/41629), TR10 (seeing international open WO 98/54202), 312C2 (seeing international open WO 98/06842) and TR11, TR11SV1 and TR11SV2 (seeing U.S. Patent application No.09/176200).
Antagonist of the present invention also comprises the antibody that is specific to TNF family receptors or Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide.Antibody of the present invention can prepare by various standard methods with Neutrokine-α of the present invention and/or Neutrokine-α SV immunogen.This Neutrokine-α and/or Neutrokine-α SV immunogen comprise respectively by Neutrokine-α and/or Neutrokine-α SV polypeptide (can comprise or not comprise leader sequence) complete shown in Figure 1A-B (SEQ ID NO:2) and Fig. 5 A-B (SEQ ID NO:19), with Neutrokine-α and/or Neutrokine-α SV polypeptide fragment, comprise for example ligand binding domains, the structural domain that TNF is conservative, ectodomain, membrane-spanning domain, and/or born of the same parents' intracellular domain, or their arbitrary combination.
Polyclone of the present invention and monoclonal antibody agonist or antagonist can be according to Tartaglia and Goeddel, journal of biological chemistry 267 (7): 4304-4307 (1992); Tartaglia etc., the method shown in cell 73:213-216 (1993) and the PCT application WO 94/09137 produces, and preferably is specific to the polypeptide of the present invention that (i.e. only combination) has SEQ ID NO:2 aminoacid sequence.Term " antibody " (Ab) or " monoclonal antibody " (mAb) be meant comprise can conjugated antigen complete molecule with and fragment) as Fb and F (ab ') fragment).Fab, and Fab ' and F (ab ') fragment deletion Fc complete segment antibody, more promptly from circulation, remove, and can have the non-specific tissue bond (Wahl etc., nucleic acid method magazine 24:316-325 (1983)) of less complete antibody.
In a preferable methods, antibody of the present invention is mAbs.This mAbs can prepare (Kohler and Millstein, natural 256:495-497 (1975) and U.S. Patent No. 4376110 with hybridoma method; Harlow etc., antibody laboratory manual, press of cold spring harbor laboratory, cold spring port, NY, 1988; Monoclonal antibody and hybridoma: the new development in the biological analysis, Plenum press, New York, NY, 1980; Campbell, " monoclonal antibody technique ", the experimental technique in biological chemistry and the molecular biology, the 13rd volume editors such as () Burdor, Elsevier, Amsterdam (1984)).
Protein and other compound in conjunction with Neutrokine-α and/or Neutrokine-α SV structural domain also are candidate's agonist of the present invention and antagonist.This binding compounds can be used yeast two-hybrid system " seizure " (Fields and Song, natural 340:245-246 (1989)).Roger Brent and colleague thereof have done yeast two-hybrid system and have modified (Gyuris, cell 75:791-803 (1993); Zervos etc., cell 72:223-232 (1993)).Preferably, yeast two-hybrid system used according to the invention is caught the ligand binding domains in conjunction with Neutrokine-α and/or Neutrokine-α SV, ectodomain, born of the same parents' intracellular domain, the compound in membrane-spanning domain and dead territory.This compound is good candidate's agonist and an antagonist of the present invention.
For example, use above-mentioned double cross analysis, the extracellular domain born of the same parents intracellular domain of Neutrokine-α and/or Neutrokine-α SV acceptor, or its part can be used for differentiating and Neutrokine-α and/or the interactional in vivo cell protein of Neutrokine-α SV acceptor.This analysis also can be used for differentiating to have Neutrokine-α and/or the interactional in vivo cell protein of Neutrokine-α SV acceptor.This analysis also can be used for differentiating to have Neutrokine-α and/or the potential excitement of Neutrokine-α SV function of receptors or the part of antagonistic activity.The cytoplasmic region interacting proteins before be used to differentiate with mouse TNF-RII is analyzed in this screening, and differentiates the albumen that two kinds of acceptors are relevant.Rothe etc., cell 78:681 (1994).This protein and aminoacid sequence in conjunction with the cytoplasmic region of Neutrokine-α and/or Neutrokine-α SV acceptor are good candidate agonist of the present invention and antagonist.
Other screening method comprises that using the cell (as the Chinese hamster ovary celI of transfection) of expressing polypeptide of the present invention to measure the outer pH of the born of the same parents that caused by receptor activation changes, as described in science 246:181-296 (1989).Whether in another embodiment, potential agonist or antagonist can contact with the cell of expressing polypeptide of the present invention, and can measure the second messenger and reply as signal transduction, effective to determine potential antagonist or agonist.
Agonist of the present invention comprise natural generation with the synthetic compound, as TNF family ligand peptide fragment, transforming growth factor, neurotransmitter (as L-glutamic acid, Dopamine HCL, N-methyl-D-aspartate), tumor-inhibiting factor (p53), cytolytic T lymphocyte and metabolic antagonist.Preferred agonist comprises chemotherapeutics and cis-platinum, Zorubicin, bleomycin, cytosine arabinoside, mustargen, methotrexate and vincristine(VCR).Other agonist comprises ethanol and starch peptide (science 267:1457-1458 (1995)).
Preferred agonist is to stimulate lymphocyte (as the B cell) propagation, differentiation and/or activatory Neutrokine-α of the present invention and/or Neutrokine-α SV fragment.Further preferred agonist comprises anti-Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide, or the polyclone and the monoclonal antibody of its fragment generation.The agonist antibody that this anti-TNF family receptors produces sees Tartaglia etc., and institute of American Academy of Sciences reports 88:9292-9296 (1991); With Tartaglia etc., shown in the journal of biological chemistry 267:4304-4307 (1992).It is described also to see PCT application WO 94/09137.
In another embodiment, immune modulatory molecules such as IL2, IL3, II4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-γ and TNF-α can be used as the agonist of Neutrokine-α of the present invention and/or Neutrokine-α SV polypeptide, it stimulates lymphocyte (as the B cell) propagation, differentiation and/or activation.In a special embodiment, IL4 and/or IL10 are used to breed the B cell proliferation of Neutrokine-α and/or Neutrokine-α SV mediation.
In another embodiment of the present invention, with the cell with expression polypeptide of the present invention of through engineering approaches, or the cell of not expressing polypeptide of the present invention of through engineering approaches (as rejecting) is applied to patient in vivo.This cell can derive from patient's (be animal, comprise the people), or the compatible donor of MHC, and can comprise but non-inoblast, medullary cell, hemocyte (as lymphocyte), adipocyte, myocyte, the endotheliocyte etc. of being limited to.Cell is genetically engineered in the external use recombinant DNA method, in encoding sequence transfered cell, perhaps destroy encoding sequence relevant and/or endogenous and regulate sequence, as (using virus vector by transduction with polypeptide of the present invention with polypeptide of the present invention, and the preferred carrier that transgenosis is integrated into cellular genome), or transfection method, comprise but non-being limited to used plasmid, clay, YACs, naked DNA, electroporation, liposomes etc. carry out.The encoding sequence of polypeptide of the present invention can place under the control of strong constitutive promoter or inducible promoter or promotor/proliferator, to express preferred secretion polypeptide of the present invention.But the genetically engineered cell general ground of expression and preferred secretion polypeptide of the present invention is as importing in patient's body by circulation or peritoneal injection.
Perhaps, but also implanted in the body in the cell doped matrix, a part that can be used as skin graft as genetically engineered inoblast is implanted; Genetically engineered endotheliocyte can be used as the part of lymphatic vessel or blood vessel graft and implants and (for example to see Anderson etc., U.S. Patent No. 5399349; With Mulligan and Wilson, U.S. Patent No. 5460959 is all incorporated reference in full into).
When the cell cell right and wrong of using self or that non-MHC is compatible, they can be used the method that the cell that imports produces immunne response with the host that prevents that know.For example cell can capsule form import, and component is exchanged in born of the same parents' external environment immediately, but the cell of importing is discerned by host immune system.
In another embodiment of the present invention, the activity of Neutrokine-α and/or Neutrokine-α SV polypeptide can be reduced by " dominant ".For this reason, coding Neutrokine-α of defective and/or Neutrokine-α SV polypeptide are as the construct of the mutant that lacks all or part of TNF conserved domain, can be used in the gene therapy, to weaken Neutrokine-α and/or Neutrokine-α SV activity suitable target cell.For example, instruct the nucleotide sequence of host cell expression Neutrokine-α and/or Neutrokine-α SV polypeptide can import in monocyte or other cell or tissue (by in vivo or be derived from intravital gene therapy or other method known in the art), the structural domain that all or part of TNF is conservative in the described nucleotide sequence changes or lacks.Perhaps, can utilize directed homologous recombination, so that this deletant or mutant are imported among the endogenous Neutrokine-α and/or Neutrokine-α SV gene that is tried in the individual monocyte.The cell of through engineering approaches will express non-functional Neutrokine-α and/or Neutrokine-α SV polypeptide (be part (as polymer), its can in conjunction with but can not inducement signal transduce).
Chromosome analysis
Nucleic acid molecule of the present invention also can be used for karyomit(e) and differentiates.The directed specific position that also can hybridize individual human chromosome in this sequence specific ground.In addition, often need special site on the differential staining body.Minority chromosome marking reagent based on true sequence data (repetition polymorphism) is suitable for the marker chromosomes position.The chromosomal DNA mapping of the present invention is to determine these sequences important the first step relevant with disease related gene.
In some preferred embodiments, cDNA shown in this article and/or polynucleotide are used to clone the genomic dna of Neutrokine-α and/or Neutrokine-α SV gene.Carry out in these available various methods of knowing and commercially available library.The method that this genomic dna is used for knowing is then carried out the original position chromosome mapping.
In addition, in some cases, can sequence be located at karyomit(e) by preparation PCR primer (preferred 15-25bp) from cDNA.To the Computer Analysis that 3 ' the untranslated district of gene carries out, be used for being chosen in rapidly crossing in the genome and be no more than an exon, otherwise the primer that makes amplification become complicated.These primers are used for the somatic hybridization body that the PCR screening contains individual human chromosome then.CDNA clone is used in the step the fluorescence in situ hybridization (FISH) of Metaphase Chromosome smear accurate chromosomal localization is provided.This method can use the weak point of taking from cDNA as 50 or the probe of 60bp.Verma etc. is seen in elaboration about this method, human body chromosome basic fundamental handbook, Pergamon press, New York (1988).
In case sequence accurately is positioned on the karyomit(e), the physical location of sequence can be relevant with the genetic map data on the karyomit(e).This data are seen for example V.McKusick, and MendelianInheritance In Man is online available from Johns Hopkins university, Welch medical library.The gene and the relation between disease that have been positioned identical chromosomal region are differentiated (nature is in the associating hereditary feature of neighbour gene) by pedigree analysis then.
Next need to determine the difference of cDNA between the ill and not ill individuality or genome sequence.Do not have sudden change if observe sudden change in some or all diseased individuals in normal individual, then this sudden change promptly is the generation reason of disease.
By the resolution of physics figure and genetic map method, the accurate location of cDNA on karyomit(e) is relevant with 50～500 kinds of potential Disease-causing gene associated diseases.(supposing gene of 1 megabasse figure resolving power and every 20kb).
Utilize aforesaid method, the chromosome position to Neutrokine-α and/or Neutrokine-α SV is used in combination somatic cell hybrid and radiation hybrid, can determine on karyomit(e) 13g34 very surely.
Embodiment
The present invention will be convenient to understand with reference to following examples, and embodiment just illustrates and unrestricted the present invention.Many in following examples is special Neutrokine-α polynucleotide of the present invention and polypeptide of setting forth.Each embodiment in fact also is applicable to and produces and/or detect Neutrokine-α SV polynucleotide of the present invention and polypeptide.Those skilled in the art can be easy to carry out embodiment about Neutrokine-α SV by following examples.Embodiment 1a: use bacterial expression vector pQE9 (pD10) to carry out bacterial expression in this embodiment at expression in escherichia coli and purifying " the His mark " Neutrokine-α.
(QIAGEN company is as preceding).The pQE9 amicillin resistance (" Ampr ") of encoding, and contain a bacterium and duplicate origin (" ori "), an IPTG inducible promoter, a ribosome bind site (" RBS "), 6 codons of encoding histidine residue, with suitable single restriction enzyme cutting site, described histidine residues can carry out affinity purification with the affine resin of nickel-nitrilotriacetic acid(NTA) (Ni-NTA) that QIAGEN company is sold.Arrange these factors, the dna fragmentation of the insertion of coded polypeptide coding polypeptide like this with 6 histidine residues (being 6X His mark), these 6 histidine residues covalently are connected in the aminoterminal of this polypeptide.
Coding comprises the dna sequence dna of the required part of extracellular domain sequence of N eutrokine-α albumen, be from the cDNA of preservation clone with the amplification of PCR Oligonucleolide primers, the PCR Oligonucleolide primers is annealed to 3 ' of cDNA encoding sequence in the amino terminal sequence of the required part of protein and the preservation construct sequence respectively.To contain and promote in the pQE9 carrier that the extra Nucleotide of clone's restriction site adds respectively in 5 ' and the 3 ' primer sequence.
For cloning proteinic extracellular domain, the sequence of 5 ' primer is 5 '-GTG GGA TCCAGC CTC CGG GCA GAG CTG-3 ' (SEQ ID NO:10), it contains the Bam HI restriction site of underscore, 18 Nucleotide of the aminoterminal encoding sequence of the extracellular domain of sequence shown in back map interlinking 1A and the 1B.Certainly, those skilled in the art will know that the initial position of 5 ' primer can change in the protein coding sequence, with the DNA section of the proteic any required part of the complete Neutrokine-α of amplification coding, this complete Neutrokine-α albumen is longer or short than extracellular domain.The sequence of 3 ' primer is 5 '-GTG AAG CTT TTA TTACAG CAG TTT CAA TGC ACC-3 ' (SEQ ID NO:11), it contains the Hind III restriction site of underscore, after connect 2 terminator codons and be complementary to Figure 1A and 1B shown in dna sequence dna 3 ' end encoding sequence 18 Nucleotide.
The dna fragmentation of amplification and carrier pQE9 link together the DNAs that digests then with Bam HI and Hind III digestion.Be inserted in the pQE9 carrier of restriction DNA with protein coding region place the IPTG inducible promoters the downstream and with initial AUG and 6 same frames of Histidine codon.
Mixture be will connect with standard method and method therefor such as Sambrook etc., molecular cloning laboratory manual, second edition will be converted in the suitable Bacillus coli cells; Press of cold spring harbor laboratory, the cold spring port, NY (1989) is described.The coli strain M15/rep4 that contains the plasmid pREP4 of a plurality of copies is used to carry out the illustrational embodiment of this paper, and described plasmid pREP4 expresses the lac repressor and gives kalamycin resistance (Kan r).This bacterial strain only is to be suitable for a kind of in many bacterial strains of marking protein, and it can be available from QIAGEN company.Transformant is differentiated there being the energy for growth on the LB flat board of penbritin and kantlex by it.Isolated plasmid dna is also by restriction analysis from resistance clone, and PCR and dna sequencing are determined clone's DNA.The clone who contains required construct, grow overnight (O/N) in the liquid LB substratum of adding penbritin (100 μ g/ml) and kantlex (25 μ g/ml).Inoculate big culture with this O/N culture, extent of dilution is approximately 1: 25 to 1: 250.Cell grows to optical density(OD) (OD600) at 600nm between 0.4-0.6.
Add then IPTG to final concentration be 1mM, with by deactivation lac repressor, from the responsive promotor inducible transcription of lac repressor.Then with cell incubation 3-4 hour again.Pass through centrifugal collecting cell then.
Then with cell 4 ℃ at the 6M Guanidinium hydrochloride, stirred 3-4 hour among the pH8.Through the centrifugal cell debris of removing, with the supernatant application of sample in the affine resin chromatography column of nickel-nitrilotriacetic acid(NTA) (Ni-NTA) (available from QIAGEN company).Protein with 6 * His mark combines with Ni-NTA resin high-affinity, and available easy one step process purifying (see QIAexpressionist for details, 1995, QIAGEN company).In brief, in the 6M Guanidinium hydrochloride, in the chromatography column among the pH8, this post is at first used the 6M Guanidinium hydrochloride of 10 volumes with the supernatant application of sample, the pH6 flushing, and use the 6M Guanidinium hydrochloride at last, pH5 wash-out Neutokine-α and Neutokine-α SV polypeptide.
By using phosphate buffered saline (PBS) (PBS) or 50mM sodium acetate, the pH6 damping fluid adds 200mM NaCl dialysis and renaturation to the protein of purifying then.Perhaps, protein can be fixed on the Ni-NTA chromatography column by the folding again of success simultaneously.The condition that is suitable for is as follows: containing the 500mM NaCl of proteinase inhibitor, 20% glycerine is among the 20mMTris/HCl pH7.4, with linear 6M-1M urea gradient renaturation.Renaturation should be carried out 1.5 hours or the longer time.After renaturation, protein can pass through to add the 250mM imidazoles and wash-out.Remove imidazoles by add the last dialysis of 200mM NaCl with PBS or 50mM sodium acetate pH6 damping fluid.The protein of purifying is stored in 4 ℃ or be chilled in-80 ℃.
Embodiment 1b: at expression in escherichia coli and purifying Neutokine-α SV
Carry out bacterial expression with bacterial expression vector pQE60 in this embodiment.(QIAGEN company, 9259 Eton Avenue, Chatsworth, CA, 91311).The pQE60 amicillin resistance (" Ampr ") of encoding, and contain a bacterium and duplicate origin (" ori "), an IPTG inducible promoter, a ribosome bind site (" RBS "), 6 codons of encoding histidine residue, with suitable single restriction enzyme cutting site, described histidine residues can carry out affinity purification with the affine resin of nickel-nitrilotriacetic acid(NTA) (Ni-NTA) that QIAGEN company is sold.The arrangement of these elements is to make the dna fragmentation of coded polypeptide insert the polypeptide that the back coding has 6 histidine residues (being 6X His mark), and these 6 histidine residues covalently are connected in the aminoterminal of this polypeptide.Yet, in this embodiment, insert polypeptid coding sequence, prevent 6 Histidine codons translations thus, and thereby produce the polypeptide that does not have 6 histidine marks.
Coding comprises the dna sequence dna of the required part of protein of extracellular domain sequence, be from the cDNA of preservation clone with the amplification of PCR Oligonucleolide primers, the PCR Oligonucleolide primers is annealed to 3 ' of cDNA encoding sequence in the sequence of the amino terminal sequence of the required part of protein and preservation construct respectively.To contain and promote in the pQE9 carrier that the extra Nucleotide of clone's restriction site adds respectively in 5 ' and the 3 ' primer sequence.
For cloning proteinic extracellular domain, the sequence of 5 ' primer is 5 '-GTG TCA TGAGCC TCC GGG CAG AGC TG-3 ' (SEQ ID NO:12), it contains the Bsp HI restriction site of underscore, 17 Nucleotide of the aminoterminal encoding sequence of the extracellular domain of sequence shown in back map interlinking 1A and the 1B.Certainly, those skilled in the art will know that the initial position of 5 ' primer can change in the protein coding sequence, with any required part of amplification coding whole protein, this whole protein is longer or short than extracellular domain.The sequence of 3 ' primer is 5 '-GTG AAG CTTTTA TTA CAG CAG TTT CAA TGC ACC-3 ' (SEQ ID NO:13), it contains the Hind III restriction site of underscore, after connect 2 terminator codons and be complementary to Figure 1A and 1B shown in dna sequence dna 3 ' end encoding sequence 18 Nucleotide.
The dna fragmentation of amplification and carrier pQE60 link together the DNAs that digests then with Bsp HI and Hind III digestion.Be inserted in the pQE60 carrier of restriction DNA with protein coding region place the IPTG inducible promoters the downstream and with initial AUG and 6 same frames of Histidine codon.Relevant terminator codon prevents 6 Histidine codon translations in the downstream, insertion point.
Mixture be will connect with standard method and method therefor such as Sambrook etc., molecular cloning laboratory manual, second edition will be converted in the suitable Bacillus coli cells; Press of cold spring harbor laboratory, the cold spring port, NY (1989) is described.The coli strain M15/rep4 that contains a plurality of copies of plasmid pREP4 is used to carry out the illustrational embodiment of this paper, and this plasmid pREP4 expresses the lac repressor and gives kalamycin resistance (Kan r).This bacterial strain is to be suitable for a kind of in many bacterial strains of marking protein, and it can be available from QIAGEN company.Transformant is differentiated there being the energy for growth on the LB flat board of penbritin and kantlex by it.Isolated plasmid dna is also by restriction analysis from resistance clone, and PCR and dna sequencing are determined clone's DNA.
Those skilled in the art recognize many bacterial expression vectors any all can be used for replacing used pQE9 and the pQE60 of expression method among this embodiment.For example, new pHE4 bacterial expression vector series, especially the pHE4-5 carrier can be used for bacterial expression (the ATCC No.209311 among this embodiment; And variant).The plasmid DNA that is called pHE4-5/MPIFD23 of ATCC preserving number 209311 is the vector plasmid DNA that contain the insertion body of the another kind of ORF that encodes.This construct is deposited in American type culture collection on September 30th, 1997, and this is centered close to the road 20110-2209 of Manassas, Virginia city university, postcode 10801.Use is inserted the Nde I and Asp 718 restriction sites of body both sides at incoherent MPIF ORF, and those skilled in the art are easy to use current molecular biology method, with incoherent ORF in the Neutrokine-α ORF displacement pHE4-5 carrier of the present invention.
The pHE4-5 bacterial expression vector comprises the neomycin phosphotransferase gene of selecting, an intestinal bacteria replication orgin, a T5 bacteriophage promoter sequences, two lac operon sequence, Shine-Delgarno sequence and lactose operon repressor thing gene (1acIq).The arrangement of these elements is to make the dna fragmentation of coded polypeptide insert the back to express the polypeptide with 6 histidine residues (i.e. 6 * His mark) that these 6 histidine residues covalency are connected in this polypeptide aminoterminal.The promotor of pHE4-5 carrier and operon sequence are synthetic the generations.The synthetic production method of well known nucleotide sequence (CLONETECH 95/96 Catalog, p215-216, CLONETECH, 1020 East Meadow Circle, Palo Alto, CA94303).
The clone who contains required Neutokine-α SV construct, grow overnight (O/N) in the liquid LB substratum of adding penbritin (100 μ g/ml) and kantlex (25 μ g/ml).Inoculate big culture with this O/N culture, extent of dilution is approximately 1: 25 to 1: 250.Cell grows to optical density(OD) (OD600) at 600nm between 0.4-0.6.Add then IPTG to final concentration be 1mM, with by deactivation lac repressor, from the responsive promotor inducible transcription of lac repressor.Then with cell incubation 3-4 hour again.Pass through centrifugal collecting cell then.
Then with cell 4 ℃ at the 6M Guanidinium hydrochloride, stirred 3-4 hour among the pH8.Through the centrifugal cell debris of removing, the supernatant that will contain Neutrokine-α is dialysed with the 50mM sodium acetate pH6 damping fluid of adding 200mM NaCl.Perhaps, protein can be with the 500mM NaCl that contains proteinase inhibitor, 20% glycerine, and 25mM Tris/HCl pH7.4 dialysis, and successfully folding again.After renaturation, protein can be by ion-exchange, hydrophobic interaction and size exclusion chromatography and purifying.Perhaps, affinity chromatography such as antibody chromatography column can be used for obtaining the protein of purifying.The protein of purifying is stored in 4 ℃ or be chilled in-80 ℃.
In some embodiments, preferably produce, with one or more of 3 cysteine residues in the sudden change Neutrokine-α peptide sequence as the described expression construct of present embodiment.Cysteine residues in the Neutrokine-α peptide sequence is positioned at 147 shown in the SEQ ID NO:2,232, with 245, and be arranged in 213 and 226 shown in the SEQ ID NO:19 (Neutrokine-α SV peptide sequence is not equivalent to the halfcystine of Neutrokine-α peptide sequence Cys-147, because there is not the 143-160 amino acids residue of Neutrokine-α peptide sequence in the Neutrokine-α SV peptide sequence).
Embodiment 2: clone in baculovirus expression system, express and purifying Neutrokine-α albumen
In this embodiment, plasmid shuttle vectors pA2GP is used for the coded protein extracellular domain, lack in its natural relevant born of the same parents and stride the clone's of film sequence DNA, insert in the baculovirus, to express the proteic extracellular domain of Neutrokine-α, use the baculovirus leader sequence and as Summers etc., baculovirus and insect cell cultural method handbook, the described method of TexasAgricultural Experimental Station Bulletin No.1555 (1987).This expression vector contains the strong polyhedrin promotor of AcMNPV, back extension bar shape virus proteic secreting signal peptide of gp67 (leader sequence) and conventional restriction site such as BamHI, Xba I and Asp 718 restriction sites.The polyadenylation site of simian virus 40 (SV40) is used for effective polyadenylation.For be easy to select recombinant virus, plasmid contain in the same way under weak fruit bat promotor control from colibacillary beta-galactosidase gene, after connect the polyadenylation signal of polyhedron gene.The gene that inserts is positioned at the both sides of virus sequence, to carry out cell-mediated homologous recombination with wild-type virus DNA, produces the live virus of the polynucleotide of cloning by expression.
Many other baculovirus vectors can be used for replacing above-mentioned carrier, as pAc373, and pVL941, and pAcIM1, as long as those skilled in the art recognize that this construct provides transcribing of appropriate location, translation, signals such as secretion comprise AUG in required signal peptide and the frame.This carrier is Luckow etc. for example, and virusology 170:31-39 (1989) is described.
The coding N-terminal lacks the Neutrokine-α albumen extracellular domain of form among the preservation clone, disappearance AUG initiator codon, interior and the membrane spaning domain sequence of natural relevant born of the same parents, with the cDNA sequence of Gln-73 to Leu-79 amino acids shown in Figure 1A and the 1B (SEQ ID NO:2), be to increase with the PCR Oligonucleolide primers that is equivalent to gene 5 ' and 3 ' sequence.5 ' primer sequence is 5 '-GTG GGA TCCCCG GGC AGA GCTGCA GGG C-3 ' (SEQ ID NO:14), it contains the Bam HI restriction enzyme sites of underscore, 18 Nucleotide of the extracellular domain of Neutrokine-α albumen shown in back map interlinking IA and 1B sequence, the N-terminal of the protein extracellular foreign lands shown in arising from.3 ' primer sequence is 5 '-GTG GGA TCCTTA TTA CAG CAG TTT CAA TGC ACC-3 ' (SEQ ID NO:15), it contains the Bam HI restriction enzyme sites of underscore, after connect two terminator codons and be complementary to Figure 1A and 1B shown in 18 Nucleotide of 3 ' encoding sequence.
At some in other the embodiment, the construct of the extracellular domain that preferred expression Neutrokine-α all infers (being Gln-73 to Leu-285 amino acids residue).Polynucleotide and peptide sequence that those skilled in the art can use SEQ ID NO:1 and SEQ ID NO:2 to provide respectively, design polynucleotide primer is to produce this clone.
In an embodiment preferred, Leu-112 to the Leu-285 amino acids residue of Neutrokine-α peptide sequence shown in the pA2GP expression construct coding SEQ ID NO:2.
In another embodiment preferred, Ser-78 to the Leu-285 amino acids residue of Neutrokine-α peptide sequence shown in the pA2GP expression construct coding SEQ ID NO:2.
(La Jolla Ca.), separates from 1% sepharose the fragment of amplification for Geneclean, BIO 101 companies with the test kit that is purchased.Digest this fragment with BamHI then, and purifying in 1% sepharose again.This fragment is called F1 at this.
Plasmid digests with restriction enzyme BamHI, and randomly with ordinary method known in the art calf small intestine Phosphoric acid esterase dephosphorylation.(La Jolla Ca.), separates from 1% sepharose for Geneclean, BIO 101 companies with the test kit that is purchased with DNA then.This carrier DNA is called V1 at this.
Fragment F1 is connected with the T4 dna ligase with dephosphorylized plasmid V1.(Statagene Cloning Systems, La Jolla Ca.), and coats on the culture plate to connect mixture transformed into escherichia coli HB101 or other suitable escherichia coli host such as XL-1 Blue cell with this.By digest the DNA of each bacterium colony with BamHI, reach and then pass through the gel electrophoresis analysis digestion product, differentiate the bacterium that contains plasmid with human body gene.Determine the sequence of cloned sequence by dna sequencing.This plasmid is called pA2GP-Neutrokine-α at this.
Linearized baculovirus dna (the BaculoGold that plasmid pA2GP-Neutrokine-α and the 1.0 μ g of 5 μ g are purchased TMBaculovirus DNA, Pharmingen, San Diego, CA) cotransfection uses as Felgner etc., and institute of American Academy of Sciences reports the described fat of 84:7413-7417 (1987) to dye method.BaculoGold with 1.0 μ g TMThe plasmid pA2GP-Neutrokine-α of viral DNA and 5 μ g, (Life Technologies company, Gaithersburg mix in the aseptic hole of microtitre flat board MD) at serum-free Grace ' the s substratum that contains 50 μ l.Afterwards, add 10 μ l Lipofectin and 90 μ l Grace ' s substratum, mix and be incorporated in the room temperature incubation 15 minutes.Then transfection mixture is dropped in the Sf9 insect cell (ATCC CRL 1711), the Sf9 insect cell is planted in the 35mm tissue culture plate that contains 1ml serum-free Grace ' s substratum.Then with this flat board 27 ℃ of incubations 5 hours.From flat board, remove transfection liquid then, and add 1ml Grace ' the s insect substratum of adding 10% calf serum.Continue to cultivate 4 days at 27 ℃ then.
After 4 days, collect supernatant and carry out the plaque analysis, as described in Summers and Smith.Use has " Blue Gal ", and (sepharose Maryland) is convenient to differentiate and is separated the gal cloning by expression that produces the blue plaque that dyes for Life Technologies company, Rockville.(about elaborating of this kind of plaque analysis, also be found in Life Technologies company, Rockville, the insect cell of Maryland is cultivated and baculovirus distribution instruction, p9-10 is described).Behind suitable incubation, the plaque that dyes with the most advanced and sophisticated picking indigo plant of micropipet (as Eppendorf).The agar resuspending that will contain recombinant virus then is in the micro-centrifuge tube that contains 200 μ lGrace ' s substratum, and the suspension that will contain recombinant baculovirus is used for infecting and plants the cell in the Sf9 of 35mm plate.After 4 days, collect the supernatant in these culture dish, then 4 ℃ of storages.This recombinant virus is called V-Neutrokine-α.
For check Neutrokine-α expression of gene, the Sf9 cell is grown in adding Grace ' the s substratum of 10% heat-inactivated FBS.This cell with recombinant baculovirus V-Neutrokine-α, is approximately in infection multiplicity (MOI) under 2 the situation and infects.Radiolabeled if desired protein is removed this substratum after 6 hours, and (available from Life Technologies company, Rockville Maryland) replaces with the SF900 II substratum that deducts methionine(Met) and halfcystine.After 42 hours, add 5 microcuries 35S-methionine(Met) and 5 microcuries 35S-halfcystine (available from Amersham).After cell incubation 16 hours, by centrifugal collection.Protein in the supernatant and intracellular protein are then analyzed by radioautograph (if radiolabeled) by SDS-PAGE.
Can use the N-terminal aminoacid sequence of protein purification is carried out the micrometering preface, with the amino terminal sequence of definite protein extracellular foreign lands, and the length of therefore definite cleavage site and secreting signal peptide.
In a special embodiment, reorganization Neutrokine-α purifying is from the Sf9 of baculovirus infection cell conditioned medium, and is as described below.Insect cell is grown in the EXCEL401 substratum (JRH Scientific) with 1% (v/v) calf serum.After infecting 92 hours, with the supernatant collected by centrifugal, then by clarifying in the 0.45m depth type filtration at 18000 * g.Also can use the degreasing filtration step, removing the lipid pollutent, and improve the proteic initial seizure of Neutrokine-α thereupon.
With supernatant at random application of sample in a series of Poros HS-50/H-50.Perhaps, can use Toyopearl QAE, Toyopearl Super Q (Tosohass), Q-Sepharose (Pharmacia) and resin of equal value.This step is used as negative purification step to remove reinforcing yin essence ionic bond pollutent.The HS/HQ of the raw material of flowing through is adjusted to pH7.5 with 1M Tris-HCl pH8, and with the Tris-HCl pH8 dilution of isopyknic 50mM, and application of sample is in porous PI-20 or PI-50 chromatography column.The PI chromatography column at first with 75mM NaCl flushing among the 50mM Tris-HCl pH7.5 of 4 volumes, is used 300mM among the 50mM Tris-HCl pH7.5 of 3-5 column volume, 750mM, 1500mM NaCl wash-out then.Neutrokine-α albumen presents a 17KD band on the SDS-PAGE that simplifies, and is present in the 0.75M-1.5M NaCl part.
This PI is partly passed through Sephacryl S100 HR (Pharmacia) size exclusion chromatography post be further purified this chromatography column 0.15M NaCl, 50mM sodium acetate, pH6 balance.It is 3M that the S200 part is mixed into final concentration with NaCl, and application of sample is in ToyopearlHexyl 650C (Tosohass) chromatography column.Hexyl chromatography column 5-10 volume, the NaCl wash-out of the 3M-0.05M linear gradient in 50mM sodium acetate pH6.The sulfuric acid amine of the 1M-0M gradient in the Hexy1 chromatography among the also available 50mM sodium acetate of this NaCl gradient pH6 replaces.Combination contains the part of analyzing the Neutrokine-α of purifying through SDS-PAGE, and with containing 150mM NaCl, 50mM sodium acetate, pH6 dialysis.
Neutrokine-α albumen at the final purifying of rhabdovirus system expression as herein described has had the N-terminal sequence from the Ala-134 amino-acid residue of SEQ ID NO:2.RP-HPLC analyzes the single peak value that is higher than 95% purity is shown.Level of endotoxin is below the detection limit value that LAL analyzes.
In another embodiment, reorganization Neutrokine-α purifying is from the Sf9 cell that contains the baculovirus infection of 0.25% bovine serum, and is as described below.
The Sf9 supernatant passes through in the centrifugal collection of 18000 * g.Then with supernatant 10mMCaCl 2, under the weak base condition, handled 10-15 minute, then carry out centrifugal and 0.22 μ m depth type filtration.Then gained Sf9 cell conditioned medium is diluted 2 times, and application of sample is in Poros PI-50 chromatography column (available from the PE biosystem).This chromatography column 50mM Tris (pH=7.4) balance.With of 50mM Tris (pH=7.4) flushing of PI-50 chromatography column, use the 1.5M NaCl wash-out among the above 50mM NaOAc (pH=6) of 3CV then with 1CV.With PI part application of sample in 50mM NaOAc (pH=6), on the 125mM NaCl equilibrated SephacrylS200 chromatography column.It is 0.7M ammonium sulfate and 0.6MNaCl that the S200 part is mixed to final concentration with salt, and application of sample is in Toyopearl Hexyl 650C chromatography column (available from Toso Haas), this chromatography column is with containing 0.6M NaCl among the 50mM NaOAc (pH=6), the damping fluid balance of 0.7M ammonium sulfate.Then with of the same buffer flushing of this chromatography column with 2CV.Reorganization Neutrokine-α uses 50mM NaOAc (pH=6) wash-out of 3CV, then 20% of usefulness 2CV alcohol flushing then.Neutrokine-α albumen recombinate then at the terminal wash-out of ammonium sulphate gradient (0.3-0M).Gather suitable part, and, pass through porous 50HQ chromatography column then with the damping fluid dialysis that contains 50mM NaOAc (pH=6).With 4 times of HQ merchantable thing dilutions, and application of sample is used the 25mM Trisodium Citrate, 125M NaCl wash-out then in Toyopearl DEAD 650M chromatography column.
In another embodiment, reorganization Neutrokine-α is with baculovirus vector system expression in the Sf+ insect cell and purifying.
At first, polynucleotide with Ser-78 to the Leu-285 amino-acid residue (equaling Ser-78 to the Leu-285 amino-acid residue of Neutrokine-α peptide sequence shown in the SEQ ID NO:2) of Neutrokine-α peptide sequence shown in Figure 1A and the 1B of encoding, subclone is gone into baculovirus and is shifted among the construct PSC, to produce the baculovirus expression plasmid.PA2GP transfer vector derived from pVL941 contains the gp67 signal peptide, and the multiple clone site of modification and be cloned in the lac gene in Drosophila heat-shocked promotor downstream is dyed plaque to select indigo plant.Use Neutrokine-α (SEQ ID NO:2) sequence, with pA2GP carrier sequence, design a kind of cloning process, the PSC signal coding sequence is closely merged on the Ala-134 of Neutrokine-α encoding sequence (SEQ ID NO:2 and Figure 1A and 1B), and be inserted in the PSC baculovirus transferring plasmid.This method comprises uses twice polymerase chain reaction (PCR).At first, the primer of design amplification Neutrokine-α sequence.5 ' primer is by coding Ala-134 and following residue (5 '-GGT CGC CGT TTC TAA CGC GGCCGT TCA GGG TCC AGA AG-3 '; SEQ ID NO:31) sequence is formed, and is positioned at before the sequence of coding PSC signal peptide C-terminal.3 ' primer (5 '-CTG GTTCGG CCC AAG GTA CCA AGC TTG TAC CTT AGA TCT TTT CTAGAT C-3 '), reverse complementary sequence by the pA2GP carrier sequence that is positioned at Neutrokine-α encoding sequence downstream is formed, and is positioned at Kpn I restriction endonuclease sites and intervening sequence (rendeing a service to improve Kpn I cutting) before.Carry out PCR, gained PCR product standard method purifying with the pA2GP that contains Neutrokine-α plasmid template and primer O-1887 and O-1888.
Carry out another PCR reaction with PSC baculovirus transferring plasmid pMGS12 as template.This pMGS12 plasmid is made up of AcNPV EcoRI " I " fragment of inserting pUC8, and the polyhedrin encoding sequence after the ATG initiator codon is replaced with a PSC signal peptide and a polylinker site.The PCR reaction is made template with pMGS12,5 ' primer (5 '-CTGGTA GTT CTT CGG AGT GTG-3 '; SEQ ID NO:33) in the AcNPV ORF603 of unique N goMIV and upstream, EcoRV site, anneals 3 ' primer (5 '-CGCGTT AGA AAC GGC GAC C-3 '; SEQ ID NO:34) is annealed to 3 ' end of the sequence of coding PSC signal peptide.
For producing the PCR product that PSC signal peptide wherein closely is blended in Neutrokine-α encoding sequence Ala-134,, and carry out again the PCR circulation with this PCR product and PSC signal peptide-polyhedrin upstream PCR product combination.Owing to 3 ' terminal 5 ' end with the Neutrokine-α PCR product of using primer O-1887/O-1888 preparation of PSC signal peptide PCR product (pMGS12/O-959/O-1044) is overlapping, make up these two PCR products and pass through the overlapping extension of PCR with primer O-959 and O-1888.
The PCR product that will contain the overlapping extension of gained of the PSC signal peptide that is blended in Neutrokine-α sequence inserts among the baculovirus transferring plasmid pMGS12.This PCR product also is connected among the pMGS12 of NgoM IV-Kpn I cutting with NgoM IV and Kpn I digestion, purifying fragment.After connecting mixture transformed competence colibacillus e.colidh5 with this, picking colony and micropreparation plasmid DNA.Some positive colonies of each ligation are differentiated by the restrictive diges-tion analysis of plasmid DNA, and (pAcC9669, pAcC9671 pAcC9672) carry out the plasmid in large scale purifying to select 3 clones.The gained plasmid DNA is carried out dna sequence analysis to determine and order-checking Neutrokine-α insertion body.
Following steps have been set forth from the Sf+ insect cell and to have been reclaimed and the method for purification of Recombinant Neutrokine-α.Unless specialize, this method is carried out at 2-8 ℃.
Reclaim
The first step: CaCl 2Handle
By at the centrifugal collection of 8000 * g Sf+ cell conditioned medium.To reclaim damping fluid-1 (1MCaCl 2) add in the supernatant CaCl thus 2Final concentration be 10mM.(in an embodiment preferred, use 1M ZnCl 2Replace 1M CaCl 2).The pH of this solution is adjusted to 7.7 with reclaiming damping fluid-2 (1M Tris pH8 (± 0.2)) ±.With this solution incubation 15 minutes, centrifugal at 8000 * g then.
Purifying
The first step: chromatography on porous PI-50 chromatography column
On porous PI-50 chromatography column (PE Biosystem), this chromatography column is with PI-1 damping fluid (50mM Tris, 50mM NaCl, pH7.4 (± 0.2)) balance with Sf+ cell conditioned medium application of sample.With of the PI-1 damping fluid flushing of PI-50 chromatography column, use PI-2 damping fluid (50mM Trisodium Citrate, pH6 (± 0.2)) then through the 3CV linear gradient elution with 1-2CV.The monitoring elutriant is in ultraviolet ray (UV) absorbancy of 280nm.Collection is crossed the component of wash-out peak value and is analyzed by SDS-PAGE.Gather suitable component.
Second step: chromatography on Toyopearl Hexyl 650C chromatography column
The PI and the salt of set are mixed to (NH 4) 2SO 4Final concentration be 0.7M, and application of sample is on Toyopearl Hexyl 650C chromatography column, this chromatography column is with HIC-1 damping fluid (50mMNaOAc, 0.6M NaCl, 0.7M (NH 4) 2SO 4PH6 (± 0.2)) balance.Then with of the HIC-1 damping fluid flushing of this chromatography column with 2CV.Then, reorganization Neutrokine-α uses 20% alcohol flushing of 2CV then with 3-5CV HIC-2 damping fluid (50mM NaOAc pH6 (± 0.2)) wash-out.The monitoring elutriant is in ultraviolet ray (UV) absorbancy and the conductivity of 280nm.Collect the component of wash-out peak value and analyze by SDS-PAGE.Gather suitable component.
The 3rd step: chromatography on SP sepharose FF
Dialysis Hexy1 component is also used SP-1 damping fluid (50mM sodium acetate, pH4.5 (± 0.2)) be 4.5 with pH regulator, dilute 4 times, and application of sample is in SP sepharose (cationite, Pharmacia) chromatography column, this chromatography column SP-1 damping fluid (50mM sodium acetate, pH4.5 (± 0.2)) balance.Use then SP-2 damping fluid (50mM sodium acetate, pH5.5 (± 0.2)) at pH5.5 from SP chromatography column wash-out reorganization Neutrokine-α albumen.Monitor ultraviolet ray (UV) absorbancy of elutriant then at 280nm.Collection is crossed the component of wash-out peak value and is analyzed by SDS-PAGE.Gather suitable component.
The 4th step: dialysis reorganization Neutrokine-α
Place 6-8kd to block film device the SP component, dialysis or diafiltration are spent the night in dialysis buffer liquid (10mM Trisodium Citrate, 140mM NaCl, pH6 (± 0.2)) then.
The 5th step: filter and fill
The protein concn of the reorganization Neutrokine-α solution in the 6th step is determined by dicinchonine acid (BCA) protein analysis.Be adjusted to whole protein concn with the suitable damping fluid Neutrokine-α that will recombinate, and under the condition of control, filter.Filtrate is stored in the suitable sterile chamber below-20 ℃.
In a special embodiment, it is 1-5mg/ml that the Neutrokine-α albumen of the present invention that produces as previously mentioned is adjusted to whole protein concn, and uses the 10mM Trisodium Citrate, 140mM NaCl, pH=6.0 (± 0.4) buffering, and be stored in the 1 type vial below-20 ℃.
During chromatography, monitoring is in ultraviolet ray (UV) absorbancy of 280nm.Work as where applicable, test chromatography intermediate specific conductivity, pH also passes through SDS and/or the RP-HPLC monitoring.
Cleaning chromatography column and purifier apparatus, and with 0.2 or 0.5M NaOH and washed with de-ionized water are then with 0.1 or the cleaning of 0.5M acetate.With chromatography column and purifier apparatus deionized water rinsing, if desired, be stored in the suitable stock solution.Before using, with this equipment with suitable damping fluid (damping fluid as described herein or well known in the art) balance.
In an embodiment preferred, in the first step of above-mentioned recovery, use 1M ZnCl 2Replace 1M CaCl 2Equally, in this embodiment, can use the ZnCl of combination 2And CaCl 20.1M ZnCl 2With 0.9M CaCl 2Some combinations can be used for recombinating in the proteic recovery method of Neutrokine-α, for example but the non-0.1M ZnCl that is limited to 2With 0.9M CaCl 2, 0.2M ZnCl 2With 0.8M CaCl 2, 0.3M ZnCl 2With 0.7M CaCl 2, 0.4M ZnCl 2With 0.6M CaCl 2, 0.5M ZnCl 2With 0.5M CaCl 2, 0.6M ZnCl 2With 0.4M CaCl 2, 0.7M ZnCl 2With 0.3M CaCl 2, 0.8M ZnCl 2With 0.2M CaCl 2, 0.9M ZnCl 2With 0.1M CaCl 2Deng.Yet the existence of EDTA will suppress removal process.In addition, there is ZnCl in the recovery damping fluid 1 2And/or CaCl 2To guide a large amount of high molecular Neutrokine-α polymers to form.
Embodiment 3: clone and expression Neutrokine-α in mammalian cell
Typical mammalian expression vector contains the promotor factor of mediation mRNA transcription initiation, protein coding sequence and be Transcription Termination and the required signal of transcript polyadenylation.Other element comprises enhanser, and Kozak sequence and both sides are the intervening sequence of RNA donor splicing site and acceptor site.With the early stage and late promoter of SV40, retroviral length terminal repetition (LTRs) is as RSV, HTLVI, and the early promoter of HIVI and cytomegalovirus (CMV) can reach efficiently and transcribe.Yet, also can use cytokine (as the human actin promotor).Be used for suitable expression vector of the present invention for example comprise pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).Spendable mammalian host cell comprises people HeLa, 293, and H9 and Jurkat cell, mouse NIH 3T3 and C127 cell, Cos 1, Cos 7 and CV1, frightening QC1-3 cell, mouse Lcell, Chinese hamster ovary (CHO) cell and HEK 293 cells.
Perhaps, gene can be expressed in suitable clone, and this clone contains and is integrated into chromosomal gene.With selectable mark such as dhfr, gpt, Xin Meisu, Totomycin cotransfection can be differentiated and separate cells transfected.
The gene of transfection also can increase to express a large amount of encoded protein matter.DHFR (Tetrahydrofolate dehydrogenase) mark is used to show the clone of the copy that carries hundreds of even several thousand corresponding gene.Another kind of effective choice mark is glutamine synthase (GS) (Murphy etc., journal of biological chemistry 227:277-279 (1991); Bebbington etc., biology/technology 10:169-175 (1992)).Use these marks, cell is grown in the substratum of selecting, and selects the cell of high resistance.These clones contain the gene that is integrated into chromosomal amplification.Chinese hamster ovary (CHO) cell and NSO cell are generally used for producing protein.
Expression vector pC1 and pC4 contain the fragment (Boshart etc., cell 41:521-530 (1985)) that the sarcoma viral strong promoter of Rous (LTR) (Cullen etc., molecule and cytobiology, 438-447 (in March, 1985)) adds the CMV-enhanser.Multiple clone site is as having restriction enzyme cutting site BamHI, XbaI and Asp718, the clone of promotion corresponding gene.Carrier contains 3 ' intron, polyadenylation and the termination signal of mouse proinsulin protogene in addition.
Embodiment 3 (a): clone and expression in the COS cell
Expression plasmid pNeutrokine-α-HA is by the part with the cDNA of the preservation of the extracellular domain of coded protein, is cloned among expression vector pcDNAI/Amp or the pcDNAIII (it can derive from Invitrogen company) and produces.For producing the polypeptide of soluble secreted form, extracellular domain is blended in the secretion leader sequence of people IL-6 gene.
Expression vector pcDNAI/Amp contains: the intestinal bacteria replication orgin that effectively breed in intestinal bacteria and other prokaryotic cell prokaryocyte (1); (2) selection contains the ampicillin resistance gene of the prokaryotic cell prokaryocyte of plasmid; The SV40 replication orgin of (3) in eukaryotic cell, breeding; (4) CMV promotor, polylinker, SV40 intron; (5) some codons of coding hemagglutinin fragment (promptly promoting the HA mark of purifying), after connect terminator codon and polyadenylation signal, cDNA can place under the expression control of CMV promotor easily thus, and operably is connected in SV40 intron and polyadenylation signal by the restriction site in the polylinker.This HA mark is equivalent to the epi-position derived from influenza hemagglutinin protein, and as Wilson etc., cell 37:767 (1984) is described.The fusion of HA mark and target protein makes the recombinant protein that is easy to detect and reclaim the antibody with identification HA epi-position.In addition, pcDNAIII contains selectable neomycin marker.
The dna fragmentation of the extracellular domain of coding Neutrokine-α polypeptide is cloned into the polylinker zone of carrier, expresses so that recombinant protein is instructed by the CMV promotor.The plasmid construction strategy is as follows.With preservation clone's Neutrokine-α cDNA, with the primer amplification that contains conventional restriction site, most as above in intestinal bacteria as described in the method for the carrier of construction expression Neutrokine-α.Suitable primer comprise following be used for this embodiment primer.The BamHI site of containing underscore, the Kozak sequence, AUG initiator codon, the sequence of the secretion leading peptide of coding people IL-6 gene, 5 ' primer with 18 Nucleotide of 5 ' encoding sequence of Neutrokine-α albumen extracellular domain has following sequence: 5 '-GCG GGA TCCGCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTCGGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTGCCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GACCTG GCC AGC-3 ' (SEQ ID NO:16).Contain the BamHI restriction site of underscore and be complementary to 3 ' primer of 18 Nucleotide of 3 ' encoding sequence before terminator codon, have following sequence: 5 '-GTG GGA TCCTTA CAG CAG TTTCAA TGC ACC-3 ' (SEQ ID NO:17).
With the dna fragmentation and the carrier pcDNAI/Amp of pcr amplification, connect then with BamHI digestion.This is connected mixture be transformed into coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and this is transformed culture be plated on the ampicillin medium, this flat board of incubation makes the amicillin resistance colony growth then.Isolated plasmid dna from the resistance bacterium colony, and have situation by the fragment that restriction analysis or other method detect coding Neutrokine-α extracellular domain.
Be express recombinant Neutrokine-α, with the COS cell with as above-mentioned expression vector, use DEAE-DEXTRAN to carry out transfection, Sambrook etc. for example, the molecular cloning laboratory manual, press of cold spring harbor laboratory, the cold spring port, New York (1989) are described.Cell is being passed through incubation under the condition of vector expression Neutrokine-α.
Neutrokine-α-HA Expression of Fusion Protein detects by radio-labeled and immunoprecipitation, uses as Harlow etc., antibody laboratory manual, second edition; Press of cold spring harbor laboratory, cold spring port, the described method in New York (1988).In transfection two days later, with cell by containing 35Incubation 8 hours in the substratum of S-halfcystine and mark.Collecting cell and substratum are with RIPA damping fluid flushing that contains stain remover and lysing cell: 150mMNaCl, 1%NP-40,0.1%SDS, 1%NP-40,0.5%DOC, 50mM TRIS, pH7.5, as described in above-mentioned Wilson etc.From cell lysate and substratum, use HA monoclonal antibody specific precipitating proteins.Then with sedimentary protein by SDS-PAGE and radioautographic analysis.The expression product of expectation size sees in the cell lysate, does not see in negative control.
Embodiment 3 (b): clone and expression in Chinese hamster ovary celI
Carrier pC4 is used to express Neutrokine-α albumen.Plasmid pC4 is the derivative of plasmid pSV2-dhfr (ATCC preserving number No:37146).For producing the polypeptide of soluble secreted form, extracellular domain is blended in the secretion leader sequence of people IL-6 gene.Vector plasmid contains the mouse DHFR gene under the control of SV40 early promoter.With the active Chinese hamster ovary of disappearance dihydrofolic acid or other cell of these plasmid transfections, can by with cell the selective medium of adding the chemotherapeutics methotrexate (α deducts MEM, LifeTechnologies) in the growth and selected.The amplification of DHFR gene in the cell that methotrexate (MTX) is had resistance fully confirms (to see for example Alt, F.W.Kellems, R.M.Bertino, J.R. and Schimke, R.T.1978, journal of biological chemistry 253:1357-1370, Hamlin, J.L. and Ma, C.1990, biological chemistry and biological physiology journal 1097:107-143, Page, M.J. and Sydenham, M.A.1991, biotechnology 9:64-68).Be grown in the cell among the MTX that concentration increases, produce directed enzyme DHFR, DHFR gene amplification as a result and medicine is produced resistance by excessive.If another kind of gene is connected in the DHFR gene, it is expressed with crossing by coamplification usually.This method known in the art can be used for producing the clone that copies more than 1000 of the gene that carries amplification.Then, after methotrexate is removed, obtain to contain the clone of the gene that is integrated into the amplification in one or more karyomit(e) of host cell.
Plasmid pC4 contains the strong promoter (Cullen etc. of the sarcoma viral length of the Rouse that expresses corresponding gene terminal repetition (LTR), molecule and cytobiology, in March, 1985: 438-447), add the fragment (Boshart etc., cell 41:521-530 (1985)) of separation from the enhanser of the immediate early gene of human cytomegalic inclusion disease virus (CMV).The downstream of promotor is restriction enzyme cutting site a: BamHI of following permission gene integration, XbaI, and Asp718.After these cloning sites, plasmid contains the 3 ' intron and the polyadenylation site of mouse proinsulin protogene.Other efficient promoter also can be used for expressing, people's beta-actin promotor for example, and the early stage or late promoter of SV40, or the length of other retrovirus such as HIV and HTLVI is terminal repetition.Clontech ' s Tet-Off and Tet-On gene expression system and similar system are used in the mammalian cell and express Neutrokine-α (Gossen, M.﹠amp in the mode of regulating; Bujard, H.1992, institute of American Academy of Sciences reports 89:5547-5551).Be other signal of polyadenylation mRNA, but for example end user's tethelin or globulin gene.Carry the stable cell lines that is integrated into chromosomal corresponding gene, also can select on the basis of G418 or Totomycin cotransfection with selectable mark such as gpt.When beginning, use more than one mark to add that as G418 methotrexate is favourable.
Plasmid pC4 with restriction enzyme BamHI digestion, is passed through the means known in the art dephosphorylation with the calf intestinal Phosphoric acid esterase then.Carrier of separating from 1% sepharose then.
The dna sequence dna of coding Neutrokine-α albumen extracellular domain is to use the PCR Oligonucleolide primers of 5 ' and the 3 ' sequence that is equivalent to gene to increase.The BamHI site of containing underscore, the Kozak sequence, AUG initiator codon, the sequence of the secretion leading peptide of coding people IL-6 gene, 5 ' primer with 18 Nucleotide of 5 ' encoding sequence of Neutrokine-α albumen extracellular domain has following sequence: 5 '-GCG GGA TCCGCC ACC ATGAAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTCTCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCTGCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3 ' (SEQID NO:16).Contain the BamHI restriction site of underscore and be complementary to 3 ' primer of 18 Nucleotide of 3 ' encoding sequence before terminator codon, have following sequence: 5 '-GTG GGA TCCTTA CAG CAG TTT CAA TGC ACC-3 ' SEQ IDNO:17).
The fragment of amplification digests with endonuclease BamHI, repurity on 1% sepharose then.This isolating fragment is connected with the T4 dna ligase then with dephosphorylized carrier.Transformed into escherichia coli HB101 or XL-1 Blue cell then, and differentiate and contain the segmental bacterium that inserts plasmid pC4, for example differentiate with the restriction enzyme analysis method.
The Chinese hamster ovary cell that lacks active DHFR gene is used for transfection.The expression plasmid pC4 of 5 μ g and the plasmid pSVneo of 0.5 μ g are dyed method cotransfection (Felgner etc. are as preceding) with fat.Plasmid pSV2-neo contains a dominant selectable marker, and the enzyme that coding is given antibiotics resistance comprises the neo gene of the Tn5 of G418.Cell seeding is subtracted in the MEM substratum in the α that adds 1mg/ml G418.After 2 days,, plant in adding 10,25, or the α of 50ng/ml methotrexate and 1mg/ml G418 subtracts among the hybridoma clone dull and stereotyped (Greiner, Germany) in the MEM substratum the cell tryptic digestion.Approximately after 10-14 days, with each clone of tryptic digestion, and with the methotrexate of different concns (50,100,200,400,800Nm) plant in 6 hole culture dish or 10ml culturing bottle.The clone that will grow in the maximum concentration methotrexate moves in the new 6 hole flat boards that contain greater concn methotrexate (1,2,5,10,20 μ M) then.Repeat identical step until the clone who obtains in the growth of 100-200 μ M concentration.By for example SDS-PAGE and Western trace or analyze the expression of required gene product by reverse hplc.
The inventor has produced at least 6 Neutrokine-alpha expression constructs, produces Neutrokine-α and/or Neutrokine-α SV polypeptide in some systems to promote reaching of different size.This expression construct is as follows: (1) pNa.A71-L285 (expressing Ala71-Leu285 amino acids residue), (2) pNa.A81-L285 (expressing Ala81-Leu285 amino acids residue), (3) pNa.L112-L285 (expressing Leu112-Leu285 amino acids residue), (4) pNa.A134-L285 (expressing Ala134-Leu285 amino acids residue), (5) pNa.L147-L285 (expressing Leu147-Leu285 amino acids residue), (6) pNa.G161-L285 (expressing Gly161-Leu285 amino acids residue).
In preferred embodiments, expression construct is used to express bacterium, the mutain of the various Neutrokine-α of baculovirus and mammlian system.
In other embodiment preferred, this construct is expressed in the Neutrokine-α polypeptide fragment that N or C-terminal are blended in heterologous polypeptide, heterologous polypeptide for example is the signal peptide of people IL-6, the signal peptide of CK-β 8 (-21 to-1 amino acids of CK-β 8 sequences that PCT publication PCT/US95/09058 is disclosed), or human IgG Fc district.Known other the operable sequence of those skilled in the art.
The tissue distribution that embodiment 4:Neutrokine-α mRNA expresses
Carry out the Northern engram analysis to detect Neutrokine-α expression of gene in tissue, use as method as described in the Sambrook etc.The cDNA probe that contains Neutrokine-α albumen (SEQID NO:1) complete nucleotide sequence uses rediprime TMDna marker system (Amersham Life Science) uses according to the producer's guidance 32The P mark.After mark, with probe CHROMA SPIN-100 TMChromatography column (ClontechLaboratories company) is according to the producer's scheme PT1200-1 and purifying.Then the probe of the mark of purifying is used to detect the Neutrokine-α and/or the Neutrokine-α SV mRNA of various tissues.
What contain various tissues (H) or human immune system tissue (IM) organizes Northern (MTN) trace more, derives from Clontech, uses ExpressHyb TMHybridization solution (Clontech) is according to the producer's the scheme PT1190-1 probe in detecting with mark.After hybridization and flushing, trace spent the night at-70 ℃ to be exposed on the film, and film develops the color according to standard method.
For determining the expression pattern of Neutrokine-α and/or Neutrokine-α SV, detect organizing the Northern trace one group more.Show 2.6kb mRNA blood leukocytes around, spleen is obviously expressed in lymphoglandula and the marrow, at placenta, and heart, lung, fetus liver can detect expression in thymus gland and the pancreas.One group of clone is carried out analysis revealed, and Neutrokine-α and/or Neutrokine-α SV express at HL60 cell camber, can detect expression in K562, but at Raji, HeLa, or do not express in the MOLT-4 cell.All analyses show that all Neutrokine-α and/or Neutrokine-α SV mRNA are expressed in enrichment in the immunity system.
Embodiment 5: carry out gene therapy with endogenous Neutrokine-α gene
According to another kind of gene therapy of the present invention, comprise by homologous recombination endogenous Neutrokine-α sequence and promotor are operably united, as the US Patent No of authorizing on June 24th, 1997: 5641670; International open WO published on September 26th, 96/29411,1996; International open WO published on August 4th, 94/12650,1994; Koller etc., institute of American Academy of Sciences report 86:8932-8935 (1989); With Zijlstra etc., natural 342:435-438 (1989) is described.This method comprises activating and is present in the target cell, but do not express in cell or the gene of low expression level.Generation contains the polynucleotide constructs of promotor and targeting sequence, and it is with the 5 ' non-coding sequence that comes from the endogenous Neutrokine-α of promotor both sides.Targeting sequence is near the 5 ' end of Neutrokine-α, and promotor operably is connected with the endogenous sequence by homologous recombination thus.Promotor and targeting sequence can be used pcr amplification.Preferably, the promotor of amplification contains unique restriction enzyme sites at 5 ' and 3 ' end.Preferably, 3 ' end of first targeting sequence contains 5 ' terminal identical restriction enzyme sites with the promotor that increases, and 5 ' end of second targeting sequence contains 3 ' terminal identical restriction enzyme sites with the promotor that increases.
The promotor of amplification and the targeting sequence of amplification are handled with the calf intestinal Phosphoric acid esterase then with suitable restriction enzyme digestion.Under the situation that has the T4 dna ligase, the promotor of digestion and the targeting sequence of digestion are added together.The gained mixture is remained under the condition that is suitable for these two fragments connections.Construct is carried out fractional separation by size on sepharose, then by phenol extraction and ethanol sedimentation and purifying.
In this embodiment, polynucleotide constructs is used by electroporation as naked polynucleotide.Yet polynucleotide constructs also can be used with transfection promotor, as liposome, and virus sequence, virion, precipitation agent etc.This conveying known in the art.
In case cell is transfected, carry out homologous recombination, make promotor operably be connected in endogenous Neutrokine-α sequence.This expresses Neutrokine-α in cell.Expression can detect by immunostaining or any other method known in the art.
Inoblast derives from the subject's skin biopsy.The gained tissue is placed the DMEM+10% foetal calf serum.To use tryptic digestion by inoblast stationary phase exponential growth or early stage, and with nutritional medium with it under the plastic cement surface washing.Shift out a part of cell suspending liquid and count, remaining cell carries out centrifugal.The sucking-off supernatant, with granular pellet resuspended in 5ml electroporation damping fluid (20mM HEPES pH7.3,137mM NaCl, 5mM KCl, 0.7mM Na 2HPO 4, 6mM glucose) in.Cell is centrifugal again, and the sucking-off supernatant is resuspended to cell in the electroporation liquid that contains the acetylizad bovine serum albumin of 1mg/ml.Whole cell suspending liquid contains about 3 * 10 6Individual cell/ml.Electroporation should carry out after resuspension at once.
Prepare plasmid DNA according to standard method.For example, for structure is oriented to the plasmid of Neutrokine-α locus, (MBI Fermentans, Amherst NY) digests with HindIII with plasmid pUC18.The CMV promotor is made it to contain the XbaI site and contain the BamHI site at 3 ' end at 5 ' end by pcr amplification.By two Neutrokine-α of pcr amplification non-coding sequence: a Neutrokine-α non-coding sequence (Neutrokine-α fragment 1) amplification back contains the HindIII site and contains the XbaI site at 3 ' end at 5 ' end; Another Neutrokine-α non-coding sequence (Neutrokine-α fragment 2) amplification back contains the BamHI site and contains the HindIII site at 3 ' end at 5 ' end.(the CMV promotor is with XbaI and BamHI with suitable enzymic digestion for CMV promotor and Neutrokine-α fragment; Neutrokine-α fragment 1 is used XbaI; Neutrokine-α fragment 2 usefulness BamHI digest), and link together.Gained is connected product digest, and connect with the pUC18 plasmid of HindIII digestion with HindIII.
The plasmid DNA adding is had in the sterile test tube (Bio-Rad) of 0.4cm electrode gap.Whole DNA concentration is at least 120 μ g/ml.Cell suspending liquid with 0.5ml (contains about 1.5 * 10 then 6Individual cell) adds in the test tube, and cell suspending liquid and dna solution are slightly mixed.(Bio-Rad) carries out electroporation with the Gene-Pulser instrument.Electric capacity and voltage are made as 960 μ F and 250-300V respectively.Along with voltage improves, cell survival reduces, but the percentage that stably DNA that imports is mixed its genomic survivaling cell obviously improves.Given these parameters should observe burst length of about 14-20 millisecond.
The cell of electroporation was kept about 5 minutes in room temperature, and the content with test tube shifts out gently with aseptic transfer pipet then.Cell is directly added in the nutritional medium (DMEM) of the pre-temperature of 10ml in the 10cm plate with 15% calf serum, and at 37 ℃ of incubations.Second day, the sucking-off substratum was also used the displacement of 10ml fresh culture, incubation 16-24 hour again.
Then the genetically engineered inoblast is injected in the host, injects after being paved with cytodex 3 microcarrier beads separately or growing to.Inoblast produces protein now.This inoblast can be imported in the patient body, as mentioned above then.
Embodiment 6:Neutrokine-α, a kind of newcomer of the tumour necrosis factor ligand family as the bone-marrow-derived lymphocyte stimulator
In the cDNA library of people's neutrophilic granulocyte/monocyte derived, identify a kind of 285 amino acid whose protein, outside the born of the same parents that it is inferred in the receptor-ligand binding domains, with APRIL (28.7%) (Hahne, M. etc., experimental technique magazine 188,1185-90 (1998)), TNF-α (16.2%) (Pennica, D. etc., nature 312,724-729 (1984)) and LT-α (14.1%) (Gray, nature 312,721-724 (1984)) present obvious homology (Fig. 7 A).We are called Neutrokine-α (based on its biological activity, also being called bone-marrow-derived lymphocyte stimulator (BlyS)) with this cytokine.The hydrophobicity analysis of Neutrokine-α protein sequence shows, before a potential membrane spaning domain between the 47-73 amino acids residue is positioned at non-hydrophobic amino acid, point out the same with other tnf ligand family, Neutrokine-α is a kind of II type embrane-associated protein (Cosman, D. stem cell 12:440-55 (1994)).152 the amino acid whose soluble forms (Fig. 7 A) that had from the N-terminal sequence of the 134th L-Ala are differentiated in the expression of this cDNA in mammalian cell (HEK293 and Chinese hamster ovary cell) and Sf9 insect cell.To rebuilding of quality and electric charge ratio, the molecular weight of determining Neutrokine-α is 17038 Daltons, and this numerical value is consistent with these 152 the amino acid whose proteinic molecular weight (17037.5 Daltons) with a disulfide linkage of inferring.
End user/hamster somatic cell hybrid and the mapping of radiation hybrid, the gene and the mark SHGC-36171 that find coding Neutrokine-α are chain, it is positioned human chromosome 13q34, the incoherent zone of any other member (Cosman, D. stem cell 12:440-55 (1994)) with the TNF superfamily of gene.
The expression pattern of Neutrokine-α is determined by Northern trace (Fig. 7 B) and fluidic cell quantitative analysis (table 5 and Fig. 8).Neutrokine-α finds its blood leukocytes around, spleen, high level expression in lymphoglandula and the marrow by the mRNA coding of a 2.6kb.At placenta, heart, lung, fetus liver detects low expression level in thymus gland and the pancreas.In this group clone, in HL-60 and K562, detect Neutrokine-α mRNA, but at Raji, HeLa, or do not detect in the MOLT-4 cell.These results use Neutrokine-alpha specific mAb 2E5 by fluidic cell quantitative analysis susceptible of proof.As shown in Figure 5, the Neutrokine-alpha expression does not detect on T or B clone, but is limited to the cell of marrow origin.To nearly step analysis revealed of normal plasma cell, on the immobilized monocyte, obviously express, with cellular exposure after IFN-γ (100U/ml) is following 3 days, just regulating about 4 times (Fig. 8 A).What also detect Neutrokine-alpha specific mRNA follows increase (Fig. 8 B).On the contrary, Neutrokine-α is at new isolating blood granulocyte on every side, and the T cell is not expressed in B cell or the NK cell.
The reorganization Neutrokine-α (rNeutrokine-α) that determines purifying is in various analyses based on cell, and the inducing cell activation is bred, differentiation or dead ability, described cell comprises the B cell, T cell, monocyte, the NK cell, the cell in hemopoietic progenitor cell and various endothelium and epithelium source.In these are analyzed, the special Neutrokine-of discovery α stimulates in the analysis in standard association and improves B cell proliferation, the tonsilla B cell of purifying wherein, cultivate (Sieckmann at the streptococcus aureus Cowan I (SAC) that has formalin fixed or the anti-people IgM of fixed under as the situation of initiator, D.G. etc., experimental technique magazine 147:814-29 (1978); Ringden, O. etc., Scand.J.Immunol.6:1159-69 (1977)).Shown in Fig. 9 A, reorganization Neutrokine-α induces tonsilla B cell dosage dependency propagation.This replys and is similar to rIL2 replying at the 0.1-10000ng/ml dosage range.When cultivating with the common irritation cell of the anti-IgM antibody of fixed, Neutrokine-α also induces B cell proliferation (Fig. 9 B).Under the situation of IL2 that has fixed concentration or rNeutrokine-α, be easy to observe dosage along with the increase of linking agent quantity and rely on replying of form.
In the trial that will connect the special biological activity and the expression of receptor of B cell, the Neutrokine-α of purifying is biotinylated.In the standard B cell proliferation was analyzed, the biotinylated Neutrokine-α of gained kept biological function.The pedigree specificity analyses of complete blood cell shows around the human body, and biotinylated Neutrokine-α is combined in the T cell, and monocyte does not detect on NK cell and the granulocyte, and this is respectively by CD3, CD14, and CD56 and CD66b determine (Figure 10 A).On the contrary, blood CD20 around the biotinylated Neutrokine-α combination +The B cell.At the B cell tumour is REH, ARH-77, and Raji, Namalwa also detects expression of receptor among RPMI8226 and the IM-9, but comprises THP-1 in the clone of the bone marrow derived of any test, and HL-60 does not detect among K-562 and the U-937.The representative fluidic cell metering pattern of myeloma cell line IM-9 and tissue lines U-937 is shown in Figure 10 B.Neutrokine-α albumen with bioactive FLAG mark replaces the biotinylated Neutrokine-α of chemically modified can obtain analog result.These results confirm that Neutrokine-α all shows tangible B cytotropism in its acceptor distribution and biological activity.Whether these results also illustrate cell activation can induce Neutrokine-α acceptor hemocyte around, expresses in the clone of other normal cell or foundation.
For the species specificity of test Neutrokine-α, mice spleen B cell is cultivated under the condition that has people Neutrokine-α and SAC.The result shows that rNeutrokine-α induces mouse spleen B cell at in-vitro multiplication, and is combined in cell surface receptor on these cells.Interesting is, separates the negative B cell precursor from the jejune surperficial Ig of mouse marrow, neither replys Neutrokine-α and breeds, also the debond part.
For determining the activity in vivo of rNeutrokine-α, with BALB/c mouse (3/group) injection every day (i.p.) twice damping fluid, or 0.08mg/kg, 0.8mg/kg, the rNeutrokine-α of 2mg/kg or 8mg/kg.Mouse was carried out this processing in continuous 4 days, put to death afterwards, collect various tissues and serum and analyze.In another embodiment, BALB/c mouse is injected the rNeutrokine-α of (i.p.) twice any dosage in the 0.01-10mg/kg scope every day.In an embodiment preferred, (particularly preferred in this embodiment dosage comprises but the non-0.01mg/kg of being limited to, 0.02mg/kg with the rNeutrokine-α of injection BALB/c mouse every day (i.p.) twice any dosage in the 0.01-3mg/kg scope, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg, 2.1mg/kg, 2.2mg/kg, 2.3mg/kg, 2.4mg/kg, 2.5mg/kg, 2.6mg/kg, 2.7mg/kg, 2.8mg/kg, 2.9mg/kg, and 3.0mg/kg).In another embodiment preferred, (particularly preferred in this embodiment dosage comprises but the non-0.02mg/kg of being limited to, 0.03mg/kg with the rNeutrokine-α of injection BALB/c mouse every day (i.p.) twice any dosage in the 0.02-2mg/kg scope, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/g, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/g, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg).
Use the effect of Neutrokine-α, microscopically with HE painted and with the spleen tissue slice of the mAb immunohistochemical staining that is specific to CD45R (B220) in very obviously (Figure 11 A).Normal spleen structure changes, and obviously extend the white matter marginarium, and the cellularstructure of red pulp obviously increases (Figure 11 A).Extend the marginarium is because due to the lymphocyte quantity increase of expression B cell marking CD45R (B220).In addition, profit is also invaded by proper C D45R (B220) positive cell in the periarterial lymphatic sheath that the T cell is intensive (PALS) zone.It is because due to the increase of B cell quantity that this prompting white matter changes.Usually filling in red pulp intensive tytosis group do not dyeed by CD45R (B220).Need carry out other test with in addition qualitative, and further determine 0.08mg/kg all types cell that comprised, 0.8mg/kg, 2mg/kg changes the mechanism of spleen structure.
Show that to taking from the fluidic cell quantitative analysis that the mouse boosting tissue that handles with 2mg/kg Neutrokine-α carries out compare with the contrast mouse, Neutrokine-α improves ripe (CD45R (B220) Dull, ThB Bright) nearly 10 times of B cell proportion (Figure 11 B).The wherein mouse that carries out in addition is to use damping fluid, 0.08mg/kg, 0.8mg/kg, 2mg/kg, or the analysis revealed of the Neutrokine-α of 8mg/kg processing compare with the contrast mouse, 0.08mg/kg 0.8mg/kg, the Neutrokine-α of 2mg/kg all improve maturation (CD45R (B220) Dull, ThB Bright) nearly 10 times of B cell proportion, and the Neutrokine-α of damping fluid and 8mg/kg produces the mature B cell of about same ratio.See Table 4.
Table 4: the facs analysis of mice spleen B cell mass
Neutrokine-α(mg/kg) Mature B cell percentage (R2) CD45R positive percentage (R1)
Contrast (damping fluid) 0.08mg/kg 0.8mg/kg 2mg/kg 8mg/kg 1.26 16.15 18.54 16.54 1.24 52.17 56.53 57.56 57.55 61.42
The potential result that cylinder mature B cell expressivity increases is that serum I g titration increases relatively.Therefore, serum IgA between the mouse that contrast damping fluid and Neutrokine-α handle, IgG and IgM level (Figure 11 C).Use Neutrokine-α and make that IgA and IgM level increase by 2 and 5 times respectively in the serum.In addition the people gazes at is that the cyclical level of IgG does not increase.
In addition, in handle 4 days the serum IgA titration of mouse with the Neutrokine-α of various quantity, observe dose-dependently and reply, and the Neutrokine-α that uses same amount handles and do not observe obvious dose-dependently in 2 days.Using under 4 days the situation, use 8,2,0.8,0.08 and the Neutrokine-α of 0mg/kg due to the serum IgA titration be approximately 800 μ g/ml, 700 μ g/ml, 400 μ g/ml, 200 μ g/ml and 200 μ g/ml.Promptly use 8,2,0.8 and the Neutrokine-α of 0.08mg/kg after 4 days, compare with only using background or the basic I gA serum level that damping fluid observes, the IgA serum level improves about 4,3.75,2 and minimum multiple respectively.In other embodiments, these tests can be used on the interior any amount of rNeutrokine-α of 0.01-10mg/kg scope and carry out.In an embodiment preferred, (in this embodiment, particularly preferred dosage for example comprises but the non-0.01mg/kg of being limited to, 0.02mg/kg to use the interior Neutrokine-α of 0.01-3mg/kg scope, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg, 2.1mg/kg, 2.2mg/kg, 2.3mg/kg, 2.4mg/kg, 2.5mg/kg, 2.6mg/kg, 2.7mg/kg, 2.8mg/kg, 2.9mg/kg, 3.0mg/kg).In another embodiment preferred, (in this embodiment, particularly preferred dosage for example comprises but the non-0.02mg/kg of being limited to use the interior Neutrokine-α of 0.02-2mg/kg scope, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg).
Argument shown in this paper is illustrated the newcomer that Neutrokine-α is a TNF-part superfamily, and it all induces B cell proliferation and differentiation with external in vivo.By its monocyte specific gene/protein expression pattern, distribute and biological activity with its specific receptors to bone-marrow-derived lymphocyte, Neutrokine-α is different from other B cell growth and differentiation factor such as IL2 (Metzger, D.W. etc., immunology research 146:499-505 (1995)), IL4 (Armitage, R.J. etc., biological experimental method progress 292:121-30 (1991); Yokota, T. etc., institute of American Academy of Sciences reports 83:5894-98 (1986)); (institute of American Academy of Sciences reports 84:4234-38 (1987) to IL5 for Takatsu, K. etc.; Bertolini, J.N. etc., European Journal of Immunology 23:398-402 (1993)), IL6 (Poupart, P. etc., EMBO magazine 6:1219-24 (1987); Hirano, T, natural 324:73-76 (1986)), (institute of American Academy of Sciences reports 86:302-06 (1989) to IL7 for Goodwin, R.G. etc.; Namen, A.E. etc., natural 333:571-73 (1988)), IL13 (Punnonen, J. etc., transformation reactions 49:576-86 (1994)), IL15 (Armitage, R.J. etc., Journal of Immunology 154:483-90 (1995)), CD40L (Armitage, R.J. etc., natural 357:80-82 (1992); Van Kooten, C. and Banchereau, J.Int, Arch.Allergy.Immunol113:393-99 (1997)) or CD27L (CD70) (Oshima, H. etc., Int.Immunol.10:517-26 (1998); Lens, S.M. etc., Semin.Immunol.10:517-26 (1998)).These arguments prompting Neutrokine-α is contained in the handshaking between the filial generation of B cell and monocyte or their differentiation.Although all B cells can utilize this signal mode, the expression pattern of this restriction and Ig secretion prompting Neutrokine-α are activating CD5 +Or the effect in " unconventional " B cell response.These B cells provide inborn immune important component, and provide the pathogenic agent of resisting in the environment (Pennell, C.A. etc., European Journal of Immunology 19:1289-95 (1989) by the IgM and the IgA antibody of its secretion multiple reactionness; Hayakawa, K. etc., institute of American Academy of Sciences reports 81:2494-98 (1984)).Perhaps, Neutrokine-α can be used as the conditioning agent that T cell dependent/non-dependent is replied, and this replys response mode identical (Eertwegh, A.J. etc., the experimental technique magazine 178:1555-65 (1993) in the activation of T cell dependence antigen with CD40 and CD40L; Grabstein, K.H. etc., Journal of Immunology 150:3141-47 (1993)).Like this, Neutrokine-α, its acceptor or relevant antagonist are used for the treatment of and autoimmunity, the B cell disease that tumorigenesis and/or immune deficiency syndromes are relevant.
Method
Mouse: BALB/cAnNCR (6-8 week) is available from Charles River laboratory, and according to the standard (National Research Council of recommending, instruct (1999) with the use laboratory animal), at the septulum of paper washer with recycling from cage (Harlan Sprague Dawley company, Indianapolis, IN) raise in, and coccoid rodent food (Harlan SpragueDawley company) is provided, and arbitrarily place bottled drinking water in the bottom.The animal scheme of in this research, using be with reference to and check and approve through the HGS system of the animal rearing and the council of use.
The cDNA that separates total length Neutrokine-α: use blast program to detect Human Genome Sciences Inc's expressed sequence mark (EST) database, seek receptors bind structural domain homologous sequence with TNF family.Total length Neutrokine-α clone is differentiated, check order and be committed to gene pool (registration number AF132600).Neutrokine-α open reading frame utilizes 5 ' primer and 3 ' primer through pcr amplification, 5 ' primer (5 '-CAGACT GGA TCC GCC ACC ATG GAT GAC TCC ACA GAA AG-3 ') is in predetermined initiator codon annealing, and 3 ' primer (5 '-CAG ACT GGT ACC GTCCTG CGT GCA CTA CAT GGC-3 ') is in predetermined downstream terminator codon annealing.Gained amplicon BamHI and Asp718 restriction site tailing, and subclone is gone in the mammalian expression vector.Neutrokine-α also expresses in p-CMV-1 (Sigma Chemicals).
The purifying of recombinant human Neutrokine-α: the full-length cDNA subclone of the Neutrokine-α that will encode is gone among the rhabdovirus expression vector pA2, and is transfected into (Pater.V.P. etc., expression method magazine 185:1163-72 (1997)) in the Sf9 insect cell.The cell conditioned medium of reorganization Neutrokine-α purifying self-infection after 92 hours is used in combination anionresin, methods such as size exclusion and hydrophobic interaction chromatography.The protein of purifying is containing 0.15M NaCl, and 50mM NaOAc prepares in the damping fluid of pH6, sterile filtration and be stored in 4 ℃ until take.SDS-PAGE and RP-HPLC analyze and show that all rNeutrokine-α is 95% above purifying.Level of endotoxin be lower than detection limit value in LAL analyzes (Associatesof Cape Cod, Falmouth, MA).The sequence of the proteic N-terminal of Neutrokine-α of final purifying is Ala-Val-Gln-Gly-Pro.This is equivalent to the sequence derived from the soluble Neutrokine-α of the Chinese hamster ovary celI system that uses the transfection of total length Neutrokine-α stable gene.
Produce monoclonal antibody:, then in incomplete Freund ' s adjuvant, attack 2 times with of the Neutrokine-alpha immunization inoculation of BALB/cAnNCR mouse with the His mark of the 50 μ g that are suspended in complete Freund ' s adjuvant.As state the preparation hybridoma and singly restrain flight of steps leading to a palace hall antibody (Gefter, M.L. etc., Somatic.Cbll Genet.3:231-36 (1977); Akerstrom, B. etc., Journal of Immunology 135:2589-92 (1985)).
Clone: everyone clone available from ATCC (American type culture collection, the Manassas, VA).
Facs analysis: the Neutrokine-alpha expression is the human cell line, new isolating normal blood karyocyte on every side, monocyte with vitro culture, mouse anti human Neutrokine-α mAb 2E5 (IgG1), the F that puts together with the PE of mouse IgG (ab ') 2 goat antibodies (CALTAG laboratory, Burlingame CA) goes up assessment.(CA) analysis of cells is got rid of dead cell with iodate third ingot for BectonDickinson immunocyte metering system, San Jose with FACScan.Neutrokine-α in conjunction with use through N-hydroxy-succinamide vitamin H reagent (Pierce, Rockford, IL) and the streptavidin puted together of PE (Denmark) biotinylated rNeutrokine-α determines for Dako Corp, Glostrup.
Chromosome mapping: for determining the chromosome position of Neutrokine-α gene, one group of monosomic somatic cell hybrid (Quantum Biotechnology of individual chromosome will be kept, Canada), with Neutrokine-α special primer by PCR screening (5 ' primer: 5 '-TGG TGT CTT TCT ACC AGG TGG-3 '), 3 ' primer: 5 '-TTTCTT CTG GAC CCT GAA CGG-3 ').The PCR product of the 233bp that infers only detects in human chromosome 13 hybrids.Use one group of 83 radiation hybrid (ResearchGenetics, St.Louis, MO) and the stanford human genome center database ( Http:// www.shgc.stanford.edu.RH/rhserver).Find that the SHGC-36171 mark on Neutrokine-α and the karyomit(e) 13 is chain.Cell generation figure with human chromosome 13 determines that to this figure overdetermination position people Neutrokine-α is on chromosome band 13q34.
The bone-marrow-derived lymphocyte proliferation assay: human tonsil B cell is by exhausting the CD3 positive cell and purifying with magnetic bead (MACS).The gained cell mass determines that by the expression of CD19 and CD20 it is the B cell more than 95%.With people rNeutrokine-α or the various dilutions of reference protein recombinant human IL2 each hole of placing 96 hole flat boards, be suspended in 10 in the nutrient solution to wherein adding 5Individual B cell, cumulative volume are 150 μ l, and used nutrient solution is for containing 10%FBS, 5 * 10 -5The 2ME of M, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and 10 -5The RPMI1640 of Pansorbin (SAC) or anti-IgM antibody.After the above-mentioned factor of adding 72 hours, by 3Pulses in 20 hours of H-thymidine (6.7Ci/Mm) (1 μ Ci/ hole) are carried out quantitatively propagation.
Histologic analysis: spleen is organized in the formalin of 10% neutral buffered fixing, be embedded in the paraffin wax, be cut into the section of 5 μ m, place on the slide glass, and with HE dyeing or indirect immunohistochemical method dyeing (Hilbert, the European Journal of Immunology 23:2412-18 (1993) of D.M.) by CD45R (B220) being carried out enzyme labelling.
The cell surface expression of table 5:Neutrokine-α
Clone Morphocytology Neutrokine--α cell surface expression
Monocytic series U-937 BL-60 K-562 THP-1 Lymphoma, histocyte/scavenger cell leukemia, acute cellulous leukemia of bone marrow, chronic cellulous leukemia of bone marrow, acute monocytic + + + +
T clone Jurkat SUP-T13 MOLT-4 Leukemia, T Lymphocytic leukemia, T lymphoblastic leukemia, T lymphoblast - - -
B clone Daudi Namalwa Raji Reh ARH-77 IM9 RPMI 8226 Burkitt ' s, lymphoblast Burkitt ' s, lymphocyte Burkitt ' s, Lymphocytic leukemia, Lymphocytic leukemia, plasma cell myelomatosis myelomatosis - - - - - - -
Embodiment 7: detect the analysis that stimulates or suppress B cell proliferation and differentiation
The generation of functional humoral immunoresponse(HI) requires solvable with the related signal between B clone and microenvironment thereof.Signal can give a positive stimulus, makes the B cell line cell continue its program and grows, or give negative a stimulation, instructs cell to suppress its current development pathway.Have now found that many stimulations and suppress effect of signals B cell response, comprise IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL14 and IL15.Interesting is, these signals be by self faint effector but can with the combination of various stimulatory protein(SP) altogether, inducing the activation of B cell mass, propagation, differentiation is gone back to the nest, tolerance and dead.One of best B cell co-stimulatory albumen is the TNF superfamily.Found the CD40 in this family, CD27 and CD30 and part CD154 separately thereof, CD70 and CD153 regulate various immunne responses.Detecting and/or observe the propagation of these B cell masses and precursor thereof and the analysis of differentiation, is very useful what determine that range protein can be to these B cell masses between propagation and differentiation phase on.What below list is the differentiation that detects B cell mass and precursor thereof, propagation or two kinds of analyses that suppress.
Analyzed in vitro: determine Neutrokine-α and/or Neutrokine-α SV albumen or its clipped form of purifying, induce the activation of B cell mass and precursor thereof, propagation, differentiation or inhibition and/or dead ability.Neutrokine-α and/or Neutrokine-α SV albumen are to the effect of the human tonsil B cell of purifying, quantitative assay in the 0.1-10000ng/ml dosage range, in stimulating altogether, the standard bone-marrow-derived lymphocyte determines, wherein there is the streptococcus aureus Cowan I (SAC) of formalin fixed in the tonsilla B cell of purifying, or cultivates under the condition of the anti-human IgM antibody of fixed as initiator.Collaborative SAC of second signal such as IL-2 and IL-15 and IgM be crosslinked to excite B cell proliferation to contain the tritium thymidine and measure by mixing.New synergist uses this analysis can be easy to differentiate.This analysis comprises by magnetic bead (MACS) exhausts the CD3 positive cell and separation of human tonsilla B cell.The gained cell mass determines more than 95% it is the B cell by the expression of CD45R (B220).The various dilutions of each sample are placed each holes of 96 hole flat boards, be suspended in 10 in the nutrient solution to wherein adding 5Individual B cell, cumulative volume are 150 μ l, and used nutrient solution is for containing 10%FBS, 5 * 10 -5The 2ME of M, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and 10 -5The RPMI 1640 of Pansorbin (SAC).After the above-mentioned factor of adding 72 hours, by 3Pulses in 20 hours of H-thymidine (6.7Ci/Mm) (1 μ Ci/ hole) are carried out quantitatively propagation.Positive and negative control group is respectively IL2 and substratum.
Agonist (comprising Neutrokine-α and/or Neutrokine-α SV polypeptide fragment) proves that when the result who is observed compared, B cell proliferation increased when contacting with the initiator of same concentrations with the B cell of similar number.Antagonist according to the present invention presents B cell proliferation when comparing with control group reduces, control group contains the B cell of similar number, the initiator of same concentrations, with the same concentrations soluble form excite the active Neutrokine-α that improves of the B cell proliferation (71-285 of the polypeptide of Neutrokine-α shown in the SEQ ID NO:2 for example, 81-285,112-285 or 134-285 amino acids), there is not antagonist.
The body inner analysis: with BALB/c mouse injection every day (i.p.) twice damping fluid, or the Neutrokine-α of 2mg/kg and/or Neutrokine-α SV albumen, or its clipped form.Mouse was accepted this processing in continuous 4 days, put to death then, and collect various tissues and serum to analyze.Contrast the spleen tissue that normal HE tissue slice and Neutrokine-α and/or Neutrokine-α SV albumen are handled, differentiate Neutrokine-α and/or Neutrokine-α SV albumen result to the effect of splenocyte, diffusion as periarterial lymphatic sheath, and/or the karyoblast structure in red pulp district obviously increases, and can show the activation of differentiation of B cell mass and propagation.Use the B cell marking, the immunohistochemistry research of anti-CD45R (B220), whether be used for any physiological change such as the change of spleen weave construction of definite splenocyte, be owing to invade due to the interior B cell expressivity raising of the loose definite B cellular regions band that moistens the T cellular regions of setting up.
The fluidic cell quantitative analysis that the spleen tissue of the mouse of handling through Neutrokine-α and/or Neutrokine-α SV albumen is carried out, be used to show with the contrast mouse and compare, the raising ThB+ whether Neutrokine-α and/or Neutrokine-α SV be special, the ratio of the empty B cell of CD45R (B220).
In addition, the result that infers of mature B cell expressivity raising improves in serum I g titration relatively in vivo.Therefore, serum IgM and IgA level between the mouse of contrast damping fluid and Neutrokine-α and/or Neutrokine-α SV albumen processing.
Embodiment 8:Neutrokine-α and agonist thereof the treatment graft to relevant mouse lymph atrophy of versus-host disease and the effect in the underdevelopment
Use Neutrokine-α treatment, prevention and/or mouse lymph atrophy and the undergrown analysis relevant to versus-host disease (GVHD) of diagnosis graft are undertaken by using the C57BL/6 parental generation to migrate in (BALB/c X C57BL/6) F1 (CBF1) mouse model.This parental generation that enters the F1 mouse model is fully qualitatively, is the renewable animal model of GVHD in the bone marrow transplantation patient, this be well known to those skilled in the art (seeing Gleichemann etc., modern immunology 5:324,1984).Bone-marrow-derived lymphocyte propagation and differentiation are induced in soluble Neutrokine-α expection, and are corrected in lymph atrophy and underdevelopment (Piguet etc., the J.Exp.Med.166:1280 (1987) that observes in the animal model of GVHD; Hattori etc., blood 90:542 (1997)).
It is by with about 1-5 * 10 that GVHD begins 8The splenocyte of individual C57BL/6, through intravenous injection go in (BALB/c X C57BL/6) F1 mouse (all available from Jackson Lab, Bar Harbor, Maine).Behind the injection parental cell, when lymph atrophy and underdevelopment are slight (about 5 days), when moderate (about 12 days) or severe (about 20 days), begin the Neutrokine-α of intramuscular or intradermal injection 0.1-5.0mg/kg or contrast damping fluid with 6-8 mouse intraperitoneal every day.Neutrokine-α is to the lymph atrophy and the undergrown effect of spleen, and a plurality of time points (3-4) between 10-30 days are by FACS and histopathological analysis.In brief, from normal CBF1, GVHD, or prepare splenocyte in the mouse of Neutrokine-α processing, and with the anti-H-2Kb antibody that the fluorescein phycoerythrin is puted together, the anti-H-2Kd antibody of biotin-conjugated, the anti-CD 4 antibodies of puting together with FITC-, anti-CD8 antibody, or anti-B220 antibody, then the avidin dyeing of puting together with CyChrome.All these antibody all can available from PharMingen (San Diego, CA).(Becton Dickinson, San Jose, CA) analysis of cells on FACScan then.Acceptor and donor lymphocyte are differentiated respectively and are H-2Kb+Kd+ and H-2Kb+Kd-cell.The CD4+T of acceptor or donor, the number of CD8+T and B220+B cell is to calculate from the splenocyte sum that reclaims, the percentage of each subgroup is determined by trichromatic analysis.Histological evaluation to the relative extent of tissue injury in the relevant organ (liver, skin and small intestine) of other GVHD carries out after putting to death animal.
At last, the animal that Neutrokine-α and damping fluid are handled every other day carries out clinical evaluation one time, determines emaciation, body weight and lethality.
In this Acute GVHD mouse model, also can test agonist and the antagonist of Neutrokine-α.
Embodiment 9: the antibody fragment that separates anti-Neutrokine-α polypeptide from the scFvs library
With the big library of separation from the gene constructed one-tenth antibody fragment of V of the natural generation of people PBLs, the reactivity of anti-Neutrokine-α and/or Neutrokine-α SV is contained in this library, it can contact or not contact donor (see United States Patent (USP) 5885793, incorporate reference in full at this).
The rescue in library
The scFvs library construction is from the RNA of people PBLs, as described in the WO 92/01047 (this incorporate in full with reference to).Be rescue phage displaying antibody fragment, about 10 9Individual intestinal bacteria of carrying phagemid are used to inoculate 2 * TY (2 * TY-AMP-GLU), and shake that to grow to O.D. be 0.8 of the 50ml that contains 1% glucose and 100 μ g/ml penbritins.This culture of 5ml is used to inoculate 2 * TY-AMP-GLU of 50ml, adds 2 * 10 8 δ gene 3 auxiliary genes of TU (M13 δ gene III sees WO 92/01047), and culture do not shaken incubation 45 minutes at 37 ℃, shook incubation 45 minutes at 37 ℃ then.With this culture centrifugal 10 minutes, and throw out is resuspended among 2 * TY of the 2L that contains 100 μ g/ml penbritins and 50 μ g/ml kantlex grow overnight at 4000r.p.m..As described in WO 92/01047, prepare phage.
M13 δ gene III is prepared as follows: M13 δ gene III helper phage is encoding gene III albumen not, yet phage (grain) shows that antibody fragment has the ability of stronger conjugated antigen.The M13 δ gene III particle that infects is by in the morphology of phages between the emergence period, helper phage is grown in carrying the cell that the proteic pUC19 derivative of wild type gene III is provided and produces.This culture is not shaken incubation 1 hour at 37 ℃, shook incubation 1 hour at 37 ℃ then.With cell reversing (IEC-Centra 84000revs/ branch, 10 minutes), be resuspended to the 300ml 2 * TY meat soup that contains 100 μ g/ml penbritins and 25 μ g/ml kantlex and (in 2 * TY-AMP-KAN), shake grow overnight at 37 ℃.By twice PEG precipitation (Sambrook etc., 1990), purifying and concentrated phage particle are resuspended among the 2ml PBS from substratum, and through 0.45 μ m filter membrane (MinisartNML; Sartorius) filter, to final concentration be about 10 13Individual transduced unit/ml (amicillin resistance clone).
The elutriation library
Immune test tube (Nunc) is spent the night with the 100 μ g/ml of 4ml or the polypeptide bag of the present invention of 10 μ g/ml in PBS.Test tube was blockaded 2 hours at 37 ℃ with 2%Marvel-PBS, then with PBS flushing 3 times.In test tube, add about 10 13The phage of TU, and room temperature upset incubation 30 minutes, static then 1.5 hours.Test tube with PBS0.1%Tween-20 flushing 10 times, is reached with PBS flushing 10 times.Add the 100mM triethylamine wash-out bacteriophage of 1ml, rotated up and down 15 minutes, use the 1.0MTris-HCl of 0.5ml afterwards immediately, the pH7.4 neutralization.Then by with bacterium in the phage of 37 ℃ of incubation wash-outs 30 minutes, and phage is used to infect the mid-log phase e. coli tg1 of 10ml.Intestinal bacteria are plated on the TYE flat board that contains 1% glucose and 100 μ g/ml penbritins.Above-mentioned δ gene 3 helper phage elutriations are used in gained bacterium library then, carry out ensuing selection with the preparation phage.Repeat this step totally 4 times, will use PBS, the affinity purification of 0.1%Tween-20 flushing test tube increases to 20 times, with PBS flushing 20 times.
Qualitative binding substances
Phage through the wash-out selected for third and fourth time is used for ehec infection HB2151, and produces soluble scFv (Marks etc., 1991) from single bacterium colony.Be used in the 50mM supercarbonate, the microtitre flat board of the polypeptide bag of the present invention of the 10pg/ml among pH9.6 quilt carries out ELISAs.Positive colony among the ELISA by PCR fingerprinting further qualitative (seeing WO 92/01047), is checked order then.
Embodiment 10: with in the anti-Neutrokine-alpha monoclonal antibodies and Neutrokine-α/Neutrokine-α receptor interaction
Produce the proteic monoclonal antibody of anti-Neutrokine-α according to following method.In brief, the Neutrokine-α of the His mark of the 50 μ g that the method for mouse subcutaneous injection by embodiment 2 produced, described Neutrokine-α are in the complete Freunds adjuvant of 100 μ l among the emulsive 100 μ l PBS.Two peritheliums are injected once 25 μ g Neutrokine-α in non-complete Freunds adjuvant down at interval, inject altogether three times.With animal tranquillization one month, after intraperitoneal advances the Neutrokine-α in PBS of 25 μ g.Put to death animal after four days, extracting spleen cell merges.
" fusion " process is to be undertaken by the cell that will take from spleen and 2 * 10E7 P3X63Ag8.653 plasmoma cytogamy, use PEG1500 (Boehringer Mannheim), carry out (seeing Gefter according to the producer's guidance, M.L. etc., Somatic Cell Genet3:231-36 (1977); Boehringer Mannheim PEG1500 (Cat.No.783641), product description).
After merging, cell is resuspended in the 400ml HAT substratum of adding 20%FBS and 4% hybridoma tonic (Boehringer Mannheim), and with the density distribution in 200 μ l/ holes in 96 hole flat boards.After merging 7 days, with the substratum sucking-off of 100 μ l and change fresh culture with 100 μ l.After merging 14 days, the screening hybridoma produces to carry out antibody.
The hybridoma supernatant is screened by ELISA, with the Neutrokine-α protein binding that is fixed on the flat board.Is that the Neutrokine-α in 2 μ g/mls 100 μ l/ holes among PBSs spend the night wrap quilt with Neutrokine-α by incubation concentration with flat board.Is 1: 10 with the hybridoma supernatant with the PBS dilution, places each hole of the flat board of Neutrokine-α bag quilt, and is incubated overnight at 4 ℃.Second day, flat board is washed 3 times with the PBS that contains 0.1%Tween-20, and carry out color reaction with anti-mouse IgG ABC system (Vector Laboratories).The 2M H that adds the 25ml/ hole 2SO 4The color development stopping reaction.Read dull and stereotyped at 450nm.
Detect the Ig isotype of hybridoma supernatant with Isostrips.Method by restricted dilution on the HT substratum is cloned.About 3 * 10E6 injection cell in the HBSS of 0.9ml is gone in the mouse of pristane initiation.After 7-9 days, collect ascites with the 19g syringe.All antibody pass through protein g affinity chromatography and purifying with Acta FPLC system (Pharmacia).
Begin and twice successive subcutaneous injection after, three all mouse all present strong immune response; Determine that by on the flat board of Neutrokine-α bag quilt, carrying out ELISA the serum titration is 10E-7.
In a test, use the elementary hybridoma of splenocyte generation more than 1000 of positive mouse.Screening 917 anti-Neutrokine-Alpha antibodies of generation wherein.Supernatant with dilution in 1: 1 screens to detect all positive colonies.In 917 hybridomas of screening, finding 76 is male, and wherein 17 is the producer of IgG.Behind affine test and clone, select wherein 9 to further expand and purifying.
The monoclonal antibody of all purifying all can be in conjunction with multi-form Neutrokine-α (comprising protein (seeing embodiment 2) the His mark and that originate from rhabdovirus system) in Western engram analysis and ELISA.6 among these 9 clones can also be at the THP-1 cell surface in conjunction with Neutrokine-α.Yet the antibody capable of none test is caught Neutrokine-α from solution.
Produce the anti-Neutrokine-alpha monoclonal antibodies of high-affinity, it is identified in the Neutrokine-α of cell surface expression, but can not be identified in the Neutrokine-α in the solution, described antibody is used in intravital neutralization research and at external monocyte and B cell analysis.These antibody also are used at the Western engram analysis Neutrokine-α being carried out sensitivity and detect.
In a test, use the elementary hybridoma of splenocyte generation more than 1000 of positive mouse.Screening 729 anti-Neutrokine-Alpha antibodies of generation wherein.Supernatant with dilution in 1: 10 screens under stringent condition, with the only clone of picking than high-affinity.In 729 hybridomas of screening, 23 is male, comprises 16 IgM and 7 IgG producers (have 4 among the latter strong IgM background is provided).In this test, the isotype of IgG hybridoma distributes and is partial to the IgG2 subclass.7 IgG hybridomas have 3 antibody that produce the IgG2a subclass, and 2 antibody that produce the IgG2b subclass are arranged, and remaining 2 is the IgG1 producer.
The supernatant of all positive hybridomas that test produces in second test suppresses the ability of the alpha mediated B cell proliferation of Neutrokine-.In first shaker test, detect two hybridomas that produce IgG neutralizing antibody (these are antibody 16C9 and 12C5).In another experiment, determine among the IgG of hybridoma (being 16C9 and 12C5) and active that persistent erection and the supernatant of two hybridoma 15C10 and 4A6 are not differentiated in addition.
Subsequently these 3 clones are expanded in vivo (clone is that 15C10 also expands in the tubular fibre system), affinitive layer purification antibody is provided.All these three clones all can be at the THP-1 cell surface in conjunction with Neutrokine-α, and also can be from solution in conjunction with (promptly catching) Neutrokine-α.
Especially, test, determine during whether antibody and Neutrokine-α/Neutrokine-α receptors bind with the anti-Neutrokine-alpha monoclonal antibodies described in above-mentioned second test.In brief, (Pierce, Rockford IL) carry out biotinylation to Neutrokine-α albumen to the TNHS-vitamin H reagent that connects with EZ.Then biotinylated Neutrokine-α is used to differentiate cell surface protein in conjunction with Neutrokine-α.Tentative experiment shows that Neutrokine-α is in conjunction with the acceptor on the bone-marrow-derived lymphocyte.
In the anti-Neutrokine-Alpha antibodies that in above-mentioned second test, produces and the combining of Neutrokine-α and Neutrokine-α acceptor.In a special embodiment, among the anti-Neutrokine-Alpha antibodies 15C10 and the combining of Neutrokme-α and Neutrokine-α acceptor.
Therefore, the anti-Neutrokine-Alpha antibodies that in above-mentioned second test, produces (especially antibody 15C10) identification and binding film bonded and soluble Neutrokine-α albumen, and in external and the combining of Neutrokine-α and Neutrokine-α acceptor.
Should know that the present invention can be different from the described especially and embodiment of preamble and practical application.According to above instruction and within the scope of the appended claims, can be in addition various modifications of the present invention.
All publications of being quoted (comprising patent, patent application, magazine article, laboratory manual, books or other document) are incorporated reference in full at this.
In addition, in the sequence table of this submission, and the sequence table of submitting among the PCT/US96/17957 of the US 60/036100 of the application submission of submitting on January 12nd, 1998 on January 14th, 09/005874,1997 and submission on October 25th, 1996, all incorporate reference into.
Sequence table
＜110〉Human Genome Sciences Inc
＜120〉Neutrokine-α and Neutrokine-alpha splice variant
<130>PF343PCT2
＜140〉do not provide
<141>2000-02-22
<150>60/122,388
<151>1999-03-02
<150>60/124,097
<151>1999-03-12
<150>60/126,599
<151>1999-03-26
<150>60/127,598
<151>1999-04-02
<150>60/130,412
<151>1999-04-16
<150>60/130,696
<151>1999-04-23
<150>60/131,278
<151>1999-04-27
<150>09/255,794
<151>1999-02-23
<150>60/131,673
<151>1999-04-29
<150>60/136,784
<151>1999-05-28
<150>60/142,659
<151>1999-07-06
<150>60/145,824
<151>1999-07-27
<150>60/167,239
<151>1999-11-24
<150>60/168,624
<151>1999-12-03
<150>60/171,108
<151>1999-12-16
<150>60/171,626
<151>1999-12-23
<150>60/176,015
<151>2000-01-14
<160>38
<170>PatentIn Ver. 2.1
<210>1
<211>1100
<212>DNA
<213>Homo sapiens
<220>
<221>CDS
<222>(147)..(1001)
<400>1
aaattcagga taactctcct gaggggtgag ccaagccctg ccatgtagtg cacgcaggac 60
atcaacaaac acagataaca ggaaatgatc cattccctgt ggtcacttat tctaaaggcc 120
ccaaccttca aagttcaagt agtgat atg gat gac tcc aca gaa agg gag cag 173
Met Asp Asp Ser Thr Glu Arg Glu Gln
1 5
tca cgc ctt act tct tgc ctt aag aaa aga gaa gaa atg aaa ctg aag 221
Ser Arg Leu Thr Ser Cys Leu Lys Lys Arg Glu Glu Met Lys Leu Lys
10 15 20 25
gag tgt gtt tcc atc ctc cca cgg aag gaa agc ccc tct gtc cga tcc 269
Glu Cys Val Ser Ile Leu Pro Arg Lys Glu Ser Pro Ser Val Arg Ser
30 35 40
tcc aaa gac gga aag ctg ctg gct gca acc ttg ctg ctg gca ctg ctg 317
Ser Lys Asp Gly Lys Leu Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu
45 50 55
tct tgc tgc ctc acg gtg gtg tct ttc tac cag gtg gcc gcc ctg caa 365
Ser Cys Cys Leu Thr Val Val Ser Phe Tyr Gln Val Ala Ala Leu Gln
60 65 70
ggg gac ctg gcc agc ctc cgg gca gag ctg cag ggc cac cac gcg gag 413
Gly Asp Leu Ala Ser Leu Arg Ala Glu Leu Gln Gly His His Ala Glu
75 80 85
aag ctg cca gca gga gca gga gcc ccc aag gcc ggc ctg gag gaa gct 461
Lys Leu Pro Ala Gly Ala Gly Ala Pro Lys Ala Gly Leu Glu Glu Ala
90 95 100 105
cca gct gtc acc gcg gga ctg aaa atc ttt gaa cca cca gct cca gga 509
Pro Ala Val Thr Ala Gly Leu Lys Ile Phe Glu Pro Pro Ala Pro Gly
110 115 120
gaa ggc aac tcc agt cag aac agc aga aat aag cgt gcc gtt cag ggt 557
Glu Gly Asn Ser Ser Gln Asn Ser Arg Asn Lys Arg Ala Val Gln Gly
125 130 135
cca gaa gaa aca gtc act caa gac tgc ttg caa ctg att gca gac agt 605
Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser
140 145 150
gaa aca cca act ata caa aaa gga tct tac aca ttt gtt cca tgg ctt 653
Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu
155 160 165
ctc agc ttt aaa agg gga agt gcc cta gaa gaa aaa gag aat aaa ata 701
Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile
170 175 180 185
ttg gtc aaa gaa act ggt tac ttt ttt ata tat ggt cag gtt tta tat 749
Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr
190 195 200
act gat aag acc tac gcc atg gga cat cta att cag agg aag aag gtc 797
Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val
205 210 215
cat gtc ttt ggg gat gaa ttg agt ctg gtg act ttg ttt cga tgt att 845
His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile
220 225 230
caa aat atg cct gaa aca cta ccc aat aat tcc tgc tat tca gct ggc 893
Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly
235 240 245
att gca aaa ctg gaa gaa gga gat gaa ctc caa ctt gca ata cca aga 941
Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg
250 255 260 265
gaa aat gca caa ata tca ctg gat gga gat gtc aca ttt ttt ggt gca 989
Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala
270 275 280
ttg aaa ctg ctg tgacctactt acaccatgtc tgtagctatt ttcctccctt
1041
Leu Lys Leu Leu
285
tctctgtacc tctaagaaga aagaatctaa ctgaaaatac caaaaaaaaa aaaaaaaaa
1100
<210>2
<211>285
<212>PRT
<213>Homo sapiens
<400>2
Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu
1 5 10 15
Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro
20 25 30
Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu
35 40 45
Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val
50 55 60
Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg
65 70 75 80
Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly
85 90 95
Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu
100 105 110
Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn
115 120 125
Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln
130 135 140
Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys
145 150 155 160
Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser
165 170 175
Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr
180 185 190
Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met
195 200 205
Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu
210 215 220
Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu
225 230 235 240
Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly
245 250 255
Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu
260 265 270
Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu
275 280 285
<210>3
<211>233
<212>PRT
<213>Homo sapiens
<400>3
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala
1 5 10 15
Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe
20 25 30
Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe
35 40 45
Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro
50 55 60
Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser
65 70 75 80
Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro
85 90 95
Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu
100 105 110
Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser
115 120 125
Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
130 135 140
Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala
145 150 155 160
Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro
165 170 175
Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu
180 185 190
Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu
195 200 205
Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly
210 215 220
Gln Val Tyr Phe Gly Ile Ile Ala Leu
225 230
<210>4
<211>205
<212>PRT
<213>Homo sapiens
<400>4
Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Arg Gly Thr Thr
1 5 10 15
Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala
20 25 30
Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala
35 40 45
Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala
50 55 60
Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg
65 70 75 80
Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn
85 90 95
Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln
100 105 110
Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro
115 120 125
Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe
130 135 140
His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln
145 150 155 160
Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr
165 170 175
Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val
180 185 190
Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu
195 200 205
<210>5
<211>244
<212>PRT
<213>Homo sapiens
<400>5
Met Gly Ala Leu Gly Leu Glu Gly Arg Gly Gly Arg Leu Gln Gly Arg
1 5 10 15
Gly Ser Leu Leu Leu Ala Val Ala Gly Ala Thr Ser Leu Val Thr Leu
20 25 30
Leu Leu Ala Val Pro Ile Thr Val Leu Ala Val Leu Ala Leu Val Pro
35 40 45
Gln Asp Gln Gly Gly Leu Val Thr Glu Thr Ala Asp Pro Gly Ala Gln
50 55 60
Ala Gln Gln Gly Leu Gly Phe Gln Lys Leu Pro Glu Glu Glu Pro Glu
65 70 75 80
Thr Asp Leu Ser Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro
85 90 95
Leu Lys Gly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe
100 105 110
Leu Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro
115 120 125
Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg
130 135 140
Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg
145 150 155 160
Ser Ser Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu
165 170 175
Leu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro Ala
180 185 190
Arg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly
195 200 205
Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser
210 215 220
His Pro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly Ala
225 230 235 240
Val Met Val Gly
<210>6
<211>281
<212>PRT
<213>Homo sapiens
<220>
＜223〉description of Zu He DNA/RNA molecule: n equals
A, t, g, or c
<400>6
Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp
1 5 10 15
Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys
20 25 30
Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro
35 40 45
Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro
50 55 60
Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly
65 70 75 80
Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly
85 90 95
Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala
100 105 110
Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu
115 120 125
Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg
130 135 140
Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu
145 150 155 160
Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr
165 170 175
Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr
180 185 190
Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Ash Leu Pro Leu Ser
195 200 205
His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met
210 215 220
Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala
225 230 235 240
Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His
245 250 255
Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser
260 265 270
Gln Thr Phe Phe Gly Leu Tyr Lys Leu
275 280
<210>7
<211>337
<212>DNA
<213>Homo sapiens
<220>
＜223〉description of Zu He DNA/RNA molecule: n equals
A, t, g, or c
<220>
＜221〉misc_ feature
<222>(3)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(58)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(67)..(71)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(212)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(255)
＜223〉n equals a, t, g or c
<220>
＜221〉misc_ feature
<222>(297)
＜223〉n equals a, t, g or c
<220>
＜221〉misc_ feature
<222>(300)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(320)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(335)
＜223〉n equals a, t, g, or c
<400>7
ggntaactct cctgaggggt gagccaagcc ctgccatgta gtgcacgcag gacatcanca 60
aacacannnn ncaggaaata atccattccc tgtggtcact tattctaaag gccccaacct 120
tcaaagttca agtagtgata tggatgactc cacagaaagg gagcagtcac gccttacttc 180
ttgccttaag aaaagagaag aaatgaaact gnaaggagtg tgtttccatc ctcccacgga 240
aggaaagccc ctctntccga tcctccaaag acggaaagct gctggctgca accttgntgn 300
tggcattgtg ttcttgctgn ctcaaggtgg tgttntt 337
<210>8
<211>509
<212>DNA
<213>Homo sapiens
<220>
＜223〉description of Zu He DNA/RNA molecule: n equals
A, t, g, or c
<220>
＜221〉misc_ feature
<222>(10)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(13)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(209)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(315)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(322)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(325)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(334)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(343)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(347)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(351)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(356)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(409)..(410)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(416)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(422)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(424)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(426)..(427)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(429)
＜222〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(431)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(433)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(438)..(439)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(443)..(444)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(446)..(447)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(449)..(450)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(452)..(453)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(458)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(461)..(462)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(466)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(469)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(471)..(472)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(474)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(478)..(481)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(496)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(498)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(504)
＜223〉n equals a, t, g, or c
<400>8
aattcggcan agnaaactgg ttactttttt atatatggtc aggttttata tactgataag 60
acctacgcca tgggacatct agttcagagg aagaaggtcc atgtctttgg ggatgaattg 120
agtctggtga ctttgtttcg atgtattcaa aatatgcctg aaacactacc caataattcc 180
tgctattcag ctggcattgc aaaactggna ggaaggagat gaactccaac ttgcaatacc 240
aggggaaaat gcacaattat cactgggatg gagatgttca cattttttgg gtgccattga 300
aactgctgtg acctncttac ancangtgct gttngctatt ttncctncct nttctntggt 360
aacctcttag gaaggaagga ttcttaactg ggaaataacc caaaaaaann ttaaangggt 420
angngnnana ngnggggnng ttnncnngnn gnnttttngg nntatnttnt nntngggnnn 480
ngtaaaaatg gggccnangg gggnttttt 509
<210>9
<211>497
<212>DNA
<213>Homo sapiens
<220>
＜221〉misc_ feature
<222>(168)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(213)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(288)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(325)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(346)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(406)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(415)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(419)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(437)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(442)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(467)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(473)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(476)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(481)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(483)..(484)
＜223〉n equals a, t, g, or c
<220>
＜221〉misc_ feature
<222>(494)
＜223〉n equals a, t, g, or c
<400>9
aattcggcac gagcaaggcc ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa 60
tctttgaacc accagctcca ggagaaggca actccagtca gaacagcaga aataagcgtg 120
ccgttcaggg tccagaagaa acagtcactc aagactgctt gcaactgntt gcagacagtg 180
aaacaccaac tatacaaaaa ggctcccttc tgntgccaca tttgggccaa ggaatggaga 240
gatttcttcg tctggaaaca ttttgccaaa ctcttcagat actctttnct ctctgggaat 300
caaaggaaaa tctctactta gattnacaca tttgttccca tgggtntctt aagttttaaa 360
aggggagtgc ccttaggagg aaaaggggat aaatattggc caaggnactg gttantttnt 420
aaatatggtc aggtttntat anctggtagg cctcgccatg ggcattnatt canggngagg 480
ncnntctttt gggntga 497
<210>10
<211>27
<212>DNA
<213>Homo sapiens
<220>
＜223〉description of Zu He DNA/RNA molecule: x=
Hypoxanthine deoxyriboside
<400>10
gtgggatcca gcctccgggc agagctg 27
<210>11
<211>33
<212>DNA
<213>Homo sapiens
<400>11
gtgaagcttt tattacagca gtttcaatgc acc 33
<210>12
<211>26
<212>DNA
<213>Homo sapiens
<400>12
gtgtcatgag cctccgggca gagctg 26
<210>13
<211>33
<212>DNA
<213>Homo sapiens
<400>13
gtgaagcttt tattacagca gtttcaatgc acc 33
<210>14
<211>28
<212>DNA
<213>Homo sapiens
<400>14
gtgggatccc cgggcagagc tgcagggc 28
<210>15
<211>33
<212>DNA
<213>Homo sapiens
<400>15
gtgggatcct tattacagca gtttcaatgc acc 33
<210>16
<211>129
<212>DNA
<213>Homo sapiens
<400>16
gcgggatccg ccaccatgaa ctccttctcc acaagcgcct tcggtccagt tgccttctcc 60
ctggggctgc tcctggtgtt gcctgctgcc ttccctgccc cagttgtgag acaaggggac 120
ctggccagc 129
<210>17
<211>30
<212>DNA
<213>Homo sapiens
<400>17
gtgggatcct tacagcagtt tcaatgcacc 30
<210>18
<211>903
<212>DNA
<213>Homo sapiens
<220>
<221>CDS
<222>(1)..(798)
<400>18
atg gat gac tcc aca gaa agg gag cag tca cgc ctt act tct tgc ctt 48
Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu
1 5 10 15
aag aaa aga gaa gaa atg aaa ctg aag gag tgt gtt tcc atc ctc cca 96
Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro
20 25 30
cgg aag gaa agc ccc tct gtc cga tcc tcc aaa gac gga aag ctg ctg 144
Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu
35 40 45
gct gca acc ttg ctg ctg gca ctg ctg tct tgc tgc ctc acg gtg gtg 192
Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Lcu Thr Val Val
50 55 60
tct ttc tac cag gtg gcc gcc ctg caa ggg gac ctg gcc agc ctc cgg 240
Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg
65 70 75 80
gca gag ctg cag ggc cac cac gcg gag aag ctg cca gca gga gca gga 288
Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly
85 90 95
gcc ccc aag gcc ggc ctg gag gaa gct cca gct gtc acc gcg gga ctg 336
Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu
100 105 110
aaa atc ttt gaa cca cca gct cca gga gaa ggc aac tcc agt cag aac 384
Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn
115 120 125
agc aga aat aag cgt gcc gtt cag ggt cca gaa gaa aca gga tct tac 432
Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr
130 135 140
aca ttt gtt cca tgg ctt ctc agc ttt aaa agg gga agt gcc cta gaa 480
Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu
145 150 155 160
gaa aaa gag aat aaa ata ttg gtc aaa gaa act ggt tac ttt ttt ata 528
Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile
165 170 175
tat ggt cag gtt tta tat act gat aag acc tac gcc atg gga cat cta 576
Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu
180 185 190
att cag agg aag aag gtc cat gtc ttt ggg gat gaa ttg agt ctg gtg 624
Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val
195 200 205
act ttg ttt cga tgt att caa aat atg cct gaa aca cta ccc aat aat 672
Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn
210 215 220
tcc tgc tat tca gct ggc att gca aaa ctg gaa gaa gga gat gaa ctc 720
Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu
225 230 235 240
caa ctt gca ata cca aga gaa aat gca caa ata tca ctg gat gga gat 768
Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp
245 250 255
gtc aca ttt ttt ggt gca ttg aaa ctg ctg tgacctactt acaccatgtc 818
Val Thr Phe Phe Gly Ala Leu Lys Leu Leu
260 265
tgtagctatt ttcctccctt tctctgtacc tctaagaaga aagaatctaa ctgaaaatac 878
caaaaaaaaa aaaaaaaaaa aaaaa 903
<210>19
<211>266
<212>PRT
<213>Homo sapiens
<400>19
Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu
1 5 10 15
Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro
20 25 30
Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu
35 40 45
Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val
50 55 60
Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg
65 70 75 80
Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly
85 90 95
Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu
100 105 110
Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn
115 120 125
Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr
130 135 140
Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu
145 150 155 160
Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile
165 170 175
Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu
180 185 190
Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val
195 200 205
Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn
210 215 220
Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu
225 230 235 240
Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp
245 250 255
Val Thr Phe Phe Gly Ala Leu Lys Leu Leu
260 265
<210>20
<211>136
<212>PRT
<213>Homo sapiens
<400>20
His Ser Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp
1 5 10 15
Ser Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg
20 25 30
Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val
35 40 45
Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr Met
50 55 60
Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr Leu Phe
65 70 75 80
Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala Tyr Asn Ser
85 90 95
Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile Leu Ser
100 105 110
Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro His Gly
115 120 125
Thr Phe Leu Gly Phe Val Lys Leu
130 135
<210>21
<211>462
<212>DNA
<213>Homo sapiens
<400>21
atggctgttc agggtccgga agaaaccgtt actcaggact gccttcagct gatcgcagac 60
tctgaaactc cgaccatcca gaaaggttct tacacctttg ttccttggct gctttctttc 120
aaacgtggtt ctgccctgga agagaaagaa aacaaaatcc tggttaaaga aactggttac 180
ttctttatct acggtcaggt tctttacact gataagacct acgccatggg tcacctgatt 240
cagcgtaaga aagttcacgt tttcggtgac gagctgtctc tggttactct gtttcgctgc 300
attcagaaca tgccggaaac tcttcctaac aactcctgct actctgctgg catcgcaaaa 360
ctggaagagg gtgatgaact gcagctggca attcctcgtg aaaacgcaca aatttctctg 420
gacggtgatg taaccttctt tggtgcactg aaacttctgt aa 462
<210>22
<211>1040
<212>DNA
<213>Homo sapiens
<220>
<221>CDS
<222>(1)..(468)
<400>22
cgc gtg gta gac ctc tca gct cct cct gca cca tgc ctg cct gga tgc 48
Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys
1 5 10 15
cgc cat tct caa cat gat gat aat gga atg aac ctc aga aac aga act 96
Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg Thr
20 25 30
tac aca ttt gtt cca tgg ctt ctc agc ttt aaa aga gga aat gcc ttg 144
Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu
35 40 45
gag gag aaa gag aac aaa ata gtg gtg agg caa aca ggc tat ttc ttc 192
Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe
50 55 60
atc tac agc cag gtt cta tac acg gac ccc atc ttt gct atg ggt cat 240
Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His
65 70 75 80
gtc atc cag agg aag aaa gta cac gtc ttt ggg gac gag ctg agc ctg 288
Val Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu
85 90 95
gtg acc ctg ttc cga tgt att cag aat atg ccc aaa aca ctg ccc aac 336
Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn
100 105 110
aat tcc tgc tac tcg gct ggc atc gcg agg ctg gaa gaa gga gat gag 384
Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu
115 120 125
att cag ctt gca att cct cgg gag aat gca cag att tca cgc aac gga 432
Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly
130 135 140
gac gac acc ttc ttt ggt gcc cta aaa ctg ctg taa ctcacttgct 478
Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu
145 150 155
ggagtgcgtg atccccttcc ctcgtcttct ctgtacctcc gagggagaaa cagacgactg 538
gaaaaactaa aagatgggga aagccgtcag cgaaagtttt ctcgtgaccc gttgaatctg 598
atccaaacca ggaaatataa cagacagcca caaccgaagt gtgccatgtg agttatgaga 658
aacggagccc gcgctcagaa agaccggatg aggaagaccg ttttctccag tcctttgcca 718
acacgcaccg caaccttgct ttttgccttg ggtgacacat gttcagaatg cagggagatt 778
tccttgtttt gcgatttgcc atgagaagag ggcccacaac tgcaggtcac tgaagcattc 838
acgctaagtc tcaggattta ctctcccttc tcatgctaag tacacacacg ctcttttcca 898
ggtaatacta tgggatacta tggaaaggtt gtttgttttt aaatctagaa gtcttgaact 958
ggcaatagac aaaaatcctt ataaattcaa gtgtaaaata aacttaatta aaaaggttta
1018
agtgtgaaaa aaaaaaaaaa aa
1040
<210>23
<211>155
<212>PRT
<213>Homo sapiens
<400>23
Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys
1 5 10 15
Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg Thr
20 25 30
Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu
35 40 45
Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe
50 55 60
Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His
65 70 75 80
Val Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu
85 90 95
Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn
100 105 110
Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu
115 120 125
Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly
130 135 140
Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu
145 150 155
<210>24
<211>26
<212>DNA
<213>Homo sapiens
<400>24
ccaccagctc caggagaagg caactc 26
<210>25
<211>19
<212>DNA
<213>Homo sapiens
<400>25
accgcgggac tgaaaatct 19
<210>26
<211>23
<212>DNA
<213>Homo sapiens
<400>26
cacgcttatt tctgctgttc tga 23
<210>27
<211>657
<212>DNA
<213>Homo sapiens
<400>27
taccaggtgg cggccgtgca aggggacctg gccagcctcc gggcagagct gcagggccac 60
cacgcggaga agctgccagc aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120
gctgtcaccg caggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt 180
cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat tcaagactgc 240
ttgcaactga ttgcagacag tgaaacacca actatacaaa aaggatctta cacatttgtt 300
ccatggcttc tcagctttaa aaggggaagt gccctagaag aaaaagagaa taaaatattg 360
gtcaaagaaa ctggttactt ttttatatat ggtcaggttt tatacactga taagacctat 420
gccatgggac atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg 480
gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa ttcctgctat 540
tcagctggca ttgcaaaact ggaagaagga gatgaacttc aacttgcaat accacgagaa 600
aatgcacaaa tatcactgga tggagatgtc acattttttg gtgccctcaa actgctg 657
<210>28
<211>219
<212>PRT
<213>Homo sapiens
<400>28
Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu
1 5 10 15
Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro
20 25 30
Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile
35 40 45
Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg
50 55 60
Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys
65 70 75 80
Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser
85 90 95
Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu
100 105 110
Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe
115 120 125
Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His
130 135 140
Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu
145 150 155 160
Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn
165 170 175
Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu
180 185 190
Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly
195 200 205
Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu
210 215
<210>29
<211>657
<212>DNA
<213>Homo sapiens
<400>29
taccaggtgg cggccgtgca aggggacctg gccagcctcc gggcagagct gcagagccac 60
cacgcggaga agctgccagc aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120
gctgtcaccg cgggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt 180
cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat tcaagactgc 240
ttgcaactga ttgcagacag tgaaacacca actatacaaa aaggatctta cacatttgtt 300
ccatggcttc tcagctttaa aaggggaagt gccctagaag aaaaagagaa taaaatattg 360
gtcaaagaaa ctggttactt ttttatatat ggtcaggttt tatacactga taagacctat 420
gccatgggac atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg 480
gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa ttcctgctat 540
tcagctggca ttgcaaaact ggaagaaggg gatgaacttc aacttgcaat accacgagaa 600
aatgcacaaa tatcactgga tggagatgtc acattttttg gtgccctcaa actgctg 657
<210>30
<211>219
<212>PRT
<213>Homo sapiens
<400>30
Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu
1 5 10 15
Leu Gln Ser His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro
20 25 30
Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile
35 40 45
Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg
50 55 60
Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys
65 70 75 80
Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser
85 90 95
Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu
100 105 110
Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe
115 120 125
Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His
130 135 140
Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu
145 150 155 160
Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn
165 170 175
Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu
180 185 190
Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly
195 200 205
Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu
210 215
<210>31
<211>38
<212>DNA
<213>Homo sapiens
<400>31
ggtcgccgtt tctaacgcgg ccgttcaggg tccagaag 38
<210>32
<211>49
<212>DNA
<213>Homo sapiens
<400>32
ctggttcggc ccaaggtacc aagcttgtac cttagatctt ttctagatc 49
<210>33
<211>21
<212>DNA
<213>Homo sapiens
<400>33
ctggtagttc ttcggagtgt g 21
<210>34
<211>19
<212>DNA
<213>Homo sapicns
<400>34
cgcgttagaa acggcgacc 19
<210>35
<211>22
<212>DNA
<213>Homo sapiens
<220>
＜221〉misc_ feature
<222>(7)
＜223〉n equals Hypoxanthine deoxyriboside
<220>
＜221〉misc_ feature
<222>(12)
＜223〉n equals Hypoxanthine deoxyriboside
<220>
＜221〉misc_ feature
<222>(16)
＜223〉n equals Hypoxanthine deoxyriboside
<400>35
taccagntgg cngccntgca ag 22
<210>36
<211>22
<212>DNA
<213>Homo sapiens
<220>
＜221〉misc_ feature
<222>(3)
＜223〉n equals Hypoxanthine deoxyriboside
<220>
＜221〉misc_ feature
<222>(14)
＜223〉n equals Hypoxanthine deoxyriboside
<220>
＜221〉misc_ feature
<222>(16)..(17)
＜223〉n equals Hypoxanthine deoxyriboside
<400>36
gtnacagcag tttnanngca cc 22
<210>37
<211>866
<212>DNA
<213>Mus musculus
<400>37
atggatgagt ctgcaaagac cctgccacca ccgtgcctct gtttttgctc cgagaaagga 60
gaagatatga aagtgggata tgatcccatc actccgcaga aggaggaggg tgcctggttt 120
gggatctgca gggatggaag gctgctggct gctaccctcc tgctggccct gttgtccagc 180
agtttcacag cgatgtcctt gtaccagttg gctgccttgc aagcagacct gatgaacctg 240
cgcatggagc tgcagagcta ccgaggttca gcaacaccag ccgccgcggg tgctccagag 300
ttgaccgctg gagtcaaact cctgacaccg gcagctcctc gaccccacaa ctccagccgc 360
ggccacagga acagacgcgc cttccaggga ccagaggaaa cagaacaaga tgtagacctc 420
tcagctcctc ctgcaccatg cctgcctgga tgccgccatt ctcaacatga tgataatgga 480
atgaacctca gaaacatcat tcaagactgt ctgcagctga ttgcagacag cgacacgccg 540
gccttggagg agaaagagaa caaaatagtg gtgaggcaaa caggctattt cttcatctac 600
agccaggttc tatacacgga ccccatcttt gctatgggtc atgtcatcca gaggaagaaa 660
gtacacgtct ttggggacga gctgagcctg gtgaccctgt tccgatgtat tcagaatatg 720
cccaaaacac tgcccaacaa ttcctgctac tcggctggca tcgcgaggct ggaagaagga 780
gatgagattc agcttgcaat tcctcgggag aatgcacaga tttcacgcaa cggagacgac 840
accttctttg gtgccctaaa actgct 866
<210>38
<211>177
<212>DNA
<213>Mus musculus
<400>38
mdsaktcccs kgdmkvgydt kgawgcrdgr aatassstam syaaadmnrm syrgsataaa 60
gatagvktaa rhnssrghrn rragtdvdsa acgcrhshdd ngmnrndcad sdtaknkvvr 120
tgyysvytda mghvrkkvhv gdsvtrcnmk tnnscysaga rgdarnasrn gddtgak 177

Claims (76)

1. isolated nucleic acid molecule comprises and has and be selected from the polynucleotide that the nucleotide sequence of at least 95% homogeny is arranged with the sequence of next group:
(a) coding has the nucleotide sequence of the Neutrokine-α polypeptide of complete amino acid sequence shown in Figure 1A and the 1B (SEQ ID No:2);
(b) coding has the nucleotide sequence by the Neutrokine-α polypeptide of the complete amino acid sequence of the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768;
(c) nucleotide sequence of coding Neutrokine-α polypeptide extracellular domain;
(d) nucleotide sequence of coding Neutrokine-α polypeptide membrane-spanning domain
(e) nucleotide sequence of coding Neutrokine-α polypeptide born of the same parents internal area;
(f) coding comprises extracellular domain and born of the same parents' internal area, but the nucleotide sequence of the soluble Neutrokine-α polypeptide of disappearance membrane-spanning domain; With
(g) be complementary to above-mentioned (a), (b), (c), (d), the nucleotide sequence of any nucleotide sequence (e) or (f).
2. the nucleic acid molecule of claim 1, wherein said polynucleotide have the complete nucleotide sequence shown in Figure 1A and the 1B (SEQ ID No:1).
3. the nucleic acid molecule of claim 1, wherein said polynucleotide have the nucleotide sequence that the coding shown in Figure 1A and the 1B (SEQ ID No:1) has the Neutrokine-α polypeptide of complete amino acid sequence shown in Figure 1A and the 1B (SEQ ID No:2).
4. the nucleic acid molecule of claim 1, wherein said polynucleotide have the nucleotide sequence that coding comprises the soluble Neutrokine-α polypeptide of extracellular domain shown in Figure 1A and the 1B (SEQ ID No:2).
5. isolated nucleic acid molecule comprises and has and be selected from the polynucleotide that the nucleotide sequence of at least 95% homogeny is arranged with the sequence of next group:
(a) coding has the nucleotide sequence of the polypeptide of the aminoacid sequence of being made up of the n-285 position residue of SEQ ID No:2, and wherein n is an integer between the 2-190;
(b) coding has the nucleotide sequence of the polypeptide of the aminoacid sequence of being made up of the 1-m position residue of SEQ ID No:2, and wherein m is an integer between the 274-284;
(c) coding has the nucleotide sequence of polypeptide of the aminoacid sequence of being made up of the n-m position residue of SEQ ID No:2, and wherein n and m are defined integers as above-mentioned (a) and (b) respectively; And
(d) nucleotide sequence of the polypeptide be made up of the part of the complete Neutrokine-alpha amino acid sequence of the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768 of coding, wherein said a part of sequence does not comprise 1-11 amino acid of the N-terminal 1-190 of a described complete amino acid sequence amino acid and C-terminal.
6. the nucleic acid molecule of claim 1, wherein said polynucleotide have the contained cDNA clone's of the preservation thing of ATCC preserving number 97768 complete nucleotide sequence.
7. the nucleic acid molecule of claim 1, wherein said polynucleotide have the nucleotide sequence of coding Neutrokine-α polypeptide, and this Neutrokine-α polypeptide has the complete amino acid sequence by the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768.
8. the nucleic acid molecule of claim 1, wherein said polynucleotide have the nucleotide sequence of the soluble Neutrokine-α polypeptide of coding, and this polypeptide comprises the extracellular domain by the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768.
9. an isolated nucleic acid molecule is included under the stringent hybridization condition, with (a) that have with claim 1, (b), (c), (d), (e) or the polynucleotide of the multi-nucleotide hybrid of the identical nucleotide sequence of the nucleotide sequence (f).
10. isolated nucleic acid molecule comprises (a) that coding has claim 1, (b), and (c), (d), (e) or the polynucleotide of the aminoacid sequence that carries the epi-position part of the Neutrokine-α polypeptide of the aminoacid sequence (f).
11. the isolated nucleic acid molecule of claim 10, its coding are selected from and carry the epi-position part with the Neutrokine-α polypeptide of next group: comprise about Phe-115 to the about polypeptide of Leu-147 (SEQ ID NO:2) amino-acid residue; Comprise the polypeptide of about Ile-150 to about Tyr-163 (SEQ ID NO:2) amino-acid residue; Comprise the polypeptide of about Ser-171 to about Phe-194 (SEQ ID NO:2) amino-acid residue; The polypeptide that comprises about Glu-223 to Tyr-246 (SEQ ID NO:2) amino-acid residue; The polypeptide that comprises about Ser-271 to Phe-278 (SEQ ID NO:2) amino-acid residue.
12. a method of producing recombinant vectors comprises the isolated nucleic acid molecule of claim 1 is inserted a carrier.
13. recombinant vectors of producing by the method for claim 12.
14. a method of producing recombinant host cell comprises that the recombinant vectors with claim 13 imports a host cell.
15. recombinant host cell of producing by the method for claim 14.
16. a recombination method of producing Neutrokine-α polypeptide is included in the recombinant host cell of cultivating claim 15 under the condition that described polypeptide expressed, and reclaims described polypeptide.
17. an isolating Neutrokine-α polypeptide comprises and is selected from the aminoacid sequence that at least 95% homogeny is arranged with the sequence of next group:
(a) has the Neutrokine-α amino acid sequence of polypeptide of complete amino acid sequence shown in Figure 1A and the 1B (SEQ ID NO:2);
(b) has Neutrokine-α amino acid sequence of polypeptide by the complete amino acid sequence of the contained cDNA clones coding of the preservation thing of ATCC preserving number 97768;
(c) aminoacid sequence of Neutrokine-α polypeptide extracellular domain;
(d) aminoacid sequence of Neutrokine-α polypeptide membrane-spanning domain;
(e) aminoacid sequence of Neutrokine-α polypeptide born of the same parents internal area;
(f) comprise the soluble Neutrokine-α amino acid sequence of polypeptide of structural domain; And
(g) (a), (b), (c) (d) aminoacid sequence that carries the epi-position part of arbitrary polypeptide (e) or (f).
18. the isolated polypeptide of claim 17 comprises the proteic part of carrying epi-position of Neutrokine-α, wherein said part is selected from next group: the polypeptide that comprises about Phe-115 to Leu-147 (SEQ ID NO:2) amino-acid residue; The polypeptide that comprises about Ile-150 to Tyr-163 (SEQ ID NO:2) amino-acid residue; The polypeptide that comprises about Ser-171 to Phe-194 (SEQ ID NO:2) amino-acid residue; Comprise about Glu-223 to Tyr-246 SEQ ID NO:2) polypeptide of amino-acid residue; The polypeptide that comprises about Ser-271 to Phe-278 (SEQ ID NO:2) amino-acid residue.
19. isolated antibody with the Neutrokine-α polypeptide specific combination of claim 17.
20. one kind comprises the polypeptide of claim 17 and the pharmaceutical composition of pharmacology acceptable carrier.
21. the proteic isolating polynucleotide of Neutrokine-α of encoding and modifying, wherein except at least one conserved amino acid replaced, the peptide of described modification had and is selected from the identical aminoacid sequence of the sequence of next group:
(a) the 1-285 amino acids of SEQ ID NO:2;
(b) the 2-285 amino acids of SEQ ID NO:2;
(c) the 1-46 amino acids of SEQ ID NO:2;
(d) the 47-72 amino acids of SEQ ID NO:2; With
(e) the 73-286 amino acids of SEQ ID NO:2.
22. the Neutrokine-α peptide molecule of a modification, wherein except at least one conserved amino acid replaced, the peptide of described modification had and is selected from the identical aminoacid sequence of the sequence of next group:
(a) the 1-285 amino acids of SEQ ID NO:2;
(b) the 2-285 amino acids of SEQ ID NO:2;
(c) the 1-46 amino acids of SEQ ID NO:2;
(d) the 47-72 amino acids of SEQ ID NO:2; With
(e) the 73-286 amino acids of SEQ ID NO:2.
23. an isolated nucleic acid molecule comprises and has and be selected from the polynucleotide that the sequence of at least 95% homogeny is arranged with the sequence of next group:
(a) nucleotide sequence of SEQ ID NO:7:
(b) nucleotide sequence of SEQ ID NO:8:
(c) nucleotide sequence of SEQ ID NO:9:
(d) nucleotide sequence of the part of sequence shown in Figure 1A and the 1B (SEQ ID No:1), wherein said part comprises at least 30 continuous nucleotides of 1-2442 position Nucleotide, but the sequence that does not comprise 1387-1421 position Nucleotide, the sequence of 9-382 position Nucleotide, the sequence of 1674-1996 position Nucleotide, the sequence of 1401-1784 position Nucleotide, the sequence of 900-1237 position Nucleotide, and be positioned at any fragment of these sequences; And
(e) be complementary to above-mentioned (a), (b), the nucleotide sequence of any nucleotide sequence (c) or (d).
24. an isolated nucleic acid molecule comprises and has and be selected from the polynucleotide that the nucleotide sequence of at least 95% homogeny is arranged with the sequence of next group:
(a) coding has the nucleotide sequence of the Neutrokine-α SV polypeptide of complete amino acid sequence shown in Fig. 5 A and the 5B (SEQ ID NO:19);
(b) coding has the nucleotide sequence by the Neutrokine-α SV polypeptide of the complete amino acid sequence of the contained cDNA clones coding of the preservation thing of ATCC preserving number 203518;
(c) nucleotide sequence of coding Neutrokine-α SV polypeptide extracellular domain;
(d) nucleotide sequence of coding Neutrokine-α SV polypeptide membrane-spanning domain;
(e) nucleotide sequence of coding Neutrokine-α SV polypeptide born of the same parents internal area;
(f) coding comprises extracellular domain and born of the same parents' internal area, but the nucleotide sequence of the soluble Neutrokine-α SV polypeptide of disappearance membrane-spanning domain; And
(g) be complementary to above-mentioned (a), (b), (c) (d), the nucleotide sequence of any nucleotide sequence (e) or (f).
25. the isolated antibody of claim 19, wherein said isolated antibody suppress the albumen of SEQ IDNO:2 and combining of Neutrokine-α acceptor.
26. an isolating protein comprises the Neutrokine-α albumen that is blended in toxin protein, wherein said Neutrokine-α albumen comprises the aminoacid sequence that is selected from next group:
(a) aminoacid sequence of the 134-285 amino acids residue of SEQ ID NO:2;
(b) aminoacid sequence of at least 90% homogeny is arranged with the 134-285 amino acids residue of SEQ ID NO:2; With
(c) aminoacid sequence of the polypeptide fragment of SEQ ID NO:2, wherein said segmental length is at least 30 amino acid, and described fragment can stimulate propagation, differentiation or the survival of B cell.
27. an in vitro method that kills bone-marrow-derived lymphocyte, it comprises the protein that makes described bone-marrow-derived lymphocyte contact claim 26.
28. the protein of claim 26 is used for killing the purposes of the medicine of bone-marrow-derived lymphocyte in preparation.
29. a method for the treatment of B cell tumour or autoimmune disorder, it comprises the described protein to the proteinic patient's administering therapeutic significant quantity that needs claim 26.
30. the method for claim 29, wherein said B cell tumour is selected from:
(a) non-Hodgkin ' s lymphoma;
(b) multiple myeloma;
(c) lymphocytic leukemia (CLL);
(d) acute lymphoblastic leukemia (ALL); With
(e) plasmoma.
31. the method for claim 29, wherein said autoimmune disorder is selected from:
(a) rheumatoid arthritis;
(b) systemic lupus erythematous;
(c) multiple sclerosis;
(d) myasthenia gravis;
(e) Sjogren ' s syndrome;
(f) type 1 diabetes;
(g) idiopathic thrombocytopenic purpura;
(h) Gullian-Barre syndrome;
(i) Hashimoto ' s thyroiditis; With
(j) Graves ' disease.
32. the protein of claim 26, wherein said toxin protein is selected from:
(a) ribosome inactivating protein;
(b) cytotoxin; With
(c) cellular toxicity prodrug.
33. an in vitro method that kills bone-marrow-derived lymphocyte, it comprises makes the described protein of described bone-marrow-derived lymphocyte contact claim 32.
34. the protein of claim 32 is used for killing the purposes of the medicine of bone-marrow-derived lymphocyte in preparation.
35. a method for the treatment of B cell tumour or autoimmune disorder, it comprises the described protein to the proteinic patient's administering therapeutic significant quantity that needs claim 32.
36. the method for claim 35, wherein said B cell tumour is selected from:
(a) non-Hodgkin ' s lymphoma;
(b) multiple myeloma;
(c) lymphocytic leukemia (CLL);
(d) acute lymphoblastic leukemia (ALL); With
(e) plasmoma.
37. the method for claim 35, wherein said autoimmune disorder is selected from:
(a) rheumatoid arthritis;
(b) systemic lupus erythematous;
(c) multiple sclerosis;
(d) myasthenia gravis;
(e) Sjogren ' s syndrome;
(f) type 1 diabetes;
(g) idiopathic thrombocytopenic purpura;
(h) Gullian-Barre syndrome;
(i) Hashimoto ' s thyroiditis; With
(i) Graves ' disease.
38. the protein of claim 26, wherein said toxin protein are thymidine kinase, endonuclease, RNA enzyme, alpha toxin, Ricin, abrin, ETA, diphtheria toxin, saporin, monordin, Bai Shusu, Pokeweed antiviral protein, α sarcina or Toxins,exo-, cholera.
39. an in vitro method that kills bone-marrow-derived lymphocyte, it comprises the protein that makes described bone-marrow-derived lymphocyte contact claim 38.
40. the protein of claim 39 is used for killing the purposes of the medicine of bone-marrow-derived lymphocyte in preparation.
41. a method for the treatment of B cell tumour or autoimmune disorder, it comprises the described protein to the proteinic patient's administering therapeutic significant quantity that needs claim 38.
42. the method for claim 41, wherein said B cell tumour is selected from:
(a) non-Hodgkin ' s lymphoma;
(b) multiple myeloma;
(c) lymphocytic leukemia (CLL);
(d) acute lymphoblastic leukemia (ALL); With
(e) plasmoma.
43. the method for claim 41, wherein said autoimmune disorder is selected from:
(a) rheumatoid arthritis;
(b) systemic lupus erythematous;
(c) multiple sclerosis;
(d) myasthenia gravis;
(e) Sjogren ' s syndrome;
(f) type 1 diabetes;
(g) idiopathic thrombocytopenic purpura;
(h) Gullian-Barre syndrome;
(i) Hashimoto ' s thyroiditis; With
(j) Graves ' disease.
44. an isolating protein comprises the Neutrokine-α albumen that is blended in toxin protein, wherein said Neutrokine-α albumen comprises the aminoacid sequence that is selected from next group:
(a) aminoacid sequence of the 134-285 amino acids residue of SEQ ID NO:2;
(b) aminoacid sequence of at least 90% homogeny is arranged with the 134-285 amino acids residue of SEQ ID NO:2; With
(c) aminoacid sequence of the polypeptide fragment of SEQ ID NO:2, wherein said segmental length is at least 30 amino acid, and described fragment can stimulate propagation, differentiation or the survival of B cell; And wherein said toxin protein comprises the aminoacid sequence that is selected from next group:
(i) comprise the polypeptide of total length Bai Shusu polypeptide;
The polypeptide that (ii) comprises the 47-297 amino acids of total length Bai Shusu polypeptide;
(iii) comprise the polypeptide of the 47-297 amino acids of total length Bai Shusu polypeptide, wherein 297 aspartic acid becomes halfcystine; With
(iv) with (i), (ii) or polypeptide (iii) have the polypeptide of 90% homogeny at least.
45. an in vitro method that kills bone-marrow-derived lymphocyte, it comprises the protein that makes described bone-marrow-derived lymphocyte contact claim 44.
46. the protein of claim 44 is used for killing the purposes of the medicine of bone-marrow-derived lymphocyte in preparation.
47. a method for the treatment of B cell tumour or autoimmune disorder, it comprises the described protein to the proteinic patient's administering therapeutic significant quantity that needs claim 44.
48. the method for claim 47, wherein said B cell tumour is selected from:
(a) non-Hodgkin ' s lymphoma;
(b) multiple myeloma;
(c) lymphocytic leukemia (CLL);
(d) acute lymphoblastic leukemia (ALL); With
(e) plasmoma.
49. the method for claim 47, wherein said autoimmune disorder is selected from:
(a) rheumatoid arthritis;
(b) systemic lupus erythematous;
(c) multiple sclerosis;
(d) myasthenia gravis;
(e) Sjogren ' s syndrome;
(f) type 1 diabetes;
(g) idiopathic thrombocytopenic purpura;
(h) Gullian-Barre syndrome;
(i) Hashimoto ' s thyroiditis; With
(j) Graves ' disease.
50. an antibody, it is by the hybridoma secretion of ATCC preserving number PTA-1159 or PTA-1158.
51. an antibody or its part, it comprises the VH and the VL structural domain of the antibody of claim 50.
52. an antibody or its part, it comprises all the VH complementary determining regions and the VL complementary determining region of the antibody of claim 50, and described antibody or its part specificity are in conjunction with proteic film combining form or the soluble form of SEQ IDNO:2.
53. an antibody or its part, it is with the antibody and the proteic film combining form of SEQ ID NO:2 or combining of soluble form of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% competitive inhibition claim 50.
54. each antibody or its part of claim 50-53, it is selected from:
(a) monoclonal antibody;
(b) chimeric antibody;
(c) humanized antibodies;
(d) people's antibody;
(e) single-chain antibody;
(f) Fab fragment; With
(g) F (ab ') 2 fragments.
55. each antibody or its part of claim 50-54, it is a mark.
56. the antibody of claim 55 or its part, wherein said mark is selected from:
(a) enzyme labelling;
(b) radio isotope;
(c) fluorescent mark; With
(d) vitamin H.
57. the antibody of claim 56 or its part, the wherein said radio isotope that is selected from next group that is labeled as:
(a) 125I；
(b) 121I；
(c) 131I；
(d) 112In; With
(e) 99Tc。
58. each antibody or its part of claim 50-54, wherein said antibody or its part are coupled to the treatment factor or cytotoxic factor.
59. the antibody of claim 58 or its part, the wherein said treatment factor or cytotoxic factor are selected from:
(a) metabolic antagonist;
(b) alkylating agent;
(c) microbiotic;
(d) somatomedin;
(e) cytokine;
(f) anti-angiogenesis;
(g) the antimitotic factor;
(h) anthracycline;
(i) toxin; With
(j) the apoptosis factor.
60. a pharmaceutical composition, it comprises each antibody or its part of claim 50-59.
61. a diagnosis composition, it comprises each antibody or its part of claim 50-59.
62. an isolated cells, it produces each antibody of claim 50-54.
63. a hybridoma, it produces the monoclonal antibody of claim 54.
64.ATCC the hybridoma of preserving number PTA-1159 or PTA-1158.
65. the proteic method of Neutrokine-α in the detection of biological sample, it comprises:
(a) make each antibody or its part of described biological sample contact claim 50-59; With
(b) detect described Neutrokine-α albumen.
66. each antibody or its part of claim 50-59 is used for the treatment of, prevents in preparation or diagnose purposes in the pharmaceutical composition of disease of immune system or immune system disorder.
67. each antibody or its part of claim 50-59 is used for the treatment of, prevents in preparation or diagnose purposes in the pharmaceutical composition of disease of immune system or immune system disorder, described disease or disorder are selected from:
(a) inflammatory diseases or disorder;
(b) leukemia;
(c) tumour;
(d) metastatic tumo(u)r; With
(e) lymphadenopathy.
68. each antibody or its part of claim 50-59 is used for the treatment of, prevents in preparation or diagnose purposes in the pharmaceutical composition of graft versus host disease (GVH disease) (GVHD).
69. each antibody or its part of claim 50-59 is used for the treatment of in preparation, the purposes in prevention or diagnosis of autoimmune disease or the disorderly pharmaceutical composition.
70. each antibody or its part of claim 50-59 is used for the treatment of in preparation, the purposes in prevention or diagnosis of autoimmune disease or the disorderly pharmaceutical composition, described disease or disorder are selected from:
(a) rheumatoid arthritis;
(b) systemic lupus erythematous;
(c) multiple sclerosis;
(d) Sjogren ' s syndrome;
(e) IgA nephropathy;
(f) glomerulonephritis;
(g) diabetes; With
(h) myasthenia gravis.
71. each antibody or its part of claim 50-59 is used for the treatment of, prevents in preparation or diagnose purposes in the pharmaceutical composition of immune deficiency.
72. each antibody or its part of claim 50-59 is used for the treatment of, prevents in preparation or diagnose purposes in the pharmaceutical composition of immune deficiency, described immune deficiency is selected from:
(a) common mutability immune deficiency (CVID);
(b) acquired immune deficiency syndrome (AIDS) (AIDS);
(c) Reconstruction in Sever Combined Immunodeciency (SCID);
(d) the very few disease of gamma-globulin in the blood; With
(e) Wiskott-Aldrich syndrome.
73. a pharmaceutical composition that is used for the treatment of the B cell tumour, it comprises each antibody or its part or claim 26,32,38 and 44 each protein of claim 50-53.
74. the pharmaceutical composition of claim 73, wherein the B cell tumour is selected from:
(a) non-Hodgkin ' s lymphoma;
(b) multiple myeloma;
(c) lymphocytic leukemia (CLL);
(d) acute lymphoblastic leukemia (ALL); With
(e) plasmoma.
75. a pharmaceutical composition that is used for the treatment of autoimmune disorder, it comprises each antibody or its part or claim 26,32,38 and 44 each protein of claim 50-53.
76. the pharmaceutical composition of claim 75, wherein said autoimmune disorder is selected from:
(a) rheumatoid arthritis;
(b) systemic lupus erythematous;
(c) multiple sclerosis;
(d) myasthenia gravis;
(e) Sjogren ' s syndrome;
(f) type 1 diabetes;
(g) idiopathic thrombocytopenic purpura;
(h) Gullian-Barre syndrome;
(i) Hashimoto ' s thyroiditis; With
(j) Graves ' disease.
CN2007101537856A 2000-01-14 2000-02-22 Neutrokine-alpha and Neutrokine-alpha splice variants Expired - Lifetime CN101210244B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US17601500P 2000-01-14 2000-01-14
US60/176,015 2000-01-14

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CNB008065411A Division CN100439499C (en) 1999-02-23 2000-02-22 Neutrokine-alpha and Neutrokine-alpha splice variant

Publications (2)

Publication Number Publication Date
CN101210244A true CN101210244A (en) 2008-07-02
CN101210244B CN101210244B (en) 2013-01-23

Family

ID=39610500

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2007101537856A Expired - Lifetime CN101210244B (en) 2000-01-14 2000-02-22 Neutrokine-alpha and Neutrokine-alpha splice variants

Country Status (2)

Country Link
CN (1) CN101210244B (en)
HK (1) HK1120077A1 (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1234833A (en) * 1996-10-25 1999-11-10 人体基因组科学有限公司 Nuetrokine 'alpha'
CA2232743A1 (en) * 1997-04-02 1998-10-02 Smithkline Beecham Corporation A tnf homologue, tl5

Also Published As

Publication number Publication date
CN101210244B (en) 2013-01-23
HK1120077A1 (en) 2009-03-20

Similar Documents

Publication Publication Date Title
US8071092B1 (en) Methods of inhibiting B lymphocytes using antibodies to Neutrokine-alpha
AU2005200237B2 (en) Neutrokine-alpha and neutrokine-alpha splice variant
US8212004B2 (en) Neutrokine-alpha fusion proteins
US8211649B2 (en) Methods of diagnosing and prognosing hodgkin&#39;s lymphoma
CA2476675A1 (en) Neutrokine-alpha and neutrokine-alpha splice variant
CA2419661A1 (en) Neutrokine-alpha and neutrokine-alpha splice variant
US20020115112A1 (en) Neutrokine-alpha and Neutrokine-alpha splice variant
CN101210244B (en) Neutrokine-alpha and Neutrokine-alpha splice variants
CN100439499C (en) Neutrokine-alpha and Neutrokine-alpha splice variant
KR100716444B1 (en) Neutrokine-alpha and neutrokine-alpha splice variant
MXPA01008565A (en) Neutrokine-alpha and neutrokine-alpha splice variant

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1120077

Country of ref document: HK

C14 Grant of patent or utility model
GR01 Patent grant
REG Reference to a national code

Ref country code: HK

Ref legal event code: GR

Ref document number: 1120077

Country of ref document: HK

CX01 Expiry of patent term
CX01 Expiry of patent term

Granted publication date: 20130123