WO2010043582A1 - Method for the treatment of cancer - Google Patents

Abstract

The present invention relates to combination therapies for the treatment of cancer using LNA oligomers targeting Bcl-2 in conjunction with CD-20 inhibitors, such as Rituximab, or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 11B8, 7D8, 2C6, Veltuzumab, AME-133v Ibritumomab Tiuxetan, Tositumomab, TRU-015, 2H7.vl6, Ocrelizumab, Pro131921, R7159, and GA-101.

Description

METHOD FOR THE TREATMENT OF CANCER

FIELD OF INVENTION

The present invention relates to combination therapies for the treatment of cancer using LNA oligomers targeting Bcl-2 mRNA in conjunction with CD-20 inhibitors.

BACKGROUND

Rituximab is sold under the trade names Rituxan, MabThera and Reditux, is a chimeric monoclonal antibody against the human protein CD-20. Rituximab is used in the treatment of B cell non-Hodgkin's lymphoma, B-cell leukemias and some autoimmune disorders.

Human Bcl-2 is a protein which is closely associated with the process of programmed cell death (apoptosis). Apoptosis is an active, tightly regulated physiological process involved in development, normal cell turnover, and hormone-induced tissue athropy. Lack of programmed cell death plays an important role in cancer and other hyperproliferative diseases. In contrast to most normal tissues, Bcl-2 is often over-expressed in cancer cells. An antisense oligonucleotide drug Genasense (G3139), which also targets the first six codons of the Bcl-2 mRNA has been developed to target Bcl-2. However, after disappointing results in a melanoma trial Genasense did not receive FDA approval. Jahrsdorfer et al., (2002) J. Leukocyte Biology 72 reports on the modulation of malignant B cell activation and apoptosis by Bcl-2 antisense oligonucleotides and immunostimulatory CpG oligonucleotides, and their results suggested that modulation of B cell apoptosis by G3139 depended on its immunostimulatory properties rather than on antisense-mediated reduction of Bcl-2 expression.

Wacheck et al., 2002, antisense and nucleic acid drug development 12:359-367) reports that G3139 chemosensitizes human malignancies by down-regulating Bcl-2 and that the antitumor effect of G3139 in human SCID mouse xenotransplantation model is independent of immune stimulation.

Loomis et al. (2003 Clinical cancer Research VoI 9, reports that G3139 enhances the in vitro and in vivo response of Epstein-Barr Virus-associated Lymphoproliferative disease to Rituximab.

Smith et al (2004) MoI cancer Ther 3(12) reports on the enhanced efficacy of therapy with antisense Bcl-2 oligonucleotides plus anti-CD-20 monoclonal antibody (Rituximab) in scid mouse/human lymphoma xenographs. US20040147473 reports on the use of Bcl-2 antisense oligomers, such as G3139, to treat and prevent bcl-2 related disorders in combination with immunotherapeutics, such as Rituximab.

Ramanarayanan et al. (2004) British J of Haematology 127 reports that pro-apoptotic therapy with the oligonucleotide GenasenseTM (Oblimersen sodium) targeting Bcl-2 protein expression enhances the biological anti-tumor activity of rituximab.

Jahrsdorfer et al., (2005) J. Leukocyte Biology VoI 77 reports on immunostimulatory oligodeoxynucleotides which induce apoptosis of B cell chronic lymphocytic leukemia cells, and concludes that antisense oligonucleotides including CpG oligonucleotides can induce apoptosis of most B CLL samples

Mays et a/. (2008) J Clin Oncol 26 reports on a short IV infusion of Oblimersen in patients with solit tumors. It was concluded that Oblimersen could be safely administered by short IV infusion with weekly doses of 300-900mgs,

Pro et al., British J of Haematology (2008) reports on a clinical trial where Oblimersen sodium (Bcl-2 antisense - Genta G3139) was evaluated in relapsed/refractory B-cell non- Hodgkin lymphoma (NHL) patients as a continuous intravenous infusion at a daily dose of 3 mg/kg/day for 7 days on alternative weeks for three weeks. Rituximab was given at a weekly dose of 375 mg/m²for six doses. Oblimersen sodium appeared to be most beneficial in patients with indolent NHL. WO 2005/061710 discloses 16 nucleobase phosphorothioate LNA gapmers which comprises a target binding domain that is specifically hybridizable to a region ranging from base position No. 1459 (5') to No. 1476 (3') of the human Bcl-2 mRNA (a region corresponding to the first six codons).

There is therefore a need for improved combination therapies which target CD-20 and Bcl-2 in malignant cells.

SUMMARY OF INVENTION

The invention relates to the enhanced efficacy of therapy of anti-CD-20 antibodies, such as Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 11 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab ,

TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA-101 , with Locked nucleic acid antisense oligonucleotides targeting Bcl-2 particularly for use in the treatment of cancer.

The invention provides for the use of a LNA oligomer targeting Bcl-2 for the preparation of a medicament, wherein said medicament is for the use in the treatment of cancer in combination with an inhibitor of CD-20, and wherein the LNA oligomer targeting Bcl-2 is an LNA oligomer targeting BCL-2.

The invention provides for a medicament comprising a LNA oligomer targeting Bcl-2, wherein said medicament is for the use in the treatment of cancer in combination with an inhibitor of CD-20, and wherein the LNA oligomer targeting Bcl-2 is an LNA oligomer targeting BCL-2, wherein the CD-20 inhibitor and the LNA oligomer targeting Bcl-2 is an LNA oligomer targeting BCL-2.

The invention provides for a pharmaceutical composition comprising an inhibitor of CD-20 and an LNA oligomer targeting BCL-2, and a pharmaceutically acceptable diluent, carrier, or salt.

The invention provides for a kit for use in the treatment of cancer, said kit comprising an inhibitor of CD-20 and an LNA oligomer targeting BCL-2.

The invention provides for a method for the treatment of cancer, said method comprising the steps of i) administering an LNA oligomer targeting Bcl-2 and ii) administering an inhibitor of CD-20 to a patient who is in need for the treatment of cancer.

The invention provides for a method for the concurrent inhibition of Bcl-2 and CD-20 in a cell, said method comprising administering a LNA oligomer targeting Bcl-2 and a CD-20 inhibitor to said cell

The invention provides a method of down-regulating or inhibiting the expression of BCL-2 protein and/or mRNA and CD-20 protein in a cell which is expressing BCL-2 protein and/or CD-20 protein mRNA, said method comprising administering i) the LNA oligomer or conjugate thereof, to said cell (to down-regulate or inhibit the expression of BCL-2 protein and/or mRNA in said cell); and ii) an inhibitor of CD-20 to said cell. Suitably the cell is a mammalian cell such as a human cell. The administration may occur, in some embodiments, in vitro. The administration may occur in some embodiments, in vivo.

BRIEF DESCRIPTION OF FIGURES

Figure 1 : Treatment effects on survival time of SCID mice bearing disseminated Raji human lymphoma. The mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 (control) at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 1 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D11 , D14, D18 and D21 (TWx3). Figure 2: Mean hCD45 positive cells in bone marrow of 4 SCID mice out of 12 bearing disseminated Raji human lymphoma at day 18. The mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 (control) at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 1 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D1 1 , D14, D18 and D21 (TWx3).

Figure 3: Treatment effects on survival time of SCID mice bearing disseminated Namalwa human lymphoma. The mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 (control) at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 1 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D11 , D14, D18 and D21 (TWx3). Figure 4: Mean hCD45 positive cells in bone marrow of 4 SCID mice out of 12 bearing disseminated Namalwa human lymphoma at day 14. The mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 (control) at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 6 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D1 1 , D14, D18 and D21 (TWx3). Figure 5: LNA oligomer targeting Bcl2 Dosing schedule, e.g. for SEQ ID No 2, at a dose of at least 2 mg/kg, such as 4 - 6mg/kg is given i.v. weekly for two 5-week treatment periods with a 4-week treatment-free period in-between (each (unit) dose is indicated by an arrow and is performed over a period of about 2 hours). During the first treatment period only, patients receive a standard dose of the CD-20 inhibitor, such as 375 mg/m² Rituximab, weekly for 4 weeks beginning on Week 2.

DETAILED DESCRIPTION OF INVENTION

The LNA Oligomer

The present invention employs LNA oligomeric compounds (referred herein as LNA oligomers), for use in modulating the function of nucleic acid molecules encoding mammalian BCL-2, such as the BCL-2 nucleic acid shown in SEQ ID 1 , and naturally occurring variants of such nucleic acid molecules encoding mammalian BCL-2.

The term "LNA oligomer" in the context of the present invention, refers to a molecule formed by covalent linkage of 10 - 30, such as 10 - 20 or 12 - 18 contiguous nucleotides (i.e. an oligonucleotide) which comprises at least one LNA unit. At least one of the nucleotides in the contiguous nucleotide sequence of the LNA oligomer is an LNA nucleotide. In some embodiments the contiguous nucleotide sequence of the LNA oligomer consists of nucleotides selected from LNA nucleotides and DNA nucleotides. In some embodiments the LNA oligomer is a gapmer as described herein.

Preferred LNA oligomers are disclosed herein and include, for example SEQ ID NO 2. The LNA oligomer can, suitably, be capable of down-regulating expression of the BCL- 2 gene in a mammalian, such as a human cell. In this regards, the LNA oligomer can affect the inhibition of BCL-2, typically in a mammalian such as a human cell. In some embodiments, the LNA oligomer binds (specifically hybridises) to the target nucleic acid and effect inhibition of expression of at least 10% or 20% compared to the normal expression level, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition compared to the normal expression level. In some embodiments, such modulation is seen when using between 0.04 and 25nM, such as between 0.8 and 2OnM concentration of the LNA oligomer. In the same or a different embodiment, the inhibition of expression is less than 100%, such as less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, such as less than 70% inhibition. Modulation of expression level may be determined by measuring protein levels, e.g. by the methods such as SDS-PAGE followed by western blotting using suitable antibodies raised against the target protein. Alternatively, modulation of expression levels can be determined by measuring levels of mRNA, e.g. by northern blotting or quantitative RT-PCR. When measuring via mRNA levels, the level of down-regulation when using an appropriate dosage, such as between 0.04 and 25nM, such as between 0.8 and 2OnM concentration, is, in some embodiments, typically to a level of between 10-20% the normal levels in the absence of the LNA oligomer. The term "target nucleic acid", as used herein with respect to the LNA oligomer, refers to the DNA or RNA encoding mammalian BCL-2 polypeptide, such as human BCL-2, such as SEQ ID NO: 1. BCL-2 encoding nucleic acids or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, preferably mRNA, such as pre-mRNA, although preferably mature mRNA. The LNA oligomer according to the invention is preferably capable of hybridising to the target nucleic acid. It will be recognised that SEQ ID NO: 1 is a cDNA sequence, and as such, corresponds to the mature mRNA target sequence, although uracil is replaced with thymidine in the cDNA sequences.

The term "naturally occurring variant thereof" refers to variants of the BCL-2 polypeptide of nucleic acid sequence which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, rat, monkey, and preferably human. Typically, when referring to "naturally occurring variants" of a polynucleotide the term also may encompass any allelic variant of the BCL-2 encoding genomic DNA which are found at the Chromosome 18: 58.94 - 59.14 Mb (NM 000633) by chromosomal translocation or duplication, and the RNA, such as mRNA derived therefrom. "Naturally occurring variants" may also include variants derived from alternative splicing of the BCL-2 mRNA. When referenced to a specific polypeptide sequence, e.g., the term also includes naturally occurring forms of the protein which may therefore be processed, e.g. by co- or post- translational modifications, such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc. The LNA oligomer targeted to the target sequence (Bcl-2) may comprise or consist of a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to the equivalent region of a nucleic acid which encodes a mammalian BCL-2 (e.g., SEQ ID NO: 1 ). However, in some embodiments, the LNA oligomer targeted to Bcl-2 may tolerate one or even, in some embodiments up to two, mismatches, when hybridising to the target sequence and still sufficiently bind to the target to show the desired effect, i.e. down- regulation of the target. The LNA oligomers comprise or consist of a contiguous nucleotide sequence which corresponds to the reverse complement of the target sequence, such as corresponds to a nucleotide sequence present in SEQ ID NO: 1. However, as illustrated by SEQ ID NO 2, for example, said oligomer (or contiguous nucleotide portion thereof) may optionally have one, mismatch against said the corresponding nucleotide sequence present in target sequence.

In some embodiments, the contiguous nucleotide sequence comprises no more than a single mismatch when hybridizing to the target sequence, such as the corresponding region of a nucleic acid which encodes a mammalian BCL-2. The nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 90% homologous to the reverse complement of a corresponding sequence present in SEQ ID NO: 1 , such at least 91 %, at least 92%at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%, at least 98%, at least 99%, such as 100% homologous (identical). When determining "homology" between the oligomers of the invention (or contiguous nucleotide sequence) and the nucleic acid which encodes the mammalian BCL-2 or the reverse complement thereof, such as those disclosed herein, the determination of homology may be made by a simple alignment with the corresponding nucleotide sequence of the compound of the invention and the corresponding region of the nucleic acid which encodes the mammalian BCL-2 (or target nucleic acid), or the reverse complement thereof, and the homology is determined by counting the number of bases which align and dividing by the total number of contiguous nucleotides in the compound of the invention, and multiplying by 100. In such a comparison, if gaps exist, it is preferable that such gaps are merely mismatches rather than areas where the number of nucleotides within the gap differs between the nucleotide sequence of the invention and the target nucleic acid. The terms "corresponding to" and "corresponds to" refer to the comparison between the nucleotide sequence of the oligomer (i.e. the nucleobase or base sequence) or contiguous nucleotide sequence (a first region) and the equivalent contiguous nucleotide sequence of a sub-sequence of the reverse complement of the nucleic acid target, such as the mRNA which encodes theBCL-2 protein, such as SEQ ID NO: 1 , The terms

"corresponding nucleotide analogue" and "corresponding nucleotide" are intended to indicate that the nucleotide in the nucleotide analogue and the naturally occurring nucleotide are identical. For example, when the 2-deoxyribose unit of the nucleotide is linked to an adenine, the "corresponding nucleotide analogue" contains a pentose unit (different from 2- deoxyribose) linked to an adenine.

The terms "reverse complement", "reverse complementary" and "reverse complementarity" as used herein are interchangeable with the terms "complement", "complementary" and "complementarity".

In some embodiments, the terms "nucleoside", "nucleotide", "unit" and "monomer" are used interchangeably. It will be recognised that when referring to a sequence of nucleotides or monomers, what is referred to is the sequence of bases, such as A, T, G, C or U.

Length

The LNA oligomers comprise or consist of a contiguous nucleotide sequence of, for example a total of 12, 13, 14, 15, 16, 17, or 18 contiguous nucleotides in length.

In some embodiments, the LNA oligomers comprise or consist of a contiguous nucleotide sequence of a total of 12 - 18, such as 13 - 17 or 12 - 16, such as 13, 14, 15, 16 contiguous nucleotides in length.

In some embodiments, the LNA oligomers comprise or consist of a contiguous nucleotide sequence of a total of 10, 1 1 , 12, 13, or 14 contiguous nucleotides in length.

In some embodiments, the LNA oligomer consists of no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. In some embodiments the LNA oligomer comprises less than 18 nucleotides.

LNA The term "LNA" refers to a bicyclic nucleoside analogue, known as "Locked Nucleic

Acid". It may refer to an LNA monomer, or, when used in the context of an "LNA oligonucleotide", LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues. LNA nucleotides are characterised by the presence of a linker group (such as a bridge) between C2' and C4' of the ribose sugar ring - for example as shown as the biradical R^4* - R^2* as described below. The LNA used in the oligonucleotide compounds of the invention preferably has the structure of the general formula I

Formula 1 wherein for all chiral centers, asymmetric groups may be found in either R or S orientation; wherein X is selected from -O-, -S-, -N(R^N*)-, -C(R⁶R^6*)-, such as, in some embodiments -O-;

B is selected from hydrogen, optionally substituted Ci_-4-alkoxy, optionally substituted d-₄-alkyl, optionally substituted Ci_-4-acyloxy, nucleobases including naturally occurring and nucleobase analogues, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; preferably, B is a nucleobase or nucleobase analogue;

P designates an internucleotide linkage to an adjacent monomer, or a 5'-terminal group, such internucleotide linkage or 5'-terminal group optionally including the substituent R⁵ or equally applicable the substituent R^5*;

P^* designates an internucleotide linkage to an adjacent monomer, or a 3'-terminal group;

R^4* and R^2* together designate a bivalent linker group consisting of 1 - 4 groups/atoms selected from -C(R^aR^b)-, -C(R^a)=C(R^b)-, -C(R^a)=N-, -O-, -Si(R^a)₂-, -S-, -SO₂-, -N(R^a)-, and >C=Z, wherein Z is selected from -O-, -S-, and -N(R^a)-, and R^a and R^b each is independently selected from hydrogen, optionally substituted Ci.-i₂-alkyl, optionally substituted C_2-i₂-alkenyl, optionally substituted C_2-i₂-alkynyl, hydroxy, optionally substituted Ci.-i₂-alkoxy, C_2--_I2- alkoxyalkyl, C_2-i₂-alkenyloxy, carboxy, Ci_i₂-alkoxycarbonyl, Ci-i₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_-6-alkyl)amino, carbamoyl, mono- and di(Ci_-6- alkyl)-amino-carbonyl, amino-Ci-β-alkyl-aminocarbonyl, mono- and di(Ci.6-alkyl)amino-Ci-6- alkyl-aminocarbonyl, d-6-alkyl-carbonylamino, carbamido, Ci_-6-alkanoyloxy, sulphono, Ci_-6- alkylsulphonyloxy, nitro, azido, sulphanyl, d-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂), wherein for all chiral centers, asymmetric groups may be found in either R or S orientation, and; each of the substituents R^1*, R², R³, R⁵, R^5*, R⁶ and R^6*, which are present is independently selected from hydrogen, optionally substituted d-₁₂-alkyl, optionally substituted C_2-i₂-alkenyl, optionally substituted C_2-i₂-alkynyl, hydroxy, d-₁₂-alkoxy, C_2--_I2- alkoxyalkyl, C_2-i₂-alkenyloxy, carboxy, d-i₂-alkoxycarbonyl, d-i₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6-alkyl)amino, carbamoyl, mono- and di(d-6- alkyl)-amino-carbonyl, amino-Ci-6-alkyl-aminocarbonyl, mono- and di(Ci_-6-alkyl)amino-Ci.6- alkyl-aminocarbonyl, d-6-alkyl-carbonylamino, carbamido, Ci_-6-alkanoyloxy, sulphono, Ci_-6- alkylsulphonyloxy, nitro, azido, sulphanyl, Ci_-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene; ; wherein R^N is selected from hydrogen and Ci_-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and R^N*, when present and not involved in a biradical, is selected from hydrogen and d-₄-alkyl; and basic salts and acid addition salts thereof. For all chiral centers, asymmetric groups may be found in either R or S orientation.

In some embodiments, R^4* and R^2* together designate a biradical consisting of a groups selected from the group consisting of C(R^aR^b)-C(R^aR^b)-, C(R^aR^b)-O-, C(R^aR^b)-NR^a-, C(R^aR^b)-S-, and C(R^aR^b)-C(R^aR^b)-O-, wherein each R^a and R^b may optionally be independently selected. In some embodiments, R^a and R^b may be, optionally independently selected from the group consisting of hydrogen and ci-βalkyl, such as methyl, such as hydrogen. In some embodiments, R^1*, R², R³, R⁵, R^5* are independently selected from the group consisting of hydrogen, halogen, Ci_-6alkyl, substituted Ci_-6 alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl or substituted C_2-6 alkynyl, Ci_-6alkoxyl, substituted Ci_-6 alkoxyl, acyl, substituted acyl, d-β aminoalkyl or substituted d-βaminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation. In some embodiments, R^1*, R², R³, R⁵, R^5* are hydrogen. In some embodiments, R^1*, R², R³ are independently selected from the group consisting of hydrogen, halogen, Ci_-6alkyl, substituted Ci_-6 alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl or substituted C_2-6 alkynyl, Ci_-6alkoxyl, substituted Ci_-6 alkoxyl, acyl, substituted acyl, C_1-6 aminoalkyl or substituted C_1-6aminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation. In some embodiments, R^1*, R², R³ are hydrogen.

In some embodiments, R⁵ and R^5* are each independently selected from the group consisting of H, -CH₃, -CH₂-CH₃,- CH₂-O-CH₃, and -CH=CH₂. Suitably in some embodiments, either R⁵ or R^5* are hydrogen, where as the other group (R⁵ or R^5* respectively) is selected from the group consisting of Ci_-5 alkyl, C_2-6 alkenyl, C_2-6alkynyl, substituted Ci_-6 alkyl, substituted C_2-6 alkenyl, substituted C_2-6alkynyl or substituted acyl (- C(=O)-); wherein each substituted group is mono or poly substituted with substituent groups independently selected from halogen, Ci_-6 alkyl, substituted Ci_-6 alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl, substituted C_2-6 alkynyl, OJi, SJi, NJ₁J₂, N₃, COOJi, CN, 0-CC=O)NJ₁J₂, N(H)C(=NH)NJ,J₂ or N(H)C(=X)N(H)J₂ wherein X is O or S; and each J₁ and J₂ is, independently, H, C_1-6 alkyl, substituted C_1-6alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl, substituted C_2-6 alkynyl, C_1-6 aminoalkyl, substituted C_1-6 aminoalkyl or a protecting group. In some embodiments either R⁵ or R^5* is substituted C_1-6alkyl. In some embodiments either R⁵ or R^5* is substituted methylene wherein preferred substituent groups include one or more groups independently selected from F, NJ₁J₂, N₃, CN, OJ₁, SJ₁, O-

CC=O)NJ₁J₂, N(H)C(=NH)NJ, J₂ or N(H)C(O)N(H)J₂. In some embodiments each J₁ and J₂ is, independently H or C_1-6 alkyl. In some embodiments either R⁵ or R^5* is methyl, ethyl or methoxymethyl. In some embodiments either R⁵ or R^5* is methyl. In a further embodiment either R⁵ or R^5* is ethylenyl. In some embodiments either R⁵ or R^5* is substituted acyl. In some embodiments either R⁵ or R^5* is C(=O)NJ-|J₂. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such 5' modified bicyclic nucleotides are disclosed in WO 2007/134181 , which is hereby incorporated by reference in its entirety.

In some embodiments B is a nucleobase, including nucleobase analogues and naturally occurring nucleobases, such as a purine or pyrimidine, or a substituted purine or substituted pyrimidine, such as a nucleobase referred to herein, such as a nucleobase selected from the group consisting of adenine, cytosine, thymine, adenine, uracil, and/or a modified or substituted nucleobase, such as 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, 2'thio-thymine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6- diaminopurine. In some embodiments, R^4* and R^2* together designate a biradical selected from - C(R^aR^b)-O-, -C(R^aR^b)-C(R^cR^d)-O-, -C(R^aR^b)-C(R^cR^d)-C(R^eR^f)-O-, -C(R^aR^b)-O-C(R^cR^d)-, - C(R^aR^b)-O-C(R^cR^d)-O-, -C(R^aR^b)-C(R^cR^d)-, -C(R^aR^b)-C(R^cR^d)-C(R^eR^f)-, - C(R^a)=C(R^b)-C(R^cR^d)-, -C(R^aR^b)-N(R^c)-, -C(R^aR^b)-C(R^cR^d)- N(R^e)-, -C(R^aR^b)-N(R^c)-O-, and - C(R^aR^b)-S-, -C(R^aR^b)-C(R^cR^d)-S-, wherein R^a, R^b, R^c, R^d, R^e, and R^f each is independently selected from hydrogen, optionally substituted Ci_i₂-alkyl, optionally substituted C_2-i₂-alkenyl, optionally substituted C_2-i₂-alkynyl, hydroxy, Ci_i₂-alkoxy, C_2-i₂-alkoxyalkyl, C_2-i₂-alkenyloxy, carboxy, Ci_i₂-alkoxycarbonyl, Ci_i₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6-alkyl)amino, carbamoyl, mono- and di(Ci_6-alkyl)-amino-carbonyl, amino- d-₆-alkyl-aminocarbonyl, mono- and di(Ci.₆-alkyl)amino-Ci.₆-alkyl-aminocarbonyl, Ci_-6-alkyl- carbonylamino, carbamido, Ci_-6-alkanoyloxy, sulphono, Ci_-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci_-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂). For all chiral centers, asymmetric groups may be found in either R or S orientation.

In a further embodiment R^4* and R^2* together designate a biradical (bivalent group) selected from -CH₂-O-, -CH₂-S-, -CH₂-NH-, -CH₂-N(CH₃)-, -CH₂-CH₂-O-, -CH₂-CH(CH₃)-, - CH₂-CH₂-S-, -CH₂-CH₂-NH-, -CH₂-CH₂-CH₂-, -CH₂-CH₂-CH₂-O-, -CH₂-CH₂-CH(CH₃)-, -

CH=CH-CH₂-, -CH₂-O-CH₂-O-, -CH₂-NH-O-, -CH₂-N(CH₃)-O-, -CH₂-O-CH₂-, -CH(CH₃)-O-, and -CH(CH₂-O-CH₃)-O-, and/or, -CH₂-CH₂-, and -CH=CH- For all chiral centers, asymmetric groups may be found in either R or S orientation.

In some embodiments, R^4* and R^2* together designate the biradical C(R^aR^b)-N(R^c)-0-, wherein R^a and R^b are independently selected from the group consisting of hydrogen, halogen, Ci_-6alkyl, substituted Ci_-6 alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl or substituted C_2-6 alkynyl, Ci_-6 alkoxyl, substituted Ci_-6 alkoxyl, acyl, substituted acyl, Ci_-6 aminoalkyl or substituted Ci_-6 aminoalkyl, such as hydrogen, and; wherein R^c is selected from the group consisting of hydrogen, halogen, Ci_-6 alkyl, substituted Ci_-6 alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl or substituted C_2-6 alkynyl, Ci_-6 alkoxyl, substituted Ci_-6 alkoxyl, acyl, substituted acyl, Ci_-6 aminoalkyl or substituted Ci_-6 aminoalkyl, such as hydrogen.

In some embodiments, R^4* and R^2* together designate the biradical C(R^aR^b)-0-C(R^cR^d) -0-, wherein R^a, R^b, R^c, and R^d are independently selected from the group consisting of hydrogen, halogen, Ci_-6 alkyl, substituted Ci_-6 alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl or substituted C₂-6alkynyl, Ci_-6alkoxyl, substituted Ci_-6 alkoxyl, acyl, substituted acyl, Ci_-6 aminoalkyl or substituted Ci-₆ aminoalkyl, such as hydrogen.

In some embodiments, R^4* and R^2* form the biradical -CH(Z)-O-, wherein Z is selected from the group consisting of Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, substituted Ci_-6 alkyl, substituted C_2-6 alkenyl, substituted C_2-6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio; and wherein each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, OJi, NJ₁J₂, SJi, N₃, OC(=X)Ji, OCC=X)NJ₁J₂, NJ³C^X)NJ₁J₂ and CN, wherein each J₁, J₂ and J₃ is, independently, H or C_1-6 alkyl, and X is O, S or NJ₁. In some embodiments Z is C_1-6 alkyl or substituted C_1-6 alkyl. In some embodiments Z is methyl. In some embodiments Z is substituted C_1-6 alkyl. In some embodiments said substituent group is C_1-G aIkOXy. In some embodiments Z is CH₃OCH₂-. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in US 7,399,845 which is hereby incorporated by reference in its entirety. In some embodiments, R^1*, R², R³, R⁵, R^5* are hydrogen. In some some embodiments, R^1*, R², R^{3 *} are hydrogen, and one or both of R⁵, R^5* may be other than hydrogen as referred to above and in WO 2007/134181.

In some embodiments, R^4* and R^2* together designate a biradical which comprise a substituted amino group in the bridge such as consist or comprise of the biradical -CH₂-N( R^c)-, wherein R^c is C₁ _ ₁₂ alkyloxy. In some embodiments R^4* and R^2* together designate a biradical -Cq₃q₄-NOR -, wherein q₃and q₄ are independently selected from the group consisting of hydrogen, halogen, C_1-6alkyl, substituted C_1-6 alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl or substituted C_2-6 alkynyl, C_1-6alkoxyl, substituted C_1-6 alkoxyl, acyl, substituted acyl, C_1-6 aminoalkyl or substituted C_1-6aminoalkyl; wherein each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, OJ₁, SJ₁, NJ₁J₂, COOJ₁, CN, 0-CC=O)NJ₁J₂, N(H)C(=NH)N J₁J₂ or N(H)C(=X=N(H)J₂ wherein X is O or S; and each of J₁ and J₂ is, independently, H, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C_1-6 aminoalkyl or a protecting group. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in WO2008/150729 which is hereby incorporated by reference in its entirity. In some embodiments, R^1*, R², R³, R⁵, R^5* are independently selected from the group consisting of hydrogen, halogen, C_1-6alkyl, substituted C_1-6 alkyl, C_2-6 alkenyl, substituted C_2-6 alkenyl, C_2-6 alkynyl or substituted C_2-6 alkynyl, C_1-6alkoxyl, substituted C_1-6 alkoxyl, acyl, substituted acyl, C_1-6 aminoalkyl or substituted C_1-6aminoalkyl. In some embodiments, R^1*, R², R³, R⁵, R^5* are hydrogen. In some embodiments, R^1*, R², R³ are hydrogen and one or both of R⁵, R^5* may be other than hydrogen as referred to above and in WO 2007/134181. In some embodiments R^4* and R^2* together designate a biradical (bivalent group) C(R^aR^b)-0-, wherein R^a and R^b are each independently halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-Ci₂ alkenyl, substituted C₂-Ci₂ alkenyl, C₂-Ci₂ alkynyl, substituted C₂-Ci₂ alkynyl, CrCi₂ alkoxy, substituted CrCi₂ alkoxy, OJi SJi, SOJi, SO₂Ji, NJiJ₂, N₃, CN, C(=O)OJi, C(=O)NJiJ₂, C(=O)Ji, O-C(=O)NJiJ₂, N(H)C(=NH)NJiJ₂, N(H)C(=O)NJiJ₂ or N(H)C(=S)NJiJ₂; or R^a and R^b together are =C(q3)(q4); q₃ and q₄ are each, independently, H, halogen, Ci-Ci₂alkyl or substituted CrCi₂ alkyl; each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, d- C₆ alkyl, substituted CrC₆ alkyl, C₂- C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, OJi, SJi, NJiJ₂, N₃, CN, C(=O)OJi, C(=O)NJiJ₂, C(=O)Ji, O- C(=O)NJiJ₂, N(H)C(=O)NJiJ₂ or N(H)C(=S)NJiJ₂ and; each J₁ and J₂ is, independently, H, CI-C₆ alkyl, substituted CI-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, CI-C₆ aminoalkyl, substituted CI-C₆ aminoalkyl or a protecting group. Such compounds are disclosed in WO2009006478A, hereby incorporated in its entirety by reference.

In some embodiments, R^4* and R^2* form the biradical - Q -, wherein Q is C(qi)(q₂)C(q₃)(q₄), C(qi)=C(q₃), C[=C(qi)(q₂)]-C(q₃)(q₄) or C(qi)(q₂)-C[=C(q₃)(q₄)]; qi, q₂, q₃, q₄ are each independently. H, halogen, C_M2 alkyl, substituted C_M2 alkyl, C_2-I2 alkenyl, substituted C_M2 alkoxy, OJi, SJi, SOJi, SO₂Ji, NJiJ₂, N₃, CN, C(=O)OJi, C(=O)-NJiJ₂,

C(=O) Ji, -C(=O)NJiJ₂, N(H)C(=NH)NJiJ₂, N(H)C(=O)NJiJ₂ or N(H)C(=S)NJiJ₂; each J₁ and J₂ is, independently, H, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, Ci_-6 aminoalkyl or a protecting group; and, optionally wherein when Q is C(qi)(q₂)(q₃)(q₄) and one of q₃ or q₄ is CH₃ then at least one of the other of q₃ or q₄ or one of qi and q₂ is other than H. In some embodiments, R^1*, R², R³, R⁵, R^5* are hydrogen. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in WO2008/154401 which is hereby incorporated by reference in its entirity. In some embodiments, R^1*, R², R³, R⁵, R^5* are independently selected from the group consisting of hydrogen, halogen, Ci_-6 alkyl, substituted Ci_-6 alkyl, C_2-6 alkenyl, substituted C_2-6alkenyl, C_2-6 alkynyl or substituted C_2-6 alkynyl, Ci_-6alkoxyl, substituted Ci_-6 alkoxyl, acyl, substituted acyl, Ci_-6 aminoalkyl or substituted Ci_-6 aminoalkyl. In some embodiments, R^1*, R², R³, R⁵, R^5* are hydrogen. In some embodiments, R^1*, R², R³ are hydrogen and one or both of R⁵, R^5* may be other than hydrogen as referred to above and in WO 2007/134181 or WO2009/067647 (alpha-L- bicyclic nucleic acids analogs). In some embodiments the LNA used in the oligonucleotide compounds of the invention preferably has the structure of the general formula II:

Formula II

wherein Y is selected from the group consisting of -O-, -CH₂O-, -S-, -NH-, N(R^e) and/or - CH₂-; Z and Z^* are independently selected among an internucleotide linkage, R^H, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety (nucleobase), and R^H is selected from hydrogen and Ci_-4-alkyl; R^a, R^b R^c, R^d and R^e are, optionally independently, selected from the group consisting of hydrogen, optionally substituted Ci_i₂-alkyl, optionally substituted C_2-i₂-alkenyl, optionally substituted C_2-i₂-alkynyl, hydroxy, Ci_i₂-alkoxy, C_2-i₂-alkoxyalkyl, C_2-i₂-alkenyloxy, carboxy, Ci_i₂-alkoxycarbonyl, C_M2- alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy- carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6-alkyl)amino, carbamoyl, mono- and di(Ci_-6-alkyl)-amino-carbonyl, amino-Ci-₆-alkyl-aminocarbonyl, mono- and di(Ci.6-alkyl)amino-Ci.6-alkyl-aminocarbonyl, d-6-alkyl-carbonylamino, carbamido, Ci_-6- alkanoyloxy, sulphono, Ci_-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci_-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂); and R^H is selected from hydrogen and C-_M-alkyl. In some embodiments R^a, R^b R^c, R^d and R^e are, optionally independently, selected from the group consisting of hydrogen and

alkyl, such as methyl. For all chiral centers, asymmetric groups may be found in either R or S orientation, for example, two exemplary stereochemical isomers include the beta-D and alpha-L isoforms, which may be illustrated as follows:

Specific exemplary LNA units are shown below:

β-D-oxy-LNA

β-D-amino-LNA

The term "thio-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from S or -CH₂-S-. Thio-LNA can be in both beta-D and alpha-L- configuration.

The term "amino-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from -N(H)-, N(R)-, CH₂-N(H)-, and -CH₂-N(R)- where R is selected from hydrogen and Ci_-4-alkyl. Amino-LNA can be in both beta-D and alpha-L- configuration.

The term "oxy-LNA" comprises a locked nucleotide in which Y in the general formula above represents -O-. Oxy-LNA can be in both beta-D and alpha-L-configuration.

The term "ENA" comprises a locked nucleotide in which Y in the general formula above is -CH₂-O- (where the oxygen atom of -CH₂-O- is attached to the 2'-position relative to the base B). R^e is hydrogen or methyl.

In some exemplary embodiments LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA. RNAse recruitment

It is recognised that an oligomeric compound may function via non RNase mediated degradation of target mRNA, such as by steric hindrance of translation, or other methods, however, the preferred oligomers of the invention are capable of recruiting an endoribonuclease (RNase), such as RNase H.

It is preferable that the LNA oligomer, or contiguous nucleotide sequence, comprises of a region of at least 6, such as at least 7 consecutive nucleotide units, such as at least 8 or at least 9 consecutive nucleotide units (residues), including 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 consecutive nucleotides, which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase. The contiguous sequence which is capable of recruiting RNAse may be region B as referred to in the context of a gapmer as described herein. In some embodiments the size of the contiguous sequence which is capable of recruiting RNAse, such as region B, may be higher, such as 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units. EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. A oligomer is deemed capable of recruiting RNase H if, when provided with the complementary RNA target, it has an initial rate, as measured in pmol/l/min, of at least 1 %, such as at least 5%, such as at least 10% or ,more than 20% of the of the initial rate determined using DNA only oligonucleotide, having the same base sequence but containing only DNA monomers, with no 2' substitutions, with phosphorothioate linkage groups between all monomers in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309. In some embodiments, an oligomer is deemed essentially incapable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less than 1%, such as less than 5%, such as less than 10% or less than 20% of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309. In other embodiments, an oligomer is deemed capable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is at least 20%, such as at least 40 %, such as at least 60 %, such as at least 80 % of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309. Typically the region of the oligomer which forms the consecutive nucleotide units which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase consists of nucleotide units which form a DNA/RNA like duplex with the RNA target - and include both DNA units and LNA units which are in the alpha-L configuration, particularly preferred being alpha-L-oxy LNA.

The oligomer of the invention may comprise or consist a nucleotide sequence which comprises both DNA nucleotides and LNA nucleotide, and may be in the form of a gapmer..

Gapmer Design

Preferably, the oligomer of the invention is a gapmer. A gapmer oligomer is an oligomer which comprises a contiguous stretch of nucleotides which is capable of recruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7 DNA nucleotides, referred to herein in as region B, wherein region B is flanked both 5' and 3' by regions of affinity enhancing nucleotide analogues, such as between 1 - 6 nucleotide analogues 5' and 3' to the contiguous stretch of nucleotides which is capable of recruiting RNAse - these regions are referred to as regions A and C respectively.

Preferably the gapmer comprises a (poly)nucleotide sequence of formula (5' to 3'), A- B-C, or optionally A-B-C-D or D-A-B-C, wherein; region A (5' region) consists or comprises of at least one nucleotide analogue, such as at least one LNA unit, such as between 1-6 nucleotide analogues, such as LNA units, and; region B consists or comprises of at least five consecutive nucleotides which are capable of recruiting RNAse (when formed in a duplex with a complementary RNA molecule, such as the mRNA target), such as DNA nucleotides, and; region C (3'region) consists or comprises of at least one nucleotide analogue, such as at least one LNA unit, such as between 1-6 nucleotide analogues, such as LNA units, and; region D, when present consists or comprises of 1 , 2 or 3 nucleotide units, such as DNA nucleotides.

In some embodiments, region A consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as between 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and/or region C consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as between 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.

In some embodiments B consists or comprises of 5, 6, 7, 8, 9, 10, 11 or 12 consecutive nucleotides which are capable of recruiting RNAse, or between 6-10, or between 7-9, such as 8 consecutive nucleotides which are capable of recruiting RNAse. In some embodiments region B consists or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably between 4-12 DNA units, more preferably between 6-10 DNA units, such as between 7-10 DNA units, most preferably 8, 9 or 10 DNA units.

In some embodiments region A consist of 3 or 4 nucleotide analogues, such as LNA, region B consists of 7, 8, 9 or 10 DNA units, and region C consists of 3 or 4 nucleotide analogues, such as LNA. Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and may further include region D, which may have one or 2 nucleotide units, such as DNA units.

Further gapmer designs are disclosed in WO2004/046160 and are hereby incorporated by reference. US provisional application, 60/977409, hereby incorporated by reference, refers to

'shortmer' gapmer oligomers, which, in some embodiments may be the gapmer oligomer according to the present invention.

In some embodiments the oligomer is consisting of a contiguous nucleotide sequence of a total of 10, 1 1 , 12, 13 or 14 nucleotide units, wherein the contiguous nucleotide sequence is of formula (5' - 3'), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; A consists of 1 , 2 or 3 nucleotide analogue units, such as LNA units; B consists of 7, 8 or 9 contiguous nucleotide units which are capable of recruiting RNAse when formed in a duplex with a complementary RNA molecule (such as a mRNA target); and C consists of 1 , 2 or 3 nucleotide analogue units, such as LNA units. When present, D consists of a single DNA unit.

In some embodiments A consists of 1 LNA unit. In some embodiments A consists of 2 LNA units. In some embodiments A consists of 3 LNA units. In some embodiments C consists of 1 LNA unit. In some embodiments C consists of 2 LNA units. In some embodiments C consists of 3 LNA units. In some embodiments B consists of 7 nucleotide units. In some embodiments B consists of 8 nucleotide units. In some embodiments B consists of 9 nucleotide units. In some embodiments B comprises of between 1 - 9 DNA units, such as 2, 3, 4, 5, 6, 7 or 8 DNA units. In some embodiments B consists of DNA units. In some embodiments B comprises of at least one LNA unit which is in the alpha-L configuration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in the alpha-L-configuration. In some embodiments B comprises of at least one alpha-L-oxy LNA unit or wherein all the LNA units in the alpha-L- configuration are alpha-L-oxy LNA units. In some embodiments the number of nucleotides present in A-B-C are selected from the group consisting of (nucleotide analogue units - region B - nucleotide analogue units): 1 -8-1 , 1 -8-2, 2-8-1 , 2-8-2, 3-8-3, 2-8- 3, 3-8-2, 4-8-1 , 4-8-2, 1-8-4, 2-8-4, or; 1-9-1 , 1-9-2, 2-9-1 , 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1 , 4- 9-1 , 1-9-4, or; 1-10-1 , 1-10-2, 2-10-1 , 2-10-2, 1-10-3, 3-10-1. In some embodiments the number of nucleotides in A-B-C are selected from the group consisting of: 2-7-1 , 1-7-2, 2-7- 2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. In some embodiments both A and C consists of two LNA units each, and B consists of 8 or 9 nucleotide units, preferably DNA units. lnternucleotide Linkages The monomers of the oligomers described herein are coupled together via linkage groups. Suitably, each monomer is linked to the 3' adjacent monomer via a linkage group. The person having ordinary skill in the art would understand that, in the context of the present invention, the 5' monomer at the end of an oligomer does not comprise a 5' linkage group, although it may or may not comprise a 5' terminal group. The terms "linkage group" or "internucleotide linkage" are intended to mean a group capable of covalently coupling together two nucleotides, two nucleotide analogues, and a nucleotide and a nucleotide analogue, etc. Specific and preferred examples include phosphate groups and phosphorothioate groups.

The nucleotides of the oligomer of the invention or contiguous nucleotides sequence thereof are coupled together via linkage groups. Suitably each nucleotide is linked to the 3' adjacent nucleotide via a linkage group.

Suitable internucleotide linkages include those listed within WO2007/031091 , for example the internucleotide linkages listed on the first paragraph of page 34 of WO2007/031091 (hereby incorporated by reference). It is, in some embodiments, preferred to modify the internucleotide linkage from its normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate - these two, being cleavable by RNase H, also allow that route of antisense inhibition in reducing the expression of the target gene.

Suitable sulphur (S) containing internucleotide linkages as provided herein may be preferred. Phosphorothioate internucleotide linkages are also preferred, particularly for the gap region (B) of gapmers. Phosphorothioate linkages may also be used for the flanking regions (A and C, and for linking A or C to D, and within region D, as appropriate).

Regions A, B and C, may however comprise internucleotide linkages other than phosphorothioate, such as phosphodiester linkages, particularly, for instance when the use of nucleotide analogues protects the internucleotide linkages within regions A and C from endo-nuclease degradation - such as when regions A and C comprise LNA nucleotides.

The internucleotide linkages in the oligomer may be phosphodiester, phosphorothioate or boranophosphate so as to allow RNase H cleavage of targeted RNA. Phosphorothioate is preferred, for improved nuclease resistance and other reasons, such as ease of manufacture. In one aspect of the oligomer of the invention, the nucleotides and/or nucleotide analogues are linked to each other by means of phosphorothioate groups.

It is recognised that the inclusion of phosphodiester linkages, such as one or two linkages, into an otherwise phosphorothioate oligomer, particularly between or adjacent to nucleotide analogue units (typically in region A and or C) can modify the bioavailability and/or bio-distribution of an oligomer - see WO2008/053314, hereby incorporated by reference.

In some embodiments, such as the embodiments referred to above, where suitable and not specifically indicated, all remaining linkage groups are either phosphodiester or phosphorothioate, or a mixture thereof.

In some embodiments all the internucleotide linkage groups are phosphorothioate.

When referring to specific gapmer oligonucleotide sequences, such as those provided herein it will be understood that, in various embodiments, when the linkages are phosphorothioate linkages, alternative linkages, such as those disclosed herein may be used, for example phosphate (phosphodiester) linkages may be used, particularly for linkages between nucleotide analogues, such as LNA, units. Likewise, when referring to specific gapmer oligonucleotide sequences, such as those provided herein, when the C residues are annotated as 5'methyl modified cytosine, in various embodiments, one or more of the Cs present in the oligomer may be unmodified C residues. Oligomeric Compounds

The oligomers of the invention may, for example, be selected from the group consisting of the LNA oligomers targeting the human Bcl-2 shown in table 1. The following LNA oligomers targeting Bcl-2, shown in Table 1 , are disclosed in WO2005/061710, and US provisional application US 61/012185, which are both hereby incorporated by reference. In the present application, the oligomeric compounds are referred to by means of the specified sequence number, e.g. "SEQ ID NO: 2". The compound "SEQ ID NO: 4" is also called Oblimersen sodium and is used herein as a reference compound.

In Table 1 , capital letters denote LNA nucleotides, superscript "α" denotes that the LNA nucleotide is an alpha-L-LNA nucleotide (i.e. an LNA analogue nucleotide), and subscript "S" denotes that the neighbouring nucleotides are preferably linked by a phosphorothioate group. All LNA-C monomers are preferably methyl-C. Unless otherwise shown, LNA is preferably beta-D-oxy LNA. M is a mismatched nucleobase - as compared to the corresponding nucleobase of SEQ ID NO 1.

In some embodiments the oligomer is selected from the group consisting of 2, and 5 - 34. In some embodiments the oligomer is SEQ ID NO 2. In some embodiments the oligomer is SEQ ID NO 12. In some embodiments is selected from an oligo of SEQ ID 35 - 67. In some embodiments the oligomer is selected from the group consisting of SEQ ID NO 36, 37, 38, 42, 43, 50 and 61. In some embodiments the oligomer is SEQ ID NO 36. In some embodiments the oligomer is SEQ ID NO 37. In some embodiments the oligomer is SEQ ID NO 38. In some embodiments the oligomer is SEQ ID NO 42. In some embodiments the oligomer is SEQ ID NO 43. In some embodiments the oligomer is SEQ ID NO 50. In some embodiments the oligomer is SEQ ID NO 61.

Conjugates

In the context the term "conjugate" is intended to indicate a heterogenous molecule formed by the covalent attachment ("conjugation") of the oligomer as described herein to one or more non-nucleotide, or non-polynucleotide moieties. Examples of non-nucleotide or non- polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically proteins may be antibodies for a target protein. Typical polymers may be polyethylene glycol.

Therefore, in various embodiments, the oligomer of the invention may comprise both a polynucleotide region which typically consists of a contiguous sequence of nucleotides, and a further non-nucleotide region. When referring to the oligomer of the invention consisting of a contiguous nucleotide sequence, the compound may comprise non-nucleotide components, such as a conjugate component.

In various embodiments of the invention the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of oligomeric compounds. WO2007/031091 provides suitable ligands and conjugates, which are hereby incorporated by reference.

The invention also provides for a conjugate comprising the compound according to the invention as herein described, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound. Therefore, in various embodiments where the compound of the invention consists of a specified nucleic acid or nucleotide sequence, as herein disclosed, the compound may also comprise at least one non-nucleotide or non- polynucleotide moiety (e.g. not comprising one or more nucleotides or nucleotide analogues) covalently attached to said compound. Conjugation (to a conjugate moiety) may enhance the activity, cellular distribution or cellular uptake of the oligomer of the invention. Such moieties include, but are not limited to, antibodies, polypeptides, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. H exy l-s-trity lth iol , a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-o- hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or a polyethylene glycol chain, an adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety.

The oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. In certain embodiments the conjugated moiety is a sterol, such as cholesterol.

In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example between 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol - see WO 2008/034123, hereby incorporated by reference. Suitably the positively charged polymer, such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable inker described in WO 2008/034123.

By way of example, the following conjugate moieties may be used in the conjugates of the invention: 5'- OLIGOMER -3'

5'- OLIGOMER -S'

Activated oligomers

The term "activated oligomer," as used herein, refers to an oligomer of the invention that is covalently linked (i.e., functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to one or more conjugated moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates herein described. Typically, a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3'-hydroxyl group or the exocyclic NH₂ group of the adenine base, a spacer that is preferably hydrophilic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group). In some embodiments, this terminal group is not protected, e.g., is an NH₂ group. In other embodiments, the terminal group is protected, for example, by any suitable protecting group such as those described in "Protective Groups in Organic Synthesis" by Theodora W. Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons, 1999). Examples of suitable hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoroacetyl. In some embodiments, the functional moiety is self- cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Patent No. 7,087,229, which is incorporated by reference herein in its entirety.

In some embodiments, oligomers of the invention are functionalized at the 5' end in order to allow covalent attachment of the conjugated moiety to the 5' end of the oligomer. In other embodiments, oligomers of the invention can be functionalized at the 3' end. In still other embodiments, oligomers of the invention can be functionalized along the backbone or on the heterocyclic base moiety. In yet other embodiments, oligomers of the invention can be functionalized at more than one position independently selected from the 5' end, the 3' end, the backbone and the base. In some embodiments, activated oligomers of the invention are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers of the invention are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis. In some embodiments, the oligomers are functionalized with a hindered ester containing an aminoalkyl linker, wherein the alkyl portion has the formula (CH₂)_W, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (-0-C(O)- (CH₂)_WNH).

In other embodiments, the oligomers are functionalized with a hindered ester containing a (CH₂)_w-sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (-O-C(O)-(CH₂)_WSH)

In some embodiments, sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can be synthesized by any method known in the art, and in particular by methods disclosed in PCT Publication No. WO 2008/034122 and the examples therein, which is incorporated herein by reference in its entirety.

In still other embodiments, the oligomers of the invention are functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a functionalizing reagent substantially as described in U.S. Patent Nos. 4,962,029 and 4,914,210, i.e., a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group. Such reagents primarily react with hydroxyl groups of the oligomer. In some embodiments, such activated oligomers have a functionalizing reagent coupled to a 5'-hydroxyl group of the oligomer. In other embodiments, the activated oligomers have a functionalizing reagent coupled to a 3'- hydroxyl group. In still other embodiments, the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Patent Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such functionalizing reagents and incorporating them into monomers or oligomers are disclosed in

U.S. Patent Nos. 4,962,029 and 4,914,210.

In some embodiments, the 5'-terminus of a solid-phase bound oligomer is functionalized with a dienyl phosphoramidite derivative, followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing 2'-sugar modifications, such as a 2'-carbamate substituted sugar or a 2'-(O-pentyl-N-phthalimido)- deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer. In other embodiments, an oligomer with an amino-containing linker at the 2'-position of one or more monomers is prepared using a reagent such as, for example, 5'-dimethoxytrityl-2'-O-(e-phthalimidylaminopentyl)-2'-deoxyadenosine-3'— N, N- diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters,

1991 , 34, 7171. In still further embodiments, the oligomers of the invention may have amine- containing functional moieties on the nucleobase, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine. In various embodiments, such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis. Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co.

(Rockford, III.). Other commercially available linking groups are 5'-Amino-Modifier C6 and

3'-Amino-Modifier reagents, both available from Glen Research Corporation (Sterling, Va.).

5'-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2, and 3'-Amino-Modifier is also available from Clontech Laboratories Inc.

(Palo Alto, Calif.).

The inhibitor of CD-20

The terms "CD20" and "CD20 antigen" are used interchangeably herein, and include any variants, isoforms and species homologs of human CD20, which are naturally expressed by cells or are expressed on cells transfected with the CD20 gene. Synonyms of

CD20, as recognized in the art, include B-lymphocyte surface antigen Bl, Leu-16 and Bp35.

Human CD20 has UniProtKB/Swiss-Prot entry P11836.

The CD20 inhibitor may be a CD-20 binding molecule, such as an antibody or fragment thereof. The term "CD20 binding molecules" as used herein refer to any molecule that specifically binds to a portion of CD20 under cellular and/or physiological conditions for an amount of time sufficient to inhibit the activity of CD20 expressing cells and/or otherwise modulate a physiological effect associated with CD20; to allow detection by ELISA, western blot, or other similarly suitable binding technique described herein and/or known in the art and/or to otherwise be detectably bound thereto after a relevant period of time (for instance at least about 15 minutes, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, such as about 1-24 hours, about 1-36 hours, about 1-48 hours, about 1-72 hours, about one week, or longer). Binding molecules encompass, but are not limited to, antibodies and fragments thereof, peptides, and low molecular weight non-peptide molecules (also referred to as "small molecules") binding to the CD20 antigen. The term "immunoglobulin" as used herein refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region, CH, typically is comprised of three domains, CH1 , CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The V_H and V_L regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V_H and V_L is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRI, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. MoI. Biol. 196, 901-917 (1987)). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health The term "antibody" as used herein refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions for a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally- defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or a time sufficient for the antibody to recruit an Fc-mediated effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as CIq, the first component in the classical pathway of complement activation. The anti-CD20 antibody may be mono-, bi- or multispecific. Indeed, bispecific antibodies, diabodies, and the like, may bind any suitable target in addition to a portion of CD20. As indicated above, the term "antibody" as used herein, unless otherwise stated or clearly contradicted by the context, includes fragments of an antibody provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant techniques that retain the ability to specifically bind to an antigen.

It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length (intact) antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include, but are not limited to (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, CL and CH1 domains; (ii) F(ab)2 and F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the V_H and CH1 domains; (iv) a Fv fragment consisting essentially of the V_L and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341 , 544-546 (1989)), which consists essentially of a V_H domain and also called domain antibodies (Holt et al. (November 2003) Trends Biotechnol. 21 (1 1 ) :484-90); (vi) a camelid antibody or nanobody (Revets et al. (January 2005) Expert Opin Biol Ther. 5(1 ): 1 11 -24), (vii) an isolated complementarity determining region (CDR), such as a V_H CDR3, (viii) a UniBody^(TM)' a monovalent antibody as disclosed in WO 2007/059782, (ix) a single chain antibody or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)), (x) a diabody (a scFv dimer), which can be monospecific or bispecific (see for instance PNAS USA 90(14), 6444-6448 (1993), EP 404097 or WO 93/1 1 161 for a description of diabodies), a triabody or a tetrabody.

In some embodiment the antibody is a chimeric antibody. A chimeric antibody is intended to refer to an antibody with non-human variable regions and human constant regions, most typically rodent variable regions and human constant regions. In some embodimented the antibody is a primatized antibody. A primatized (R) antibody refers to an antibody with primate variable regions, e. g., CDR's, and human constant regions. Preferably, such primate variable regions are derived from an Old World monkey. In some embodiment the antibody is a humanized antibody. A humanized antibody refers to an antibody with substantially human framework and constant regions, and non- human complementarity-determining regions (CDRs). "Substantially" refers to the fact that humanized antibodies typically retain at least several donor framework residues (of non- human parent antibody from which CDRs are derived). In some embodiment the CD-20 inhibitor is a humanized anti-CD20 antibody, In some embodiment the CD-20 inhibitor is a fully human anti-CD20 antibody.

Methods for producing chimeric, primate, primatized, humanized and human antibodies are well known in the art. See, e. g., U. S. Patent 5,530,101 , issued to Queen et al, U. S. Patent 5,225,539, issued to Winter et al, U. S. Patents 4,816,397 and 4,816,567, issued to Boss et al, and Cabilly et al, respectively, all of which are incorporated by reference in their entirety.

The selection of human constant regions may be significant to the therapeutic efficacy of the subject anti-CD20 antibody. In the preferred embodiment, the subject anti-CD20 antibody will comprise human, gamma 1 , or gamma 3 constant regions and, more preferably, human gamma 1 constant regions. The use of gamma 1 anti-CD20 antibodies as therapeutics is disclosed in US 5,500,362, hereby incorporated by reference.

Methods for making human antibodies are also known and include, by way of example, production in SCID mice, and in vitro immunization.

As noted, one chimeric anti-CD20 antibody is Rituximab - also known by the tradenames RITUXANO®, RITUXAN®, and MabThera®, which is a chimeric gamma 1 anti- human CD20 antibody. The complete amino acid and corresponding nucleic acid sequence for this antibody may be found in US5,736,137, which is incorporated by reference in its entirety.

In the preferred embodiment, the anti-CD20 antibody will bind CD20 with high affinity, i.e., ranging from 10^"5 to 10^"9M.

Preferably, the anti-CD20 antibody will comprise a chimeric, primate, primatized, human or humanized antibody. Also, the invention embraces the use of antibody fragments, e. g.,Fab's, Fv's, Fab's, F (ab) 2, and aggregates thereof.

In some embodiments, the CD-20 inhibitor is an antibody, such as Rituximab or an antibody or antibody fragment which retains the same variable regions as Rituximab and/or specifically binds to the Rituximab epitope on CD-20. Rituximab specifically binds to an epitope of amino acids 170-173 and 182-185 on the human CD-20 protein (Genbank reference NP 068769, hereby incorporated by reference). Rituximab is also referred to as the antibody "C2B8"in US Patent No. 5,736,137 issued April 7,1998 (Anderson et al.) and in US Pat No. 5,776,456, both of which are hereby incorporated by reference.

In some embodiments of the invention the CD20 antibody is Ofatumumab (2F2) or 1 1 B8 or 7D8 or2C6, all available from Genmab A/S (DENMARK).

In some embodiments of the invention the CD20 antibody is Veltuzumab (Immunomedics Inc). In some embodiments of the invention the CD20 antibody is AME-133v (EIi Lilly).

In some embodiments of the invention the CD20 antibody is lbritumomab Tiuxetan (Biogen Idee, sold under the tradename ZEVALI N^(R)),

In some embodiments of the invention the CD20 antibody is Tositumomab (GSK, marketed as BEXXAR^(R)) In some embodiments of the invention the CD20 antibody is TRU-015 (Trubion and

Whyeth).

In some embodiments of the invention the CD20 antibody is 2H7.vl6 (Genentech).

In some embodiments of the invention the CD20 antibody is Ocrelizumab (Genetch/Roche/Biogen Idee), In some embodiments of the invention the CD20 antibody is Pro131921 (Genentech),

In some embodiments of the invention the CD20 antibody is R7159 (Roche)

In some embodiments of the invention the CD20 antibody is GA-101 (Genentech/Roche and GlicArt).

When the CD-20 inhibitor is other than Rituximab, the LNA oligomer targeting Bcl-2 may be, in some embodiments, be substituted with another oligomer targeting Bcl2, such as Oblimersen or SEQ ID NO 4, such as when the CD-20 inhibitor is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan , Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159, GA-101.

In some embodiments of the invention the CD20 binding molecule is selected from the anti-CD20 antibodies disclosed in WO2004/035607, such as ofatumumab (2F2), 11 B8, or 7D8, the CD20 antibodies disclosed in WO2005/103081 , such as 2C6, the CD20 antibodies disclosed in WO 2004/103404, AME-133 (humanized and optimized anti-CD20 monoclonal antibody, developed by Applied Molecular Evolution), the CD20 antibodies disclosed in US 2003/0118592, TRU-015 (CytoxB20G, a small modular immunopharmaceutical fusion protein derived from key domains on an anti-CD20 antibody, developed by Trubion Pharmaceuticals Inc), the CD20 antibodies disclosed in WO 2003/68821 , IMMU-106 (a humanized anti- CD20 monoclonal antibody), the CD20 antibodies disclosed in WO 2004/56312, ocrelizumab (2H7.V16, PRO-70769, R-1594), Bexxar(R) (tositumomab), and Rituxan(R) / MabThera(R) (rituximab), the CD20 antibodies disclosed in WO06133148A (fucosylated with enhanced adcc(Genentech) , the CD20 antibodies disclosed in WO06084264A (improved 2H7, Genentech, mutated VH CDR2), and the CD20 antibodies disclosed in WO2004103404. WO07059188A, hereby incorporated by reference, reports that 'several anti-CD20 antibodies, including rituximab, have been shown to induce apoptosis in vitro when crosslinked by a secondary antibody or by other means. These anti-CD20 antibodies specifically bind to the CD20 antigen of (ostensibly) both normal and malignant B cells; the antibody bound to the CD20 surface antigen may lead to the destruction and depletion of neoplastic B cells Additionally, chemical agents or radioactive labels having the potential to destroy the tumor can be conjugated to the anti-CD20 antibody such that the agent is specifically "delivered" to the neoplastic B cells. Irrespective of the approach, a primary goal is to destroy the tumor; the specific approach can be determined by the particular anti- CD20 antibody that is utilized, and thus, the available approaches to targeting the CD20 antigen can vary considerably. More recently, rituximab has been shown to have antiproliferative effects in tritiated thymidine-incorporation assays and to induce apoptosis directly, while other anti-CD 19 and anti-CD20 antibodies do not.'

In some embodiments, the CD-20 inhibitor is an antibody which does not exhibit antiproliferative effects in tritiated thymidine-incorporation assays or does not induce apoptosis directly. In some embodiments, the CD-20 inhibitor is an antibody which exhibits antiproliferative effects in tritiated thymidine-incorporation assays or does induce apoptosis directly.

In some embodiments of the invention the CD20 binding molecule is selected from one of the anti-CD20 antibodies disclosed in WO 2004/035607, hereby incorporated by reference, such as ofatumumab (2F2), 1 1 B8, or 7D8, one of the antibodies disclosed in WO 2005/103081 , hereby incorporated by reference, such as 2C6, one of the antibodies disclosed in WO 2004/103404, hereby incorporated by reference, AME-133 (humanized and optimized anti-CD20 monoclonal antibody, developed by Applied Molecular Evolution and EIi Lilly), one of the antibodies disclosed in US 2003/01 18592, , hereby incorporated by reference, TRU-015 (CytoxB20G, a small modular immunopharmaceutical fusion protein derived from key domains on an anti-CD20 antibody, developed by Trubion Pharmaceuticals Inc), one of the antibodies disclosed in WO 2003/68821 , hereby incorporated by reference, IMMU-106 (a humanized anti- CD20 monoclonal antibody), one of the antibodies disclosed in WO 2004/56312, hereby incorporated by reference, ocrelizumab (2H7.V16, PRO-70769, R-1594), Bexxar(R) (tositumomab), and Rituxan(R) / MabThera(R) (rituximab). In some embodiments, the antibody may be a Radioimmunotherapeutic, such as lbritumomab (Zevalin^®)or Tositumomab (Corixa) BEXXAR ^®. In some embodiments, the CD-20 antibody is Ofatumumab (HUMAX-CD20, Genmab, DK). In some embodiments, the CD-20 antibody is Rituximab (US 5,736,137). In some embodiments, the CD-20 antibody is Ocrelizumab, also referred to as R1594 - a humanised anti-CD20 monoclonal antibody developed by Roche. In some embodiments, the CD-20 antibody is R7159, also referred to as GA-101 , as disclosed in WO06084264 (hereby incorporated by reference) developed by Roche. R7159 (GA101 ) is a novel fully humanized IgGI -type monoclonal antibody that binds with high affinity and selectivity to the extracellular part of the human CD20 antigen on malignant and normal human B cells. In contrast to rituximab and other CD20 antibodies currently in development, R7159 recognizes a CD20 type Il epitope, resulting in enhanced direct cell death induction. Through glycoengineering R7159 also mediates enhanced induction of effector-cell-mediated ADCC. In some embodiments, the CD-20 antibody is Pro131921 developed by Genentech. In some embodiments, the CD-20 antibody is AME-133/ AME - 133 #x2122 initially developed by Applied Molecular Evolution, now being developed by EIi Lilly - which is described as a humanized and optimized version of the FDA-approved or Rituximab. In some embodiments the CD-20 antibody recognizes the CD20 type Il epitope. In some embodiments the CD-20 antibody has been glycoengineered. In some embodiments the CD-20 antibody mediates enhanced induction of effector-cell-mediated ADCC.

The following patent and patent applications, hereby incorporated by reference, provide CD20 inhibitors which may be used in the present invention, or refer to the preparation of CD20 inhibitors which may be used in the present invention: US Patent Nos. US 5,776,456, US 5,736,137, US 5,843,439, US 6,399,061 , and US 6,682,734, as well as US 2002/0197255, US 2003/0021781 , US 2003/0082172, US 2003/0095963, US 2003/0147885, US 2005/0186205, and WO 1994/11026; US 6,455,043, US 2003/0026804, US 2003/0206903, and WO 2000/09160; WO 2000/27428; US 2004/0213784 and WO 2000/27433; WO 2000/44788; WO 2001/10462; WO 2001/10461 ; WO 2001/10460; US 2001/0018041 , US 2003/0180292, US 2002/0028178, WO 2001/34194, and WO 2002/22212; US 2002/0006404 and WO 2002/04021 ; US 2002/0012665, US 2005/0180975, WO 2001/74388, and US Patent No. 6,896,885B5; US 2002/0058029; US 2003/0103971 ; US 2005/0123540; US 2002/0009444 and WO 2001/80884; WO 2001/97858; US

2005/01 12060, US 2002/0039557, and US Patent No. 6,846,476; US 2002/0128448 and WO 2002/34790; WO 2002/060955;W0 2002/096948;W0 2002/079255; US Patent Nos. 6, 171 , 586 and 6,991 , 790, and WO 1998/56418; US 2004/0191256 and WO 1998/58964; WO 1999/22764; WO 1999/51642, US Patent No. 6,194,551 , US Patent No. 6,242,195, US Patent No. 6,528,624 and US Patent No. 6,538,124; US Patent No. 7,122,637, US 2005/01 18174, US 2005/0233382, US 2006/0194291 , US 2006/0194290, US 2006/0194957, and WO 2000/42072; WO 2000/67796; WO 2001/03734; US 2002/0004587, US 2006/0025576, and WO 2001/77342; US 2002/0197256 and WO 2002/078766; US 2003/0157108 and WO 2003/035835; US 5,648,267, US 5,733,779, US 6,017,733, and US 6,159,730, and WO 1994/1 1523; US Patent Nos. 6,565,827, 6,090,365, 6,287,537,

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The invention provides for a pharmaceutical composition comprising an inhibitor of CD-20, such as Rituximab, and an LNA oligomer targeting BCL-2, such as SEQ ID NO 2 and a pharmaceutically acceptable diluent, carrier, or salt. PCT/DK2006/000512 provides suitable pharmaceutically acceptable diluent, carrier and adjuvants - which are hereby incorporated by reference. Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in PCT/DK2006/000512 - which are also hereby incorporated by reference. Alternatively, the LNA oligomer may be used in standard therapeutic antibody compositions, such as those used for Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v

Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159, GA-101.

By admixing the CD-20 inhibitor and LNA oligomer targeting Bcl-2 in the same pharmaceutical composition, it allows for the administration of the LNA oligomer and the CD- 20 inhibitor in the same dosage formulation to the subject. However, the medical practitioner may combine the LNA oligomer and the CD-20 inhibitor prior to or even during administration, or alternatively, the CD-20 and LNA oligomer may be administered to the patient independently. A kit The invention provides a kit for use in the treatment of cancer, said kit comprising an inhibitor of CD-20 and an LNA oligomer targeting BCL-2. Typically the LNA oligomer, such as SEQ ID NO 2, and the CD-20 inhibitor, such as Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA-101 , are in independent compartments within the kit. A Method for the treatment of cancer

The invention provides for a method for the treatment of cancer, said method comprising the steps of i) administering an LNA oligomer targeting Bcl-2 and ii) administering an inhibitor of CD-20 to a patient who is in need for the treatment of cancer. The administration of the CD-20 inhibitor, such as Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA-101 , and the LNA oligomer, such as SEQ ID NO 2, may be performed simultaneously or independent from one another. For simultaneous administration, the administration may be the form of a single dosage formulation of both the CD-20 inhibitor and the LNA oligomer, or may be in the form of the administration of independent dosage formulations of CD-20 inhibitor and LNA oligomer. For independent administration, the administration is performed over a time period which allows for the concurrent activity of the CD-20 inhibitor, such as Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA-101 , and the LNA oligomer, such as SEQ ID NO 2, in the patient - i.e. at the same time, the LNA oligomer is inhibiting (reducing) expression of Bcl-2, and the CD-20 inhibitor is inhibiting the function of CD-20 in the patient, preferably within (or in the case of surface bound CD-20 on the surface of) the cancer cells within the patient. The concurrent activity of the CD-20 inhibitor, such as Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 11 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA-101 , and the LNA oligomer, such as SEQ ID NO 2, therefore refers to the two active ingredients are in operation at the same time, existing in the patient at pharmacologically active concentrations at the same time.

In some embodiments, the LNA oligomer targeting LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are administered in the same dosage formulation. In some embodiments, the LNA oligomer targeting Bcl-2 and the inhibitor of CD-20 are administered separately. In some embodiments, the LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are used concurrently. In some embodiments, the LNA oligomer targeting Bcl-2 and the inhibitor of CD-20 are administered together.

The LNA oligomer targeting BCL-2 is typically administered in an effective dose. In some embodiments the CD-20 inhibitor is administered in an effective dose. In some embodiments, both the LNA oligomer and the CD-20 inhibitor are administered in effective dosages.

The Patient

The patient is a subject who is in need for the treatment of cancer. The patient (subject) may be a mammal, such as a rat, a mouse, a monkey etc., or, preferably a human being, who is suffering from cancer. The patient may have a history of unsuccessful treatment with the CD-20 inhibitor - and may, in some embodiments be a relapsed or refractory patient. In some embodiments, the patient is suffering from non-Hodgkin's lymphoma. Cancer

In some embodiments, the LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are used for treatment of a CD-20+ cancer, such as CD-20-positive B-cell malignancies. In some embodiments, the cancer is a CD-20+ solid tumor. In some embodiments, the cancer is CD-20+ leukemia. In some embodiments, the cancer is selected from the list of Lymphoma, non-

Hodgkin's Lymphoma, including but not limited to Follicular NHL, lymphoplasmacytic NHL, Waldenstrom's macroglobulinemia.

In some embodiments, the cancer is relapsed or relapsed partially sensitive to treatment with a CD-20 inhibitor. In some embodiments, the cancer is refractory and not sensitive to treatment with a CD-20 inhibitor.

A cancer or cancer sample that is "CD20+" or "CD20 positive" refers to cancer, or cancer cells, wherein at least 10%, such as 9% or 8% or 7% or 6% or 5% or 4% or 3% or 2% or such as at least 1 % of the cells stain positive for CD20 protein. Suitable protocols for determination of CD20 expression, by staining has been published previously (i.e. Tzankov et al. Clininical Cancer Research, (2003), vol. 9, p 1381-1386, hereby included by reference).

In some embodiments the cancer cell(s) is substantially CD20- or CD20-negative, i.e. less than 1 % of a population of the cancer cells stain positive for CD20 protein. In some embodiments the cancer cell is substantially insensitive to said CD20 inhibitor, for example when applied to the cell at a concentration of 0.1 , 1 , 10 or 100ug/ml. in the absence of the LNA oligomer targeting Bcl-2, but is sensitive to the CD20 inhibitor in the presence of the LNA oligomer targeting Bcl-2, such as at the concentrations referred to herein. Administration Regimes

The LNA oligomer targeting Bcl-2 may be administered at regular intervals (Dose intervals, Dl) of between 3 days and two weeks, such as 4, 5, 6, 7, 8, 9, 0, 1 1 , 12, 13 days, such as about 1 week, such as 6, 7 or 8 days. Suitably at least two doses are provides with a Dl period between the two dosages, such as 3, 4, 5, 6, 7, 8, 9 or 10 dosages, each with a dose interval (Dl) between each dose of LNA oligomer. The Dl period between each dosage may the same, such as between 3 days and two weeks, such as 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 days, such as about 1 week, such as 6, 7 or 8 days.

In some embodiments, each dose of the LNA oligomer targeting Bcl-2 may be between about 0.25mg/kg - about 10mg/kg, such as about 0.5mg/kg, about 1 mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg. In some embodiments, each does of the LNA oligomer targeting BcI- 2 may be between about 2 mg/kg - about 8mg/kg, or about 4 to about 6 mg/kg or about 4mg/kg to about 5mg/kg. In some embodiments, each does of the LNA oligomer targeting Bcl-2 is at least 2mg/kg, such as 2, 3, 4, 5, 6, 7 or 8 mg/kg, such as 6 mg/kg.

In some embodiments the dosage regime for the LNA oligomer may be repeated after an initial dosage regime, for example after a rest period where no LNA oligomer targeting Bcl-2 is administered. Such as rest period may be more than 2 weeks in duration, such as about 3 weeks or about 4 weeks, or about 5 weeks or about 6 weeks.

In some embodiments the dosage regimen for the LNA oligomer is one weekly dosage, repeated three, four or five times. This dosage regimen may then be repeated after a rest period of, for example, about 3 - 5 weeks, such as about 4 weeks. In some embodiments, the LNA oligomer targeting Bcl-2 is administered during a first dosage regimen at regular dosage intervals (Dl) of between 4 and 13 days for between 2 - 10 administrations.

In some embodiments, the CD-20 inhibitor is administered during the first dosage regimen. In some embodiments, a second dosage regimen follows the first dosage regimen after a rest period when no LNA oligomer targeting Bcl-2 is administered of between 3 - 5 weeks, wherein second dosage regimen comprises the administration of the LNA oligomer targeting Bcl-2 at regular dosage intervals (Dl) of between 4 and 13 days for between 2 - 10 administrations. The first and second dosage regimens may have the same Dl and/or the same number of administrations of the LNA oligomer targeting Bcl-2. In some embodiments, the CD-20 inhibitor is administered during the second dosage regimen. In some embodiments, the CD-20 inhibitor is administered during the first and the second dosage regimen. In some embodiments, the CD-20 inhibitor is administered within one or two weeks prior to the first and/or second dosage regimen. Administration of the LNA oligomer is typically performed by parenteral administration, such as subcutaneous, intramuscular, intravenous or intra peritoneal administration. Intravenously administration is preferred.

One advantage of the LNA oligomers targeting Bcl-2 is that they may be administered over a relatively short time period rather than continuously. This provides a marked improvement in the quality of life for the patient as they are not required to be hospital bound for long periods of time. Therefore in a preferred embodiment, the LNA oligomer targeting Bcl-2 is not administered by continuous infusion. Each dose of the LNA oligomer may therefore be administered to the patient in a time period of less than 12 hours, such as less than about 8 hours, less than about 4 hours, such as less than about 3 hours. Each dose of the LNA oligomer may therefore be administered to the patient in a time period of between about 1 and about 4 hours, such as between about 2 and about 3 hours, or about 2 hours. The LNA oligomer may be administered to the patient in a time period of at least 30 minutes such as at least 1 hour. Such administrations may be given intravenously, for example. The anti CD-20 inhibitor, typically an antibody such as those disclosed herein, including Rituximab or a CD20 antibody is selected from the group consisting of

Ofatumumab (2F2), 11 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA-101 , may be given using the standard prescribed dosages for the patient. The dosage may, for example, be about weekly dosages of about 375 mg/m². This is a standard dosage for Rituximab, for example in the treatment of NHL. Suitably the CD-20 inhibitor may be dosed in a similar regime (in terms of the number of dosages and the Dl), and may for example be four weekly dosages. In some embodiments, the first dosage of the CD-20 inhibitor is administered after the first dose of the LNA-oligomer, such as about 1 week after the first dosage of the LNA oligomer. Each dose of the CD-20 inhibitor may however be a sub-optimal dose, i.e. be a dose which is below the standard prescribed dosage for the patient. It is considered that LNA oligomers may allow for a reduced dosage of the CD-20 inhibitor, such as the CD-20 antibody, such as Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 11 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159, GA-101. This also results in reduced side effects of the CD-20 inhibitor administration. The reduced administration may be in form the form of fewer and possibly less frequent administrations, and/or a reduced dosage at each dosage administration (i.e. a reduced unit dose). In some embodiments the CD20 inhibitor is administered in a dosage that is at least 20%, below the recommended dosage of that compound when used for the cancer treatment. In some embodiments for example, the CD20 inhibitor is Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 11 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159, GA-101 and the dosage is less than 300mg/m² or less than 250mg/m² per administration. In some embodiments the CD20 inhibitor is an antibody, such as those referred to here, including Ritoximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA- 101 , and the dosage is between 20mg/m² and 250mg/m²; in some embodiment the dosage is at least 20mg/m², such as at least 50mg/m², such as at least 100mg/m²; in some embodiments the dosage is less than 300mg/ m², such as less than 250mg/ m², such as less than 150mg/ m², such as less than 100mg/ m².

As shown in Figure 5, in some embodiments, the starting dose of the LNA oligomer, such as SEQ ID No 2, is given i.v. weekly for two 5-week treatment periods with a 4-week treatment-free period in-between. During the first treatment period only, patients receive a standard dose of the CD-20 inhibitor, such as 375 mg/m² rituximab, weekly for 4 weeks beginning on Week 2. For example this may be represented as follows:

• SEQ ID NO 2 i.v. weekly for 5 weeks beginning on Week 1 (Weeks 1 to 5)

• Rituximab 375 mg/m² i.v. weekly for 4 weeks beginning on Week 2 (Weeks 2 to 5) • Optionally, a pause in dosing beginning the day after the Week 5 doses of SEQ ID NO

2 and rituximab and concluding at the beginning of SEQ ID NO 2 treatment on Week 9 (i.e. a 4 week pause).

• Optionally, SEQ ID NO 2 i.v. weekly for 5 weeks beginning on Week 9 (Weeks 9 to 13) The second dosage period of repeated weekly dosages of SEQ ID No 2 may be optional. The term about used herein may, when used in relation to a specific numerical value, discloses the absolute value of said numerical value, i.e. about 6, discloses the specific value 6.

The above administration regimens may be used, for example for the treatment for patients with cancer, such as the cancers described herein, such as patients suffering from follicular or lymphoplasmacytic Non-Hodgkin's lymphoma, and/or patients with follicular or lymphoplasmacytic Non-Hodgkin's lymphoma whose condition has now relapsed (condition has come back) after their previous chemotherapy treatment.

A method for the concurrent inhibition of Bcl-2 and CD-20 in a cell

The invention provides for a method for the concurrent inhibition of Bcl-2 and CD-20 in a cell, said method comprising administering a LNA oligomer targeting Bcl-2 and a CD-20 inhibitor to said cell. The method may be performed in vivo or in vitro. For in vivo application, the method may be part of the method of treatment as referred to herein. The cell is a mammalian cell, such as a mouse, rat, monkey cell or a human cell. The cell may be a cancer cell, such as a cancer of the types referred to herein, and typically is in the form of a population of such cancer cells. The cells express Bcl-2, such as SEQ ID NO 1 or naturally occurring variant thereof. The level of inhibition of Bcl-2 may be as referred to herein. The cell may or, in some embodiments, may not, express CD-20 prior to administration of the LNA oligomer targeting Bcl-2. Indeed, it is considered in the context of the present invention that the LNA oligomer targeting Bcl-2 may sensitize cancer cells to CD-20 inhibitors and even resensitize cancer cells to CD-20 inhibitors. The method may therefore be for sensitizing or resensitizing cancer cells to CD-20 inhibitors, Use for the Preparation of a medicament

The invention provides for the use of a LNA oligomer targeting Bcl-2 for the preparation of a medicament, wherein said medicament is for the use in the treatment of cancer in combination with an inhibitor of CD-20, and wherein the LNA oligomer targeting Bcl-2 is an LNA oligomer targeting BCL-2. The use may be to the patient as referred to herein. The use may be for the method of treatment as referred to herein. The use may be for the in vivo concurrent inhibition of Bcl-2 and CD-20 in a cell, as referred to herein. EMBODIMENTS 1. The use of a LNA oligomer targeting Bcl-2 for the preparation of a medicament, wherein said medicament is for the use in the treatment of cancer in combination with an inhibitor of CD-20, and wherein the LNA oligomer targeting Bcl-2 is an LNA oligomer targeting BCL-2.

2. The use according to embodiment 1 , wherein the CD-20 inhibitor is a CD-20 antibody or a CD-20 antibody fragment, such as a FAB.

3. The use according to embodiment 2, wherein the CD-20 inhibitor is selected from the group consisting of Rituximab, Ofatumumab (2F2), 11 B8, 7D8, 2C6, Veltuzumab, AME- 133v Jbritumomab Tiuxetan , Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab ,

Pro"! 31921 , R7159, and GA-101. 4. The use according to embodiment 3, wherein the LNA oligomer targeting Bcl-2 is an LNA gapmer targeting BCL-2.

5. The use according to embodiment 4, wherein the LNA oligomer targeting Bcl-2 is selected from the group consisting of SEQ ID NO 2, 5-34 and 35-67. 6. The use according to embodiment 4 wherein the LNA oligomer targeting Bcl-2 is SEQ ID NO: 2.

7. The use according to any one of embodiments 1 - 6, wherein the LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are administered in the same dosage formulation. 8. The use according to any one of embodiments 1-6, wherein the LNA oligomer targeting Bcl-2 and the inhibitor of CD-20 are administered separately.

9. The use according to anyone of embodiments 1-8, wherein the LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are used concurrently.

10. The use according to anyone of embodiments 1 -9, wherein the LNA oligomer targeting Bcl-2 and the inhibitor of CD-20 are administered together.

1 1. The use according to anyone of embodiments 1 -10, wherein the LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are used for treatment of a CD-20+ cancer, such as CD- 20-positive B-cell malignancies.

12. The use according to anyone of embodiments 1-11 , wherein the cancer is a CD-20+ solid tumor.

13. The use according to anyone of embodiments 1-12, wherein the cancer is CD-20+ leukemia.

14. The use according to anyone of embodiments 1-1 1 , wherein the cancer is selected from the list of Lymphoma, non-Hodgkin's Lymphoma, including but not limited to Follicular NHL, lymphoplasmacytic NHL.

15. The use according to any one of embodiments 1-14, wherein the cancer is relapsed or relapsed partially sensitive to treatment with a CD-20 antibody.

16. The use according to any one of embodiments 1-15, wherein the cancer is refractory not sensitive to treatment with a CD-20 antibody. 17. The use according to any one of embodiments 1-16, wherein the LNA oligomer targeting Bcl-2 is to be administered at a dosage in the range of 2-8 mg/kg, such as about 2, about 3, about 4, about 5, about 6, about 7 or about 8 mg/kg, such as about 4 to about 6 mg/kg. 18. The use according to any one of embodiments 1 - 17, wherein the LNA oligomer targeting Bcl-2 is administered by continuous infusion.

19. The use according to any one of embodiments 1 - 17, wherein the LNA oligomer targeting Bcl-2 is not administered by continuous infusion. 20. The use according to embodiment 19 wherein the LNA oligomer targeting Bcl-2 is to be administered with an interval between administrations of between 3 days and 2 weeks, such as about once weekly (Dose Interval, Dl).

21. The use according to any one of embodiments 1 - 20, wherein each administration of the LNA oligomer targeting Bcl-2 is performed by parenteral, such as subcutaneous, intramuscular, intravenous or intra peritoneal administration, preferably intravenous administration.

22. The use according to any one of embodiments 19 - 21 , wherein each administration of the LNA oligomer targeting Bcl-2 to the patient is performed in less than 8 hours, such as less than 6, such as less than 4, such as about 2 hours. 23. The use according to embodiment 22, wherein the LNA oligomer targeting Bcl-2 is administered during a first dosage regimen at regular dosage intervals (Dl) of between 4 and 13 days for between 2 - 10 administrations.

24. The use according to embodiment 23, wherein the CD-20 inhibitor is administered during the first dosage regimen. 25. The use according to embodiment 23 or 24, wherein a second dosage regimen follows the first dosage regimen after a rest period when no LNA oligomer targeting Bcl-2 is administered of between 3 - 5 weeks, wherein second dosage regimen comprises the administration of the LNA oligomer targeting Bcl-2 at regular dosage intervals (Dl) of between 4 and 13 days for between 2 - 10 administrations. 26. LNA oligomer targeting Bcl-2A medicament comprising a LNA oligomer targeting Bcl-2, wherein said medicament is for the use in the treatment of cancer in combination with an inhibitor of CD-20, and wherein the LNA oligomer targeting Bcl-2 is an LNA oligomer targeting BCL-2, wherein the CD-20 inhibitor and the LNA oligomer targeting Bcl-2 is as according to any one of the preceding embodiments. 27. The medicament according to embodiment 20, wherein the cancer is as according to any one of embodiments 11 - 16.

28. A pharmaceutical composition comprising an inhibitor of CD-20 and an LNA oligomer targeting BCL-2, and a pharmaceutically acceptable diluent, carrier, or salt, wherein the CD-20 inhibitor and the LNA oligomer targeting Bcl-2 is as according to any one of the preceding embodiments. 29. A kit for use in the treatment of cancer, said kit comprising an inhibitor of CD-20 and an LNA oligomer targeting BCL-2, wherein the CD-20 inhibitor and the LNA oligomer targeting Bcl-2 is as according to any one of the preceding embodiments.

30. A method for the treatment of cancer, said method comprising the steps of i) administering an LNA oligomer targeting Bcl-2 and ii) administering an inhibitor of CD-

20 to a patient who is in need for the treatment of cancer; wherein the CD-20 inhibitor and the LNA oligomer targeting Bcl-2 is as according to any one of the preceding embodiments.

31. The method according to embodiments 30, wherein the cancer is as according to any one of embodiments 11 - 16.

32. The method according to embodiment 30 or 31 , wherein the LNA oligomer targeting BcI- 2 is to be administered at a dosage in the range of 2-8 mg/kg.

33. The method according to any one of embodiments 30 - 32, wherein the LNA oligomer targeting Bcl-2 and/ or CD-20 inhibitor is administered as according to any one of embodiments 17 - 25.

34. A method for the concurrent inhibition of Bcl-2 and CD-20 in a cancer cell, said method comprising administering a LNA oligomer targeting Bcl-2 and a CD-20 inhibitor to said cancer cell, wherein said LNA oligomer targeting Bcl-2 and said CD-20 inhibitor are as according to any one of the preceding embodiments.. 35. The method according to embodiment 34, wherein said cell is a cancer cell, such as a CD20+ cancer cell.

36. The method according to embodiment 34 or 35 wherein said cell expressing both Bcl-2 and CD20.

37. The method according to embodiment 34, wherein said cell is a cancer cell which is either CD20- or is not sensitive to said CD20 inhibitor.

38. The method according to any one of embodiments 34 - 37, wherein said method is performed in vitro or in vivo.

39. The method or use according to any one of the proceeding embodiments wherein between 2 and 6, such as 3, 4 or 5 dosages of the LNA oligomer is administered to the patient, optionally followed by a pause in treatment of at least 2 weeks followed by, optionally a further round of treatment of the LNA oligomer wherein between 2 and 6, such as 3, 4 or 5 dosages of the LNA oligomer is administered by intravenous administration to the patient. EXAMPLES

Example 1 In vivo anti-tumour activity of SEQ ID NO 2 and SEQ ID NO 3 in SCID mice IV injected with Raji and Namalwa human burkitt's lymphoma cells. SEQ ID NO 2: b c b =.- ^m _r υ_s o_τ I _g o c_sc_sc_sa_sa_sc_sg_st_sg_sc_sg_s m u_s om υ_s o a-c _s,

Nucleotide sequence = CTCCCAACGTGCGCCA SEQ ID NO 3

^{5 " c}s ^Gs ^τs ⁰S³SgS¹S³S¹SgS⁰SgSAs ^As ^τs °-³ Nucleotide sequence = CGTCAGTATGCGAATC 0 Bold capitals = LNA units, superscript m = 5'methyl cytosine, superscript o = beta-D-oxy, subscript s = phosphorothioate linkage, small letters = DNA units. Oligomers SEQ ID NO 2 and SEQ ID NO 3, were prepared at 90% purity, water content: 15%, MW: 5512 g/mol and 5344 g/mol respectfully and stored at room temperature. The oligomers were dissolved in sterile saline (NaCI 0.9 %) (batch number: 5001191 01 ,5 Laboratoires Aguettant, France) at 0.5 mg/ml (SEQ ID NO 2 ) or at 1 mg/ml (SEQ ID NO 3 ). Working solutions, were aliquoted and stored at 4°C.

Rituximab (RITUXIMAB, 100 mg, 10 ml, AMM: 5606003, batch number: B2118, ROCHE) was stored at 4°C. Before the injection to mice, the Rituximab stock solution (10 mg/ml) was diluted in NaCI 0.9% at 0.1 mg/ml. 0 SEQ ID NO 2 was injected at 5 mg/kg/inj in both antitumour activity studies. SEQ ID NO 3 was injected at 10 mg/kg/inj in both antitumour activity studies. Rituximab was injected at 1 and 10 mg/kg/inj (antitumour activity study in SCID mice bearing disseminated Raji human lymphoma) and at 6 and 25 mg/kg/inj (antitumour activity study in SCID mice bearing disseminated NAMALWA human lymphoma). 5 The test and reference substances were intravenously (IV, bolus) injected into the caudal vein of mice. Test and reference substances were injected at 10 ml/kg/inj accordingly to the most recent body weight of mice.

For antitumour activity study in SCID mice bearing disseminated Raji human lymphoma, 101 healthy female CB-17 SCID scid/scid mice 6 week-old and weighing 16-22 g, were obtained0 from HARLAN (San Pietro al Natisone, Italia). For antitumour activity study in SCID mice bearing disseminated NAMALWA human lymphoma, 102 healthy female CB-17 SCID scid/scid mice 6 week-old and weighing 17-22 g, were obtained from CHARLES RIVER (L'Arbresles, France). The Raji and NAMALWA cell lines were purchased and provided by ONCODESIGN and are available from the American Type Culture Collection.

All the tumour cell lines were grown in suspension at 37°C in a humidified atmosphere (5% CO₂, 95% air). The culture medium was RPMI 1640 containing 2 mM L-glutamine (ref: BE12-702F, batch number: 7MB0161 , Lonza, Verviers, Belgium) and supplemented with 20% foetal bovine serum (ref: DE14-802F, batch number: 4SB0012, Lonza, Verviers, Belgium), 4.5 g/l of Glucose (ref: G8769, batch number: 037K2415, Sigma, France), Hepes 1OmM (ref: BE17-737E, batch number: 7MB0087, Lonza, Verviers, Belgium) and Na⁺ Pyruvate 1 mM (ref: BE13-1 15E, batch number: 7MB0045, Lonza, Verviers, Belgium). Induction of Raji lymphoma in SCID mice: Five million (5x10⁶) of Raji tumour cells in 200 μl of RPMI 1640 were intravenously injected into the caudal vein of 95 female SCID mice (DO). The tumour cells injection was performed two days after a whole body irradiation of mice (1.8 Gy, ⁶⁰Co, INRA, BRETENIERES). Six (6) irradiated mice not injected with Raji cells were used as negative control for human CD45 positive cell detection. At D4, 84 SCID mice out of 95 were weighed and randomized according to their individual body weight to form 7 groups of 12 mice each (mean body weight: 19.0 ± 0.9 g ). The mean body weight of each group was not statistically different from the others (analysis of variance). The treatment schedule was as followed:

• The mice from group 1 received one daily IV injection of saline for 14 consecutive days (Q1 Dx14, from D4 to D17),

• The mice from group 2 received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17),

• The mice from group 3 received one daily IV injection of SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17), • The mice from group 4 received 6 IV injections of Rituximab at 1 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ),

• The mice from group 5 received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17) in combination with 6 IV injections of Rituximab at 1 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ). The administration of Rituximab was performed 15 minutes before the administration of SEQ ID NO 2 ,

• The mice from group 6 received one daily IV injection of SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17) in combination with 6 IV injections of Rituximab at 1 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ). The administration of Rituximab was performed 15 minutes before the administration of SEQ ID NO 3 ,

• The mice from group 9 received 6 IV injections of Rituximab at 10 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D11 , D14, D18 and D21 ).

• The treatment schedule is summarized in the table below:

The monitoring of mice was performed as described below.

Human cell detection in mice tissues: At D18, 4 mice per group were terminated. Four not xenografted mice out of 6 were used as negative control. To study the human cells engraftment in these mice, bone marrow were collected processed and analysed by flow cytometry. Bone marrow collection: To study the human cells engraftment in mice, bone marrow (from both femurs) was collected at the time of termination. The human cells in bone marrow were detected by FACS analysis using an anti-human CD45 antibody. Briefly, the bone marrow was incubated in the dark for 15 min at room temperature with either 20 μl FITC-conjugated anti human CD45 antibody (Clone I33, lsotype IgGI , ref: A07782, batch number: 1 1 , Beckman Coulter, Villepinte, France) or 20 μl FITC-conjugated mouse IgGI isotype (Clone 679.1 Mc7, ref: A07795, batch number: 1 1 , Beckman Coulter) as negative control. After incubation, cells were washed twice with 500 μl of staining buffer, resuspended in 800 μl of staining buffer and transferred to cytometer tubes (ref: 055484, Partec, Sainte Genevieve des Bois, France). The surface fluorescence of cells was analyzed with a flow cytometer apparatus (CyFlow^® space, Partec, Sainte Genevieve des Bois, France) using 488 nm wavelength laser excitation. A total of 2x10⁴ events were collected for each sample. Induction of NAMALWA lymphomas in SCID mice

One million two hundred and fifty thousand (1.25x10⁶) NAMALWA tumour cells in 200 μl of RPMI 1640 were intravenously injected into the caudal vein of 95 female SCID mice (DO). The tumour cells injection was performed two days after a whole body irradiation of mice (1.8 Gy, ⁶⁰Co, INRA, BRETENIERES). Seven (7) irradiated mice not injected with NAMALWA cells were used as negative control for human CD45 positive cell detection. At D4, 84 SCID mice out of 95 were weighed and randomized according to their individual body weight to form 7 groups of 12 mice each (mean body weight: 19.2 ± 1.5 g ). The mean body weight of each group was not statistically different from the others (analysis of variance). The treatment schedule was chosen by SANTARIS PHARMA and was as followed:

• The mice from group 1 received one daily IV injection of saline for 14 consecutive days (Q1 Dx14, from D4 to D17),

• The mice from group 2 received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17),

• The mice from group 3 received one daily IV injection of SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17), • The mice from group 4 received 6 IV injections of Rituximab at 6 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D11 , D14, D18 and D21 ),

• The mice from group 5 received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17) in combination with 6 IV injections of Rituximab at 6 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ). The administration of Rituximab was performed 15 minutes before the administration of SEQ ID NO 2 ,

• The mice from group 6 received one daily IV injection of SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17) in combination with 6 IV injections of Rituximab at 6 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ). The administration of Rituximab was performed 15 minutes before the administration of SEQ ID NO 3 ,

• The mice from group 7 received 6 IV injections of Rituximab at 25 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D11 , D14, D18 and D21 ).

The treatment schedule is summarized in the table below:

The monitoring of mice was performed as described below.

Human cell detection in mice tissues: At D14, 4 mice per group were terminated. Four not xenografted mice out of 7 were used as negative control. To study the human cells engraftment in these mice, bone marrow were collected, processed and analysed by flow cytometry (as described previously). Animal monitoring and termination: lsoflurane Forene (Minerve, Bondoufle, France) was used to anaesthetize the animals before tumour cells injection, IV treatment and termination. The viability and behaviour of mice were recorded every day. The mice's body weight were recorded twice a week. Efficacy Parameters: The efficacy parameters were chosen to be expressed as a percent (T/C %). T was the median of the survival times of animals treated with test substances and C was the median survival times of control animals treated with saline. Survival systems indicated a degree of success when T/C percents exceed 125 (5). T/C % was expressed as follows: T/C %= [T/C] x 100

• Survival curves of mice during experiment were drawn,

• Mean survival time was calculated for each group as the mean of the day of death,

• Median survival time was calculated for each group as the median of the day of death. All statistical analyses were performed using Vivo Manager (Biosystemes, France). The statistical analyses of mean body weight the day of randomization and mean body weight changes (MBWC, %) were performed using the Bonferroni/Dunn test. Survival curves were compared using the log rank test. All groups were compared with themselves. A p value <0.0014 was considered as significant (Bonferroni/Dunn test). A p value <0.05 was considered as significant (log rank test). RESULTS

Antitumour activity study of SEQ ID NO 2 and SEQ ID NO 3 in SCID mice bearing disseminated Raji human lymphoma

Induction of Raji lymphomas : During the Raji tumour cells amplification, mycoplasma detection was performed with the Mycotect assay kit. The cells were found negative for mycoplasma contamination. Before injection to mice, the cells were counted in a haemocytometer. Their viability, assessed by a 0.25 % trypan blue exclusion was 93 %. Antitumour activity analysis

• The survival curves of SCID mice are shown in Figure 1.

• The mean percent of hCD45+ cells in bone marrow at D18 are shown in Figure 2.

• The summary results of median survival times and T/C% values are shown in Table Error! Reference source not found.2.

• The summary results of mean percent hCD45+ cells in bone marrow at D14 are shown in Table 3.

Table 2: Treatment effects on survival time of SCID mice bearing disseminated Raji human lymphoma. The mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 1 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D11 , D14, D18 and D21 (TWx3).

SD: Standard Deviation.

T: Median survival time of mice treated with drugs C: Median survival time of mice treated with saline

*At D18, 4 mice per group were terminated for human cell detection in bone marrow. These mice were excluded from survival time parameters calculation. The surviving mice were terminated at D126 (2 in group of mice treated with Rituximab at 1 mg/kg/inj, 3 in group of mice treated with SEQ ID NO 2 at 5 mg/kg/inj in combination with Rituximab at 1 mg/kg/inj, and 1 of mice treated with Rituximab at 10 mg/kg/inj). For the purpose of the median survival times calculation, these mice were considered as dead at D126 and excluded from mean survival times calculation. Table 3: Mean (±SD) CD45 positive cells in bone marrow of 4 SCID mice out of 12 bearing disseminated Raji human lymphoma at D18. The mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 1 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D11 , D14, D18 and D21 (TWx3).

At D18, no hCD45 positive cells (hCD45+) were detected in bone marrow of mice treated with Rituximab at 1 mg/kg alone or in combination with SEQ ID NO 2 at 5 mg/kg and SEQ ID NO 3 at 10 mg/kg. A slight level of hCD45+ cells were detected in bone marrow of mice treated with SEQ ID NO 2 at 5 mg/kg (1.32 %). In contrast, a similar level of hCD45+ cells were detected in bone marrow of mice treated with SEQ ID NO 3 at 10 mg/kg and mice treated with saline (19.6 and 12.4 %). Regarding the hCD45 level in bone marrow of mice treated with SEQ ID NO 3 alone (19.6 %), the hCD45 level decrease observed for mice treated in combination (0.48 %) is attributed to the effect of Rituximab alone (0.67). The median survival times of mice treated with SEQ ID NO 2 at 5 mg/kg and Rituximab at 1 mg/kg/inj alone were significantly increased when compared to saline treated mice (26 and 31 days vs 21 days, respectively). Compared to saline treated mice the corresponding T/C % parameters were 123.8 and 147.6%, respectively. In contrast, similar median survival times were observed for mice treated with SEQ ID NO 3 at 10 mg/kg and saline treated mice (21 days). The median survival time was increased significantly for mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 1 mg/kg/inj when compared to mice treated with SEQ ID NO 2 and Rituximab alone at the same respective doses (96 days vs 26 and 31 days). Compared to saline treated mice the corresponding T/C % parameters were 457.1 % vs 123.8 and 147.6%. Moreover, the median survival time improvement for mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 1 mg/kg was significantly higher when compared to mice treated with Rituximab at 10 mg/kg alone (96 and 63 days vs 33 days, respectively). At D126, 6 surviving mice were terminated (2 in group of mice treated with Rituximab at 1 mg/kg/inj, 3 in group of mice treated with SEQ ID NO 2 at 5 mg/kg/inj in combination with Rituximab at 1 mg/kg/inj, and 1 in group of mice treated with Rituximab at 10 mg/kg/inj). No hCD454+ cells were found in the bone marrow. These mice were considered as cured.

Regarding the median survival time of mice treated with SEQ ID NO 3 alone (21 days), the median survival time increase observed for mice treated in combination (29 days) is attributed to the effect of Rituximab alone (31 days). Antitumour activity study of SEQ ID NO 2 and SEQ ID NO 3 in SCID mice bearing disseminated NAMALWA human lymphoma

Antitumour activity analysis

• The survival curves of SCID mice are shown in Figure 3,

• The mean percent of hCD45+ cells in bone marrow at D14 are shown in Figure 4

• The summary results of mean and median survival time are shown in Table 4, • The summary results of mean percent hCD45+ cells in bone marrow at D14 are shown in Table 5,

Table 4: Treatment effects on survival time of SCID mice bearing disseminated NAMALWA human lymphoma. The mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 6 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D11 , D14, D18 and D21 (TWx3).

• SD: Standard Deviation.

• T: Median survival time of mice treated with drugs

• C: Median survival time of mice treated with saline

• *At D 14, 4 mice per group were terminated for human cell detection in bone marrow. These mice were excluded from survival time parameters calculation.

At D14, a slight level of hCD45+ cells were detected in bone marrow of mice treated with SEQ ID NO 2 at 5 mg/kg (0.36%). A hCD45 level increase was observed for mice treated with Rituximab at 6 mg/kg (7.90 %) similar to that observed for mice treated with Rituximab at 25 mg/kg (9.68 %). A similar level of hCD45+ cells were detected in bone marrow of mice treated with SEQ ID NO 3 at 10 mg/kg and mice treated with saline (14.52 and 16.65 %). Regarding bone marrow invasion, SEQ ID NO 2 at 5 mg/kg was found more potent than Rituximab. This lack of efficacy of Rituximab could be due to the low level of hCD20 positive cells in the NAMALWA tumour cell line. However, because of a too marked antitumour activity of SEQ ID NO 2 at 5 mg/kg, no difference was observed at D14 between groups of mice treated with SEQ ID NO 2 and Rituximab alone or in combination. The median survival times of mice treated with SEQ ID NO 2 at 5 mg/kg and Rituximab at 6 mg/kg/inj alone were significantly increased when compared to saline treated mice (31 .5 and 22 days vs 19 days, respectively). Compared to saline treated mice the corresponding T/C % parameters were 165.8 and 1 15.7 %, respectively. In contrast, similar median survival times were observed for mice treated with SEQ ID NO 3 at 10 mg/kg and saline treated mice (19 days).

The median survival time was increased for mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 6 mg/kg/inj when compared to mice treated with SEQ ID NO 2 and Rituximab alone at the same respective doses (43 days vs 31 .5 and 22 days).

Compared to saline treated mice the corresponding T/C % parameters were 226.3 % vs 165.8 and 1 15.7 %. It should be noted that, the median survival time of mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 6 mg/kg was increased significantly when compared to SEQ ID NO 2 and Rituximab administered alone at the same respective doses.

Table 5: Mean (±SD) CD45 positive cells in bone marrow of 4 SCID mice out of 12 bearing disseminated NAMALWA human lymphoma at D14. The mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 6 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D1 1 , D14, D18 and D21 (TWx3).

Repeated IV injections SEQ ID NO 2 were well tolerated by female SCID mice bearing disseminated lymphoma at the tested dose. Based upon the evaluation criteria of antitumour efficacy, SEQ ID NO 2 administered alone displayed a moderate antitumour activity against disseminated Raji tumour xenografted in SCID mice. The optimal T/C% value was 123.8 % for mice treated with SEQ ID NO 2 at 5 mg/kg. In contrast, SEQ ID NO 2 showed a marked and significant improvement of the antitumour activity when combined with Rituximab. The optimal T/C% values were 457 % vs 124 and 148% for mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 1 mg/kg/inj when compared to mice treated with SEQ ID NO 2 and Rituximab alone at the same respective doses. SEQ ID NO 2 administered alone displayed a marked antitumour activity against disseminated NAMALWA tumour xenografted in SCID mice superior to that observed for mice treated with Rituximab alone. The T/C% value was 165.8% for mice treated with SEQ ID NO 2 at 5 mg/kg. SEQ ID NO 2 showed a marked and significant improvement of the antitumour activity when combined with Rituximab. The optimal T/C% values were 226.3 % vs 165.8 and 1 15.8% for mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 6 mg/kg/inj when compared to mice treated with SEQ ID NO 2 and Rituximab alone at the same respective doses. SEQ ID NO 2 was found more potent in NAMALWA disseminated tumour model when administered alone and was found more potent in Raji disseminated tumour model when administered in combination with Rituximab. This difference may be attributed to the in vitro level of hCD20 antigen, 20 fold less express on NAMALWA cell line when compared to Raji cell line. EXAMPLE 5

Pharmacokinetic (PK) data indicated that when SEQ ID No 2 was administered by intravenous (i.v.) infusion to a human patient, the maximum plasma concentration (C_max) was reached at 2 hours (end of the infusion). It was followed by a distribution phase with a distribution half-life of 2 hours, indicating that there is rapid tissue uptake. The plasma elimination half-life [VA) was estimated to be 200 hours, representing equilibrium between tissue and plasma. Furthermore, the plasma elimination half-life can be used as a surrogate of the tissue half-life. Due to the plasma elimination half-life of 200 hours, the steady state in plasma concentration during repeated doses would be reached in 30-40 days. The volume of distribution (Vz) of SEQ ID No 2 was estimated to be 6-7 L/kg, indicating the tissue drug concentration attained was likely to be 10-20 times higher than that in plasma. Dose proportionality of SEQ ID No 2 was shown for the C_max and area under the plasma concentration/time curve (AUC) within the dose levels investigated. EXAMPLE 6

Patients suffering from relapsed follicular or lymphoplasmacytic non-Hodgkin's lymphoma are selected, and undergo the administration regimen shown in Figure 5 using SEQ ID NO 2 and Rituximab. After the administration regimen, patients are monitored to identify any toxicity, especially relating to the higher dosages of SEQ ID NO 2, and to determine the overall response rate (Complete Response [CR] + Partial Response [PR]) of the combination of SEQ ID NO 2 and rituximab in the treatment of relapsed follicular or lymphoplasmacytic non-Hodgkin's lymphoma. Other variable which are determined include: 1. the median duration of response of the combination of SEQ ID NO 2 and Rituximab in the treatment of relapsed follicular or lymphoplasmacytic, non-Hodgkin's lymphoma 2. the median progression-free survival of patients treated with SEQ ID NO 2 and rituximab in the treatment of relapsed follicular or lymphoplasmacytic, non-Hodgkin's lymphoma

3. the median time to subsequent therapy for patients treated with SEQ ID NO 2 and rituximab in patients with relapsed follicular or lymphoplasmacytic non-Hodgkin's lymphoma

4. the pharmacokinetics (PK) of SEQ ID NO 2 given as weekly doses for 10 weeks over two 5-week treatment periods

5. the changes in bcl-2 protein expression following therapy with SEQ ID NO 2 and rituximab compared with pre-therapy levels in paraffin-embedded, formalin-fixed lymph node tissue using immunohistochemical analysis

6. the correlation of response to the combination of SEQ ID NO 2 and rituximab using high-density genomic profiling technology

7. the pharmacological effect of SEQ ID NO 2 and rituximab on a molecular basis by analyzing changes in the gene expression profile pre-treatment and post-treatment on lymph node tissue

8. the presence of bcl-2 (t14;18) translocation by FISH procedure following therapy with SPC2996 and rituximab compared with pre-therapy profile on the fixed tissue obtained from the lymph node biopsy References

1. Principe d'ethique de ('experimentation animale, Directive n°86/609 CEE du 24 Nov. 1986, Decret n°87/848 du 19 Oct. 1987, Arrete d'Application du 19 Avril 1988.

2. WORKMAN P. et al., UKCCCR guideline, Br. J. Cancer, 77: 1-10, 1998.

3. KLEIN F. et al., Antimicrob. Agents Chemother. 15: 420-427, 1979. 4. PULVERTAFT JV. Lancet, 1 : 238-240, 1964.

5. BISSERY M.C. et al., Bull. Cancer, 78 :587-602, 1991.

Claims

1. The use of a LNA oligomer targeting Bcl-2 for the preparation of a medicament, wherein said medicament is for the use in the treatment of cancer in combination with an inhibitor of CD-20, and wherein the LNA oligomer targeting Bcl-2 is an LNA oligomer targeting BCL-2, wherein the LNA oligomer targeting Bcl-2 is SEQ ID NO 2.

2. The use according to claim 1 , wherein the CD-20 inhibitor is a CD-20 antibody or a CD- 20 antibody fragment, such as a FAB.

3. The use according to claim 2, wherein the CD-20 inhibitor is selected from the group consisting of Rituximab, Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan , Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 ,

R7159, and GA-101.

4. The use according to any one of claims 1-3, wherein the LNA oligomer targeting Bcl-2 and the inhibitor of CD-20 are administered separately.

5. The use according to anyone of claims 1-4, wherein the LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are used for treatment of a CD-20+ cancer, such as CD-20- positive B-cell malignancies.

6. The use according to anyone of claims 1-5, wherein the cancer is a CD-20+ solid tumor.

7. The use according to anyone of claims 1-6, wherein the cancer is CD-20+ leukemia.

8. The use according to anyone of claims 1-7, wherein the cancer is selected from the list of Lymphoma, non-Hodgkin's Lymphoma, including but not limited to Follicular NHL, lymphoplasmacytic NHL.

9. The use according to any one of claims 1-8, wherein the cancer is relapsed or relapsed partially sensitive to treatment with a CD-20 antibody.

10. The use according to any one of claims 1-9, wherein the cancer is refractory not sensitive to treatment with a CD-20 antibody.

1 1. The use according to any one of claims 1 -10, wherein the LNA oligomer targeting Bcl-2 is to be administered at a dosage in the range of 2-8 mg/kg, such as about 2, about 3, about 4, about 5, about 6, about 7 or about 8 mg/kg, such as about 4 to about 6 mg/kg.

12. The use according to any one of claims 1- 11 wherein the LNA oligomer targeting Bcl-2 is to be administered with an interval between administrations of between 3 days and 2 weeks, such as about once weekly (Dose Interval, Dl).

13. The use according to any one of claims 1 - 12, wherein each administration of the LNA oligomer targeting Bcl-2 to the patient is performed in less than 8 hours, such as less than 6, such as less than 4, such as about 2 hours.

14. A medicament comprising a LNA oligomer of SEQ ID NO 2, wherein said medicament is for the use in the treatment of cancer in combination with an inhibitor of CD-20.

15. The medicament according to claim 14, wherein the cancer is as according to any one of claims 5 - 10.