WO2014174293A1

WO2014174293A1 - Anti-fungal antibody molecules and uses thereof

Info

Publication number: WO2014174293A1
Application number: PCT/GB2014/051272
Authority: WO
Inventors: Andrew Porter; Megan LENARDON; Abhishek Saxena
Original assignee: The University Court Of The University Of Aberdeen
Priority date: 2013-04-25
Filing date: 2014-04-24
Publication date: 2014-10-30
Also published as: GB201307501D0

Abstract

Provided are antibody molecules that bind novel epitopes on fungal mannans, which are attached to glycosylated fungal cell wall proteins. The antibodies can inhibit growth of Candida spp. such as C. albicans. Such antibody molecules find use in the treatment, diagnosis and/or detection of fungal infections.

Description

Anti-fungal Antibody Molecules and Uses Thereof

This invention relates to antibody molecules that bind fungal mannans, which form part of the carbohydrate component of the fungal cell wall. It relates to antibodies that specifically bind Candida spp. mannans, and can inhibit growth of Candida spp., particularly C albicans. Such antibody molecules find use in the treatment, diagnosis and/or detection of fungal infections.

Background art

Collectively, fungi cause more than two million deaths and acutely or chronically infect 300 million people per year worldwide. In the developed world, fungi are a major problem in the intensive care unit (ICU) causing life-threatening infections in severely ill and

immunocompromised patients. These lethal mycoses often undo the good work done in the treatment of cancer, transplant recipients and other severely immunocompromised individuals. C. albicans is the most common serious fungal pathogen of humans. This fungus is part of the normal gut flora of around 50% of the population and is normally harmless but can cause superficial mucosal infections such as oral and vaginal thrush and life-threatening systemic disseminated disease in severely ill and immunocompromised individuals. In the USA and Europe, the incidence of Candida infections in the ICU is second only to

Staphylococcus aureus (including MRSA) (Vincent et al., 2009). The most recent UK study estimates the mortality attributable to candidaemia in the ICU to range from 21.5% to 34.7%, with a cost of at least £16.2 million and 683 deaths per year (Hassan et al., 2009). There are three main classes of antifungal drugs currently available for use in the clinic: the azoles (e.g. fluconazole); the polyenes (e.g. amphotericin B); and the echinocandins (e.g. caspofungin). The azoles and polyenes target the cell membrane, and the echinocandins target β(1 ,3)^Ιυο8η in the cell wall. There are problems associated with all three classes of antifungal drugs. Multiple resistance mechanisms have been described for the azoles, and the polyenes have toxic side effects (reviewed in Shapiro et al. (2011)). The echinocandins are cidal against most Candida species but are only static against other common fungal pathogens such as Aspergillus species. Antibody therapeutics against fungal infections are an alternative to chemical based drugs and function with high specificity and low toxicity. The cell wall of C. albicans comprises a carbohydrate skeleton made up of chitin and β(1 ,3)- glucan to which highly glycosylated cell wall proteins are attached through β(1 ,6)^Ιυο8η linkages (Netea et al., 2008). The cell wall proteins are decorated with both N- and O-linked mannans as they pass through the endoplasmic reticulum and Golgi. O-mannan structures vary significantly across different fungi (see e.g. Cummings and Doering (2009) and Netea et al. (2006)). In C. albicans, O-mannan is a simple linear carbohydrate consisting of a single β(1 ,2)-ηΐ3ηηο3β coupled to a series of a(1 ,2)-linked mannose units (typically 1-5 residues). As is well known in the art, the A/-mannans of C. albicans and Saccharomyces cerevisiae have a "core" A/-mannan made up of β(1 ,4)-, α(1 ,6)-, α(1 ,3)- and a(1 ,2)-mannans to which outer chain branched /V-mannan is added. The branches have an a(1 ,6)-linked "backbone" with additional a(1 ,2)- and a(1 ,3)-linked "branches". These are illustrated in Figure 13, which also shows how the backbone and branches may be absent in certain mutants (e.g. ochI ) and the branches may also be absent in other mutants (e.g. mnn2-26A). C. albicans has additional β(1 ,2)-linked mannans on the branches of the A/-mannans. The proportions of specific linkages and the sizes of the chains may differ between Candida species (see e.g. Shibata et al. (2012)).

The mannans comprise up to 40% of the dry weight of the cell wall (Netea et al., 2008). C. albicans strains are divided into two main serotypes, designated A and B (Hasenclever and Mitchell, 1961). Serotype A contains all of the antigens found in serotype B as well as additional antigens not found in serotype B (summarised in Kozel et al. (2004)). The mannan structures expressed on strains of each serotype differ (Shibata et al., 1989).

The outer mannan layer is thought to act as a glycoshield, protecting the other cell wall components from enzymes that can degrade them and compromise the integrity of the cell wall resulting in a loss of viability. The outer mannan layer is also the first point of contact of C. albicans with the cells of the innate and adaptive immune system. Mannans act as pathogen associated molecular patterns (PAMPs) and are recognised by the cells of the innate immune system through pattern recognition receptors (PRRs) including the toll-like receptors TLR4 and TLR2, and the C-type lectins MMR, MINCLE/CLEC4E, Dectin-2, galectin 3 and DC-SIGN/CD209 (Netea et al., 2006; Netea et al., 2010).

Naturally occurring antibodies reactive with specific C. albicans mannan epitopes are present in the sera of most normal individuals but vary with respect to the amount, immunoglobulin class and/or binding specificity (Lehmann and Reiss, 1980; Zhang et al., 1997; Kozel et al., 2004). Anti-mannan IgG is the primary antibody found in the sera of normal adults and can act as an opsonin without the need for complement (Kozel et al., 2004). This suggests that anti-mannan antibodies with different epitope specificities might enhance host resistance either through active or passive immunisation. Previous studies of C. albicans anti-mannan monoclonal antibodies (mAb) have shown that antibodies specific for some epitopes are protective, whereas antibodies specific for others are not. For example, Han and Cutler (1995) identified two IgM mAbs that were specific for the mannan fraction of C. albicans cell wall, B6.1 and B6. These mAbs were shown to recognise different mannan epitopes, and only B6.1 was protective in a mouse model of disseminated candidiasis (Han and Cutler, 1995). B6.1 recognises β(1 ,2)-ηΐ3ηηοίπο3β (Han et al., 1998). A mouse lgG3 mAb C3.1 which recognises the same epitope as B6.1 was also protective in a mouse model of disseminated candidaisis and vaginal infection (Han et al., 2000). In this case, protection was associated with complement-mediated phagocytosis and killing of the fungus (Han et al., 2001).

Phage display technology has been used to isolate antibody fragments that recognise the C. albicans cell surface (Haidaris et al., 2001 ; Zhang et al., 2006). Phage display is advantageous because it allows high throughput screening of libraries for antibody fragments that bind specific antigens and easy reformatting of the antibody fragments. A single chain variable fragment (scFv) A2-18 that recognises C. albicans mannans on the yeast cell surface was identified by Haidaris et al. (2001). This fragment, in combination with an anti-FLAG Ab raised in mouse and a rabbit anti-mouse IgG, could opsonise C. albicans cells and enhance phagocytosis using a human monocytic cell line (in vitro) and human monocytes and neutrophils isolated from donors (ex vivo) (Wellington et al., 2003). Zhang et al. (2006) identified an anti-C. ato/cans-mannan Fab M 1 which was then converted to a full length IgG (M 1g1) and expressed in CHO cells. M1g1 recognises C. albicans mannan from both serotype A and B strains (Zhang et al., 2006). Mice passively immunised with M1 g1 were protected from disseminated candidiasis due to the M1 g1 -mediated promotion of phagocytosis and killing of the fungus by mouse macrophages and activation of the mouse complement cascade (Zhang et al., 2006).

Anti-fungal vaccines are another alternative to chemical antifungal drugs. Several vaccines based on antigens from the fungal cell wall are in various stages of development (reviewed in Edwards (2012)), but none has yet been approved for active (or passive) immunisation in humans. Active vaccination is not a viable option in patients that are severely

immunosuppressed, i.e. those most susceptible to disseminated candidaisis. A particularly attractive alternative to active vaccination is immunotherapy/passive immunisation.

Examples of antibodies that are protective in mouse models of disseminated candidaisis are described above. Another possible therapeutic strategy is combination immunotherapy, i.e. administering a chemical antifungal drug along with an antibody. Protective anti-mannan mAbs have been shown to be effective in combination immunotherapy. For example, mAb B6.1 (Han and Cutler, 1995) is synergistic with amphotericin B (Han, 2010) and augments the therapeutic effect of fluconazole (Lee et al., 201 1) in a mouse model of disseminated candidaisis.

Disclosure of the invention The present invention relates to the unexpected finding that antibody molecules which recognise mannan can directly neutralise fungal cells, in the absence of a host immune response. The inventors isolated an anti-mannan antibody, 1A2, which surprisingly neutralises C. albicans cells in vitro, even as an antibody fragment. It was not previously known that a mannan-binding molecule could inhibit fungal growth, indicating that the inventors have found a new mannan epitope and mechanism for inhibiting fungal growth, which can be exploited as described herein to provide anti-fungal agents. Without wishing to be bound by theory, it is believed that the antibody binds to a mannosylated cell wall protein (or proteins) that is (are) critical to the fungal cell. The antibody may disrupt protein function or the cell wall itself through steric hindrance.

In other words, the antibody molecules of the invention can inhibit fungal cell growth independently of antibody effector functions, i.e. in vitro, in vivo, or in the absence of an antibody Fc domain. Such effector functions include opsonisation, enhancing phagocytosis and activation of the complement cascade. Such antibody molecules may therefore find use in the treatment, prevention and diagnosis of fungal infection. Preferably the fungus is Candida spp., even more preferably C. albicans.

Further, the anti-mannan antibody molecules described herein show a higher affinity for mannan than prior art anti-mannan antibodies. This is particularly remarkable, as the anti- mannan antibody molecules described herein were screened for specificity to fungal cell wall glycans, not mannans. Wthout being bound by theory, it is believed that the purified glycans contained trace amounts of mannan, and therefore the screening strategy serendipitously selected for antibodies with a very high affinity for trace amounts of mannan. Even in a scAb format, antibody 1A2 recognised a minimum concentration of purified C. albicans mannan that was 2.5 times lower than the minimum concentration recognised by the prior art antibody M1 g1. This is despite M 1g1 being a full-length, bivalent mAb. It is known in the art (see e.g. Grant et al. (1999)) that reformatting a monomeric scAb into a full-length IgG antibody improves affinity by an order of magnitude through the avidity effect. Reformatting a scAb according to the present invention into a full-length antibody would therefore be expected to show the same effect, and so even in terms of affinity, the anti-mannan antibody molecules described herein provide a significant improvement over the prior art. Antibody affinity

Anti-mannan antibody molecules as described herein are specific for fungal mannan and bind to this epitope with high affinity relative to other epitopes, for example epitopes from glycans, bacterial dextran, starch, C. albicans chitin or sugar monomers. The anti-mannan antibodies described herein bind to C. albicans mannan with high affinity relative to some other fungal mannans, for example those from S. cerevisiae. In one embodiment, the anti- mannan antibody molecules described herein are selective for C. albicans over

S. cerevisiae. In some embodiments, the anti-mannan antibodies described herein bind to C. albicans O-mannan with high affinity relative to other fungal mannans, optionally including C. albicans A/-mannan. For example, an anti-mannan antibody molecule of the invention may display a binding affinity for C. albicans mannan which is at least 100 fold, 500 fold, at least 1000 fold or at least 2000 fold greater than any one of purified C. albicans β(1 ,3)- glucan, pustulan ( (1 ,6)-glucan from Lichen), laminarin ( (1 ,3)-glucan from algae), S.

cerevisiae mannan, bacterial dextran, starch, purified C. albicans chitin, D-glucose, D- mannose, galactose, and A/-acetyl glucosamine. The binding affinity may be tested by indirect binding ELISA as described in Examples 9 and 15, wherein specificity for C. albicans mannan may be indicated by a positive signal of A450≥ 0.5 for C. albicans, and < 0.5 for e.g. S. cerevisiae. An anti-mannan antibody molecule of the present invention may have a half maximal inhibitory concentration (IC₅₀) for purified C. albicans mannan inhibiting binding to purified C. albicans cell wall material of less than about 10,000 ng/ml, less than about 1000 ng/ml, less than about 100 ng/ml, less than about 10 ng/ml, less than about 6 ng/ml, less than about 1 ng/ml, or less than about 0.5 ng/ml. A suitable anti-mannan antibody molecule may, for example, have an IC₅₀ of about 0.1 ng/ml to 100 ng/ml, e.g. 0.5 ng/ml to 10 ng/ml. The assay to determine the IC₅₀ may be carried out as described in Examples 9 and 15.

An anti-mannan antibody molecule may have a dissociation constant for fungal mannan of less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, or less than 1 nM. For example, an antibody molecule may have an affinity for mannan of 0.1 to

50 nM, e.g. 0.5 to 10 nM. A suitable anti-mannan antibody molecule may, for example, have an affinity for mannan of about 1 nM. Preferably the fungal mannan is purified C. albicans mannan, e.g. obtainable from NIBSC, UK, Cat No. 77/600. In some embodiments, the mannan may be prepared as described in Example 2.

Binding kinetics and affinity (expressed as the equilibrium dissociation constant K_D) of the anti-mannan antibody molecules may be determined using standard techniques, such as surface plasmon resonance e.g. using BIAcore analysis. For example, methods for determining association constants (K_a), dissociation constants (K_d) and affinity contants(K_A) are outlined in Grant et al. (1999).

An anti-mannan antibody molecule described herein may specifically bind isolated mannan, purified hyphal cell wall material, mannan that is incorporated into the fungal cell wall, or mannan that is attached to a fungal protein (i.e. mannosylated proteins). In some embodiments, an anti-mannan antibody molecule described herein specifically binds Candida spp. mannan, preferably C. albicans mannan. The C. albicans may be serotype A or serotype B. In some embodiments, the C. albicans is serotype A.

In some embodiments, C. albicans (also known as C. stellatoidea or C. stellatoidea type I) may be a variant selected from C. albicans 1 161 , C. albicans 11 C, C. albicans 11 E, C. albicans 12C, C. albicans 18E, C. albicans 18J, C. albicans 18K, C. albicans 18M, C. albicans 19F, C. albicans 220 , C. albicans 22F, C. albicans 22K, C. albicans 220, C. albicans 23B, C. albicans 23C, C. albicans 23F, C. albicans 23G, C. albicans 23K, C. albicans 23P, C. albicans 23Q, C. albicans 23R, C. albicans 24A, C. albicans 24C, C. albicans 24E, C. albicans 24F, C. albicans 25M, C. albicans 25N, C. albicans 250, C. albicans 25P, C. albicans 28A, C. albicans 28C, C. albicans 28 H, C. albicans 28I, C. a/<b/^'cans 3153, C. albicans A 123, C. albicans A155, C. albicans A20, C. albicans A203, C. albicans A48, C. albicans A67, C. albicans A84, C. albicans A92, C. albicans Ca6, C. albicans CAS15, C. albicans CHN1 , C. albicans GC75, C. albicans L26, C. albicans P34048, C. albicans P37005, C. albicans P37037, C. albicans P37039, C. albicans P57055, C. albicans P57072, C. albicans P60002, C. albicans P75010, C. albicans P75016,

C. albicans P75063, C. albicans P76055, C. albicans P76067, C. albicans P78042,

C. albicans P78048, C. albicans P87, C. albicans P94015, C. albicans SC5314, C. albicans var. stellatoidea, C. albicans WO-1 or C. albicans NGY152. Preferably the variant is C. albicans SC5314.

Antibody molecules

An anti-mannan antibody molecule as described herein may be an immunoglobulin or fragment thereof, and may be natural or partly or wholly synthetically produced, for example a recombinant molecule. Anti-mannan antibody molecules may include any polypeptide or protein comprising an antibody antigen-binding site, including Fab, Fab₂, Fab₃, diabodies, triabodies, tetrabodies, minibodies and single-domain antibodies, as well as whole antibodies of any isotype or subclass. The anti-mannan antibody molecules may also be a single-chain variable fragment (scFv) or single-chain antibody (scAb). An scFv fragment is a fusion of a variable heavy (VH) and variable light (VL) chain. A scAb has a constant light (CL) chain fused to the VL chain of an scFv fragment. The CL chain is optionally the human kappa light chain (HUCK).

A single chain Fv (scFv) may be comprised within a mini-immunoglobulin or small immunoprotein (SIP), e.g. as described in Li et al. (1997). An SIP may comprise an scFv molecule fused to the CH4 domain of the human IgE secretory isoform lgE-S2 (£_S2-CH4; Batista et al., 1996) forming an homo-dimeric mini-immunoglobulin antibody molecule. Antibody molecules and methods for their construction and use are described, in for example, Holliger and Hudson (2005).

In some preferred embodiments, the anti-mannan antibody molecule may be a whole antibody. For example, the anti-mannan antibody molecule may be an IgG, IgA, IgE or IgM or any of the isotype sub-classes, particularly lgG1 and lgG4. The anti-mannan antibody molecules may be monoclonal antibodies. The antibodies may be bi-specific, antibody-drug conjugates, dimers, trimers, have natural or non-natural Fc portions, or be glycosylated or non-glycosylated.

Anti-mannan antibody molecules may be chimeric, humanised or human antibodies.

Anti-mannan antibody molecules as described herein may be isolated, in the sense of being free from contaminants, such as antibodies able to bind other polypeptides and/or serum components. Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies may also be employed.

Anti-mannan antibody molecules of the invention may be obtained in the light of the disclosure herein using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the mannan (e.g. purified cell wall, preferably purified hyphal cell wall) or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al., 1992). Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be naive, that is constructed from

sequences obtained from an organism which has not been immunised with any of the mannans (or fragments), or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

Anti-mannan antibody molecules as described herein may lack antibody constant regions. In some embodiments, an anti-mannan antibody molecule as described herein may comprise a mannan binding site within a non-antibody molecule, normally provided by one or more CDRs e.g. a set of CDRs in a non-antibody protein scaffold. An antigen binding site may be provided by means of arrangement of complementarity determining regions (CDRs) on non-antibody protein scaffolds such as fibronectin or cytochrome B etc. (Haan and Maggos, 2004; Koide et al., 1998; Nygren and Uhlen, 1997), or by randomising or mutating amino acid residues of a loop within a protein scaffold to confer binding specificity for a desired target. Scaffolds for engineering novel binding sites in proteins have been reviewed in detail by Nygren and Uhlen (1997). Protein scaffolds for antibody mimics are disclosed in WO/0034784, in which the inventors describe proteins (antibody mimics) that include a fibronectin type III domain having at least one randomised loop. A suitable scaffold into which to graft one or more CDRs, e.g. a set of HCDRs, may be provided by any domain member of the immunoglobulin gene superfamily. The scaffold may be a human or non- human protein. An advantage of a non-antibody protein scaffold is that it may provide an antigen-binding site in a scaffold molecule that is smaller and/or easier to manufacture than at least some antibody molecules. Small size of a binding member may confer useful physiological properties such as an ability to enter cells, penetrate deep into tissues or reach targets within other structures, or to bind within protein cavities of the target antigen. Use of antigen binding sites in non-antibody protein scaffolds is reviewed in Wess (2004). Typical are proteins having a stable backbone and one or more variable loops, in which the amino acid sequence of the loop or loops is specifically or randomly mutated to create an antigen- binding site that binds the target antigen. Such proteins include the IgG-binding domains of protein A from S. aureus, transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type III domain) and lipocalins. Other approaches include synthetic "Microbodies" (Selecore GmbH), which are based on cyclotides - small proteins having intra-molecular disulphide bonds.

In some embodiments, anti-mannan antibody molecules may be produced by any convenient means, for example a method described above, and then screened for binding to fungal mannan (e.g. C. albicans mannan) relative to other fungal carbohydrates or cell wall components for example glycans, bacterial dextran, starch, chitin or sugar monomers.

Suitable screening methods are well-known in the art and described herein.

After production and/or isolation, the biological activity of an anti-mannan antibody molecule may be tested. For example, the ability of the antibody molecule to inhibit fungal growth may be determined, in vitro or in vivo. Suitable assays are well-known in the art and described herein.

Antibody molecules normally comprise an antigen binding domain comprising an

immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), although antigen binding domains comprising only a heavy chain variable domain (VH) are also possible (e.g. camelid or shark antibodies).

Each of the VH and VL domains typically comprise three complementarity determining regions (CDRs) responsible for antigen binding, interspersed by framework regions.

In some embodiments, binding to mannan may occur wholly or substantially through the VHCDR3 of the anti-mannan antibody molecule.

For example, an anti-mannan antibody molecule may comprise a VH domain comprising a HCDR3 having the amino acid sequence of SEQ ID NO: 5 or the sequence of SEQ ID NO: 5 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. The substitutions may be conservative substitutions. The HCDR3 may be the only region of the antibody molecule that interacts with a mannan epitope or substantially the only region. The HCDR3 may therefore determine the specificity and/or affinity of the antibody molecule for the mannan. The VH domain of an anti-mannan antibody molecule may additionally comprise an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or the sequence of SEQ ID NO: 4 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. The VH domain of an anti-mannan antibody molecule may further comprise an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or the sequence of SEQ ID NO: 3 with 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions.

In some embodiments, an antibody molecule may comprise a VH domain comprising a HCDR1 , a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5 respectively. For example, an antibody molecule may comprise a VH domain having the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 2 with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions in SEQ ID NO: 2.

The anti-mannan antibody molecule may further comprise a VL domain, for example a VL domain comprising LCDR1 , LCDR2 and LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9 respectively, or the sequences of SEQ ID NOs 7, 8 and 9 respectively with, independently, 1 or more, for example 2, 3, 4 or 5 or more amino acid substitutions, deletions or insertions. The substitutions may be conservative substitutions. For example, an antibody molecule may comprise a VL domain having the sequence of SEQ ID NO: 6 or the sequence of SEQ ID NO: 6 with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions in SEQ ID NO: 6. The anti-mannan antibody molecule may for example comprise one or more amino acid substitutions, deletions or insertions which improve one or more properties of the antibody, for example affinity, functional half-life, on and off rates.

The techniques required in order to introduce substitutions, deletions or insertions within amino acid sequences of CDRs, antibody VH or VL domains and antibodies are generally available in the art. Variant sequences may be made, with substitutions, deletions or insertions that may or may not be predicted to have a minimal or beneficial effect on activity, and tested for ability to bind to C. albicans mannan and/or for any other desired property. In some embodiments, an anti-mannan antibody molecule may comprise a VH domain comprising a HCDR1 , a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 3, 4, and 5, respectively, and a VL domain comprising a LCDR1 , a LCDR2 and a LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9, respectively. For example, the VH and VL domains may have the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6 respectively; or may have the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6 comprising, independently 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions. The substitutions may be conservative substitutions.

In some embodiments, an anti-mannan antibody molecule VH domain may have at least about 60% sequence identity to SEQ ID NO: 2, e.g. at least about 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2.

In some embodiments, an anti-mannan antibody molecule VL domain may have at least about 60% sequence identity to SEQ ID NO: 6, e.g. at least about 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6. In some embodiments, an antibody may comprise one or more substitutions, deletions or insertions which remove a glycosylation site.

The anti-mannan antibody molecule may be in any format, as described above, In some preferred embodiments, the anti-mannan antibody molecule may be a whole antibody, for example an IgG, such as lgG1 or lgG4, IgA, IgE or IgM. In some preferred embodiments, than anti-mannan antibody molecule is a scAb or scFv.

An anti-mannan antibody molecule of the invention may be one which competes for binding to mannan with an antibody molecule described herein, for example an antibody molecule which

(i) binds fungal, e.g. Candida spp., mannan and

(ii) comprises a VH domain of SEQ ID NO: 2 and/or VL domain of SEQ ID NO: 6; an HCDR3 of SEQ ID NO: 5; an HCDR1 , HCDR2, LCDR1 , LCDR2, or LCDR3 of SEQ ID NOS: 3, 4, 7, 8 or 9 respectively; a VH domain comprising HCDR1 , HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4 and 5 respectively; and/or a VH domain comprising HCDR1 , HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4 and 5 and a VL domain comprising LCDR1 , LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8 and 9 respectively.

Competition between antibody molecules may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one antibody molecule which can be detected in the presence of one or more other untagged antibody molecules, to enable identification of antibody molecules which bind the same epitope or an overlapping epitope. Such methods are readily known to one of ordinary skill in the art. Thus, a further aspect of the present invention provides a binding member or antibody molecule comprising an antigen-binding site that competes with an antibody molecule, for example an antibody molecule comprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of the parent antibody described above for binding to mannan. A suitable antibody molecule may comprise an antibody antigen-binding site which competes with an antibody antigen-binding site for binding to mannan wherein the antibody antigen-binding site is composed of a VH domain and a VL domain, and wherein the VH and VL domains comprise HCDR1 , HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4, and 5 and LCDR1 , LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8, and 9 respectively, for example the VH and VL domains of SEQ ID NOS: 2 and 6. An anti-mannan antibody molecule as described herein may specifically bind an epitope on C. albicans mannan which leads to inhibition of C. albicans growth in vitro or in vivo. An anti-mannan antibody molecule as described herein may specifically bind an epitope on the core A/-mannan structure of Candida species e.g. any one or more of C. albicans, C. dubliniensis, C. tropicalis or C. krusei As illustrated in the Examples below, it is believed that both 1 H6 and 1A2 bind an epitope of the core A/-mannan structure, albeit slightly different parts of the core.

An anti-mannan antibody molecule as described herein may specifically bind an epitope on the core A/-mannan structure which is partially masked by the a(1 ,6)-A/-mannan backbone and optionally branches in wild-type C. albicans cells (see also Figure 13 and legend thereto).

As illustrated in the Examples below, it is believed that the epitope recognised by antibody fragment 1A2 is partially masked by the a(1 ,6)-A/-mannan backbone (but not the branches).

An anti-mannan antibody molecule as described herein may be one which does not specifically bind to O-mannan.

An anti-mannan antibody molecule as described herein may be one which does not specifically bind to the epitope bound by anti-mannan mAb B6.1 (Han and Cutler, 1995) which is believed to be β(1 ,2)-ηΐ3ηηοίπο3β in phospho-mannan attached to the α(1 ,6)-Λ/- mannan backbone (i.e. not part of the core mannan structure). Similarly an anti-mannan antibody molecule of the invention may be one which does not compete for binding to that epitope with mAb B6.1.

Likewise an anti-mannan antibody molecule as described herein may be one which does not specifically bind to the epitope bound by the mouse lgG3 mAb C3.1 described supra and may be one which does not compete for binding to that epitope with mAb C3.1.

Likewise an anti-mannan antibody molecule as described herein may be one which does not specifically bind to the epitope bound by the_M1 g1 anti-mannan mAb (Zhang et al., 2006) and may be one which does not compete for binding to that epitope with that mAb.

In some embodiments, the epitope on C. albicans mannan specifically bound by an anti- mannan antibody molecule is not present on mannan isolated from S. cerevisiae. For example, an anti-mannan antibody molecule of the invention may have a K_D for binding S. cerevisiae mannan (e.g. obtained as described herein) of greater than about 50 nM, 100 nM, 1 ,000 nM, 5,000 nM, 0.01 mM, 0.05 mM, 0.1 mM, 1 mM or 10 mM. An anti-mannan antibody molecule as described herein may inhibit fungal growth, preferably Candida or C. albicans growth. For example, an anti-mannan antibody molecule may inhibit fungal cell population growth, e.g. by disrupting cell division or cell growth, e.g. by binding directly to the core A/-mannan structure of glycans attached to cell wall proteins, which may optionally compromise the function of vital cell wall proteins.

An anti-mannan antibody molecule as described herein may inhibit fungal growth by inhibiting one or more activities required for fungal cell growth. An antibody molecule may inhibit growth of C. albicans growing in the yeast, hyphal or pseudohyphal phase. An antibody molecule of the invention may find particular use in inhibiting growth of C. albicans in the hyphal phase.

Techniques for measuring fungal cell growth, for example by measuring the growth of a fungus in vitro, are standard in the art and are described herein.

Anti-mannan antibody molecules may be further modified by chemical modification, for example by PEGylation, or by incorporation in a liposome, to improve their pharmaceutical properties, for example by increasing in vivo half-life.

Pharmaceutical compositions

Anti-mannan antibody molecules may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the anti-mannan antibody molecule, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the anti-mannan antibody molecule.

In some embodiments, anti-mannan antibody molecules may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised antibody molecules may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

Anti-mannan antibody molecules will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the antibody molecule. Thus pharmaceutical compositions may comprise, in addition to the anti-mannan antibody molecule, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the anti-mannan antibody molecule. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

For parenteral, for example sub-cutaneous or intra-venous administration, e.g. by injection, the pharmaceutical composition comprising the anti-mannan antibody molecule may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers,

antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;

hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as

polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™,

PLURONICS™ or polyethylene glycol (PEG). Combination and other treatments

The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.

The agents (i.e. the anti-mannan antibody molecules described herein, plus one or more other agents) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g. 1 , 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being

commensurate with the properties of the therapeutic agent(s) as described herein, including their synergistic effect.

The agents (i.e. the anti-mannan antibody molecules described here, plus one or more other agents) may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.

For example, the compounds described herein may in any aspect and embodiment also be used in combination therapies, e.g. in conjunction with other agents e.g. antifungal agents. The second antifungal agent may be selected from an azole (e.g. fluconazole), a polyene

(e.g. amphotericin B), a echinocandin (e.g. caspofungin), an allylamine (e.g. terbinafine), and a flucytosine (also called 5-fluorocytosine). The skilled person will recognise that other antifungal agents may also be used. In some embodiments, the second antifungal agent is a second anti-fungal antibody or an antimicrobial peptide. In some embodiments, the anti- mannan antibody molecule described herein is conjugated to the second antifungal agent.

An anti-mannan antibody molecule as described herein may be used in a method of treatment of the human or animal body, including prophylactic or preventative treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset). The method of treatment may comprise administering an anti-mannan antibody molecule to an individual in need thereof.

Administration is normally in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann et al., 1991 ; Bagshawe et al., 1991). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of an antibody molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody, fragment) and the nature of any detectable label or other molecule attached to the antibody.

A typical antibody dose will be in the range 100 μg to 1 g for systemic applications, and 1 μg to 1 mg for topical applications. An initial higher loading dose, followed by one or more lower doses, may be administered. Typically, the antibody will be a whole antibody, e.g. the lgG1 or lgG4 isotype, and where a whole antibody is used, dosages at the lower end of the ranges described herein may be preferred. This is a dose for a single treatment of an adult patient, which may be

proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight.

Preferably the antibody or fragment will be dosed at no more than 50 mg/kg or no more than 100 mg/kg in a human patient, for example between 1 and 50, e.g. 5 to 40, 10 to 30, 10 to 20 mg/kg. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. The treatment schedule for an individual may be dependent on the pharmocokinetic and pharmacodynamic properties of the antibody composition, the route of administration and the nature of the condition being treated. Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g. about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks. Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above.

In some embodiments, anti-mannan antibody molecules as described herein may be administered as sub-cutaneous injections. Sub-cutaneous injections may be administered using an auto-injector, for example for long term prophylaxis/treatment.

In some preferred embodiments, the therapeutic effect of the anti-mannan antibody molecule may persist for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of anti-mannan antibody molecule may persist in an individual for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more. Anti-mannan antibody molecules described herein inhibit C. albicans fungal growth (and in preferred embodiments also inhibit C. dubliniensis, C. tropicalis and C. krusei growth) and may be useful in the treatment of fungal infections.

Aspects of the invention provide; an anti-mannan antibody molecule as described herein for use in a method of treatment of the human or animal body; an anti-mannan antibody molecule as described herein for use in a method of treatment of a fungal infection; the use of an anti-mannan antibody molecule as described herein in the manufacture of a medicament for the treatment of a fungal infection; and a method of treatment of a fungal infection comprising administering an anti-mannan antibody molecule as described herein to an individual in need thereof.

Inhibition of fungal growth by anti-mannan antibodies as described herein may be of clinical benefit in the treatment of any fungus-associated condition, and particularly infections caused by Candida species, i.e. candidiasis.

C. albicans is the most common serious fungal pathogen of humans, and the embodiments disclosed herein may be used in the prophylaxis or treatment of any condition related to infection caused by C. albicans. This fungus is part of the normal gut flora of around 50% of the population and is normally harmless but can cause superficial mucosal infections such as oral and vaginal thrush and life-threatening systemic disseminated disease in

immunocompromised individuals. Immunocompromised individuals may have a weakened immune system due to medical treatment (e.g. cancer treatment or organ transplant recipients), or due to a disease or disorder (e.g. HIV/AIDS, SCID, CVID). Other conditions that may be treated include lung infections in cystic fibrosis patients, mixed microbial infections.which include both bacteria (e.g. Pseudomonas spp.) and fungi, fungal infections on indwelling medical devices such as catheters, and skin and urinary tract infections.

However, as explained in the Examples, other Candida species (such as C. dubliniensis, C. tropicalis and C. krusei) may also be targeted by the anti-mannan antibodies as described herein. Embodiments described herein in relation to C. albicans will thus be understood to apply mutatis mutandis to these other targets.

For example, anti-mannan antibodies as described herein may be useful in the surgical and other medical procedures which may lead to immunosuppression, or medical procedures in patients who are already immunosuppressed.

Patients suitable for treatment as described herein include patients with conditions in which fungal infection is a symptom or a side-effect of treatment or which confer an increased risk of fungal infection or patients who are predisposed to or at increased risk of fungal infection, relative to the general population. For example, an anti-mannan antibody molecule as described herein may also be useful in the treatment or prevention of fungal infection in cancer patients. Anti-mannan antibody molecules as described herein may also be useful in in vitro testing, for example in the detection of fungus or a fungal infection, for example in a sample obtained from a patient. Anti-mannan antibody molecules as described herein may be useful for identifying

C. albicans, and/or distinguishing C. albicans from other fungi, e.g. S. cerevisiae.

The presence or absence of a fungus (e.g. C. albicans) may be detected by

(i) contacting a sample suspected of containing the fungus with an antibody molecule described herein, and

(ii) determining whether the antibody molecule binds to the sample, wherein

binding of the antibody molecule to the sample indicates the presence of the fungus.

A fungal infection, e.g. C. albicans infection, in an individual may be diagnosed by

(i) obtaining a sample from the individual;

(ii) contacting the sample with an antibody molecule as described herein, and

(iii) determining whether the antibody molecule binds to the sample, wherein

binding of the antibody molecule to the sample indicates the presence of the fungal infection. Binding of antibodies to a sample may be determined using any of a variety of techniques known in the art, for example ELISA, immunocytochemistry, immunoprecipitation, affinity chromatography, and biochemical or cell-based assays. In some embodiments, the antibody is conjugated to a detectable label or a radioisotope. Anti-mannan antibody molecules as described herein may find use in a method of detecting mannan, e.g. C. albicans mannan, the method comprising

(i) contacting a sample suspected of containing mannan with an antibody molecule described herein, and

(ii) determining whether the antibody molecule binds to the sample, wherein

binding of the antibody molecule to the sample indicates the presence of mannan.

One aspect of the invention relates to the use of an anti-mannan antibody molecule as described herein for binding mannan, e.g. C. albicans mannan e.g. the core A/-mannan structure of glycans attached to cell wall proteins of Candida spp.

A method for producing an antibody antigen-binding domain for fungal mannan, preferably C. albicans mannan, may comprise:

providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR1 , HCDR2 and HCDR3, wherein the parent VH domain HCDR1 , HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, a VH domain which is an amino acid sequence variant of the parent VH domain, and;

optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and

testing said VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain for mannan. A VH domain which is an amino acid sequence variant of the parent VH domain may have the HCDR3 sequence of SEQ ID NO: 5 or a variant with the addition, deletion, substitution or insertion of one, two, three or more amino acids. The VH domain which is an amino acid sequence variant of the parent VH domain may have the HCDR1 and HCDR2 sequences of SEQ ID NOS: 3 and 4 respectively, or variants of these sequences with the addition, deletion, substitution or insertion of one, two, three or more amino acids. A method for producing an antibody molecule that specifically binds to fungal mannan, preferably C. albicans mannan, may comprise:

providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise a HCDR1 , HCDR2 and/or HCDR3 to be replaced or lack a HCDR1 , HCDR2 and/or HCDR3 encoding region;

combining said starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDR1 , HCDR2, and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, such that said donor nucleic acid is or donor nucleic acids are inserted into the CDR1 , CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains;

expressing the nucleic acids of said product repertoire to produce product VH domains; optionally combining said product VH domains with one or more VL domains;

selecting an antibody molecule that binds fungal mannan, which antibody molecule comprises a product VH domain and optionally a VL domain; and

recovering said antibody molecule or nucleic acid encoding it.

Suitable techniques for the maturation and optimisation of antibody molecules are well- known in the art.

Antibody antigen-binding domains and antibody molecules for fungal mannan may be tested as described herein. For example, the ability to bind to mannan or mannan-containing substrates may be determined. Provided herein is a method of inhibiting growth of a fungal cell, comprising contacting the fungal cell with an agent capable of specifically binding mannan obtained from the fungal cell. Preferably, the agent is an antibody. In some embodiments, the agent is an anti- mannan antibody molecule as described herein. In some embodiments the fungus may be any species with mannan structures similar or substantially similar to C. albicans, and/or are specifically bound by an anti-mannan antibody molecule described herein. In some embodiments, the fungus is a member of the "CTG clade", which includes for example Candida albicans, Candida dubliniensis, Candida guilliermondii, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Debaryomyces hansenii and Lodderomyces elongisporus.

A method of screening for an agent capable of inhibiting growth of a fungal cell, may comprise: (i) obtaining a mannan sample from the fungal cell;

(ii) contacting the mannan sample with an agent;

(iii) determining whether the agent binds the mannan sample, wherein an agent capable of binding mannan is a candidate antifungal agent.;

(iv) selecting the candidate antifungal agent; and

(v) contacting a fungal cell with the candidate antifungal agent in vitro and determining whether the candidate antifungal agent is capable of inhibiting growth of the fungus.

In some embodiments, a method of screening for an agent capable of inhibiting growth of C. albicans (or other Candida spp.), may comprise identifying an agent capable of competing with an anti-mannan antibody molecule described herein for binding to C. albicans mannan. For example, the method may comprise

(i) measuring the binding of an agent to C. albicans mannan in the presence and absence of an anti-mannan antibody molecule described herein, wherein

a decrease in binding of the agent to the C. albicans mannan in the presence of the anti- mannan antibody molecule relative to the binding of the agent to the C. albicans mannan in the absence of the anti-mannan antibody molecule indicates the agent is a candidate agent for inhibiting growth of C. albicans;

(ii) selecting the candidate agent; and

(iii) contacting a C. albicans cell with the candidate antifungal agent and determining whether the candidate antifungal agent is capable of inhibiting growth of the fungus.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety.

Unless stated otherwise, antibody residues are numbered herein in accordance with the Kabat numbering scheme.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Thus, the features set out above are disclosed in all combinations and permutations.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and tables described below. Description of figures

Figure 1 : Schematic representation of the phagmid vector pHEN2a. Linked VH-VA fragments (scFv) were cloned into the Nco\ and Λ/ofl sites to construct the phage display library. pelB leader - leader sequence, M 13 ori - M 13 origin of replication, pUC ori - pUC plasmid origin of replication, gill - fd phage gene III, myc-tag - c-myc sequence, 6xHis tag- six histidines, Amp^r - gene encoding ampicillin resistance, Plac- lac promoter. Figure 2. Polyclonal serum from sheep immunised with purified hyphal cell walls contains IgG antibodies that predominantly bind mannan. Panels show indirect binding ELISA curves comparing the polyclonal serum IgG antibodies pre-immunisation (PI; squares) and after round 5 immunisation (R5; triangles) against target antigens. A. PI and R5 IgG response against purified hyphal cell walls (HCW). B. PI and R5 IgG response against purified mannan. C. PI and R5 IgG response against purified (1 ,3)-glucan. D. PI and R5 IgG response purified C. albicans chitin.

Figure 3: Sheep immunised with purified hyphal cell walls produced polyclonal IgG antibodies that specifically bind the C. albicans cell wall. Transmission electron micrographs of sections of wild type C. albicans cells stained with pre-immune serum/anti-sheep IgG-gold (15 nm) (A) and CTA-1 R5/anti-sheep IgG-gold (15 nm) (B). The scale bars represent 0.5 μηι.

Figure 4: C. albicans growth is inhibited by CTA-1 R5 in vitro. Specific numbers (top) of wild type C .albicans hyphal cells were treated with pre-immune serum (PI) (A), polyclonal serum for the fifth bleed of a sheep immunised with purified hyphal cell wall material (CTA-1 R5) (B) or left untreated (C) for 5 h before being plated in segmented petri dishes. Growth was assessed after overnight incubation. The results shown are representative of four independent experiments.

Figure 5: PAN6 was enriched with mannan-binding monoclonal phage. The phage supernatants from 94 rescued clones from PAN6 were assessed for their specificity to bind C. albicans mannan by ELISA. Thirty two percent of clones had an A₄₅₀ of >1.0 (arbitrary value) and were considered to be high affinity binders.

Figure 6: DNA and protein sequences of the scFvs 1A2 and 1 H6. A. scFv 1A2. B. scFv 1 H6. In both panels, the nucleotide sequence is aligned above the amino acid sequence. The positions of the start of the heavy and light chain frameworks (HFW, LFW),

complementarity determining regions of the VH (HCDR; light grey) and VA (LCDR; dark grey) chains, and the cellulase linker inserted between the VH and VA chains (LINKER; black) are indicated above the nucleotide sequence.

Figure 7: The scAbs 1A2 and 1 H6 are highly specific for C. albicans mannan. Indirect binding ELISAs performed to assess the binding of the scAbs 1A2 and 1 H6 (1/1000 dilution) to various glycans (1 μg/ml) and monosaccharides (1 μg/ml). Ca: C. albicans; Sc:

S. cerevisiae, GlcNAc: A/-acetyl glucosamine. A positive signal (A₄₅₀≥ 0.5) was only observed for purified C. albicans mannan.

Figure 8: scAb 1A2 binds C. albicans mannan more strongly than scAb 1 H6. Indirect binding ELISAs were performed with decreasing concentrations of scAbs 1A2 (squares) and 1 H6 (triangles). The concentration of scAb 1A2 required to bind 1 μg of purified C. albicans mannan (A₄₅₀≥1) was > 0.002 μg/ml. The concentration of scAb 1 H6 required to bind 1 μg of purified C. albicans mannan was 0.07 μg/ml. Figure 9: scAb 1A2 recognises a lower concentration of purified C. albicans mannan than M1 g1 or scAb 1 H6. Indirect binding ELISAs were performed with decreasing concentrations of purified C. albicans mannan and equal concentrations of scAbs 1A2, M1 g1 and scAb 1 H6. scAb 1A2 at a concentration of 1 μg/ml can recognise -0.03 μg/ml mannan (A₄₅o=0.5). M1g1 at a concentration of 1 μg/ml can recognise -0.07 μg/ml mannan. scAb 1 H6 at a

concentration of 1 μg/ml can recognise -0.2 μg/ml mannan.

Figure 10: Purified C. albicans mannan competes for the binding of the scAb 1A2 to purified hyphal cell wall material. An indirect competition ELISA was performed by mixing the scAb 1A2 (1 μg/ml) with decreasing amounts of purified C. albicans mannan (10 μg/ml to

0.009 μg/ml). The concentration of purified C. albicans mannan required to inhibit the binding of 1A2 to 1 μg of purified hyphal cell wall material (IC₅₀) is - 0.006 μg/ml, and the limit of detection (IC₂₀) is - 0.002 μg/vΓ^\.

Figure 11 : The scAb 1A2 binds to mannans in the C. albicans cell wall with a stronger affinity than 1 H6. Transmission electron micrographs of sections of wild type C. albicans cells stained with the scAb 1A2/anti-HuCK/anti-lgG-gold (40 nm) (A), scAb 1 H6/anti- HuCK/anti-lgG-gold (40 nm) (B), mannan:scAb 1A2/anti-HuCK/anti-lgG-gold (40 nm) (C) and mannan:scAb 1A2/anti-HuCK/anti-lgG-gold (40 nm) (D). The scale bars represent 0.5 μηι.

Figure 12: The scAb 1A2 inhibits the growth of C. albicans cell in vitro. Specific numbers (top) of wild type C .albicans yeast (A,C) or hyphal (B,D) cells were treated with scAbs 1A2 (50 μg/ml) (Ai, Bi), 1 H6 (50 μg/ml) (Ci, Di), an irrelevant scAb Hap 2 (which has affinity for acyl-homoserine lactone signalling molecules produced by bacteria such as Pesudomonas aeruginosa)(50 μg/ml) (Aii, Bii, Cii, Dii), or left untreated (Aiii, Biii, Ciii, Diii) for 5 h before being plated in segmented petri dishes. Growth was assessed after over night incubation. The results shown are representative of four independent experiments. Figure 13: Diagrams representing the N- and O-mannan structures present in C. albicans mannosylation mutants. The wild-type strain has a complete O-mannan chain and an N- mannan core structure to which an a(1 ,6)-mannan backbone decorated with branching mannans is attached. The och IA mutant lacks the a(1 ,6)-mannan A/-mannan backbone and branches. The mnt1-mnt2A mutant lacks a(1 ,2)-mannose in the O-mannan chain. The a(1 ,6)-mannan backbone of the A/-mannan of the mnn2-26A mutant is not decorated with any branches.

Figure 14: Treatment with 1A2 and 1 H6 prolong the survival of wax moth larvae infected with C. albicans. Galleria mellonella larvae were infected with 3 x 10⁵ yeast cells of

C. albicans strain SC5314 (except uninfected control) and immediately treated with 4.1 μg scAb (410.8 μg/ml), caspofungin (0.128 μg/ml) as a positive control, or saline as a negative control. The percentage of larvae alive in each group after 24, 48 and 72 h is shown.

Squares: 1A2; triangles: 1 H6; diamonds: uninfected control and caspofungin-treated; circles: unrelated scAb (Hap2) and saline control; The number of larvae in each group is indicated on the graph. * p<0.05, ** p<0.01 compared to the saline control.

Figure 15: 1 H6 and 1A2 kill C. albicans, C. dubliniensis, C. tropicalis and C. krusei. Yeast cells were treated with 1 H6, 1A2 or an unrelated scAb (Hap2) (100 μg/ml), caspofungin (0.128 Mg/ml), or YEPD only (untreated) for 30 min at 37°C with shaking at 200 rpm, and then for a further 4-5 h at room temperature. Cells were plated, colonies were counted and the viability determined by dividing the number of colonies by the number of cells that were treated. These values were then normalised relative to the untreated control. n=2 for each strain and treatment. Error bars are sd.

Examples - Materials and Methods:

Example 1

Preparation of C. albicans hyphal cell wall material

Cell wall material was prepared from C. albicans strain SC5314 (Gillum et al., 1984) grown in hyphae-inducing conditions (RPMI 1640, 37°C, 200 rpm) for 24 h. Hyphal cells were collected by centrifugation (3400 x g, 4°C, 10 min), washed three times and resuspended in cold dH₂0. Cells were broken in the presence of acid-washed glass beads using a FastPrep machine (10 x 45 s bursts, speed 4, with incubation on ice between bursts). Unwanted cellular debris and glass beads were pelleted by centrifugation (2300 x g, 4°C, 5 min). The supernatant containing cell wall material was removed by gentle aspiration. Cytoplasmic proteins were removed from the cell wall material by washing five times with 1 M NaCI.

Example 2

Preparation of C. albicans mannan

Mannans were extracted from C. albicans strain SC5314 (Gillum et al., 1984) grown in yeast-inducing conditions (YEPD (1 % (w/v) yeast extract, 2% (w/v) mycopeptone, 2% (w/v) D-glucose), 30°C, 200 rpm) for 48 h. Yeast cells were collected by centrifugation (3400 x g, 4°C, 15 min), resuspended in 0.2 M NaCI, autoclaved at 140°C, then freeze-dried overnight. Freeze dried material was resuspended in 2% KOH and incubated at 100°C for 2 h. Cellular debris was collected by centrifugation 2300 x g, RT, 15 min) and the supernatant containing mannosylated cell wall proteins was collected. Mannans were precipitated by adding

Fehling's reagent (7% (w/v) CuS0₄.5H₂0, 35% (w/v) potassium sodium tartrate, 10% (w/v) NaOH), and purified by repeated dissolution in 3 M HCI and precipitatation in

methanol :acetic acid (8: 1). The purified precipitated mannans were collected by

centrifugation (2300 x g, 4°C, 15 min), resuspended in dH₂0, dialysed against dH₂0 for 24 h then freeze dried.

Example 3

Immunisation of sheep with hyphal cell wall material

A Welsh breed of Suffolk sheep was immunised with a stable emulsion of hyphal cell wall material and adjuvant (100 μg of hyphal cell wall material, 2 ml PBS, 2.66 ml Freund's complete/incomplete adjuvant) on a 4 weekly basis by subcutaneous injection at six sites to ensure maximum exposure of the antigen to lymph glands. The immunisation schedule is shown in Table 1. Example 4

Analysis of serum antibody response by indirect ELISA

The wells of a Nunc-lmmuno MaxiSorp F96 well flat bottomed ELISA plate were coated with hyphal cell wall material, purified C. albicans mannan (prepared as described above), purified C. albicans β(1 ,3)-glucan (a kind gift from David Williams, East Tennessee State University, USA) or purified C. albicans chitin (a kind gift from Jeanette Wagener, University of Aberdeen, UK) at a concentration of 1 μg/well overnight at 4°C. Plates were rinsed three times with PBS supplemented with 0.1 % Tween 20 (PBS-T) and once with PBS, blocked with 2% (w/v) skimmed milk powder in PBS at 37°C for 1 h, then rinsed three times with PBS-T. Ten-fold serial dilutions of pre-immune serum or CTA-1 R5 (serum prepared from the fifth bleed of the immunised sheep) were added to the wells. After 1 h incubation at room temperature, the plates were rinsed three times with PBS-T. Anti-sheep IgG-HRP (Sigma; 1/1000 dilution) or anti-sheep IgM-HRP (KPL; 1/500 dilution) were added to the wells and incubated at room temperature for 1 h. Plates were washed with PBS-T and PBS and TMB substrate solution (Sure Blue™) was added. The reaction was stopped by the addition of 1 M H₂S0₄ when a deep blue colour had developed. The absorbance at 450 nm was measured. Example 5

Transmission electron microscopic analysis of the binding of CTA-1 R5 polyclonal serum to the C. albicans cell wall

C. abicans strain NGY152 (Brand et al., 2004) was grown in YEPD supplemented with uridine to mid-log phase at 30°C. Samples were collected by centrifugation, transferred to specimen carriers and frozen in liquid nitrogen at high pressure using a Leica EM PACT2 high-pressure freezer (Leica Microsystems) and EM RTS rapid transfer system (Leica Microsystems). Freeze-substitution was carried out using a Leica EM AFS2 automatic freeze substitution system (Leica Microsystems) and EM FSP freeze substitution processor in dried acetone for 24 h at -90°C, warmed to -50°C over 8 h in acetone, held at -50°C for 24 h in acetone, dehydrated in 100% ethanol at -50°C, embedded in increasing amounts of Lowicryl HM20 resin at -50°C over 8 h, held at -50°C for 24 h, warmed to 20°C over 14 h and held at 20°C for 48 h Ultrathin sections (90-100 nm) were cut with a diamond knife (Diatome Ltd.) using a Leica UC6 ultramicrotome (Leica Microsystems) onto copper grids, stained with colloidal gold (as described below) and imaged using a Philips CM 10 transmission electron microscope (FEI). Images were recorded using Digital Micrograph software (Gatan Inc.).

Ultra-thin sections on copper grids were stained with colloidal gold by floating the grids through drops of PBS+0.02% glycine for 10 min, PBS+1 % BSA for 5 min, CTA-1 R5

(10 Q/ml; 1/500 dilution) in PBS+1 % BSA for 1 h in the dark, PBS+1 % BSA 5 x 1 min, gold- labelled anti-sheep IgG (raised in rabbit, 15 nm gold particle, Aurion #815-155, 1/20 dilution) in PBS+1 % BSA for 30 min in the dark, PBS+1 % BSA 4 x 5 min and lastly transferred through 5 drops of dH₂0. Grids were dried and then stained with uranyl acetate and lead citrate before imaging in the TEM. A pre-immune serum control was performed by staining grids as above but with pre-immune serum instead of polyclonal antibody at an equivalent dilution (1/560) in PBS+1 % BSA. Example 6

C. albicans cell growth inhibition assay using polyclonal serum

C. albicans strain SC5314 (Gillum et al. , 1984) was grown in hyphae-inducing conditions (RPMI 1640, 37°C, 200 rpm) for 3 h. Hyphal cells were counted, and ten-fold serial dilutions in RPMI were prepared in wells of a microtitre tissue culture plate starting at 10⁵ cells.

Serially diluted hyphal cells were treated with a 1/100 dilution of pre-immune serum or CTA- 1 R5 for 30 min at 37°C with shaking at 200 rpm and then incubated for a further 5 h at room temperature. Cells from each well were plated on SD agar in segmented square petri dishes and incubated overnight at 37°C. Growth was visually assessed by imaging with a GelLogic 2200PRO imager.

Example 7 Construction of an scFv M 13 phage display library from peripheral blood lymphocytes of a sheep immunised with purified C. albicans hyphal cell wall material

Peripheral blood lymphocytes were isolated by density gradient centrifugation using

Histoplaque columns (Sigma) from the production bleed of the sheep immunised with C. albicans hyphal cell wall material (PB; Table 1 ) and stored in RNAIater (QIAGEN) at - 80°C. Total RNA was extracted from 1.4 x 10⁸ cells using an RNeasy midi kit (QIAGEN) as per the manufacturer's instructions. Total RNA was treated with amplification grade DNase I (Sigma) and used as template for cDNA synthesis using Superscript I I I RNase H reverse transcriptase (Invitrogen). An scFv library was constructed by cloning the immunoglobulin variable gene repertoire from the cDNA using the strategy similar to that described in Charlton et al. (2000). Primer sequences are listed in Table 2.

Variable heavy (VH) gene segments were PCR amplified from the cDNA template using H F Phusion mastermix (N EB) and primers OvVM BACK, OvVH2BACK, OvVH3BACK and

OvVH4BACK with a mixture of JH region FOR primers (OvJM LI NKFOR, OvJH2LI NKFOR, OvJH3LI NKFOR and OvJH4LI NKFOR). Similarly, variable light (VA) gene segments were amplified using specific OvVLI LI NKBACK, OvVL2LI NKBACK, OvVL3LI NKBACK,

OvVL4LI NKBACK and OvVL5LI NKBACK primers and a mixture of JL region FOR primers (OvJL1 FOR and OvJL2FOR). PCR products were gel extracted using a QIAquick gel extraction kit (QIAGEN), poly-A tailed using Taq polymerase, purified using the QIAquick PCR purification kit (QIAGEN), ligated into the pGEM-T Easy vector (Promega) and transformed into electrocompetent E. coli TG1 cells (Stratagene) by electroporation.

Plasmids containing the PCR products were extracted using a QIAprep spin miniprep kit (QIAGEN) and the diversity of the products was confirmed by DNA sequencing (Dundee University) of≥ 100 random clones using the primers M 13FOR and M 13 REV.

A cellulase linker was inserted between heavy (VH) and light (VA) chains by restriction- ligation creating single chain fragments (scFv). Asc\ and Mlu\ restriction sites were previously incorporated in the variable heavy and light chains respectively at the linker region by the primers OvJHLI NKBACK and OvVLLINKBACK. VH PCR products were digested Asc\ and VA PCR products were were digested with Mlu\. The digested DNA was purified using a QIAquick PCR Purification kit (QIAGEN) according to the manufacturer's instructions. Equal quantities of Ascl-digested VH DNA was ligated to M/ul-digested VA DNA with T4 DNA ligase (Promega). Asc\ and Mlu\ were added to the ligation reaction to prevent the formation of VH-VH and VA-VA products. Ligation products were gel purified using a QIAquick gel extraction kit (QIAGEN).

The gel extracted scFv fragments were PCR amplified using Phusion HF mastermix (NEB) and primers VH-A/col and JL1 -A/ofl to add Nco\ and Λ/ofl sites to the ends of the scFv fragments. PCR products were purified using a QIAquick PCR purification kit (QIAGEN), poly-A tailed using Taq polymerase, purified using the QIAquick PCR purification kit

(QIAGEN), ligated into the pGEM-T Easy vector (Promega) and transformed into

electrocompetent E. coli TG1 cells (Stratagene) by electroporation. Plasmids containing the PCR products were extracted from 20 clones using a QIAprep spin miniprep kit (QIAGEN) and the diversity of the products was confirmed by DNA sequencing (Dundee University) of 10 random clones using the primers M 13FOR and M 13 REV.

The VH-VA fragments were cloned into the phagemid vector pHEN2a via the Nco\ and Λ/ofl sites (Figure 1) and transformed into electrocompetent E. coli TG1 cells (Stratagene) by electroporation. Again, diversity of the products was confirmed by DNA sequencing (Dundee University) of 40 pHEN2a-VH-VA random clones using the primer AH 18REV. The size of the scFv (VH-VA) library in E. coli TG1 cells was 3.88 x 10¹⁰.

Example 8 Phage display generation of soluble scAb fragments to C. albicans mannan

The svFv library was screened for antibodies with specificity to C. albicans cell wall glycans using a method similar to that described in Leel et al. (2004). Three rounds of panning were performed against purified hyphal cell wall material immobilised on immunotubes (PAN 1 : 100 μg/ml; PAN2: 50 μg/ml; PAN3: 5 μg/ml), flowed by three pans against purified

C. albicans β(1 ,3)-glucan (a kind gift from David Williams, East Tennessee State University, USA) (PAN4: 100 g/ml; PAN5: 50 g/ml; PAN6: 5 g/ml). The pans were conducted in 4 ml PBS with 2% (w/v) skimmed milk powder. Individual colonies from PAN6 were grown in 96 well plates and rescued with helper phage M 13 K07. Specificity of phage supernatants for binding to C. albicans mannan was determined by ELISA. The wells of a Nunc-lmmuno MaxiSorp F96 well flat bottomed ELISA plate were coated with purified C. albicans mannan (NI BSK, UK, Cat No. 77/600 at a concentration of 1 μg/well overnight at 4°C. Plates were rinsed three times with PBS-T and once with PBS, blocked with 2% (w/v) skimmed milk powder in PBS at 37°C for 1 h, then rinsed three times with PBS-T. The supernatants from monoclonal phage in PBS with 2% (w/v) skimmed milk powder were added to the wells. After 1 h incubation at room temperature, the plates were rinsed three times with PBS-T. Anti-M 13-HRP (1/1000 dilution) was added to the wells and incubated at room temperature for 1 h. Plates were washed with PBS-T and PBS and TMB substrate solution (Sure Blue™) was added. The reaction was stopped by the addition of 1 M H₂S0₄ when a deep blue colour had developed. The absorbance at 450 nm was measured.

The scFv encoding regions of positive clones (1A2 and 1 H6) were subcloned into the Nco\ and Not] sites of plMS147 (Hayhurst and Harris, 1999) and transformed into E. coli XL1-Blue supercompetent cells (Agilent Technologies). scAbs were expressed in IPTG-treated cells and purified via the C-terminal 6xHis tag using Ni²⁺ charged immobilised metal ion chelate affinity chromatography as described in (McElhiney et al., 2000). The purified proteins were dialysed against PBS at 4°C and stored in aliquots at -20°C. Western blots were performed using anti-HuCK-HRP (Sigma; 1/5000) to confirm the expression and purification of the scAbs 1A2 and 1 H6.

Example 9 Analysis of the specificity of scAbs by ELISA

To assess the specificity of the scAbs to related glycans and monosaccharides, the wells of a Nunc-lmmuno MaxiSorp F96 well flat bottomed ELISA plate were coated with purified C. albicans β(1 ,3)-glucan (kind gift from David Williams, East Tennessee State University, USA), pustulan ( (1 ,6)-glucan from Lichen), laminarin ( (1 ,3)-glucan from algae),

Saccharomyces cerevisiae mannan, bacterial dextran, starch, purified C. albicans chitin (a kind gift from Jeanette Wagener, University of Aberdeen, UK), D-glucose, D-mannose, galactose, and A/-acetyl glucosamine at a concentration of 1 μg/well overnight at 4°C.

Pustulan was purchased from Calbiochem (Cat no. 540501), laminarin was purchased from Sigma Aldrich UK (Cat no. L9634), S. cerevisiae mannan was purchased from Sigma-Aldrich UK (Cat no. M7504), bacterial dextran was purchased from Sigma-Aldrich UK (Cat no.

D8906), starch was purchased from Sigma-Aldrich UK (Cat no. S9765), D-glucose was purchased from Fischer Chemicals (Cat no. G/0500/65), D-mannose was purchased from Sigma-Aldrich UK (Cat no. M6020), galactose was purchased from Sigma-Aldrich UK (Cat no. G5388), and A/-acetyl glucosamine was purchased from Sigma-Aldrich UK (Cat no. A3286). Wells coated with purified C. albicans mannan (NIBSC, UK) were included as a positive control. Plates were rinsed three times with PBS supplemented with 0.1 % Tween 20 (PBS-T) and once with PBS, blocked with 2% (w/v) skimmed milk powder in PBS at 37°C for 1 h, then rinsed three times with PBS-T. scAb 1A2 or 1 H6 (1/1000 dilution) was added to the wells. After 1 h incubation at room temperature, the plates were rinsed three times with PBS- T. Anti-HuCK-HRP antibody was added to the wells and incubated at room temperature for 1 h. Plates were washed with PBS-T and PBS and TMB substrate solution (Sure Blue™) was added. The reaction was stopped by the addition of 1 M H₂S0₄ when a deep blue colour had developed. The absorbance at 450 nm was measured. To compare the concentration of scAbs required to recognise the same amount of purified C. albicans mannan, indirect binding ELISAs were performed by coating the wells of a Nunc- lmmuno MaxiSorp F96 well flat bottomed ELISA plate with purified C. albicans mannan (NIBSC, UK) at a concentration of 1 μg/well overnight at 4°C. Plates were rinsed three times with PBS-T and once with PBS, blocked with 2% (w/v) skimmed milk powder in PBS at 37°C for 1 h, then rinsed three times with PBS-T. Decreasing concentrations of the scAbs 1A2 or 1 H6 (starting at 1 μg/ml) were added to the wells. After 1 h incubation at room temperature, the plates were rinsed three times with PBS-T. Anti-HuCK-HRP antibody was added to the wells and incubated at room temperature for 1 h. Plates were washed with PBS-T and PBS and TMB substrate solution (Sure Blue™) was added. The reaction was stopped by the addition of 1 M H₂S0₄ when a deep blue colour had developed. The absorbance at 450 nm was measured. To compare the minimum concentration of purified C. albicans mannan recognised by the same concentration of scAbs and a human anti-mannan IgG (M1g1 ; Zhang et al. (2006)), the wells of Nunc-lmmuno MaxiSorp F96 well flat bottomed ELISA plate were coated with purified C. albicans mannan (NIBSC, UK) in concentrations ranging from 10 to 0.004 μg/ml overnight at 4°C. Plates were rinsed three times with PBS-T and once with PBS, blocked with 2% (w/v) skimmed milk powder in PBS at 37°C for 1 h, then rinsed three times with PBS-T. 1A2, 1 H6 and M1g1 were added to the wells at a concentration of 1 μg/ml. After 1 h incubation at room temperature, the plates were rinsed three times with PBS-T. Anti-HuCK- HRP antibody was added to the wells and incubated at room temperature for 1 h. Plates were washed with PBS-T and PBS and TMB substrate solution (Sure Blue™) was added. The reaction was stopped by the addition of 1 M H₂S0₄ when a deep blue colour had developed. The absorbance at 450 nm was measured.

To assess the ability of the scAb 1A2 to recognise purified C. albicans mannan (NIBSC, UK), the wells of a Nunc-lmmuno MaxiSorp F96 well flat bottomed ELISA plate were coated with hyphal cell wall material at concentration of 1 μg/well overnight at 4°C. Plates were rinsed three times with PBS-T and once with PBS, blocked with 2% (w/v) skimmed milk powder in PBS at 37°C for 1 h, then rinsed three times with PBS-T. The scAb 1A2 (1 Mg/ml) was mixed with decreasing amounts of purified C. albicans mannan (from 10 μg/ml to 0.009 μg/ml) and incubated on ice for 1 h at 4°C. The scAb: mannan mixtures were added to wells of the ELISA plate. After 1 h incubation at room temperature, the plates were rinsed three times with PBS-T. Anti-HuCK-HRP antibody was added to the wells and incubated at room temperature for 1 h. Plates were washed with PBS-T and PBS and TMB substrate solution (Sure Blue™) was added. The reaction was stopped by the addition of 1 M H₂S0₄ when a deep blue colour had developed. The absorbance at 450 nm was measured and the IC₅₀ and IC₂o values calculated.

Example 10 Transmission electron microscopic analysis of the binding of scAbs 1H6 and 1A2 to the C. albicans cell wall

Ultrathin sections prepared as described above were stained by floating the grids through drops of PBS+0.02% glycine for 10 min, PBS+1 % BSA for 5 min, 1 H6 or 1A2 (10 Mg/ml) in PBS+1 % BSA for 1 h in the dark, PBS+1 % BSA 5 x 1 min, anti-human kappa light chain (raised in mouse, Sigma K4377, 1/50 dilution) in PBS+1 % BSA for 1 h in the dark, PBS+1 % BSA 5 x 1 min, gold-labelled anti-mouse IgG (raised in goat, 40 nm gold particle, KPL #57- 18-06, 1/50 dilution) in PBS+1 % BSA for 30 min in the dark, PBS+1 % BSA 4 x 5 min and lastly transferred through 5 drops of dH₂0. Grids were dried and then stained with uranyl acetate and lead citrate before imaging in the TEM. Competition with mannan was performed by mixing the scAbs with purified C. albicans mannan (NIBSC, UK) (10 Mg/ml 1 H6 or 1A2, 50 Mg/ml mannan in PBS+1 % BSA) and incubating for 1 h at RT before staining as described above but using scAb-mannan mixture instead of scAb.

Example 1 1

C. albicans cell growth inhibition assay using scAbs C. albicans strain SC5314 (Gillum et al., 1984) were grown in yeast inducing conditions (YEPD, 30°C, 200 rpm) or hyphae-inducing conditions (RPMI 1640, 37°C, 200 rpm) for 3 h. Cells were counted, and ten-fold serial dilutions in YEPD (yeast) or RPMI (hyphae) were prepared in wells of a microtitre tissue culture plate starting at 10⁵ cells. Serially diluted cells were treated with 1A2, 1 H6 or an unrelated scAb at a final concentration of 50 μg/ml for 30 min at 37°C with shaking at 200 rpm and then incubated for a further 5 h at room temperature. Cells from each well were plated on SD agar in segmented square petri dishes and incubated overnight at 37°C. Growth was visually assessed by imaging with a GelLogic 2200PRO imager.

Example 12

Transmission electron microscopic analysis of the binding of scAbs 1H6 and 1A2 to the cell wall of C. albicans glycosylation mutants

C. abicans strains NGY152 (wild-type; Brand et al., 2004), NGY357 (ochIA; Bates et al.,

2006) , NGY112 (mnt1 -mnt2A; Munro et al., 2005), and NGY600 (mnn2-26A; Hall et al., 2013), were grown in YEPD supplemented with uridine to mid-log phase at 30°C, collected and frozen under high pressure, freeze substituted and embedded in Lowicryl HM20 resin as described previously. Ultrathin sections were stained with 1 H6, 1 A2 and colloidal gold (as described previously) and imaged using a JEM-1400 Plus transmission electron microscope (JEOL UK Ltd.). Images were recorded using an AMT ActiveVu XR16M camera (Deben UK Ltd.). The binding of the scAbs to the cell wall was visually assessed by scoring the number of gold particles that bound to the outer fibrillar layer of the cell wall in TEM images. The average number of binding events per cell for each strain and scAb was calculated by dividing the number of gold particles by the number of cells imaged.

Example 13 Galleria mellonella model of C. albicans infection

To assess whether treatment with 1 H6 and 1A2 protected G. mellonella from killing by C. albicans, wax moth larvae in the final instar stage (Livefoods By Post Ltd., Isle of Wight, UK) were injected with 3 x 10⁵ cells of C. albicans strain SC5314 (Gillum et al., 1984) in 10 μΙ PBS into the last left proleg using a 50 μΙ Hamilton syringe, and immediately treated by injecting 10 μΙ of 1 H6, 1A2 or and unrelated scAb (410.8 μg/ml), caspofungin (0.128 μg/ml) or PBS into the last right proleg. The larvae were incubated with moisture at 35°C and assessed for signs of life after 24 h, 48 h and 72 h. Statistical analyses were carried out in IBM SPSS v20. Survival data were plotted using the Kaplan-Meier method and comparisons made between groups using the log rank test. P values of≤0.05 were deemed statistically significant.

Example 14 Determination of the viability of non-albicans Candida species treated with 1H6 and 1A2

C. albicans SC5314 (Gillum et al., 1984), C. dubliniensis SCSI 71549690 (Odds et al.,

2007) , C. tropicalis SCS501 148 (Odds et al., 2007) and C. krusei AM2007/0102 (Odds et al., 2007) were grown in YEPD overnight at 30°C with shaking at 200 rpm. Cells were counted and the culture diluted to a concentration of 2 x 10⁶ cells/ml in YEPD and ten-fold serial dilutions were prepared in YEPD. Serially diluted cells were treated with 1 H6, 1 A2 or an unrelated scAb at a final concentration of 100 μg/ml, or caspofungin at a final concentration of 0.128 μς/ηιΙ, or YEPD only in duplicate wells in a 96-well plate for 30 min at 37°C with shaking at 200 rpm, and then for a further 4-5 h at room temperature. Cells from each well were plated on YEPD plates and incubated over-night at 30°C. Colonies were counted and the viability determined by dividing the number of colonies by the expected number of cells that were added to each well.

Example - Results:

Example 15

The polyclonal serum from a sheep immunised with purified hyphal cell walls of C. albicans contains IgG antibodies that bind predominantly to mannan

Whether sheep mounted an immune response to purified hyphal cell walls of C. albicans was assessed by comparing the titres of IgG and IgM antibodies binding to purified hyphal cell walls in pre-immune serum (PI) to the titres of those in the polyclonal serum from the fifth bleed of an immunised sheep (CTA-1 R5) by indirect ELISA. No increase in the titre of IgM antibodies recognising purified hyphal cell walls in the CTA-1 R5 serum compared to PI serum was observed (data not shown). Polyclonal serum (CTA-1 R5) contained high titres (>10⁶) of IgG antibodies that bind purified hyphal cell walls (Figure 2A), indicating that an immune response was mounted. Hyphal cell walls contain mannans, glucans and chitin.

Polyclonal serum (CTA-1 R5) contained high titres (>10⁶) of IgG antibodies that bind purified C. albicans mannan (Figure 2B) and very low titres of IgG antibodies that recognise β(1 ,3)- glucan (-5000) (Figure 2C) and chitin (-1500) (Figure 2D). This indicated that the predominant antigen in purified hyphal cell walls of C. albicans is mannan.

Electron micrographs of sections of C. albicans cells stained with CTA-1 R5 and anti-sheep IgG conjugated to colloidal gold provided visual confirmation that sheep IgG antibodies in the polyclonal serum bind specifically to the C. albicans cell wall (Figure 3). Example 16

The polyclonal serum from a sheep immunised with purified hyphal cell walls of C. albicans inhibits the growth of C. albicans cells in vitro

Whether polyclonal antibodies in the serum from the fifth bleed of a sheep immunised with purified hyphal cell walls were able to inhibit the growth of C. albicans cells in vitro was assessed. Compared to untreated cells (Figure 4C), the CTA-1 R5 polyclonal serum (1/100 dilution) was able to inhibit the growth of 10⁵ C. albicans cells in 5 h (Figure 4B). Pre- immune serum did not inhibit growth (Figure 4A). It can therefore be hypothesised that mannan-binding IgG antibodies in the serum of sheep immunised with purified hyphal cell walls might be neutralising.

Example 17

Recombinant phage antibodies that recognise C. albicans mannans were identified

In order to isolate specific anti-glycan mAbs that inhibit the growth of C. albicans, an scFv library containing the immunoglobulin variable gene repertoire from the cDNA from peripheral blood lymphocytes isolated from the sheep immunised with purified hyphal cell wall material was constructed. The scFv (VH-VA only) library contained 3.88 x 10¹⁰ clones. Phage display was used to pan for recombinant phage with high affinity for C. albicans cell wall glycans. Phage antibodies were selectively bound and amplified through three separate pans against decreasing amounts of purified hyphal cell wall material (PAN1-3), followed by three pans against purified C. albicans (1 ,3)-glucan (PAN4-6). Surprisingly, PAN6 was enriched with clones with high specificity to C. albicans mannan (Figure 5) and not β(1 ,3)- glucan (data not shown), as determined by ELISA. (1 ,3)-glucan extracted from C. albicans cells contains trace amounts of contaminating mannan and this library is predominantly made up of mannan binders. It was therefore hypothesised that this panning strategy actually selected for clones with a very high affinity for the trace amounts of mannan present in the (1 ,3)-glucan preparations. Individual clones from PAN6 whose monoclonal phage showed high affinity binding to C. albicans mannan were sequenced. Two sequences were enriched, represented as 1A2 (8 of 17 clones sequenced) and 1 H6 (9 of 17 clones sequenced). The nucleotide and amino acid sequences of scFv 1A2 and 1 H6 are shown in Figure 6.

Example 18

Comparison of the ability of scAbs 1A2 and 1H6 to bind C. albicans mannan

The scFv encoding regions of clones 1A2 and 1 H6 were cloned into plMS147 and recombinant scAbs were expressed and purified and western blots were performed to confirm the expression and purification of the scAbs (data not shown). Indirect binding ELISAs showed that the scAbs recognise purified C. albicans mannan, but not glycans from other sources (e.g. pustulan, laminarin, Saccharomyces cerevisiae mannan, bacterial dextran, starch or C. albicans chitin) or sugar monomers (glucose, mannose, galactose or GlcNAc) (Figure 7).

Indirect binding ELISAs showed that the concentration of scAb 1A2 required to bind 1 μg of purified C. albicans mannan was more than 35 times lower than concentration of scAb 1 H6 required to bind 1 μg of purified C. albicans mannan (Figure 8), indicating that scAb 1A2 has a higher affinity for C. albicans mannan than scAb 1 H6. The minimum concentration of purified C. albicans mannan that is recognised by 1 μg/ml of scAb 1A2 was nearly 7 times less than the minimum concentration of purified C. albicans mannan that is recognised by 1 μg/ml of scAb 1 H6 (Figure 9). Surprisingly, the minimum concentration of purified

C. albicans mannan that is recognised by 1 μg/ml of M1g1 (the full length, bivalent anti-

C. albicans-mannan IgG mAb produced by (Zhang et al., 2006)) was nearly 2.5 times more than the minimum concentration recognised by an equivalent concentration of the scAb 1A2 (Figure 9). The scAb 1A2 can be reformatted into a full length antibody which will increase its affinity by an order of magnitude through the avidity effect. Therefore scAb 1A2 is better than previously described anti-C. albicans mannan mAbs.

The strength of binding of the scAb 1A2 was determined by performing an indirect competition ELISA (Figure 10). The concentration of mannan required to inhibit the binding of scAb 1A2 to purified hyphal cell wall material (IC₅₀) was ~ 0.006 μg/ml, which is very close to the limit of detection (IC₂₀ ~ 0.002 μg/ml) indicating that the scAb 1A2 has a very high sensitivity for purified C. albicans mannan. The IC₅₀ for the scAb 1 H2 was greater than 10 μg/ml (data not shown). Electron micrographs of sections of C. albicans cells stained with the scAbs 1A2 and 1 H6 provided visual confirmation that both scAbs bind specifically to mannosylated cell wall proteins in the C. albicans cell wall (Figure 1 1A, B). The mannans are predominantly seen as fibrils projecting from the cell wall, but additional mannans are attached to the cell wall proteins that are embedded in the chitin and (1 ,3)-glucan layer of the cell wall. Purified C. albicans mannan (50 μg/ml) was able to compete for the binding of the scAb 1 H6 but not 1A2 (both at a concentration of 10 μg/ml), providing further confirmation that 1A2 has a higher affinity for mannan than 1 H6 (Figure 1 1C, D). Example 19 scAb 1A2 but not 1H6 inhibits the growth of C. albicans cells in vitro

Whether the mannan-binding scAbs 1A2 and 1 H6 were able to inhibit the growth of

C. albicans cells in vitro was assessed. Compared to untreated cells (Figure 12Aiii, Biii) and an irrelevant scAb control (Figure 12Aiii, Bii), the scAb 1A2 at a concentration of 50 μg/ml was able to inhibit the growth of 10² yeast cells in 5 h (Figure 12Ai) and 10⁴ C. albicans hyphal cells in 5 h (Figure 12Bi). In contrast, the scAb 1 H6 was not able to inhibit growth (Figure 12C, D). This emphasises the unique properties of the scAb 1A2 as not all mannan- binding scAb are neutralising in this assay. Importantly, the assay was performed in vitro using a scAb and not a full-length mAb. Therefore the killing of the fungal cells cannot be through opsonisation or activation of the complement cascade (as for other anti-mannan antibodies) but must be via binding alone and probable inhibition by steric hindrance of a key biological function associated with growth. Example 20

TEM-based determination of the scAb specificities

We next confirmed the binding properties of the scAbs 1A2 and 1 H6 and determined the specific mannose epitopes that these scAbs bind to by visually assessing the binding of these scAbs to the cell wall of various C. albicans glycosylation mutants by TEM.

We assessed binding of the scAbs to wild-type cells, an och IA mutant which has a severe A/-mannosylation defect, an mnt1-mnt2A mutant which lacks O-mannan, and an mnn2-26A mutant in which there is no elaboration of the a(1 ,6)-A/-mannan backbone. These defects are illustrated in Figure 13.

The binding of both scAbs to the fibrillar layer of the ochIA mutant was significantly increased compared to wild-type (>40 binding event per cell of the ochIA mutant compared to <15 per cell for the wild-type; Table 3). This indicates that both 1A2 and 1 H6 recognise a portion of the core A/-mannan structure that may be partially masked by the a(1 ,6)-N- mannan backbone and branches in wild-type cells. Since the binding of 1A2 to the outer fibrillar layer of the mnn2-26A mutant is roughly equivalent to that of the wild-type, the recognition site for 1A2 appears to be partially masked by the a(1 ,6)-A/-mannan backbone (and not the branches). This is not the case for 1 H6, and strongly suggests that 1 H6 and 1 A2 recognise slightly different parts of the core A/-mannan structure. Neither scAb binds to O-mannan as there was no reduction in binding to the outer fibrillar layer of the mnt1-mnt2A mutant which does not have O-mannans.

This data also confirms that the epitope of the existing anti-mannan mAb B6.1 (Han and Cutler, 1995) is distinct from that recognised by both 1A2 and 1 H6. The epitope of B6.1 has been identified as β(1 ,2)-Γη3ηηοίπο3β in phospho-mannan (Han et al., 1997) which is attached to the a(1 ,6)-A/-mannan backbone, and is therefore not part of the core mannan structure. The binding to the core mannan structure may compromise the function of vital cell wall proteins in a way that other antibodies, that bind to more peripheral mannan structures, do not. This may in turn explain the cell killing seen with antibodies 1 H6 and 1A2, even as fragments.

Example 21

Treatment with 1H6 and 1A2 prolongs survival in an invertebrate model of C. albicans infection

We next assessed whether 1A2 and 1 H6 provide protection against C. albicans infections in vivo using an invertebrate model of C. albicans infection. As shown in Figure 14, a higher percentage of infected larvae that were treated with 1 A2 or 1 H6 were alive after 24, 48 and 72 h compared to those treated with saline. The average mass of the larvae was 0.25 g and each larva was injected with 4.1 μg of scAb. Therefore, the dose used was equivalent to approximately 16.4 mg/kg of antibody fragment. This is an excellent and surprising result as the antibody fragment alone shows protection in an in vivo model. The dose used here (approx. 16 mg/kg) is a low equivalent dose for most antibody therapeutics (typically 50-100 mg /kg in patients) and therefore these results are a good predictor that treatment with 1 H6 and 1A2 will provide protection in vivo at doses that are readily achievable in humans. In addition most therapeutic antibodies are administered not as fragments but as whole antibodies with a human Fc region. Further hyphal killing may be possible in this format through recruitment of the human immune system to the site of binding.

Example 22

Comparison of the ability of 1H6 and 1A2 to inhibit the growth of non-albicans Candida species

We also assessed whether 1 H6 and 1 A2 were able to kill yeast cells of non- albicans

Candida species. As shown in Figure 15, the percentage viability of yeast cells of clinical isolates of C. dubliniensis, C. tropicalis and C. krusei treated with 100 μg/ml of 1 H6 and 1A2 was reduced by more than 2.5-fold compared to untreated cells. This was a larger reduction in viability than was observed when C. albicans yeast cells were treated with 1 H6 and 1A2. This indicates that these scAbs will also be useful for the treatment of non- albicans Candida infections. C. krusei is resistant to caspofungin, one of the important and current antifungals used for the treatment of systemic Candida infections in man. These anti-mannan antibodies may provide a credible therapeutic alternative for the treatment of drug resistant strains of Candida spp.

Further, this information indicates that the epitopes recognised by 1 H6 and 1A2 differ from that of the existing M 1g1 anti-mannan mAb (Zhang et al., 2006). While the precise mannan epitope recognised by M1 g1 is not known, fluorescently labelled M 1g1 did not bind to yeast cells of C. krusei (Zhang et al., 2006). In contrast, our work has shown that 1 H6 and 1A2 inhibit the growth of C. krusei yeast cells (Fig. 15).

References:

Armitage, R.J., Fanslow, W.C., Strockbine, L, Sato, T.A., Clifford, K.N., Macduff, B.M.,

Anderson, D.M., Gimpel, S.D., Davis-Smith, T., Maliszewski, C.R., et al. (1992).

Molecular and biological characterization of a murine ligand for CD40. Nature 357:80- 82. Bagshawe, K.D., Sharma, S.K., Springer, C.J., Antoniw, P., Rogers, G.T., Burke, P.J.,

Melton, R. and Sherwood, R.F. (1991). Antibody-enzyme conjugates can generate cytotoxic drugs from inactive precursors at tumor sites. Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922.

Bates, S., Hughes, H.B., Munro, C.A., Thomas, W.P., MacCallum, D.M., Bertram, G., Atrih, A., Ferguson, M.A., Brown, A.J., Odds, F.C., Gow, N.A. (2006). Outer chain N- glycans are required for cell wall integrity and virulence of Candida albicans. J. Biol. Chem. 281 :90-98.

Batista, F.D., Anand, S., Presani, G., Efremov, D.G. and Burrone, O.R. (1996). The two membrane isoforms of human IgE assemble into functionally distinct B cell antigen receptors. J. Exp. Med. 184:2197-2205

Brand, A., MacCallum, D.M., Brown, A.J., Gow, N.A. and Odds, F.C. (2004). Ectopic

expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted reintegration of URA3 at the RPS10 locus. Eukaryot. Cell 3:900-909.

Charlton, K.A., Moyle, S., Porter, A.J. and Harris, W.J. (2000). Analysis of the diversity of a sheep antibody repertoire as revealed from a bacteriophage display library. J.

Immunol. 164:6221-6229.

Cummings, R.D. and Doering, T.L. (2009). Chapter 21 Fungi. In: 'Essentials of Glycobiology (2nd ed)'. Varki, A., Cummings, R.D., Esko, J.D., et al. (eds). Cold Spring Harbor

Laboratory Press: New York.

Edwards, J.E., Jr. (2012). Fungal cell wall vaccines: an update. J. Med. Microbiol. 61 :895- 903.

Gillum, A.M., Tsay, E.Y. and Kirsch, D.R. (1984). Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and

E. coli pyrF mutations. Mol. Gen. Genet. 198: 179-182.

Grant, S.D., Porter, A.J. and Harris, W.J. (1999). Comparative sensitivity of immunoassays for haptens using monomeric and dimeric antibody fragments. J. Agric. Food Chem.

47:340-345.

Haan, K. and Maggos, C. (2004). The protein engineers. BioCentury - The Bernstein Report on BioBusiness 12(5): A1-A6.

Haidaris, C.G., Malone, J., Sherrill, LA., Bliss, J.M., Gaspari, A.A., Insel, R.A. and Sullivan, M.A. (2001). Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens. J. Immunol. Methods 257: 185-202.

Hall, R.A., Bates, S., Lenardon, M.D., MacCallum, D.M., Wagener, J., Lowman, D.W.,

Kruppa, M.D., Wlliams, D.L., Odds, F.C, Brown, A.J., Gow, N.A. (2013). The Mnn2 mannosyltransferase family modulates mannoprotein fibril length, immune recognition and virulence of Candida albicans. PLoS Pathog. 9:e1003276.

Han, Y. (2010). Efficacy of combination immunotherapy of IgM MAb B6.1 and amphotericin B against disseminated candidiasis. Int. Immunopharmacol. 10: 1526-1531.

Han, Y. and Cutler, J.E. (1995). Antibody response that protects against disseminated

candidiasis. Infect. Immun. 63:2714-2719.

Han, Y., Kanbe, T., Cherniak, R. and Cutler, J.E. (1997). Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies. Infect. Immun. 65:4100-4107.

Han, Y., Kozel, T.R., Zhang, M.X., MacGill, R.S., Carroll, M.C. and Cutler, J.E. (2001).

Complement is essential for protection by an IgM and an lgG3 monoclonal antibody against experimental, hematogenously disseminated candidiasis. J. Immunol.

167:1550-1557. Han, Y., Morrison, R.P. and Cutler, J.E. (1998). A vaccine and monoclonal antibodies that enhance mouse resistance to Candida albicans vaginal infection. Infect. Immun. 66:5771-5776.

Han, Y., Riesselman, M.H. and Cutler, J.E. (2000). Protection against candidiasis by an immunoglobulin G3 (lgG3) monoclonal antibody specific for the same mannotriose as an IgM protective antibody. Infect. Immun. 68:1649-1654.

Hasenclever, H.F. and Mitchell, W.O. (1961). Antigenic studies of Candida. I. Observation of two antigenic groups in Candida albicans. J. Bacteriol. 82:570-573.

Hassan, I., Powell, G., Sidhu, M., Hart, W.M. and Denning, D.W. (2009). Excess mortality, length of stay and cost attributable to candidaemia. J. Infect. 59:360-365.

Hayhurst, A. and Harris, W.J. (1999). Escherichia coli Skp chaperone coexpression

improves solubility and phage display of single-chain antibody fragments. Protein Expr. Purif. 15:336-343.

Holliger, P. and Hudson, P.J. (2005). Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23: 1126-1136.

Koide, A., Bailey, C.W., Huang, X. and Koide, S. (1998). The fibronectin type III domain as a scaffold for novel binding proteins. J. Mol. Biol. 284: 1 141-1 151.

Kozel, T.R., MacGill, R.S., Percival, A. and Zhou, Q. (2004). Biological activities of naturally occurring antibodies reactive with Candida albicans mannan. Infect. Immun. 72:209- 218.

Ledermann, J. A., Begent, R.H., Massof, C, Kelly, A.M., Adam, T. and Bagshawe, K.D.

(1991). A phase-l study of repeated therapy with radiolabeled antibody to

carcinoembryonic antigen using intermittent or continuous administration of cyclosporin A to suppress the immune response. Int. J. Cancer 47:659-664.

Lee, J.H., Jang, E.C. and Han, Y. (201 1). Combination immunotherapy of MAb B6.1 with fluconazole augments therapeutic effect to disseminated candidiasis. Arch. Pharm. Res. 34:399-405.

Leel, V., Elrick, L.J., Solares, J., Ingram, N., Charlton, K.A., Porter, A.J. and Wright, M.C.

(2004). Identification of a truncated ratp28-related protein expressed in kidney.

Biochem. Biophys. Res. Commun. 316:872-877.

Lehmann, P.F. and Reiss, E. (1980). Comparison by ELISA of serum anti- Candida albicans mannan IgG levels of a normal population and in diseased patients. Mycopathologia

70:89-93.

Li, E., Pedraza, A., Bestagno, M., Mancardi, S., Sanchez, R. and Burrone, O. (1997).

Mammalian cell expression of dimeric small immune proteins (SIP). Protein Eng.

10:731-736.

McElhiney, J., Lawton, L.A. and Porter, A.J.R. (2000). Detection and quantification of

microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naive human phage display library. FEMS Microbiol. Lett. 193:83-88. Munro, C.A., Bates, S., Buurman, E.T., Hughes, H.B., MacCallum, D.M., Bertram, G., Atrih, A., Ferguson, M.A., Bain, J.M., Brand, A., Hamilton, S., Westwater, C, Thomson, L.M., Brown, A.J., Odds, F.C., Gow, N.A. (2005). Mntl p and Mnt2p of Candida albicans are partially redundant alpha-1 ,2-mannosyltransferases that participate in delinked mannosylation and are required for adhesion and virulence. J. Biol. Chem. 280: 1051-1060.

Netea, M.G., Brown, G.D., Kullberg, B.J. and Gow, N.A.R. (2008). An integrated model of the recognition of Candida albicans by the innate immune system. Nat. Rev.

Microbiol. 6:67-78. Netea, M.G., Gow, N.A.R., Joosten, L.A., Verschueren, I., van der Meer, J.W. and Kullberg, B.J. (2010). Variable recognition of Candida albicans strains by TLR4 and lectin recognition receptors. Med. Mycol. 48:897-903.

Netea, M.G., Gow, N.A.R., Munro, C.A., Bates, S., Collins, C, Ferwerda, G., Hobson, R.P., Bertram, G., Hughes, H.B., Jansen, T., et al. (2006). Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Tolllike receptors. J. Clin. Invest. 116: 1642-1650.

Nygren, P. A. and Uhlen, M. (1997). Scaffolds for engineering novel binding sites in proteins.

Curr. Opin. Struct. Biol. 7:463-469.

Odds, F.C., Hanson, M.F., Davidson, A.D., Jacobsen, M.D., Wright, P., Whyte, J.A., Gow, N.A., Jones B.L. (2007). One year prospective survey of Candida bloodstream infections in Scotland. J. Med. Microbiol. 56:1066-1075.Shapiro, R.S., Robbins, N. and Cowen, L.E. (2011). Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol. Mol. Biol. Rev. 75:213-267.

Shibata, N., Fukasawa, S., Kobayashi, H., Tojo, M., Yonezu, T., Ambo, A., Ohkubo, Y. and Suzuki, S. (1989). Structural analysis of phospho-D-mannan-protein complexes isolated from yeast and mold form cells of Candida albicans NIH A-207 serotype A strain. Carbohydr. Res. 187:239-253.

Shibata, N., Kobayashi, H. and Suzuki, S. (2012). Immunochemistry of pathogenic yeast, Candida species, focusing on mannan. Proc. Jpn. Acad., Ser. B. 88:250-265.

Vincent, J.L., Rello, J., Marshall, J., Silva, E., Anzueto, A., Martin, CD., Moreno, R., Lipman, J., Gomersall, C, Sakr, Y., et al. (2009). International study of the prevalence and outcomes of infection in intensive care units. JAMA 302:2323-2329.

Wess, L. (2004). Antibody trek, the next generation. BioCentury - The Bernstein Report on BioBusiness 12(42) , A1-A7.

Wellington, M., Bliss, J.M. and Haidaris, C.G. (2003). Enhanced phagocytosis of Candida species mediated by opsonization with a recombinant human antibody single-chain variable fragment. Infect. Immun. 71 :7228-7231.

Zhang, M.X., Bohlman, M.C., Itatani, C, Burton, D.R., Parren, P.W., St Jeor, S.C. and Kozel, T.R. (2006). Human recombinant antimannan immunoglobulin G1 antibody confers resistance to hematogenously disseminated candidiasis in mice. Infect. Immun.

74:362-369.

Zhang, M.X., Lupan, D.M. and Kozel, T.R. (1997). Mannan-specific immunoglobulin G

antibodies in normal human serum mediate classical pathway initiation of C3 binding to Candida albicans. Infect. Immun. 65:3822-3827.

Sequences

Amino acid sequence of scFv 1A2 (SEQ ID NO: 1). The positions of the start of the heavy and light chain frameworks (HFW, LFW), complementarity determining regions of the VH (HCDR; light grey) and VA (LCDR; dark grey) chains, and the cellulase linker inserted between the VH and VA chains (LINKER; black) are indicated above the nucleotide sequence.

>1A2 scFv (V_H, linker and V_L)

HFWl HCDRl HFW2 HCDR2 HFW3

1 QVRLQESGPS LVKPSQTLSL TCTVSGFGLT 1|1||WVRQA PGKALEWGll||ii||llll||iil|lRLSIT

HCDR3 HFW4 LINKER LFW1

71 RDTSKSQVSL SLSSVTTEDT AVYYCVRlIlllfWGRGLLVTV S EGKSSGAS GESKVDD QAV LTQPSSVSRS

LCDRl LFW2 LCDR2 LFW3

141 LGQSVSITCf ¾ FQVIPG SAPRTLIif DR FSGSRSGNTA TLTITSLQAE

LCDR3 LF 4

211 DEADYYCl IFGS GTRLTVLG Amino acid sequence of scFv 1A2 VH domain with Kabat Numbering (CDRs in grey): (SEQ ID NO: 2).

>1A2

HFWl

CAG GTG CGG CTG CAG GAG TCG GGA CCC AGC CTG GTG AAG CCC

1 Q V R L Q E S G P S L V K P

Kabat 1 2 3 4 5 6 7 8 9 10 11 12 13 14

TCA CAG ACC CTC TCC CTC ACC TGC ACA GTC TCT GGA TTC GGT

15 S Q T L S L T C T V S G F G

Kabat 15 16 17 18 19 20 21 22 23 24 25 26 27 28

HCDRl HFW2

TTA ACC ACC TATi : Ϊ| !:: 111 TGG GTC CGC CAG GCT CCA GGA

29 L T 111 111 11 W V R Q A P G

Kabat 29 30 31 32 33 34 36 37 38 39 40 41 42

AAG GCA CTG GAG TGG GTT GGT « if!i Iff!; 11 111 ill 43 K A L E W V G Ilill i| Ill ill 111 111

Kabat 43 44 45 46 47 48 49 51 52 53 54 55 56

HFW3

Hi liliiili; 111 ill 111 ^{CGG CTC AGC ATC ACT}

57 11111111 11 P ill 111 K 1 R L S I T

Kabat 57 58 59 60 61 62 63 64 65 66 67 68 69 70 AGG GAC ACC TCC AAG AGT CAA GTC TCT CTG TCA CTG AGC AGC 71 R D T S K S Q V S L S L S S

Kabat 71 72 73 74 75 76 77 78 79 80 81 82 82A 82B

HCDR3

GTG ACA ACT GAG GAT ACG GCC GTC TAC TAC TGT GTA AGG ||| 85 V T T E D T A V Y Y C V R I

Kabat 82C 83 84 85 86 87 88 89 90 91 92 93 94 95

HFW4

lllllilllll TGG GGC CGA GGA CTC CTG GTC ACC GTC TCC TCA

w G R G L L V T v s s

96 97 98 103 104 105 106 107 108 109 110 111 112 113

Amino acid sequence of scFv 1A2 HCDR1 (SEQ ID NO: 3). TYSIE

Amino acid sequence of scFv 1A2 HCDR2 (SEQ ID NO: 4). AVNNNGRTFYNPALKS

Amino acid sequence of scFv 1A2 HCDR3 (SEQ ID NO: 5). TWDV Amino acid sequence of scFv 1A2 VL domain with Kabat Numbering (CDRsin grey): (SEQ ID NO: 6).

LFW1

CAG GCT GTG CTG ACT CAG CCG

127 Q A V L T Q P

Kabat 2 3 4 5 6 7 8 10 11 12 13 14

Kabat 15 16 17 18 19 20 21 22

LFW2

Kabat 27B 27C 29 30 31 32 33 34 35 36 37 38 39

LCDR2 GCC CCC AGA ACC CTC A C ACT

169 A P R T L I T

43 44 45 46 47 48 49

LFW3

57 58 59 60 61 62 63 64 65 66 67

GGC AAC ACA GCG ACT CTT ACC ATC ACC TCG CTC CAG GCT GAG

197 G N T A T L T I T S L Q A E Rabat 68 69 70 71 72 73 74 75 76 77 78 79 80 81

LCDR3

Rabat 82 83 84 85 92 93 94 95

LFW4

T

Rabat 95A 99 100 101 102 103 104 105 106 107 108 109 Amino acid sequence of scFv 1A2 LCDR1 (SEQ ID NO: 7). SGSSSNIGSWNYVD

Amino acid sequence of scFv 1A2 LCDR2 (SEQ ID NO: 8).

AATSRTS

Amino acid sequence of scFv 1A2 LCDR3 (SEQ ID NO: 9). AAWDRSNSKI

Amino acid sequence of scFv 1 H6 (SEQ ID NO: 10). The positions of the start of the heavy and light chain frameworks (HFW, LFW), complementarity determining regions of the VH (HCDR; light grey) and VA (LCDR; dark grey) chains, and the cellulase linker inserted between the VH and VA chains (LINKER; black) are indicated above the nucleotide sequence.

>1H6 scFv (V_H, linker and V_L)

HFWl HCDRl HFW2 HCDR2 HFW3

1 QVKLQESGPS LVKPSQTLSL TCTVSGFSLT !!i§fciVRQA PGKVPEWLGA IKNDERTYYN ?AL SRLSIT

HCDR3 HFW4 LINKER

RDTSKSQVSL ALSSVTTEDS AVYYCARNSA WYRGAAYSS WSGVDVWGP GLLVTVS

LFWl LCDR1 LFW2 LCDR2 LF 3

141 ΒΒΐΒΙθΆλ/Τ,ΤΟΡ SSVSGSPGQR VSITCI ¾JF QQLPGSGLRT ΙΙΥ^^^β TVPDRFSGSR

LCDR3 LFW4

211 SGNTATLTIS SLQAEDEADY FC SFGSGTRLT VLG

Amino acid sequence of scFv 1 H6 VH domain with Kabat Numbering (CDRs in grey): (SEQ ID NO: 11).

HFWl

CAG GTC AAG CTG CAG GAG TCG GGA CCC AGC CTG GTG AAG CCC

1 Q V K L Q E S G P S L V K P

Kabat 1 2 3 4 5 6 7 8 9 10 11 12 13 14

TCA CAA ACC CTC TCC CTC ACC TGC ACG GTC TCT GGA TTC TCA

15 S Q T L S L T C T V S G F S

Kabat 15 16 17 18 19 20 21 22 23 24 25 26 27 28

HCDRl HFW2

TTG ACC 11! TGG GTC CGC CAG GCT CCA GGA

29 L T ill W V R Q A P G

Kabat 29 30 32 33 34 36 37 38 39 40 41 42 HCDR2

AAG GTG CCG GAG TGG CTT GGT m mmmmmmm mm

43 K V P E W L G ^^^ m m

Kabat 43 44 45 46 47 48 49 50 51 52 53 54 55 56 HFW3

^^^^^^^^^^^^^^^^^^^^^^B ^{CGG CTC AGC ATC ACC}

57 ^^^^^^^^^^^^^^^^^^^^^M R L S I T

Kabat 57 58 59 60 61 62 63 64 65 66 67 68 69 70 AGA GAC ACC TCC AAG AGC CAA GTC TCC CTG GCA CTG AGC AGC 71 R D T S K S Q V S L A L S S

Kabat 71 72 73 74 75 76 77 78 79 80 81 82 82A 82B

HCDR3

GTG ACA ACT GAG GAC TCG GCC GTG TAT TAC TGT GCA AGA |§§§ 85 V T T E D S A V Y Y C A R I

Kabat 82C 83 84 85 86 87 88 89 90 91 92 93 94 95 ββιιβιβ ιιιβιβιιβιββΐίΐ ιβιβιβ

99 m m m m mmmm m m m

Kabat 96 97 98 99 100 100A100B100C100D100E100F100G100H100I

HFW4

^^^^^^^^^^^H ^{TGG GGC CCA GGA CTC CTG GTC ACC GTC} 113 ^^^^^^^^^^H W G P G L L V T V

Kabat 100J100K100L101 102 103 104 105 106 107 108 109 110 111

TCC TCA

127 S S

Kabat 112 113

Amino acid sequence of scFv 1H6 HCDR1 (SEQ ID NO: 12). SNNVG

Amino acid sequence of scFv 1H6 HCDR2 (SEQ ID NO: 13). AIRNDERTYYNPALKS

Amino acid sequence of scFv 1H6 HCDR3 (SEQ ID NO: 14). NSAWYRGAAYSDVYVHGVDV

Amino acid sequence of scFv 1H6 VL domain with Kabat Numbering (CDRs in grey): (SEQ ID NO: 15).

LFWl

CAG GCT GTG CTG ACT CAG CCG TCC TCC GTG TCC

141 Q A V L T Q P S S V S

Kabat 2 3 4 5 6 7 8 9 10 11 12

LCDR1

GGG TCC CCG GGC CAG AGG GTC TCC ATC ACC TGC

155 G S P G Q R V S I T C

Rabat 13 14 15 16 17 18 19 20 21 22 23 24 25 26

LFW2

Rabat 27 27A 35 36 37 38

LCDR2

Rabat 39 40 41 42 43 44 45 46 47 48 49 50 5

LFW3

ACT GTC CCG GAC CGA TTC TCC GGC TCC AGG

Rabat 53 54 55 56 57 58 59 60 61 62 63 64 65 66

TCT GGC AAC ACG GCC ACC CTG ACC ATC AGC TCG CTC CAG GCT

211 S G N T A T L T I S S L Q A Rabat 67 68 69 70 71 72 73 74 75 76 77 78 79 80

LCDR3

GAG GAC GAG GCC GAT TAT TTC TGT 111111

225 E D E A D Y F C

Rabat 81 82 83 84 85 86 87 88 89 9 93 94

LFW4

GGT

253 G

Rabatl09

Amino acid sequence of scFv 1 H6 LCDR1 (SEQ ID NO: 16).

SGSSRNVGRFGVG

Amino acid sequence of scFv 1 H6 LCDR2 (SEQ ID NO: 17). DTNSRPS Amino acid sequence of scFv 1 H6 LCDR3 (SEQ ID NO: 18).

ASYDKNSGTL

Amino acid sequence of scAb 1A2 (SEQ ID NO: 19)

>1A2 scAb (PelB leader, V_H, linker, V_L, HUCK and 6xHis tag)

PelB leader HFW1 HCDR1 HF 2

1 MKYLLPTAAA GLLLLAAQPA MAQVRLQESG PSLVKPSQTL SLTCTVSGFG LT|||1|WVR QAPGKALEWV

HCDR2 HFW3 HCDR3 HFW4 LINKER

71 GA HiiNSR P YNf&LKSRLS ITRDTSKSQV SLSLSSVTTE DTAVYYCVR1111§WGRGLLV TVS£

LFW1 LCDR1 LFW2 LCDR2 LFW3

141 g SGESKVDDQ AVLTQPSSVS RSLGQSVSIT elf IWFQVI PGSAPRTLIT SGVP

LCDR3 LFW4 HUCK

211 DRFSGSRSGN TATLTITSLQ AEDEADYYCj IF GSGTRLTVLG AAAAPSVFIF PPSDEQLKSG

281 TASWCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH

6xHis

351 QGLSSPVTKS FNRGESHHHH HH

Amino acid sequence of scAb 1 H6 (SEQ ID NO: 20)

>1H6 scAb (PelB leader, V_H, linker, V_L, HUCK and 6xHis tag)

PelB leader HFW1 HCDR1 HFW2

1 MKYLLPTAAA GLLLLAAQPA MAQVKLQESG PSLVKPSQTL SLTCTVSGFS LTlltlflwVR QAPGKVPEWL

HCDR2 HFW3 HCDR3 HFW4

71 GAIRKDE.RTY YifPAL SRLS ITRDTSKSQV SLALSSVTTE DSAVYYCARN SAWYRGAAYS DVYVH.GVDYW

LINKER LFW1 LCDR1 LFW2

141 GPGLLVTVSS EGKSSGASGE SKVDDQAVLT QPSSVSGSPG QRVSITCl WFQQLPGSGL

LCDR2 LFW3 LCDR3 LFW4 HUCK IITVPDRFSG SRSGNTATLT ISSLQAEDEA DYFCI IFGSGTR LTVLGAAAAP

281 SVFIFPPSDE QLKSGTASW CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSK

6xHis

351 ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE SHHHHHH Table 1 : Immunisation schedule

SP: sample preparation and primary immunisation (hyphal cell wall material and Freund' complete adjuvant)

R: re-immunisation (hyphal cell wall material and Freund's incomplete adjuvant) B: bleed/serum sample preparation

PB: production bleed (3 d after R5)

G: grazing

Table 2: Primers

Ovine heavy chain constant region 3' primer

OvCHFOR 5'-GAC TTT CGG GGC TGT GGT GGA GGC-3'

Ovine kappa chain constant region 3' primer

OvCKFOR 5'-GA TGG TTT GAA GAG GGA GAC GGA TGG CTG AGC-3'

Ovine lambda chain constant region 3' primer

OvCLFOR 5'-A CAG GGT GAC CGA GGG TGC GGA CTT GG-3'

Ovine heavy chain variable region 5' primers (Degenerate Nucleotide code (M = A/C, R = A/G, W = All, S = G/C, Y : C/T, K = G/T)

OvVH1 BACK 5'-CAG GTK CRR CTG CAG GRG TCG GG-3'

OvVH2BACK 5'-CAG GTK CAG YTK CAG GAG TCG GG-3'

MuVHI BACK 5'-SAG GTS MAR CTG CAG SAG TCW GG-3'

Hu4aBACK 5'-CAG GTG CAG CTG CAG GAG TCG GG-3'

Ovine heavy chain variable region 3' primers

OvJH1 LINKFOR 5'-CTC AGA AGO CGC GCC TGA GGA GAC GGT GAC CAG GAG TCC-3' OvJH2LINKFOR 5'-CTC AGA AGG CGC GC' TGA GGA GRC GGW GAY YAG KAG TCC-3' OvJH3LINKFOR 5'-CTC AGA AGG CGC GCC TGA GGA GAY RGT RAS CAG GAS TCC-3' OvJH4LINKFOR 5'-CTC AGA AGG CGC GC' TGA AAG AAC GCT GAT CAG GAG-3'

Ovine lambda chain variable region 5' primers

OWL1 LINKBACK 5'-AG TCA AAC GCG TCT GC ::AC CAG GCT GTG CTG ACT CAG CCG 3' OWL2LINKBACK 5'-AG TCA AAC GCG TCT GC SAC CAR GCT GTG CTG ACY CAR CYG-3' OWL3LINKBACK 5'-AG TCA AAC GCG TCT G< SAC CAG GCY STG STG ACT CAG CCR-3' OWL4LINKBACK 5'-AG TCA AAC GCG TCT GC SAC MRG GTC RTG CKG ACT CAR CCG-3' OWL5LINKBACK 5'-AG TCA AAC GCG TCT G< 5AC CAG KCT GYS CTG ACT CAG CCK-3'

Ovine lambda chain variable region 3' primers

OvJL1 FOR 5'-ACC CAG GAC GGT CAG CCT GGT CC-3'

OvJL2FOR 5'-ACC CAG GAC GGT CAG YCK RGW CC-3'

Ovine kappa chain variable region 5' primers

OWK1 LINKBACK 5'-AG TCA AAC GCG TCT GGC GAG TCT AAA GTG GAT GAC GAC ATC CAG GTG ACC CAG TCT CCA-3'

OWK2LINKBACK 5'-AG TCA AAC GCG TCT GGC GAG TCT AAA GTG GAT GAC GAC ATC CAG CTC ACC CAG TCT CCA-3'

Ovine kappa chain variable region 3' primer

OvJK1 FOR 5'-CCG TTT GAT TTC CAC GTT GGT CC-3'

OvJK2FOR 5'-CCT TTT GAT CTC TAG TTT GGT TCC-3'

OvJK3FOR 5'-CCT TTT GAT CTC TAC CTT GGT TCC-3'

Cloning primers (Degenerate Nucleotide code (M = A C, R = A G, W = A/T, S = G/C, Y = C/T, K = G/T)

OvVH1 BACK-Sft 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTK CRR CTG CAG GRG TCG GG- 3'

OvVH2BACK-Sft 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTK CAG YTK CAG GAG TCG GG-3' MuVH-I BACK-S 5'-CAT GCC ATG ACT GCG GCC CAG CCG GCC ATG GCC SAG GTS MAR CTG CAG SAG TCW GG- 3'

Hu4aBACK-Sft 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG CAG CTG CAG GAG TCG GG-

3'

OvJL1 FOR-Wof 5'-GAG TCA TTC TCG ACT TGC GGC CGC ACC CAG GAC GGT CAG CCT GGT CC-3'

OvJL2FOR-Wof 5'-GAG TCA TTC TCG ACT TGC GGC CGC ACC CAG GAC GGT CAG YCK RGW CC-3'

OvJK1 FOR- Not 5'-GAG TCA TTC TCG ACT TGC GGC CGC CCG TTT GAT TTC CAC GTT GGT CCC-3'

OvJK2FOR-Wof 5'-GAG TCA TTC TCG ACT TGC GGC CGC CCT TTT GAT CTC TAG TTT GGT TCC-3'

OvJK3FOR-Wof 5'-GAG TCA TTC TCG ACT TGC GGC CGC CCT TTT GAT CTC TAC CTT GGT TCC-3'

Sequencing primers

AH18REV 5'-AAA TAC CTA TTG CCT ACG GCA GCC GCT GG-3'

GIN 5'-GAA TTT TCT GTA TGA GGT TTT GC-3'

M13FOR 5'-GTA AAA CGA CGG CCA GTG-3' M13REV 5'-GGA AAC AGC TAT GAC CATG-3'

Table 3: Binding of 1 H6 and 1 A2 to the cell wall of C. albicans glycosylation mutants scAb Strain Gold particles bound to Cells Average gold

outer fibrillar layer particles per cell

1 H6 wild-type 420 37 1 1

1 H6 ochIA 704 17 41

1 H6 mnt1-mnt2A 515 19 27

1 H6 mnn2-26A 395 19 21

1A2 wild-type 492 33 15

1A2 ochIA 690 15 46

1A2 mnt1-mnt2A 561 19 30

1A2 mnn2-26A 281 23 12

Claims

1. An isolated antibody molecule which specifically binds to fungal mannan.

2. An antibody molecule according to claim 1 which inhibits fungal growth.

3. An antibody molecule according to claim 1 or 2 which specifically binds to Candida spp, preferably C. albicans.

4. An antibody molecule according to any one of claims 1 to 3 which specifically binds to the core A/-mannan structure.

5. An antibody molecule according to any one of claims 1 to 4 wherein the antibody molecule comprises an HCDR3 having the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or more amino acid substitutions, deletions or insertions.

6. An antibody molecule according to claim 5 wherein the antibody molecule comprises an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of

SEQ ID NO: 4 with one or more amino acid substitutions, deletions or insertions.

7. An antibody molecule according to claim 5 or claim 6 wherein the antibody molecule comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one or more amino acid substitutions, deletions or insertions.

8. An antibody molecule according to any one of claims 1 to 6 wherein the antibody molecule comprises a VH domain having the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 with one or more amino acid substitutions, deletions or insertions.

9. An antibody molecule according to any one of claims 1 to 8 wherein antibody molecule comprises LCDR1 , LCDR2 and LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9 respectively, or the sequences of SEQ ID NOs 7, 8 and 9 respectively, with one or more amino acid substitutions, deletions or insertions.

10. An antibody molecule according to any one of claims 1 to 9 wherein the antibody molecule comprises a VL domain having the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 with one or more amino acid substitutions, deletions or insertions.

11. An antibody molecule according to any one of claims 1 to 10 comprising a VH domain comprising a HCDR1 , HCDR2 and HCDR3 having the sequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising a LCDR1 , LCDR2 and LCDR3 having the sequences of SEQ ID NOs 7, 8 and 9, respectively, or the sequences of SEQ ID NOs 3, 4, 5, 7, 8 and 9 respectively, with one or more amino acid substitutions, deletions or insertions..

12. An antibody molecule according to claim 11 comprising a VH domain having the amino acid sequence of SEQ ID NO: 2 and a VL domain having the amino acid sequence of SEQ ID NO: 6, or the sequences of SEQ ID NOs 2 and 6 respectively, with one or more amino acid substitutions, deletions or insertions.

13. An antibody molecule according to any one of claims 1 to 12 which is a whole antibody or a scAb.

14. An antibody molecule according to any one of claims 1 to 13 which has a dissociation constant (K_D) for C. albicans mannan of less than about 50 nM.

15. An antibody molecule according to any one of claims 1 to 14 which inhibits fungal growth independently of antibody effector functions. 16. An antibody molecule which competes with an antibody molecule according to any one of claims 5 to 15 for binding to mannan.

17. A pharmaceutical composition comprising an antibody molecule according to any one of claims 1 to 15 and a pharmaceutically acceptable excipient.

18. An antibody molecule according to any one of claims 1 to 16 for use in a method of treatment of the human or animal body.

19. An antibody molecule according to any one of claims 1 to 16 for use in a method of treatment of a fungal infection.

20. Use of an antibody molecule according to any one of claims 1 to 16 in the

manufacture of a medicament for use in treating or preventing a fungal infection. 21. A method of treatment of a fungal infection comprising administering an antibody molecule according to any one of claims 1 to 16 to an individual in need thereof.

22. An antibody molecule according to claim 19, use according to claim 20 or method according to claim 21 wherein the fungal infection is a Candida infection, preferably a C. albicans, C. dubliniensis, C. tropicalis or C. krusei infection.

23. An antibody molecule, use or method according to claim 22 wherein the C. albicans infection is in a hyphal or yeast phase. 24. An antibody molecule according to any one of claims 19, 22 or 23, use according to any one of claims 20, 22 or 23 or method according to any one of claims 21 , 22 or 23, wherein the treatment comprises administering a second antifungal agent, wherein the second antifungal agent is optionally an azole, a polyene or an echinocandin. 25. An antibody molecule according to any one of claims 19, 22, 23 or 24, use according to any one of claims 20, 22, 23 or 24 or method according to any one of claims 21 , 22, 23 or

24. wherein the fungal infection is in an immunosuppressed individual.

26. An antibody molecule according to any one of claims 19, 22, 23, 24 or 25, use according to any one of claims 20, 22, 23, 24 or 25 or method according to any one of claims 21 , 22, 23, 24 or 25, wherein the antibody is dosed at between 1 and 50 mg/kg. 27. A method for detecting the presence or absence of a fungus, the method comprising

(i) contacting a sample suspected of containing the fungus with an antibody molecule according to any one of claims 1 to 15, and

(ii) determining whether the antibody molecule binds to the sample, wherein

28. A method for diagnosing a fungal infection in an individual, the method comprising

(i) contacting a biological sample obtained from the individual with an antibody molecule according to any one of claims 1 to 15, and

(ii) determining whether the antibody molecule binds to the biological sample, wherein binding of the antibody molecule to the biological sample indicates the presence of a fungal infection.

29. The method of claim 27 or claim 28, wherein the fungus or fungal infection is caused by Candida spp, optionally C. albicans.

30. A method of inhibiting growth of a fungal cell, comprising contacting the fungal cell with an agent capable of specifically binding mannan obtained from the fungal cell.

31. A method of screening for an agent capable of inhibiting growth of a fungal cell, the method comprising:

(i) obtaining a mannan sample from a fungus;

(ii) contacting the mannan sample with an agent;

(iv) selecting the candidate antifungal agent; and

32. A method for producing an antibody antigen-binding domain for fungal mannan, the method comprising;

(i) providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR1 , HCDR2 and HCDR3,

wherein the parent VH domain HCDR1 , HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, a VH domain which is an amino acid sequence variant of the parent VH domain,

(ii) optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and

(iii) testing said VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain for the fungal mannan.

33. A method for producing an antibody molecule that specifically binds to a fungal mannan epitope, which method comprises:

recovering said antibody molecule or nucleic acid encoding it.