WO2009030884A2 - Mannosylated butyrophilin tumour markers - Google Patents

Abstract

This invention relates to the finding that butyrophilins (BTNs) are mannosylated when expressed by cancer cells. Methods and compositions relating to the detection and treatment of cancer cells expressing mannosylated butyrophilins (BTNs) are provided.

Description

Mannosylated Butyroph.ilin Tumour Markers

This invention relates to tumour markers which may be useful, for example, in detecting, imaging or targeting cancer cells.

The butyrophilins (BTNs) belong to the immunoglobulin superfamily (IgSF) , and are encoded in a cluster of seven genes in the extended MHC class I region on chromosome 6 (1, 2) . BTNl, BTN2 and BTN3 subfamilies share -50% amino acid identity. The extracellular domains of the three members within each subfamily are more closely related and share 88% (BTN2A1, -A2 and -A3) and 95% (BTN3A1, -A2 and -A3) identity, respectively. BTNl, the prototype of the family, is a major component of the milk fat globule membrane and is regulated by lactogenic hormones . BTNl has a crucial function in the secretion of lipids into milk (3) . Collectively, BTN2 and BTN3 are cell surface transmembrane glycoproteins, transcripts of which are ubiquitously expressed at a low to intermediate level (2) . The extracellular IgV and IgC folds of BTN are related to myelin oligodendrocyte glycoprotein (MOG) , a molecule confined to the CNS and a potential autoantigen in Multiple Sclerosis, to B-G molecules of the chicken MHC and to the B7 (CD80/86) costimulatory molecules (1, 4) . Indeed, a T cell regulatory function is likely as BTN3A1 bound to a ligand on T cells. Similarly, a related mouse gene, butyrophilin-like 2 (BTNL2) functioned as an inhibitor of T cell activation (5-7) . A heptad repeat of a 7-aa sequence encoded by a single exon and a B30.2/SPRY domain at the C-terminus are not found on other B7- like molecules (2) . The B30.2/SPRY domain is also part of a large set of TRIM proteins, that include TRIM5α which has a function in defense against retroviral infections (8) .

DCs are professional APCs that have a pivotal role in controlling immune responses, directing them towards immune activation or tolerance (9) . An important family of antigen receptor involved in recognition and uptake of glycan structures are the C-type lectin receptors (CLRs) (10) . DC-SIGN (DC-specific ICAM-3 grabbing nonintegrin) functions as an internalization receptor for HIV-I, HCV, Mycobacterium tuberculosis and other pathogens and also mediates cellular interactions with T cells and endothelial cells (11) . Recently, evidence has emerged that DC-SIGN recognizes carbohydrate structures on CEACAM-I/Mac -1 and carcinoembryonic antigen (CEA) , specifically expressed on neutrophils and tumor tissues, respectively (12, 13) .

The present inventors have recognised that butyrophilins (BTNs) , such as BTN2 , are differently glycosylated on tumour cells relative to normal cells. BTN expressed by tumour cells comprises high mannose carbohydrate moities which are not present when BTN is expressed by normal cells. Differentially glycosylated BTN may therefore be useful as a tumour marker.

One aspect of the invention provides a method of identifying a tumour cell comprising: determining the presence or amount of mannosylated BTN polypeptide on the surface of a cell, wherein an increased amount of mannosylated BTN polypeptide on the cell relative to control cells is indicative that the cell is a tumour cell .

In some embodiments, a method of identifying a tumour cell may comprise : determining the presence or absence of mannosylated BTN polypeptide on the surface of a cell, wherein the presence of mannosylated BTN polypeptide is indicative that the cell is a tumour cell.

A BTN polypeptide may be a lactogenic BTN polypeptide, such as BTNl, or a nqn- lactogenic polypeptide, such as BTN2 or BTN3 , or an allele or variant of one of any of these polypeptides. The public database entries for the sequences of these BTN polypeptides are listed in Table 1. For example, a BTNl polypeptide may comprise the amino acid sequence of BTNlAl as shown in Table 1 or an allele or variant thereof . An allele or variant of a BTNl polypeptide may comprise an amino acid sequence having at least 80% preferably at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the BTNlal sequence shown in Table 1.

A BTN2 polypeptide may comprise the amino acid sequence of any one of the BTN2A1V1, BTN2Alv2 , BTN2A2vl, BTN2A2v2 or BTN2A3 sequences shown in Table 1 or an allele or variant of any of these sequences. An allele or variant of a BTN2 polypeptide may comprise of an amino acid sequence having at least 80% preferably at least 85%, at least 90%, at least 95% or at least 98% sequence identity with one of these sequences .

A BTN3 polypeptide may comprise the amino acid sequence of any one of the BTN3A1, BTN3A2 or BTN3A3 sequences shown in Table 1 or an allele or variant of any of these sequences. An allele or variant of a BTN3 polypeptide may comprise an amino acid sequence having at least at least 80%, preferably at least 85%, at least 90%, at least 95% or at least 98% sequence identity with one of these sequences.

In preferred embodiments, the BTN polypeptide may be a BTN2 polypeptide, more preferably a BTN2A1 polypeptide.

Amino acid identity is generally defined with reference to the algorithm GAP (GCG Wisconsin Package™, Accelrys, San Diego CA) . GAP uses the Needleman & Wunsch algorithm (J. MoI. Biol. (48) : 444-453 (1970) ) to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST or TBLASTN (which use the method of Altschul et al. (1990) J. MoI. Biol. 215: 405-410) , FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444- 2448) ,or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197) , generally employing default parameters.

Particular amino acid sequence alleles or variants may differ from that a given sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30 or 30-50 amino acids

A mannosylated BTN polypeptide is a BTN polypeptide which is glycosylated at one or more glycosylation sites with a carbohydrate moiety containing a high proportion of mannose residues (i.e. a high mannose moiety) . For example, a mannosylated BTN polypeptide may comprise a carbohydrate moiety having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% mannose residues .

Suitable carbohydrate moieties bind to the mannose-specific lectin Galanthus nivalis agglutinin (GNA) , which binds terminal mannose residues of carbohydrate moieties.

Unlike non-mannosylated BTN polypeptides, mannosylated BTN polypeptides bind to SIGN receptors, such as DC-SIGN. SIGN receptors bind to high mannose moieties which comprise an internal core of high-mannose oligosaccharides . Suitable high mannose moieties are described in more detail in Feinberg et al . , Science 294, 2001. SIGN receptors are described in more detail below.

A cell for use in the methods described herein may be within a sample of cells which have been obtained from an individual, for example a cancer patient or an individual suspected of suffering from cancer. For example, a cell may be comprised in a biopsy obtained from the individual . A tumour cell identified using a method described herein may be a cancer cell from any type of solid cancer or malignant lymphoma and especially leukaemia, sarcomas, skin cancer, bladder cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer, stomach cancer and cerebral cancer. In some preferred embodiments, the tumour cell may be breast, ovary, pancreas or prostate cancer cell.

In some embodiments, the presence or amount of mannosylated BTN polypeptide on a cell may be determined using a specific binding member which binds specifically to mannosylated BTN polypeptide i.e. a specific binding member which binds preferentially to mannosylated BTN polypeptide relative to non-mannosylated BTN polypeptide. For example, a method may comprise;

(i) contacting the cell with a specific binding member which specifically binds mannosylated BTN polypeptide and

(ii) determining the binding of the specific binding member to the cell, wherein the presence or amount of binding being indicative of the presence or amount of mannosylated BTN polypeptide on the cell .

Suitable controls include normal, non-cancer cells. Preferably, the control cells are the same cell type as the test cell.

In some embodiments, binding of the specific binding member to the sample may be determined relative to the binding of the specific binding member to a known cancerous or non-cancerous sample, for example, a sample from a healthy individual or a cancer patient. A method may comprise determining the binding of the specific binding member to a test sample obtained from the individual relative to a normal control sample, an increase in the binding of the specific binding member to the test sample relative to the normal control being indicative that sample contains one or more tumour cells and the individual has a cancer condition. Alternatively, the sample obtained from the individual may comprise one or more regions suspected of being cancerous (i.e. regions which show histological signs of being cancerous) and one or more normal regions (i.e. regions which are histologically normal or noncancerous) . A method may comprise determining the binding of a specific binding member reactive with mannosylated BTN polypeptide to a putative tumour region of said sample relative to a normal region of said sample, an increase in the binding of said specific binding member to the putative tumour region relative to the normal region being indicative that the putative tumour region is cancerous and the individual has a cancer condition.

Specific binding members which specifically bind mannosylated BTN polypeptide include antibodies and antibody fragments or derivatives.

Specific binding members which specifically bind mannosylated BTN polypeptide may bind preferentially to mannosylated BTN polypeptide relative to non-mannosylated BTN polypeptide. Preferably, the specific binding member shows no binding or substantially no binding to non-mannosylated BTN polypeptide or to other molecules, in particular proteins, lipids and carbohydrates, found on the mammalian cell surface.

Techniques for producing suitable specific binding members are well known in the art and described in more detail below.

In other embodiments, the presence or amount of mannosylated BTN polypeptide on the cell may be determined using a specific binding member which binds specifically to BTN polypeptide i.e. a specific binding member which binds to both mannosylated and non-mannosylated BTN polypeptide. Preferably, the specific binding member shows no binding or substantially no binding to other molecules, in particular proteins, lipids and carbohydrates, found on the mammalian cell surface. For example, a method may comprise; (i) contacting the cell with a specific binding member which binds BTN polypeptide and

(ii) determining the amount of binding of the specific binding member to mannosylated BTN.

Binding of the specific binding member to a BTN polypeptide may be determined as described below. For example, the amount of binding of the specific binding member to mannosylated BTN polypeptide may be determined by any suitable technique, including, for example, immunoprecipitation-western, isoelectric focussing (IEF) , immunoprecipitation-IEF and immunoprecipitation-mass spectrometry.

For example, the amount of binding of the specific binding member to mannosylated BTN polypeptide may be determined by precipitation of mannosylated BTN using an mannose-binding member followed by immunodetection of BTN. Alternatively, BTN may be immunoprecipitated and mannosylated BTN detected by using a mannose binding member or by isoelectric focusing. Suitable mannose binding members include plant lectins such as GNA and SIGN receptors such as DC-SIGN.

Suitable specific binding members include antibodies and antibody fragments or derivatives. Antibodies which specifically bind to a BTN polypeptide or to a mannosylated BTN polypeptide may be produced 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 protein 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) Nature 357: 80-82) . Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal. Suitable antibodies may also be isolated from the serum of human cancer patients.

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 proteins (or fragments) , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

Antibodies may be modified in a number of ways. Indeed, the term "antibody" should be construed as covering any binding substance having a binding domain with the required specificity. Thus the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.

The term * specific' refers to the situation in which an antibody molecule will not show any significant binding to molecules displayed on the surface of a mammalian, preferably a human, cell other than its target antigen. The term is also applicable where the antibody molecule is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

In other embodiments, the presence or amount of mannosylated BTN polypeptide on a cell may be determined using a SIGN (specific ICAM grabbing non-integrin) receptor. For example, a method may comprise;

(i) contacting the cell with a SIGN (specific ICAM grabbing non- integrin) receptor, and

(ii) determining the binding of the SIGN receptor to mannosylated BTN polypeptide on the cell.

SIGN receptors are shown herein to bind to mannosylated BTN polypeptides expressed by tumour cells.

A SIGN receptor may be a polypeptide comprising an amino acid sequence set out in database accession numbers AAG13814.1, AAG13848.2, or NP__066978.1, a sequence having at least 50%, at least 60% or at least 70%, at least 80%, at least 90% or at least 95% amino acid sequence identity to one of these amino acid sequences, or a fragment of one of these sequences, in particular a fragment comprising the extracellular domain.

The extra cellular domain of a SIGN receptor may be determined using the Tmpred program that identifies membrane- spanning regions and their orientation. The algorithm is based on the statistical analysis of TMbase, a database of naturally occuring transmembrane proteins (Hofmann & W. Stoffel (1993) Biol. Chem. Hoppe-Seyler 374,166) .

Suitable SIGN receptors may include DC-SIGN, L-SIGN and CD23 receptors. In particular, a SIGN receptor may comprise the extracellular domain of a DC-SIGN or L-SIGN receptor.

A DC-SIGN receptor may comprise the amino acid sequence of AAG13814.1 GI: 10179610 or NP_066978.1 GI : 10863957 , or a sequence having at least 70% amino acid sequence identity to the sequence of AAG13814.1 GI: 10179610 or NP_066978.1 GI: 10863957, preferably at least 80%, at least 90% or at least 95% sequence identity. A L-SIGN receptor may comprise the amino acid sequence of database accession number AAG13848.2 GI: 12084797, or a sequence having greater than 70% amino acid sequence identity to the sequence of AAG13848.2 GI: 12084797, preferably at least 80%, at least 90% or at least 95% sequence identity. A CD23-SIGN receptor may comprise the amino acid sequence of database accession number P06734 GI: 119862, or a sequence having greater than 70% amino acid sequence identity to the sequence of P06734 GI: 119862, preferably at least 80%, at least 90% or at least 95% sequence identity.

A SIGN receptor may be bound to a membrane, for example on a cell surface or may be soluble. A soluble SIGN receptor may comprise the extracellular domain of a sequence set out above, or an allele or variant of such a sequence .

The binding of the SIGN receptor to mannosylated BTN polypeptide on the cell may be determined by standard techniques, such as immunoprecipitation-western, western blotting, flow cytometry, isoelectric focussing (IEF) , immunoprecipitation- IEF, mass spectrometry and immunoprecipitation-mass spectrometry.

For example, a cell may be contacted with immobilised SIGN receptor and probed with an antibody which binds to the BTN polypeptide or a cell may be contacted with immobilised antibody which binds to the BTN polypeptide and probed with a soluble SIGN receptor or lectin, such as GNA.

A method may comprise; (i) determining the binding of a SIGN receptor to a cell, wherein the presence or amount of binding is indicative of the presence or amount of mannosylated BTN polypeptide on the cell .

Determining the binding of the SIGN receptor to the cell may comprise bringing the cell and the SIGN receptor into contact and determining binding as described below.

Binding of a specific binding member or SIGN receptor to a BTN polypeptide may be determined by any appropriate means . Tagging with individual reporter molecules is one possibility. The reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.

Linkage of detectable labels to a specific binding member or SIGN receptor may be direct or indirect, covalent, e.g. via a peptide bond, or non-covalent . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding specific binding member (e.g. antibody) and label molecule. Linkage via a non-covalent bond may be a result of a binding between a biotinylated specific binding member and a streptavidin/avidin linked label molecule.

Labels include fluorochromes such as fluorescein, rhodamine, phycoerythrin, Europium, Alexa 488, Alexa 647 and Texas Red, chromogenic dyes such as diaminobenzidine, macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded, for example in a FACS, ELISA, Western blot, TRFIA such as DELFIA™ (dissociation enhanced lanthanide fluorescence immunoassay) , immunohistochemistry, or lateral flow assay.

Biologically or chemically active agents include enzymes, which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed. Further examples include horseradish peroxidase and chemiluminescence. For example, the specific binding member or SIGN receptor may be labelled with a fluorophore such as FITC or rhodamine, a radioisotope, or a non-isotopic-labelling reagent such as biotin or digoxigenin,- specific binding members containing biotin may be detected using "detection reagents" such as avidin conjugated to any desirable label such as a fluorochrome. In some embodiments, one or more antibodies may be used to detect the binding of the specific binding member or SIGN receptor .

The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge. Suitable approaches include flow cytometry (e.g. FACS), immunohistochemical staining, immunocytochemical staining, Western Blotting, immunofluorescence, enzyme linked immunosorbent assays (ELISA) , radioimmunoassays (RIA) , immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA) , including sandwich assays using monoclonal and/or polyclonal antibodies. All of these approaches are well known in the art .

Other aspects of the invention relate to the in vivo imaging of tumours. A method of imaging tumour tissue may comprise: administering an agent which specifically binds to mannosylated BTN to an individual with a tumour, and detecting or imaging tissue which is bound by the agent, said tissue being tumour tissue.

An agent which specifically binds to mannosylated BTN may comprise a binding moiety and a detection moiety. The binding moiety may comprise a specific binding member such as an antibody, SIGN receptor or lectin which binds mannosylated BTN, for example a plant lectin such as GNA.

The detection moiety may comprise a label such as a radioisotope or paramagnetic particle which is detectable by in vivo imaging techniques such as radiography, fluoroscopy, MRI, bioluminescence imaging (BLI) or positron emission tomography (PET) .

Alternatively, the detection moiety may comprise a tag. A second conjugate comprising a label which is detectable by in vivo imaging techniques and a specific binding member which binds the tag may be used to detect or image tissue which is bound by the agent. Suitable tags are described in more detail below.

Other aspects of the invention relate to the use of mannosylated BTN polypeptide to deliver targetted therapies to tumour cells. A method of treating tumour tissue in an individual may comprise: administering a specific binding member which specifically binds to mannosylated BTN polypeptide to an individual in need thereof .

The specific binding member may have a direct therapeutic effect or may be attached to an anti-cancer agent in a conjugate.

A specific binding member which specifically binds to mannosylated BTN polypeptide may be an antibody or SIGN receptor. A specific binding member which binds to mannosylated BTN polypeptide may be monoclonal antibody specific for oligomannose or a high mannose carbohydrate group of the BTN polypeptide. Such a specific binding member may be an antibody capable of distinguishing between mannosylated and non-mannosylated BTN polypeptide, or between BTN polypeptides that are or are not mannosylated. The production of carbohydrate-specific antibodies that bind oligomannose type sugars has been demonstrated (Calarese et al . , Science, 300: 2065-2071, 2003) .

An anti-cancer agent is an agent which has a cytotoxic effect on cancer cells. Suitable agents include radioisotopes and chemotherapeutic compounds such as inhibitors of topoisomerase I and II activity, such as camptothecin, drugs such as irinotecan, topotecan, bleomycin, gemcitabine, yondelis and rubitecan, alkylating agents such as temozolomide and DTIC (dacarbazine) , and platinum agents like cisplatin, cisplatin-doxorubicin- cyclophosphamide, carboplatin, and carboplatin-paclitaxel or analogues, derivatives or salts of any of these. Other suitable anti-cancer agents include doxorubicin- cyclophosphamide, capecitabine, cyclophosphamide-methotrexate-5-fluorouracil, docetaxel, 5-flouracil-epirubicin- cyclophosphamide, paclitaxel, vinorelbine, etoposide, PEGylated liposomal doxorubicin, topotecan and analogues, derivatives and salts of any of these.

In other embodiments, the specific binding member may be attached to a tag moiety. A second conjugate comprising an anti- cancer agent and a specific binding member which specifically binds to the tag moiety is then administered to the individual.

Suitable tag moieties are well known in the art and include biotin and epitopes which are bound by antibody molecules.

A tag sequence may for example consist of at least 2, 4, 6, or 8 amino acid residues. A tag sequence may consist of 25 or less, 20 or less, 15 or less or preferably 10 or less amino acid residues.

Various suitable tag sequences are known in the art, including, for example, MRGS (H) ₆, DYKDDDDK (FLAG™), T7-, S- (KETAAAKFERQHMDS) , poly-Arg (R_5-6) , poly-His (H_2-I0) , poly-Cys (C₄) poly-Phe (Fi₁) poly- Asp (D_5-16), Strept-tag II (WSHPQFEK), c-myc (EQKLISEEDL) , Influenza- HA tag (Murray, P. J. et al (1995) Anal Biochem 229, 170-9), GIu- Glu-Phe tag (Stammers, D. K. et al (1991) FEBS Lett 283, 298-302) , Tag.100 (Qiagen; 12 aa tag derived from mammalian MAP kinase 2), Cruz tag 09™ (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.) and Cruz tag 22™ (MRDALDRLDRLA, Santa Cruz Biotechnology Inc.) . Known tag sequences are reviewed in Terpe (2003) Appl . Microbiol. Biotechnol . 60 523-533. Another aspect of the invention provides a pharmaceutical composition as described above, comprising a specific binding member which binds a BTN polypeptide, as described above and a pharmaceutically acceptable excipient, vehicle or carrier. The formulation and administration of pharmaceutical compositions comprising specific binding members and conjugates is described in more detail below.

The present inventors have also recognised that BTNs mediate various cellular effects through binding to SIGN receptors and may be induced by inflammatory cytokines . BTN polypeptides may therefore have a range of therapeutic applications.

Another aspect of the invention provides a composition comprising a BTN polypeptide for use in a method of treatment of the human or animal body .

A BTN polypeptide for use in a method of treatment may comprise the amino acid sequence of a BTNl, BTN2 or BTN3 polypeptide as shown in Table 2 or a sequence having at least 50% amino acid sequence identity, for example at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to the amino acid sequence of the BTN polypeptide or a fragment of any one of these sequences comprising the extracellular domain .

The extra cellular domain of a BTN polypeptide may be determined using the Tmpred program that identifies membrane-spanning regions and their orientation. The algorithm is based on the statistical analysis of TMbase, a database of naturally occuring transmembrane proteins (Hofmann & W. Stoffel (1993) Biol. Chem. Hoppe-Seyler 374,166) .

A suitable BTN polypeptide binds specifically to a SIGN receptor. SIGN receptors are described in more detail above. A BTN polypeptide may comprise one or more glycosylation sites and is preferably glycosylated. In some embodiments, the BTN polypeptide may be glycosylated with a carbohydrate group comprising one or more mannose residues. For example, the BTN polypeptide may be mannosylated as described above.

Glycosylation sites within BTN polypeptides may be recognised using the NetNglyc server 1.0, which predicts N-glycosylation sites in human proteins using artificial neural networks that examine the sequence context of Asn-Xaa-Ser/Thr sequins (R. Gupta, efc al in preparation, 2002) . Glycosylation sites in the BTN sequences are shown in Table 2. Preferably, a BTN polypeptide is post- translationally glycosylated at one or more, for example two, three or four glycosylation sites in a mammalian expression system, for example, a human cell culture expression system.

Related aspects of the invention provide a BTN polypeptide as described above for use in the modulation of the immune response of an individual, the use of a BTN polypeptide in the preparation of a medicament for use in the modulation of the immune response of an individual and a method of modulation of the immune response of an individual comprising administering a BTN polypeptide to the individual .

An individual may have a condition associated with an aberrant immune response, for example an allergy, an autoimmune disease, or a transplant rejection. In some embodiments, an aberrant immune response may include an impaired immune response to pathogens, leading to infectious disease or an impaired immune response to abnormal host cells, leading to a cancer condition.

A BTN polypeptide may be used, for example, to block the binding of a ligand to a SIGN receptor. Ligands whose binding may be blocked include physiological ligands, such as ICAM-2 and ICAM- 3, and non- physiological ligands, such as pathogenic antigens.

The binding of a BTN polypeptide to a SIGN receptor, such as DC- SIGN, may reduce or inhibit the binding of the SIGN receptor to other mediators of the immune response, such as ICAM-2 (Genbank accession number NM_000873) and ICAM-3 (Genbank accession number NP_002153) . This binding plays an important role in the activation of T lymphocytes by dendritic cells and the inhibition of binding may reduce or inhibit the activation of T lymphocytes by dendritic cells, thereby reducing or inhibiting T cell mediated immune responses. This may be useful in the treatment of autoimmune disorders, transplantation rejection, and/or allergic reactions.

The invention encompasses a BTN polypeptide as described above for use in inhibiting the immune response of an individual, in particular the T-cell mediated immune response, the use of a BTN polypeptide in the preparation of a medicament for use in inhibiting the immune response of an individual, in particular the T cell mediated immune response and a method of inhibiting the immune response of an individual, in particular the T cell mediated immune response, comprising administering a BTN polypeptide to the individual .

The interaction of BTN and DC-SIGN is also shown herein to activate dendritic cells, in particular to stimulate the maturation of immature dendritic cells. Mature dendritic cells are known to express high levels of MHC and co- stimulatory molecules and are responsible for the initiation of primary T cell mediated immune responses. Thus, BTN polypeptides may be useful in the stimulation of immune responses, including T cell immune responses.

The invention encompasses a BTN polypeptide as described above for use in stimulating the immune response of an individual, the use of a BTN polypeptide in the preparation of a medicament for use in stimulating the immune response of an individual, and a method of stimulating the immune response of an individual, comprising administering a BTN polypeptide to the individual. An immune response may be a T-cell immune response.

The interaction of a BTN polypeptide with DC-SIGN at a cell surface is shown herein to induce the expression of cytokines which are characteristic of the ThI response. A BTN polypeptide as described herein may therefore be used to stimulate a ThI immune response in an individual .

A ThI immune response is an immune response that has a major ThI component. More preferably, a ThI immune response is predominantly or substantially ThI. It will be appreciated that complete polarization of an immune response in a mammal into either ThI or Th2 is virtually impossible, so a minor proportion of any ThI immune response which is elicited by an antigen will be Th2. ThI immune responses are characterised by the production of cytokines such as TNFα, IL-I, IL- 2, IL- 12 or IFN-γ.

The presence of a ThI immune response in an individual may be determined by means of a delayed hypersensitivity skin-test response after intradermal injection. In a delayed hypersensitivity response to soluble antigen there is swelling and induration that peaks at 48-72 hours.

Alternatively, the existence of a major ThI component in a patient's immune response may be determined by culturing peripheral blood mononuclear cells obtained from the spleen, blood or lymph node of a patient in vitro with a suitable antigen, and determining or measuring the production of ThI cytokines, for example, using enzyme- 1inked immunoabsorbent assay or by reverse transcriptase polymerase chain reaction. A suitable indicator of a ThI response is release of interferon gamma (IFNy) , TNFα, IL-I, IL-2 or IL-12. The invention encompasses a BTN polypeptide as described above for use in stimulating a ThI immune response in an individual, the use of a BTN polypeptide in the preparation of a medicament for use in stimulating a ThI immune response in an individual, in particular the ThI immune response, and a method of stimulating a ThI immune response in an individual, comprising administering a BTN polypeptide to the individual .

Stimulation of a ThI immune response in an individual may be useful in the treatment of a disease condition associated with eosinophilia, such as asthma and allergic rhinitis asthma, systemic lupus erythematosis, Ommen's syndrome (hypereosinophilia syndrome) , parasitic infections, such as cutaneous and systemic leishmaniasis, toxoplasma infection and trypanosome infection, fungal infections, such as candidiasis and histoplasmosis, and intracellular bacterial infections, such as leprosy and tuberculosis.

In other embodiments, the binding of a BTN polypeptide may reduce or inhibit the binding of DC-SIGN to a pathogen antigen. Binding to DC- SIGN is an early stage in the infection process of many pathogens and blocking the interaction using a BTN polypeptide may thus inhibit infection process of the pathogen. Examples of viral antigens that bind to DC-SIGN are HIV envelope glycoprotein gpl20, Ebola virus glycoproteins, Hepatitis C virus glycoprotein E2 and Cytomegalovirus envelope glycoprotein B. Examples of other pathogens that bind to DC-SIGN are M. tuberculosis, Helicobacter pylori, Leishmanla mexicana, Schistosoma mansoni and Candida albicus.

The monoclonal antibody IBlO is shown herein to block the binding of BTN to DC-SIGN. This monoclonal has previously been shown to block the binding of HIV and CMV to dendritic cells and this provides further evidence that BTN polypeptides may be used to reduce or inhibit the binding of DC-SIGN to pathogens, in particular viral pathogens such as HIV and CMV. A BTN polypeptide may be used to reduce or inhibit the infectivity of a pathogen that binds to DC-SIGN as part of its infection cycle. Pathogens that bind DC-SIGN are described above. A BTN polypeptide may therefore be useful in the prophylactic or therapeutic treatment of pathogen infection.

Other aspects of the invention provide a BTN polypeptide for use in the prophylactic or therapeutic treatment of a pathogen infection, the use of a BTN polypeptide in the preparation of a medicament for the prophylactic or therapeutic treatment of a pathogen infection in an individual and a method of treating a pathogen infection in an individual comprising administering a composition comprising a BTN polypeptide to the individual .

As described above, a BTN polypeptide may be active against pathogenic infection by specifically blocking the interaction of the pathogen with DC- SIGN and/or by stimulating the immune response of an individual against the pathogen.

In view of its immunostimulatory activity, a BTN polypeptide may be used as an adjuvant to promote an immune response to an antigen. The antigen is preferably co-administered and therefore may be present in the same composition as the BTN polypeptide. BTN polypeptides as described herein may be useful in a vaccine . The term 'adjuvant' as used herein means a substance that increases the immune response to an antigen.

An 'antigen' is a substance that can specifically bind an antibody molecule . It includes an agent that , when introduced into an immunocompetent animal, stimulates the production of a specific antibody that binds with the agent or a specific immune response against the agent. The term 'antigen' also includes a substance that can combine specifically with an antibody but is not itself able to stimulate antibody production unless bound to an immunogenic carrier or combined with adjuvant. An antigen may be a protein, peptide, nucleic acid or other component derived from a pathogen, such as a bacteria, virus or lower eukaryote. It may be isolated from a pathogen, or may be recombinant. An antigen may for example be a viral coat protein or a fragment thereof .

In other embodiments, a BTN polypeptide may be used to target agents such as antigens to dendritic cells. This may be useful for example, in generating a T-cell mediated immune response to a specific antigen.

BTN polypeptide bound to DC-SIGN is shown herein (for example see figure 19) to be internalised into the DC-SIGN expressing dendritic cell . A BTN polypeptide may be fused to an antigen to which an immune response is desired. The antigen-BTN fusion is targeted to the dendritic cell via DC-SIGN and internalised. Epitopes from the antigen may then be displayed by an MHC molecule on the cell surface and presented to a T cell to evoke an immune response.

A BTN polypeptide useful in such embodiments may be joined or fused to a heterologous amino acid sequence (i.e. a non-BTN sequence to which BTN is not naturally linked) .

A heterologous amino acid sequence may, for example, be an antibody or antibody fragment, e.g. IgG, or an antigen as described above.

Antigen-BTN fusions can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook "Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press 3^rd Edition (2001) , and Ausubel et al . , Current Protocols in Molecular Biology, John Wiley and Sons, (1994)) .

Another aspect of the invention provides a pharmaceutical composition for use in the modulation of immune responses or the prophylactic treatment of a pathogen infection, as described above, comprising a BTN polypeptide as described above and a pharmaceutically acceptable excipient, vehicle or carrier.

A pharmaceutically acceptable excipient, vehicle or carrier is preferably non-toxic and does not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier, such as gelatin, or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will 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 included, as required.

Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of medical practitioners.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.

Preferred and exemplary embodiments of such methods of treatment are as explained above .

Another aspect of the invention provides a method of producing a BTN polypeptide comprising; expressing a nucleic acid encoding a BTN polypeptide in a host cell, and; determining the interaction of the expressed polypeptide with a SIGN receptor.

A host cell may be an isolated or cultured cell, preferably a mammalian or human cell .

The interaction of the expressed polypeptide with a SIGN receptor may be determined by contacting the host cell with a cell expressing a SIGN receptor such as DC- SIGN or L-SIGN. Interaction may be detected as described above.

BTN polypeptides for use in therapy may be expressed in vivo within an individual from encoding nucleic acid to achieve a therapeutic effect. Another aspect of the invention provides a composition comprising a nucleic acid encoding a BTN polypeptide for use in a method of treatment of the human or animal body Compounds which induce or enhance BTN expression may be used to increase the level of BTN polypeptide in an individual and thereby modulate immune responses and/or ameliorate pathogen infection.

The invention encompasses a BTN inducing compound for use in modulating the immune system of an individual, the use of a BTN inducing compound in the preparation of a medicament for modulating the immune system of an individual and a method of modulating the immune system of an individual comprising administering a composition comprising a BTN inducing compound to the individual.

A BTN inducing compound increases the expression and/or activity of a BTN polypeptide, for example a BTNl, BTN2 or BTN3 polypeptide. Suitable BTN inducing compounds include inflammatory cytokines such as IFNγ.

BTN inducing compounds are described in more detail above. A BTN inducing compound may be formulated into a pharmaceutical composition as described above prior to administration to an individual .

Modulating the immune system may include increasing or enhancing immune responses, for example, ThI immune responses or reducing or inhibiting immune responses, as described above.

Related aspects of the invention provide methods for identifying and/or obtaining additional BTN inducing compounds which are candidate agents for modulating immune responses.

A method of identifying/obtaining an immunmodulatory agent, may comprise; contacting a cell with a test compound; and, determining the expression of a BTN polypeptide by said cell, an increase in the expression of the BTN polypeptide by said compound is indicative that the test compound is a candidate immunomodulatory agent .

A suitable cell may be a cultured cell, preferably a mammalian cell, for example from a human cell line.

The expression of a BTN polypeptide may be determined using standard techniques such as Northern or Western Blotting, flow cytometry and immunoassays .

Suitable test compounds are described in more detail below and may, in particular, include inflammatory cytokines, such as IFNγ, and analogues, derivatives and mimetics thereof.

The data set out herein show that the binding of a ligand to a DC- SIGN receptor can activate dendritic cells and stimulate immune responses. Further aspects of the invention provide methods of identifying DC-SIGN receptor ligands which have immunomodulatory, in particular immunostimulatory, activity.

A method of identifying/obtaining an immunostimulatory agent, may comprise; contacting a test compound with a DC- SIGN receptor polypeptide; and, determining binding of the DC-SIGN receptor polypeptide by the test compound, wherein binding of the DC-SIGN receptor polypeptide by the test compound is indicative that the test compound is a candidate immunostimulatory agent .

A method may further comprise determining the immunostimulatory activity of the test compound. Immunostimulatory activity may include the induction of ThI immune responses, for example the production of cytokines characteristic of the ThI response, and/or the activation of dendritic cells. The extent of activation of a dendritic cell may be determined by determining up-regulation of CD86 and secretion of TNF-α, for example using the methods described herein.

A suitable test compound may comprise or consist of a polysaccaride having one or more mannose groups, a BTN polypeptide or an analogue, derivative or mimetic thereof.

The findings set out herein demonstrate that BTN polypeptides can modulate immune responses through interaction with SIGN receptors . Immune responses mediated by the interaction of BTN with SIGN receptors may include immune responses mediated by SIGN presenting cells, such as dendritic cells, and immune responses mediated by BTN presenting cells. Methods of identifying agents that modulate BTN activity are provided by further aspects of the invention.

A method of identifying/obtaining an immunomodulatory agent may comprise; contacting a test compound with a BTN polypeptide; and determining binding of the BTN polypeptide by the test compound, wherein binding of the BTN polypeptide by the test compound is indicative that the test compound is a candidate immunomodulatory agent .

The method may comprise determining the ability of the test compound to reduce or inhibit the interaction of a BTN polypeptide with a SIGN receptor and/or the ability of the test compound to modulate BTN polypeptide activity. BTN polypeptide activity may include induction of ThI cytokine activity, inhibition of DC-SIGN/ICAM- 3/ICAM-2 interaction, inhibition of DC-SIGN/pathogen interaction and/or activation of dendritic cells. In other embodiments, a method of identifying/obtaining an immunomodulatory agent may comprise; contacting a SIGN receptor and a BTN polypeptide in the presence of a test compound; and determining binding of the BTN polypeptide to the SIGN receptor and/or BTN polypeptide activity.

A change in binding and/or activity in the presence relative to the absence of test compound is indicative that the test compound is a candidate immunomodulatory agent

The findings set out herein also demonstrate that mannosylated BTN polypeptides expressed specifically by tumour cells. Methods of identifying agents that target tumour cells by binding mannosylated BTN polypeptides are provided by further aspects of the invention.

A method of identifying/obtaining a compound which binds specifically to tumour cells may comprise; determining the binding of a test compound to mannosylated BTN polypeptide and non-mannosylated BTN polypeptide, wherein an increase in binding of the test compound to mannosylated BTN polypeptide relative to non-mannosylated BTN polypeptide is indicative that the test compound binds specifically to tumour cells.

Test compounds may be natural or synthetic chemical compounds used in drug screening programmes . Extracts of plants that contain several characterised or uncharacterised components may also be used.

Combinatorial library technology (Schultz, JS (1996) Biotechnol. Prog. 12:729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide. Suitable test compounds may be based on BTN polypeptides i.e. BTN2 or BTN3 derivatives, analogues or fragments. Another class of potential test compounds include antibody- molecules. Antibody molecules may, for example, elicit an immunomodulatory effect by blocking the interaction of a BTN polypeptide with a SIGN receptor and thereby reducing or preventing the induction of immunomodulatory effects associated with this interaction or by mimicking the effect of SIGN receptor binding on BTN and triggering BTN-mediated immunomodulatory effects in the BTN presenting cell .

Alternatively, antibodies may bind preferentially to mannosylated BTN polypeptide relative to non-mannosylated BTN polypeptide and therefore be useful in targeting cancer cells. Preferred antibody molecules may bind to an epitope within the carbohydrate moiety of the BTN polypeptide or an epitope which is formed by the both carbohydrate and peptidyl moieties of the BTN polypeptide. The production of suitable antibodies for use as test compounds is described in more detail above .

Further aspects of the invention provide antibody molecules which specifically bind to BTN polypeptides, antibody molecules which specifically bind to BTN polypeptides for use in a method of treatment of the human or animal body and the use of such an antibody molecule in the manufacture of a medicament for use in modulating the immune system, for example in the treatment of a condition associated with an aberrant immune response. Also provided are antibody molecules which specifically bind to mannosylated BTN polypeptides (i.e. bind preferentially to mannosylated BTN polypeptides relative to non-mannosylated BTN polypeptides and show no significant binding to other molecules present on the surface of mammalian cells) , antibody molecules which specifically bind to mannosylated BTN polypeptides for use in a method of treatment of the human or animal body and the use of such an antibody molecule in the manufacture of a medicament for use in targeting cancer cells, for example in the treatment or diagnosis of cancer. Another class of potential test compounds includes agents which bind to or disrupt the carbohydrate moieties of glycosylated BTN polypeptide. Such agents may include lectins, antibody molecules and polypeptides with glycosyl transferase activity.

Another class of potential test compounds include carbohydrates and polysaccarides, in particular carbohydrates comprising one or more mannose groups, for example mannan.

The amount of test substance or compound which may be employed in a method will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.1 to 100 μM concentrations of putative inhibitor compound may be used, for example from 1 to 10 μM.

The test compound may be identified as an immunomodulatory agent and, optionally, isolated and/or purified.

The test compound may be synthesised, manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e.g. for any of the purposes discussed elsewhere herein.

Following identification of a compound as described above, a method may further comprise modifying the compound to optimise the pharmaceutical properties thereof .

Such a method may comprise determining the ability of the modified compound to bind to a SIGN receptor, and optionally determining one or more of: the ability of the modified compound to activate a DC, the ability of the modified compound to block ICAM-2 and/or ICAM-3 binding, the ability of the modified compound to block pathogen binding . For example, a method may comprise; modifying a BTN polypeptide to produce a derivative; and, determining the SIGN receptor binding activity of said derivative .

A test compound may be modified by covalent attachment of further moieties, and/or deletion or substitution of existing moieties. Modifying may also comprise mutating the sequence by addition, deletion and/or substitution. Modifying may also involve adapting and optimising the test compound for pharmaceutical use e.g. by altering its toxicological properties and/or its solubility, as described below.

The modification of a ^λlead' compound identified as biologically active is a known approach to the development of pharmaceuticals. Modification of a known active compound (such as a BTN polypeptide) may be used to avoid randomly screening large number of molecules for a target property.

Modification of a 'lead' compound to optimise its pharmaceutical properties commonly comprises several steps. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. These parts or residues constituting the active region of the compound are known as its "pharmacophore".

Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR.

Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the modified compound is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the activity of the lead compound. The modified compounds found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Modified compounds include mimetics of the lead compound .

Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.

In another aspect, the invention provides a method of identifying an immune response inhibitor comprising: contacting a SIGN receptor ligand polypeptide and a SIGN polypeptide in the presence of a BTN polypeptide; and determining binding of the SIGN polypeptide by the SIGN receptor ligand, a decrease in binding in the presence relative to the absence of BTN polypeptide being indicative that the BTN polypeptide is a candidate immune response inhibitor.

A SIGN receptor ligand may include ICAM2. Other suitable SIGN receptor ligands include ICAM3 expressed by polymorphonuclear (PMN) cells.

As described above, a method may comprise; modifying a BTN polypeptide, and; determining binding of the SIGN polypeptide by the SIGN receptor ligand, in the presence of said modified BTN polypeptide. In another aspect, the invention provides a method of identifying an agent for use in the prophylactic treatment of an individual against infection by a pathogen, comprising: contacting a pathogenic agent and a SIGN polypeptide in the presence of a BTN polypeptide; and determining binding of the SIGN polypeptide by the pathogenic agent , a decrease in binding in the presence relative to the absence of BTN polypeptide being indicative that the test compound is a candidate agent for prophylactic treatment of an individual against infection by a pathogen

Preferred BTN polypeptides preferentially inhibit or reduce the binding of DC-SIGN to a pathogenic agent relative to the binding of DC-SIGN to an endogenous ligand, such as ICAM2 or PMN expressed ICAM3.

A method may comprise the additional steps of: contacting a SIGN ligand and a SIGN polypeptide in the presence of a BTN polypeptide; determining binding of the SIGN polypeptide by the SIGN ligand; and, determining the effect of said BTN polypeptide on the binding of pathogenic agent relative to the SIGN ligand.

Suitable SIGN ligands include ICAM2. Other suitable SIGN receptor ligands include ICAM3 expressed by polymorphonuclear (PMN) cells.

A BTN polypeptide may be optimised to improve its efficacy or pharmaceutical properties.

For example, a method may comprise modifying the BTN polypeptide and determining binding in the presence relative to the absence of modified BTN polypeptide. The effect on binding of the modified BTN polypeptide may be compared with the effect of the unmodified BTN polypeptide .

A 'pathogenic agent' is a substance from a pathogen. It includes the entire pathogen, and fragments of the pathogen such as membrane fragments, proteins and glycoproteins or fragments thereof, or surface antigens of the pathogen. When the pathogen is a virus, the pathogenic agent may be a virus particle, viral coat protein or surface antigen of the virus . It may be a fragment of a virus or viral protein. Generally, a pathogenic agent is selected because it binds to DC-SIGN. Examples of viral antigens that bind to DC-SIGN are HIV envelope glycoprotein gpl20, Ebola virus glycoproteins, Hepatitis C virus glycoprotein E2 and Cytomegalovirus envelope glycoprotein B. Examples of other pathogens that bind to DC-SIGN are Mycobacteria such as M. tuberculosis, Heliobacter such as H. pylori, Leishmania such as L. mexicana, Schistosoma such as S. mansoni and Candida such as C. albicans.

A method may be used to identify an agent that blocks binding of a pathogenic agent to a DC- SIGN polypeptide more effectively than a BTN2 polypeptide having the sequence of SEQ ID NO: 1. Reduction of binding of the DC-SIGN polypeptide by the pathogenic agent is an indication that the BTN polypeptide may be useful as an agent for prophylactic treatment of an individual against infection by a pathogen .

The pathogen may be any pathogen that binds to DC-SIGN. For example, the pathogen may be viral , bacterial , fungal , lower eukaryote or other parasite. Examples of pathogens are described above. Thus, the present invention comprises a method of identifying an anti- pathogenic agent .

A test compound may be identified as an immune response inhibitor or anti -pathogen agent using a method as described above and, optionally, isolated and/or purified. The test compound may be synthesised and/or produced using standard synthetic or recombinant techniques and, optionally, used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. The formulation of medicaments comprising an active agent is well-known in the art. A medicament may be administered to individuals, e.g. for any of the purposes discussed elsewhere herein.

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.

The skilled person is readily able to devise and perform suitable control experiments for the methods described herein.

"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.

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

Figure 1 shows the screening of a panel of cells and cell lines for binding to soluble BTN2A1-Ig (10 μg/ml) by flow cytometry. Figure IA shows that BTN2A1 does not interact with untreated or PMA- activated MOLT-4 cells, or with NK-cells or B-LCL YT or Raji. Figure IB shows that BTN2A1 binds to MoDCs , but not to LCs . Binding of soluble BTN2A1-Ig (10 μg/ml, open histogram) to MoDCs and LCs is shown versus binding to hFc protein (black filled histogram) . The phenotype of MoDCs used was HLA-DR ^high, CDl ^high and CD14 ^low. Figure 1C shows that BTN2A1-Ig is concentration-dependent. BTN2A1 concentrations were 5, 12, 25 μg/ml (dotted, broken lines, solid lines, respectively) . Background binding of hFc (14, 35, 70 μg/ml) and hlgG (7, 18, 35 μg/ml) used as control proteins was low.

Figure 2 shows that putative BTN2Al-counter-receptor expression is IL-4 -dependent and is downregulated on mDCs . Figure 2A shows that MoDCs were activated with LPS for 48h and stained for B7.2 expression using MαCD86-RPE. Mo-derived imDC (top panel) and mDCs

(bottom panel) binding to BTN2A1-Ig or CTLA-4 -Ig was analyzed by- flow cytometry. Specific binding is shown as open histograms versus binding of hlgG (filled histograms) . Figure 2B shows the incubation of monocytes, isolated by plastic adherence, for 72h in the presence of IL-4 (top panel) or a combination of IL-4/GM-CSF (bottom panel) . Cells were stained with soluble BTN2A1-Ig, MαCDla or RatαDC-SIGN- RPE. Specific antibody or fusion protein binding is shown as open histogram versus isotype control or hlgG binding, respectively

(filled histogram) .

Figure 3 shows that the BTN2A1- counter-receptor is a C-type lectin and rapidly internalized. Figure 3A shows that BTN2A1 protein is rapidly internalized after ligation. DCs were labeled with BTN2A1-Ig (10 μg/ml) on ice and incubated at 37⁰C for 0 min (solid line) , 5 min (dotted line) and 15 min (scattered line) . Internalization was stopped at 4⁰C and bound BTN2A1-Ig detected by flow cytometry. Percent DC-BTN2Al-Ig binding was calculated as follows: [(MFl of sample minus MFI of negative control)/ MFI of positive control minus MFI of negative control] x 100. Figure 3B shows binding of BTN2A1 protein to DCs is Ca-dependent . DCs were preincubated on ice for 20 min in the presence of 1 mM Ca²⁺ with increasing concentrations of Ca²⁺-chelator, before soluble BTN2A1-Ig (10 μg/ml) fusion protein was added. Binding without inhibitor (solid line) was compared to binding in the presence of 5 mM or 20 mM EDTA and EGTA (dotted or scattered lines, respectively. Figure 3C shows binding of BTN2A1-Ig is blocked by mannan and inhibition is dose -dependent. MoDCs were preincubated as for Figure 3B with increasing doses of mannan and analyzed for binding of BTN2A1-Ig by flow cytometry (see also inset) . Figure 3D shows BTN2A1 binding is abrogated by Endo H digestion. BTN2A1-Ig was treated with Endo H (1 mϋ/μg) at RT overnight and binding to DCs was analyzed by flow cytometry.

Figure 4 shows that BTN2A1 binds to DC-SIGN-transfectants with high affinity and binding is blocked by DC-SIGN-specific antibody. Figure 4A shows binding of soluble BTN2A1-Ig (10 μg/ml) and hFc (10 μg/ml) to HEK293T cells transfected with DC-SIGN and blocking of BTN2A1 binding by a DC-SIGN-blocking antibody (IBlO, 35 μl/ml) or an isotype control (IgG2a, 35 μg/ml, BD Biosciences, Oxford, UK) . The number of cells that were positive for "Zenon Alexa Fluor 488"- labeled BTN2A1-Ig was analyzed by flow cytometry. The data are representative of three independent experiments. Figure 4B shows BTN2A1 binding and inhibition of binding to DCs by IBlO as for 4A. Figure 4C shows binding of BTN2A1 (10 μg/ml) and inhibition of binding to DC-SIGN-transfectants by HIVgpl20.

Figure 5 shows DC-SIGN binds to BTN2A1 on human tumor cell lines but not on HUVECs and HFF. Figure 5A shows HEK293T lysate immunoprecipitated with DC-SIGN-FLAG or a FLAG-BAP-protein bound to MαFLAG-agarose was analyzed on a PVDF membrane using rat mab to BTN2A1 B30.2 domain. A full-length BTN2A1/2/3 protein at ~69kD was detected in the cell- lysate and precipitate, but not in the control . Figure 5B shows PMNs, HEK293T cells and PBLs analyzed for sDC-SIGN binding. SDC-SIGN binding (open histogram) is shown versus binding of a BAP-FLAG protein (black filled histogram) .

Figure 5C shows a panel of human cells and cell lines transiently transfected with BTN2A1- or BTN3A3-GFP. Relative binding of DC-SIGN- FLAG was plotted against the level of BTN2A1-GFP expression (FITC Log) . An arbitrary baseline was set to 100% binding for the mean FL 6-fluorescence intensity of DC-SIGN binding to untransfected cells. Percentage of normalized binding was calculated as the mean FL 6 ratio of transfected to untransfected cells.

Figure 6 shows that GNA inhibits binding of DC-SIGN and binds to BTN2A1 on HEK293T, not HUVECs. Figure 6A shows the binding of HEK293T transiently transfected with BTN2A1-GFP to DC-SIGN-FLAG in the presence and absence of GNA (100 μg/ml) . Relative binding was normalized as described in Fig. 5C. Figure 6B shows the binding of HEK293T and HUVECs transiently transfected with BTN2A1- and BTN3A3- GFP as described in Fig. 5C. Relative binding of GNA (20 μg/ml) was plotted against the level of BTN2A1-GFP expression (FITC Log) and evaluated as described in Fig. 5C.

Examples

Materials and Methods

RNA analysis, cloning and transfeetion

BTN2A1 RNA expression was analyzed by RT-PCR of a human multiple tissue cDNA panel (Clontech, Mountain View, CA) . Human BTN2A1 was amplified with forward primer BTN2Alf, 5¹- CACCTCGTAGTGGCAGGACTA-3 ^■ and reverse primer BTN2r, B'-TGGGCATAAAGGATTCTGGA-S', designed to bridge two adjacent exons of the IgC domain. The GAPDH primers used were GAPDHf, 5 ' -ACAACAGCCTCAAGATCATCAG-3 ' , GAPDHr, 5'- GGTCCACCACTGACACGTTG-3 ' . For RT-PCR analysis and cDNA cloning, total cellular RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany) . RNA was reverse transcribed using the Prostar kit (Stratagene, La Jolla, CA) and random hexamers. Full-length DC-SIGN cDNA was amplified by RT-PCR with BIO-X-ACT DNA polymerase (Bioline, Luckenwalde, Germany) from total human MoDC cDNA, cloned into pcDNA5/Frt/TOPO (Invitrogen, Groningen, The Netherlands) , and confirmed by DNA sequencing. Human embryonic kidney HEK293T cells were transfected with pcDNA5/Frt/TOPO-DC-SIGN using effectene (Qiagen, Hilden, Germany) and analyzed after 48h.

To create a DC-SIGN-FLAG fusion protein, a second PCR insert of DC- SIGN was generated using sense oligonucleotide 5'- CCCAGCTCCATAAGTCAGGAA- 3' and antisense primer 5'-

AAGTTCTGCTACGCAGGAGG- 3' corresponding to the C-terminus, and ligated in frame into BamHI pFLAG -CMV-3 (Sigma-Aldrich, St. Louis, MO) . Full-length human BTN2A1 cDNAs were cloned into mammalian expression vectors as described (2) and transfected into a panel of primary cells and cell lines using Fugene (Roche Applied Science, Indianapolis, IN) . BTN2A1 fusion proteins (referred to as BTN2A1-Ig) were prepared by cloning the extracellular domain in- frame with the hinge -CH2 - CH3 domain of human IgGl (14) . To produce fusion proteins, HEK293T cells were transfected using effectene. BTN2A1-Ig, hFc and DC-SIGN-FLAG fusion proteins were purified from culture supernatant using protein A-Sepharose or MαFLAG-agarose (Sigma-Aldrich, St. Louis, MO) .

Irmunohistochemistry, Immunoprecipitation and Western Blot Formalin- fixed sections of normal colon (Imgenex, San Diego, CA) were stained for BTN2A1 expression using the avidin-biotin- peroxidase system (Vectastain, Vector Laboratories, and Peterborough, UK) . Sections were blocked with 2% normal rabbit serum and the tissue culture supernatant of a monoclonal rat anti BTN2A1 antibody was applied followed by biotinylated rabbit anti -rat IgG and the avidine-biotin-horseradish peroxidase (HRP) complex. Secondary antibodies were preabsorbed with 10% human AB serum before application. HRP activity was developed with diaminobenzidine (DAB) and sections were counterstained with Carrazzi hematoxylin.

HEK293T cells were washed with PBS and suspended in lysis buffer containing 50 mM Tris-HCL, pH 7.2, 150 mM NaCl, ImM Ca²⁺, 1 mM Mg²⁺, 1% Triton X-100 and a mixture of protease inhibitors (Roche Applied Science, Indianapolis, IN) at 4°C for 1 h. sDC-SIGN-FLAG tissue culture supernatant was coupled to MαFLAG -agarose at 4°C for 12 h, washed with lysis buffer and subsequently used for precipitation of HEK293T lysates at 4°C, overnight. The eluate in SDS sample buffer was separated by 10% SDS-PAGE under reducing conditions, transferred onto Immobilon P membrane (Millipore, Billerica, MA) and probed with tissue culture supernatant of a rat anti BTN2A1-B30.2 antibody followed by HRP-rabbit anti rat Ig (Dako, Glostrup, Denmark) . Western blots were developed using ECL system (Amersham Biosciences, Uppsala, Sweden) .

Cells, antibodies and reagents

PBMCs were obtained from buffy coats of healthy donors by Ficoll gradient centrifugation. Monocytes were prepared from plastic adherent PBMC and incubated for 72h in the presence of IL-4 (500 U/ml) or for 5 d in IL-4 and GM-CSF (500 U/ml and 100 ng/ml, R&DSystems, Minneapolis, MN) . At day 5, the phenotype of cultured MoDCs was confirmed by flow cytometry and typically was CD14 ^low, HLA-DR ^high and CDIa ^high with moderate levels of CD86. MoDCs were activated in the presence of 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO, L2654) for 48 h. Langerhans cells (LCs) were generated as described (15) .

HUVECs (human umbilical vein endothelial cells) were grown in Endothelial Growth Medium (Promocell, Heidelberg, Germany) and IMR- 90 (human lung embryonic fibroblasts) were grown in D-MEM with glutamax, glucose 1000 mg/1, sodium pyruvate. MOLT-4 (human acute lymphoblastic leukemia) , MeIJuSo (human melanoma cell line) , HeIa (cervical carcinoma cells) , HEK293 (embryonal kidney carcinoma cells) and BAF/3 (mouse B cell lymphoma) were grown in RPMI 1640 supplemented with 2 mM glutamine, penicillin/ streptomycin (100 U/ml, Invitrogen, Groningen, The Netherlands) and 10% FCS (Harlan, Indianapolis, IN) . The medium for BAF/3 was also supplemented with raIL-3. T cells were separated by a Dako MoFIo cell sorter by negative selection from PBL stained with MαCD14- (Diatec, Oslo, Norway) , MαCD19-FITC and MαCD56-RPE (Dako, Glostrup, Denmark) and treated with PHA/IL-2 (5 μg/ml/100 U/ml ; R&D Systems, Minneapolis, MN) , PMA/calciumionophore (10/100 ng/ml) or immobilized UCHT-I anti CD3 ab (Dako, Glostrup, Denmark) .

The following mouse anti-human monoclonal antibodies (mAbs) were used: NA1/34-HLK (CDIa, Insight Biotechnology) ; IT2.2 (CD86-R-PE; L243 (HLA-DR) ; DCN46 (DC-SIGN, BD Biosciences, Oxford, UK) ; Mablδl

(DC-SIGN, R&DSystems, Minneapolis, MN) ; IBlO (DC-SIGN, F. Arenzana-

Seidedos) ;

The following rat anti -human mAbs were used: RatαDC-SIGN-RPE

(eBioscience, San Diego, CA) ; monoclonal anti BTN2A1 antibody was raised by immunization of rats with the recombinant B30.2 domain of the BTN2A1 protein and does not crossreact with BTN3 family members but may also recognize other BTN2 molecules.

Binding of soluble fusion proteins by flow cytometry To analyze BTN2Al-counter-receptor expression, cells were incubated on ice with soluble BTN2A1-Ig (10 μg/ml, unless indicated otherwise) in FACS binding buffer, 1% FCS, 0.02% sodium azide in D-PBS with Ca²⁺ and Mg²⁺) . After 20 min cells were washed and stained with FITC- conjugated goat F(ab')₂ anti-human IgG (Caltag, Burlingame, CA) or PE- conjugated goat anti-human IgG (Jackson ImmunoResearch, Newmarket, UK) . HIgG (Sigma-Aldrich, St. Louis, MO) , hFc and CTLA-4- Ig were used at 10 μg/ml. For inhibition assays, MoDCs or DC-SIGN- transfectants were preincubated on ice for 20 min with increasing concentrations of mannan, Ca²⁺-chelator or rHIV-l_SF2 gpl20 (from Dr. L. Williams, NIBSC Centralised Facility for AIDS Reagents supported by EU Programme EVA and the UK Medical Research Council) before fusion proteins were added. To study carbohydrate-dependency of binding, BTN2A1 was treated with Endo H (1 mU/μg; Roche Applied Science, Indianapolis, IN) at RT for 15 h. For antibody blocking experiments, DCs and DC-SIGN-transfectants were preincubated at 4°C for 20 min with the anti DC-SIGN IBlO mAb (35 μg/ml) , before directly labeled fusion proteins were added. BTN2A1-Ig was labeled with the Zenon Alexa Fluor 488 human IgG labeling kit (Molecular Probes, Eugene, OR) . Cells were analyzed on a FACScan (Becton Dickinson) using CELLQUEST software. To analyze binding of DC-SIGN- FLAG to a panel of cells transfected with BTN2A1-GFP, a two-step detection protocol was employed. An anti DC-SIGN ab (DCN46, BD Biosciences, Oxford, UK) was used to bridge bound sDC-SIGN-FLAG (5 μg/ml) to a goat anti mouse Alexa 647 (Molecular Probes, Eugene, OR) . Using Alexa 647 had the advantage that cells could be analyzed using SUMMIT 4.2 software on a Cyan ADP (Dako, Glostrup, Denmark) without using compensation. For lectin blocking experiments, HEK293T-DC-SIGN transfectants were preincubated at 4⁰C for 20 min with 100 μg/ml Galanthus nivalis agglutinin (GNA, Vector Laboratories, Burlingame, CA) , before the two-step staining was conducted. Binding of biotinylated GNA was detected using streptavidin-R-Phycoerythrin (Sigma-Aldrich, St. Louis, MO) .

To examine internalization, DCs were labeled with soluble BTN2A1-Ig, washed, and subsequently incubated at 37°C. Aliquots were removed at 5 min and 15 min and internalization stopped by metabolic fixation (D-PBS with Ca2+ and Mg2+ and sodium azide) at 4⁰C. Bound BTN2A1-Ig was analyzed by a FITC- conjugated goat F(ab')2 anti- human IgG.

Cytokine ELISA

On day 4-5.5 DCs were seeded at 2xlO⁴ cells/well in 96-well plates (flat bottom, Falcon-Becton Dickinson) . BTN-Ig fusion proteins (20 μg/ml), LPS (1 μg/ml) , pIC (Calbiochem, 100 μg/ml) and G28.5 (10 μg/ml) were added and incubated at 37⁰C for 40-48h. Culture supernatants were collected and frozen at -20⁰C. Cytokines were analyzed with monoclonal antibodies and recombinant cytokine standards .

Results

Regulation of BTN2 and BTN3 expression by inflammatory mediators BTN2 and BTN3 are type I transmembrane glycoproteins which are ubiquitously expressed, although transcript levels are low in most tissues .

Using Western blotting, BTN protein expression was found to be low or even undetectable in cells from a range of different tissues, including HeIa cells, PBMCs, monocytes, PMBC-derived naϊve B and T cells and PAF cells. However, in the presence of inflammatory cytokines like IFNγ, BTN3 proteins were found to be induced and also transported to the cell surface.

HeIa cells were found to induce two BTN3 proteins, a long and a short version, either representing two different BTN3 genes or different splice variants. Induction was shown to be tissue dependent, since monocytes up-regulated the 67K protein only.

Another stimulus, PMA/CI, did not increase BTN3 receptor expression and BTN3 protein level was observed to drop to even lower levels in PBMCs treated with PMA/CI. However, the expression of BTN2 was observed to increase in PBMC treated with PMA/CI . Three induced BTN2s were found, a 35K, a 46K and a 67K protein, corresponding with the theoretical molecular weight of different BTN2 genes.

The effects of the inflammatory stimuli IFNγ and TNF-α were analysed further using RT-PCR. Monocytes, isolated by plastic adherence, were stimulated with IFNγ (100 U/ml) and the combination of phorbol myristate acetate (PMA) with ionomycin (CI) , a protein kinase C activator. RT-PCR was performed using BTN2 and BTN3 subfamily- specific primers. The PCR-data was normalized to GAPDH and titrated the amount of cDNA used, so the data are semi -quantitative. Before induction, transcript levels of most BTNs were low.

BTN3A2 and BTN3A3 were highly induced by IFNγ as was an isoform containing the B30.2 domain that could be either BTN3A1 or BTN3A3. BTN2 products, including both splice variants of BTN2A2 were also induced but at a lower level. BTN2A1 levels were low even in stimulated monocytes after 35 cycles. Interestingly, PMA/CI did not up-regulate any BTN2- or BTN3- transcripts to an appreciable level. BTN3 and, to a lesser extent BTN2, transcripts were also induced by IFNγ in the epithelium-derived tumor cell line HeIa under conditions where other IFNγ inducible proteins, such as HLA-DM, were induced. Treatment of HUVECs with TNF-α (10 ng/ml) , but not with LPS (1 μg/ml) , triggered BTN2 and BTN3 mRNA up-regulation. BTN2 and particularly BTN3 transcripts are therefore induced by inflammatory cytokines .

The BTN2 expression of PMBC-derived naϊve B and T cells was found to be very low. However, B and T cell lines, including Raji and Jurkat, were found to express significantly higher levels of BTN2.

CD40 crosslinking up-regulates BTN2 and BTN3 on B cells. The effect of CD40 ligation was studied using early passage group I BL lines L3055 (EBV-) and Ramos cells. Ramos-BL were triggered by an α-CD40 antibody (G28.5) for 20h under conditions that induced Fas on the cell surface. Full length BTN2A2 and a splice variant missing the IgV domain were up-regulated with this treatment, as was Fas. The low constitutive levels of BTN2A1 mRNA were only marginal affected by CD40 crosslinking. cDNA titration was used to semi- quantify BTN2A2 induction in comparison to Fas. In another experiment L3055 cells, which expressed low levels of both BTN2 and 3, were stimulated by soluble CD40L for 6h. After CD40 ligation, two BTN2A2 splice variants were strongly induced along with the Fas control . BTN2A1 was hardly detectable in stimulated and non- stimulated B cells. BTN3 mRNA was also induced, at lower level than BTN2A2 by CD40 ligation. CD40 ligation therefore induces to different levels expression of some but not all isotypes of BTN2 and BTN3.

BTN protein is induced on the cell surface by IFNγ A polyclonal antiserum (GMBTN3) to human BTN3 was generated by- immunizing rats with a soluble BTN3A2 protein. GMBTN3 was observed by flow cytometry to stain CHO cells transfected with BTN3A2-GFP but not cells that were transfected with BTN2A1-GFP (Figure 1) . Soluble BTN3A2 protein blocked binding of the antiserum. GMBTN3 is therefore specific for BTN3 protein.

Using GMBTN3 , BTN3 protein was undetectable on Raji B cells and MelJuso melanoma cells. Molt -4 T cells and MoDCs expressed low levels of BTN3 (Figure 2) . BTN3 expression on DCs was unchanged after LPS treatment .

Among the cells we analyzed, we found the highest constitutive level of BTN3 expressed on lymphocytes (B-, T- and NK-cells) . HeIa cells treated with IFNγ were observed to up-regulate BTN3 cell surface expression. A time-course experiment showed optimal induction at 48h (figure 3) . Treatment with IFNγ, but not LPS or TNF-α, triggered BTN3 expression on PAF cells (figure 4) . Thus, BTN3 molecules are inducible cell surface receptors consistent with the data on BTN3- transcript induction.

Expression of BTN2A1

Using oligonucleotides specific for BTN2A1, RT-PCR analysis indicated that BTN2A1 mRNA was ubiquitously expressed. Immunohistochemistry (IHC) using a BTN2A1- specific mab localized a high level of expression of BTN2A1 to epithelial cells whereas lower levels were also found in leukocytes . Western Blot analysis confirmed BTN2A1 expression (~69 kDa protein band) on a variety of primary cells and cell lines, such as HUVECs, IMR- 90, HEK293T and Jurkat, whereas expression on freshly isolated leukocytes, such as PBLs and monocytes was low. This is in contrast to other B7-like molecules, as well as the related set of BTJSJ3 butyrophilins. BTN3 molecules are preferentially expressed on T cells, T cell lines and at lower levels on other PBMCs and some tumor cell lines (5) . BTN2A1-Ig binds to monocyte-derived dendritic cells (MoDCs) , not to Langerhans cells

To identify the counter-receptor for BTN2A1, we constructed fusion protein comprising its ectodomain with the Fc portion of human IgGl. The purified recombinant fusion protein was used to detect the presence of a counter-receptor by flow cytometry. First, we studied lymphocyte subsets including T cells, as BTN3A1 bound these cells (5) . PMA/CI-activated and non-activated MOLT-4 T cells did not bind to BTN2A1, nor did NK-cells or B cells (Fig. IA) . Other tissues tested, including HeIa cells, were also negative. In contrast, MoDCs, but not epidermal Langerhans cells (LCs) bound the BTN2A1 fusion protein with high intensity (Fig IB) . Titration experiments showed only minimal binding of an hFc protein, hlgG or hBTN3A3-Ig, a close homolog of BTN2A1, whereas BTN2A1-Ig bound in a dose-dependent manner (Fig. 1C) .

BTN2A1 counter-receptor expression is up-regulated by IL-4 and down- regulated on mature DCs

Immature MoDCs (imMoDCs) express low levels of B7 costimulatory molecules such as B7.2 (Fig. 2A) . When triggered by microbial stimuli, pattern recognition receptors (PRRs) mediate maturation into immunogenic DCs that express high levels of MHC class II molecules such as HLA-DR and costimulatory molecules such as B7.1 and B7.2 (Fig. 2A) . To determine how DC activation affects BTN2A1 counter-receptor expression, we compared imMoDCs to LPS-treated mature MoDCs (mMoDCs) . After LPS-activation, BTN2A1- counter-receptor expression diminished significantly (Fig. 2A) . Down-regulation of the putative BTN2A1 counter-receptor was also observed on DCs activated by TNF-α. As expected, a CTLA-4-Ig fusion protein showed a reciprocal staining pattern, bright on mMoDCs and low on imMoDCs, in accordance with the regulation of its ligands B7.1 and B7.2 (Fig.2A) . Human monocytes differentiated into a homogenous population of CDIa ^high, CD14 ^low imDCs and expressed the BTN2A1 counter-receptor (Fig. 2B) . To determine the factors responsible for the induction of the counter-receptor on differentiating DCs, monocytes were isolated and treated with IL-4. CD14 expression was low, as was CDIa expression, in contrast to IL-4/GM-CSF treated cells (Fig. 2B) . However, IL-4 stimulated monocytes acquired significant levels of the BTN2Al-counter-receptor . Counter-receptor expression levels were similar on IL-4/GM-CSF treated cells, suggesting that expression was primarily IL-4 mediated. Expression of the counter-receptor was high at 48h after IL-4 stimulation, indicating that it is acquired early during MoDC differentiation (Fig. 2B) .

The counter-receptor for BTN2A1 is an endocytic C- type lectin MoDCs express a variety of cell surface receptors that exhibit dual functions as antigen receptors and as cellular adhesion receptors. To help distinguish between them we tested whether the BTN2A1 counter-receptor could function as an endocytic receptor. MoDCs were incubated with BTN2Al~Ig under saturating conditions and transferred from 4°C to 37°C. Receptor internalization was stopped at 4°C at various time points. Cell surface binding of BTN2A1-Ig was analyzed by flow cytometry using a FITC- conjugated goat F(ab')₂ anti- human IgG. After 5 min more than 80% of the ligand had been removed from the cell surface (Fig. 3A) . External loss of bound BTN2A1-Ig correlated with appearance of internalized BTN2-Ig as observed in permeabilized DCs. Thus, bound BTN2A1 was rapidly internalized from the cell surface, consistent with endocytosis. Together, the IgV- and the IgC- like domains of the BTN2A1 monomer contain four potential N-linked glycosylation sites. To evaluate the role of sugars in BTN2A1 binding to DCs we conducted a series of inhibition experiments. Interaction of BTN2A1 -proteins with DCs required Ca²⁺, as cation chelators such as EDTA and EGTA were inhibitory in a dose- dependent manner (Fig. 3B) . Whereas monosaccharides blocked BTN2A1- Ig binding only weakly, mannan was a potent, dose-dependent inhibitor (Fig. 3C) . The significance of carbohydrates for binding, in particular high-mannose-type oligosaccharides, was further corroborated by Endoglycosidase H (Endo H) digestion. Endo H- treated BTN2A1-Ig failed to bind to DCs (Fig 3D) . To rule out binding to high-mannose- carbohydrate on the Fc-domain, we also tested binding of hFc and hBTN3A3-Ig. HFc and hBTN3A3 , expressed in HEK293T like BTN2A1, did not bind (Fig. 1) . Thus, the counter- receptor recognized mannose moieties on BTN2A1, most likely branched mannose- structures . Taking these data together, the profile of BTN2A1 binding to MoDCs indicates that its counter-receptor belongs to the C-type lectin family.

BTN2A1 binds DC-SIGN transfectants and binding is blocked by- specific antibodies and HIVgpl20

DC-SIGN was first identified as a C-type lectin that binds to HIV envelope glycoprotein gpl20 (16) . This molecule is predominantly expressed on DCs, including MoDCs, but not LCs (17) . Its expression is IL-4 -dependent and is negatively regulated by LPS and TNF-α (18) . We confirmed that DC-SIGN is IL-4 inducible and its induction peaks at 48 h (Fig. 2) . Thus DC-SIGN has several hallmarks exhibited by the BTN2A1 counter-receptor. To test this, we cloned and expressed the full-length DC-SIGN cDNA in HEK293T cells. Staining with a specific Mab confirmed transient DC-SIGN expression on a majority of the HEK293T transfectants. BTN2A1 bound to DC-SIGN-transfectants, but not to non-transfected cells (Fig. 4A) . Interaction was specific for the BTN2A1 domain of the chimera since a control Fc protein did not bind. We also assayed the interaction in the presence of an inhibitory antibody. As shown in Figure 4A, binding of BTN2A1 was inhibited by preincubation with the DC-SIGN-blocking antibody IBlO but was not affected by an isotype control . We then addressed the question of whether DC-SIGN is the only receptor on DCs or whether there are other C-type lectins interacting with BTN2A1. The IBlO antibody completely abrogated binding to DCs (Fig. 4B) , suggesting that DC-SIGN is the exclusive receptor for BTN2A1 on DCs. Thus, the biochemistry, Ig-fusion protein binding and antibody blocking studies, were all consistent with DC-SIGN as a counter-receptor of BTN2A1. DC-SIGN is an HIV receptor and we explored whether BTN2A1 and HIVgpl20 compete for binding. Both molecules bind to DC-SIGN via high-mannose oligosaccharides and we found that binding of BTN2A1-Ig to DC-SIGN-transfectants was blocked by recombinant gpl20 in a dose- dependent manner (Fig. 4C) . Thus recombinant BTN2A1 is a candidate to inhibit transmission of HIV to DCs.

Tissue- and/or tumor-specific glycosylation of native BTN2A1 is recognized by DC-8IGN

We demonstrated that a soluble BTN2A1- fusion protein binds to DC- SIGN on MoDCs . In order to obtain direct evidence for the interaction of DC-SIGN to native BTN2A1, we used DC-SIGN-FLAG for immunoprecipitation. Lysates of HEK293T expressing BTN2A1 endogenously were precipitated and separated by SDS-PAGE. By western blotting with an antibody to the recombinant B30.2 domain of BTN2A1, full length BTN2 was detected, whereas a control immunoprecipitation was negative (Fig. 5A) .

BTN2A1 is expressed in most tissues and we wondered therefore why DC-SIGN has not been reported to exhibit widespread binding. DC-SIGN has been shown to bind selective oligosaccharide residues on various proteins. Therefore, binding of BTN2A1 to DC-SIGN could vary if glycosylation of the butyrophilin molecule is cell -type specific. To address this, we studied binding of DC-SIGN-FLAG to cells by flow cytometry. As a positive control, we first confirmed strong binding of the DC- SIGN fusion protein to MAC-I and CEACAM-I on neutrophils (Fig. 5B, (13)) . We then showed that HEK293T cells also bound sDC- SIGN, in accordance with our immunoprecipitation data. However, we could not detect PBL binding to DC-SIGN (Fig. 5B) .

A potential problem with clearly demonstrating that cell -surface BTN2A1 binds DC- SIGN is that the lectin has been assigned a number of different cellular ligands (13, 17, 19) . To overcome this problem, GFP-labeled BTN2A1 was transfected into a range of cells and DC- SIGN binding was assayed in relation to the level of BTN2A1 expression. BTN3A3, a homologue to BTN2A1 that does not bind to DC- SIGN (Fig.

5) was used as a negative control. The cells expressed some BTN2A1 endogenously so the increase in binding of DC- SIGN after transfection of BTN2A1 was measured. There was a marked increase of DC-SIGN binding to HEK293T, HeIa and MeIJuSo, as BTN2A1 expression increased (Fig. 5C) . Interestingly, binding to transfected HUVECs, HFFs, on the other hand, was unchanged with increasing BTN2A1-GFP expression levels (Fig. 5C) . This provides indication that BTN2A1 on these cells does not contain the appropriate carbohydrate moieties for binding to DC-SIGN. Together, the data confirm that BTN2A1 is recognized by DC-SIGN, but only on certain tissues.

GNA inhibits binding of DC-SIGN and binds to BTN2A1 on tumor cells such as HEK293T, not on HOVECs

BTN2A1-GFP transfectants (Fig.5) were used to study binding of DC- SIGN in the presence of Galanthus nivalis agglutinin (GNA) . GNA, a strictly mannose-binding plant lectin, blocked binding of DC-SIGN to HEK293T. Binding was inhibited by -80% (Fig. 6A) . Thus, mannose carbohydrates are mandatory in binding although we cannot rule out other carbohydrates being involved. Next, we used GNA to assess mannose-moieties of BTN2A1. GNA bound strongly to BTN2A1 on HEK293T and not to the related BTN3A3. However, GNA binding to BTN2A1 on HUVECs was low (Fig.6B) . Taking these data together, we demonstrated that BTN2A1 is differentially decorated with high-mannose moieties that determine binding to DC-SIGN.

BTN3-Ig binds DC-SIGN

BTN2 binds to DC-SIGN with high affinity and binding can be inhibited by mannan. To investigate the binding of BTN3, we prepared 293T cells, which express high levels of DC-SIGN. Transfection efficiency was verified by monoclonal antibodies specific for DC- SIGN and was usually higher than 50%. BTN3-Ig did not bind to 293T cells. However, BTN3 did stain DC-SIGN-transfectants . Fewer cells were positively stained compared to BTN2-Ig. No binding of hlgG control proteins was detected to either transfectant . Thus, BTN3 is a cellular ligand for DC-SIGN.

BTN2 and BTN3 interact with DC-SIGNR (L-SIGN)

The DC-SIGN homologue DC-SIGNR functions as an HIV-I trans-receptor similarly to DC-SIGN but is not expressed by DCs. Instead, DC-SIGNR is localized to certain endothelial cell populations (Bashirova, Geijtenbeek et al . 2001) . In the same way as described above for DC- SIGN, the binding of BTN2- and BTN3 -fusion proteins to DC-SIGNR- transfectants was analysed. An antibody specific for DC-SIGN and DC- SIGNR confirmed a transfection rate usually higher than 50%. Both fusion proteins, BTN2 and BTN3 interacted with DC-SIGNR-293T cells, not with mock- transfected 293T cells. Interaction is very similar to DC-SIGN, in that BTN2-FC stains a higher cell number than BTN3-FC does. These findings provide indication that the higher binding affinity of BTN2-FC for DC-SIGN and DC-SIGNR is higher than that of BTN3-FC and is reminiscent the interaction between DC-SIGN and its other cellular ligands ICAM2 and ICAM3 (Geijtenbeek, Krooshoop et al. 2000) .

The butyrophilin family member BTN3A1 and mouse butyrophilin-like 2 (BTNL2) were recently shown to interact with T cells. The murine BTNL2 functioned as an inhibitor of T cell activation (5, 6) . Using an Ig-fusion protein, produced in HEK293T cells, we demonstrated that, rather than interacting with T cells, BTN2A1 is recognized by immature MoDCs. Biochemical 'fingerprinting' and antibody-blocking studies revealed that DC- SIGN was the receptor expressed on immature MoDCs interacting with BTN2A1. A monoclonal antibody directed against the DC-SIGN carbohydrate-recognition domain (CRD) blocked binding of BTN2A1 (20) . Other C- type lectins were not involved in the interaction of BTN2A1 with DC-SIGN.

Crystal structures of the carbohydrate recognition domain, in combination with binding studies, revealed that DC-SIGN has a dual binding specificity and selectively recognizes endogenous high- mannose oligosaccharides in addition to fucose- containing glycans (21, 22) . These studies predicted binding of DC-SIGN to other cell surface or soluble glycoproteins with appropriately displayed high- mannose oligosaccharides. Interaction of DC-SIGN with endogenous glycans and HIVgpl20 results from high affinity binding to a characteristic internal feature of high-mannose oligosaccharides (21) . Our studies are consistent with BTN2A1 on some cells, but not all, having appropriate high-mannose moieties for binding to DC- SIGN.

We did not detect binding of DC-SIGN to PBLs. This could be explained by their low level of BTN2A1 expression. However, PBLs have high levels of ICAM-3, an alternative DC-SIGN ligand that was shown to support primary immune responses (17) . Bogoevska et al . showed DC-SIGN binding to ICAM-3 on PMNs but not on T cells (23) . ICAM-3 on T cells is not fucosylated and lacks the appropriate mannose structures for binding, whereas PMNs bind DC-SIGN via fucosylated ICAM-3 (23) . Our data are consistent with these observations .

DC-SIGN bound to BTN2A1 expressed on HEK293T, HeIa and MeIJuSo cells. In contrast, primary cells such as HUVECs and HFF failed to bind, in spite of similar BTN2A1 expression levels. However, we found that BTN2A1 of HEK293T have more high-mannose moieties in comparison to HUVECs and those high-mannose moieties are instrumental for binding to DC-SIGN. DC-SIGN did not bind to BTN2A1 expressed on any normal tissues we have studied so far. This is reminiscent of the carcinoembryonic antigen (CEA) , a tumor- associated antigen, reported to bind to DC-SIGN via Lewis^x and Lewis^y carbohydrates. CEA-glycosylation is dysregulated in a number of malignant tissues and DCs recognize the tumor- specific glycosylation on colorectal cancer cells through DC-SIGN (12) . Thus there is a precedent for binding of DC-SIGN molecules being restricted to tumor tissues. It is also possible that glycosylation of specific tissue- types is responsible for binding to tumor cells.

We, and others, cloned a molecule related to DC-SIGN called L-SIGN or DC-SIGNR, which is expressed in liver sinusoids, placental capillaries and the endothelium of lymph node sinuses (25, 26) . Both SIGN-receptors bind to pathogens like HIV, CMV₇ Ebola, mycobacteria and selectively recognize endogenous high-mannose oligosaccharides. Since DC-SIGN and L-SIGN share the structural basis for selective recognition of high-mannose oligosaccharides it is reasonable to assume that BTN2A1 also interacts with L-SIGN, implying a functional interaction of BTN2A1 beyond DCs (21) .

Table 1

Table 2 References

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5. Compte, E. et al 2004. Bur J Immunol 34:2089-2099.

6. Nguyen, T. et al 2006. J^" Immunol 176:7354-7360.

7. Arnett, H. A. et al 2007. J Immunol 178:1523-1533.

8. Bieniasz, P. D. 2004. Nat Immunol 5:1109-1115.

9. Banchereau, J., and R. M. Steinman. 1998. Nature 392:245-252.

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12. van Gisbergen, K. P. et al 2005. Cancer Res 65:5935-5944.

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14. Fawcett, J. et al 1992. Nature 360:481-484.

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16. Curtis, B. M. et al 1992 Proc Natl Acad Sci U S A 89:8356- 8360.

17. Geijtenbeek, T. B. et al . 2000. Cell 100:575-585.

18. Relloso, M., et al 2002. J Immunol 168:2634-2643.

19. Geijtenbeek, T. B. et al 2000. Nat Immunol 1:353-357.

20. Halary, F. et al 2002. Immunity 17:653-664.

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23. Bogoevska, V. et al 2007. Glycobiology 17:324-333.

24. Bogoevska, V. et al . 2006. Glycobiology 16 : 197-209.

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Claims

Claims :

1. A method of identifying a tumour cell comprising: determining the presence or amount of mannosylated BTN polypeptide on the surface of a cell, wherein an increased amount of mannosylated BTN polypeptide on the cell relative to controls is indicative that the cell is a tumour cell .

2. A method according to claim 1 wherein the mannosylated BTN polypeptide is a mannosylated BTN2 polypeptide.

3. A method according to claim 2 wherein the mannosylated BTN2 polypeptide comprises a sequence which has at least 80% sequence identity with the BTN2Alvl sequence of NP_008980.1 GI: 5921461.

4. A method according to any one of the preceding claims wherein the cell is comprised within a sample of cells obtained from an individual .

5. A method according to any one of the preceding claims wherein the presence or amount of mannosylated BTN polypeptide on the cell is determined by;

(i) contacting the cell with a specific binding member for mannosylated BTN polypeptide and

(ii) determining the binding of the specific binding member to the cell, wherein the binding being indicative of the presence or amount of mannosylated BTN polypeptide on the cell .

6. A method according to claim 5 wherein the specific binding member is an antibody.

7. A method according to claim 5 wherein the specific binding member is a SIGN receptor.

8. A method according to claim 7 wherein the SIGN receptor comprises the extracellular domain of an amino acid sequence having at least 50% to the amino acid sequence set out in database accession numbers AAG13814.1, AAG13848.2, or NP_066978.1.

9. A method according to any one of claims 1 to 4 wherein the presence or amount of mannosylated BTN polypeptide on the cell is determined by,-

(i) contacting the cell with a specific binding member which binds BTN polypeptide and

(ii) determining the amount of binding of the specific binding member to mannosylated BTN.

10. A method according to claim 9 wherein the amount of binding of the specific binding member to mannosylated BTN is determined by isoelectric focussing.

11. A composition comprising a BTN polypeptide for use in a method of treatment of the human or animal body.

12 A composition according to claim 11 wherein the BTN polypeptide comprises an amino acid sequence having at least 45% sequence similarity with the extracellular domain of the BTN2A1 polypeptide as shown in Table 2.

13. A composition according to claim 11 or claim 12 , wherein the BTN polypeptide is a BTNl polypeptide which comprises an amino acid sequence having at least 80% sequence similarity with the extracellular domain of the BTNlal polypeptide shown in Table 2.

14. A composition according to claim 11 or claim 12, wherein the BTN polypeptide is a BTN2 polypeptide which comprises an amino acid sequence having at least 80% sequence similarity with the extracellular domain of the BTN2al polypeptide shown in Table 2.

15. A composition according to claim 1 or claim 2, wherein the BTN polypeptide is a BTN3 polypeptide which comprises an amino acid sequence having at least 80% sequence similarity with the extracellular domain of the BTN3al polypeptide shown in Table 2.

16. A composition according to any one of claims 11 to 15 for use in the prophylactic treatment of pathogen infection.

17. A composition according to claim 16 wherein the pathogen is selected from the group consisting of HIV, Ebola virus, Hepatitis C virus, Cytomegalovirus, Mycobacteria, Heliobacter, Leishmania, Schistosoma and Candida.

18. A composition according to any one of claims 11 to 15, for use as an adjuvant in a vaccine preparation.

19. A composition according to claim 18 wherein the composition further comprises an antigen.

20. A composition according to any one of claims 11 to 19, wherein the composition further comprises a pharmacologically acceptable excipient .

21. Use of a composition comprising a BTN polypeptide in the preparation of a medicament for prophylactic treatment of a pathogen infection.

22. Use according to claim 21 wherein the pathogen is selected from the group consisting of HIV, Ebola virus, Hepatitis C virus, Cytomegalovirus, Mycobacteria, Helicobacter, Leishmania, Schistosoma and Candida.

23. Use of a composition comprising a BTN polypeptide in the preparation of a medicament for stimulating or inhibiting the immune system of an individual .

24. Use according to claim 23 wherein the medicament is for stimulating or inhibiting a ThI response .

25. Use according to any one of claims 21 to 24 wherein the BTN polypeptide comprises an amino acid sequence having at least 45% sequence similarity with the extracellular domain of the BTN2A1 polypeptide set out in database accession number NP_008980.

26. An antibody molecule which specifically binds to a BTN polypeptide for use in a method of treatment of the human or animal body.

27. An antibody molecule which specifically binds to a mannosylated BTN polypeptide for use in a method of treatment of the human or animal body.

28. Use of an antibody molecule which specifically binds to a BTN polypeptide in the manufacture of a medicament for use in modulating the immune system of an individual .

29. Use of an antibody molecule which specifically binds to a mannosylated BTN polypeptide in the manufacture of a medicament for use the treatment or diagnosis of cancer.

30. A method of identifying/obtaining an immunomodulatory agent comprising: contacting a test compound with a DC-SIGN polypeptide, and; determining binding of the polypeptide by the test compound, binding of the DC-SIGN polypeptide being indicative that the test compound is a candidate immunomodulatory agent.

31. A method according to claim 30, comprising determining the ability of said test compound to activate a dendritic cell.

32. A method of identifying/obtaining an immunmodulatory agent comprising; contacting a cell with a test compound; and, determining the expression of a BTN polypeptide by said cell, a difference in the expression of the BTN polypeptide in the presence relative to the absence of said compound being indicative that the test compound is a candidate immunomodulatory agent.

33. A method of identifying/obtaining an immunomodulatory agent comprising; contacting a test compound with a BTN polypeptide; and, determining binding of the BTN polypeptide by the test compound, wherein binding of the BTN polypeptide by the test compound is indicative that the test compound is a candidate immunomodulatory agent .

34. A method of identifying/obtaining a compound which binds specifically to tumour cells comprising; determining the binding of a test compound to mannosylated BTN polypeptide and non-mannosylated BTN polypeptide, wherein an increase in binding of the test compound to mannosylated BTN polypeptide relative to non-mannosylated BTN polypeptide is indicative that the test compound binds specifically to tumour cells.

35. A method of identifying/obtaining an immunomodulatory agent comprising; contacting a SIGN receptor and a BTN polypeptide in the presence of a test compound; and determining binding of the BTN polypeptide to the SIGN receptor and/or BTN polypeptide activity.

36. A method according to claim 32, 33 or 35 comprising identifying the test compound as an immunomodulatory agent .

37. A method according to claim 34 comprising identifying the test compound as a compound which binds specifically to tumour cells.

38. A method according to claim 36 or 37 comprising modifying the test compound to optimise its pharmaceutical properties.

39. A method according to any one of claims 36 to 38 comprising formulating the test compound with a pharmaceutically acceptable excipient .