MXPA01007200A

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Publication number: MXPA01007200A
Application number: MXPA/A/2001/007200A
Authority: MX
Inventors: David P Andrew; Brian A Zabel; Paul D Ponath
Original assignee: Millennium Pharmaceuticals Inc
Priority date: 1999-03-11
Filing date: 2001-07-16
Publication date: 2002-03-26

Abstract

The invention relates to an antibody or antigen-binding fragment thereof which binds to the CC chemokine receptor GPR-9-6 and blocks the binding of a ligand (e.g, TECK) to the receptor. The invention also relates to a method of identifying agents (molecules, compounds) which can bind to GPR-9-6 and inhibit the binding of a ligand (e.g., TECK) and/or modulate a function of GPR-9-6. The invention further relates to a method of modulating a function of GPR-9-6, and to the use of the antibodies, antigen-binding fragments and agents identified by the method of the invention in research, therapeutic, prophylactic and diagnostic methods.

Description

ANTI-GPR-9-6 AND ANTI-TECK ANTIBODIES AND METHODS TO IDENTIFY GPR-9-6 AND TECK FUNCTION MODULATORSBACKGROUND OF THE INVENTION Chemokines are a large and growing family of almost forty 6-14 kD (non-glycosylated) proteins that bind to heparin and that mediate a wide range of biological functions (Taub, DD and Openheim, JJ, Ther. Immunol., 1: 229-246 (1994)). The chemokines can be divided into families based on the position of four cysteine residues that form two disulfide bonds (Kelner, GS, et al., Science, 265: 12395-1399 (1994); Bazan, JF, et al. , Nature, 385: 640-644 (1997); Pin, Y., et al., Nature, 385: 611-617 (1997)). Chemokine receptors can also be divided into families based on the type of chemokine to which they bind, although no clear structural differences have been identified that distinguish the subfamilies from the receptors (Mackay, CR, J.) Exp. Med. ., 184: 799-802 (1996).) In addition, there are several so-called "orphan" chemokine receptors (eg, GPR-9-6) that share sequence homology with well-characterized chemokine receptors. Biological functions and specific agonists of orphan receptors remain unknown Chemokines play a vital role in the adhesion and extravasation of leukocytes For example, in several in vitro assays, chemokines can induce chemotaxis or transendothelial migration of leukocytes (Taub, DD and Openheim, JJ, Ther.Immunol., 1: 229-246 (1994)), while in vivo injection (Taub, DD, et al., J. Clin. Invest., 97: 1931- 1941 (1996)) or overexpression of chemokines (Fuentes, M.E., et al., J. Immunol. , 155: 5769-5776 (1995)) may result in the accumulation of leukocytes at the site of injection or expression of the chemokines. Chemokine antagonists can prevent leukocyte trafficking (Bargatze, RF and Butcher, EC, "Exp. Med., 178: 367-372 (1993)) and may have beneficial effects in various models of acute and chronic inflammation (Sekido , N., et al., Nature, 365: 654-657 (1993); Karpus, .J., Et al., ". Im unol., 155: 5003-5010 (1995)). It has also been reported that chemokines modulate angiogenesis (Gupta, SK, et al., Proc. Nati, Acad. Sci. USA, 92: 7799-7803 (1995)), hematopoiesis (Taub, DD and Openheim, JJ, Ther. Immunol., 1: 229-246 (1994)) as well as the activation of T lymphocytes (Zhou, Z., et al., J. Immunol., 151: 4333-4341 (1993); Taub, DD, and col., J. I unol., 156: 2095-2103 (1996)). In addition, several chemokine receptors act as co-receptors, together with CD4, for the entry of tropic M-tropic HIV-1 (Choe, H. et al., Cell, 85: 1135-1148 (1996); Feng, Y., et al., Science, 272: 872-877 (1996)). Several subsets of CD4 lymphocytes can be defined on the basis of their expression of several adhesion molecules that are known to traffic to different physiological sites (Mackay, C.R., Curr Opin. Immunol., 5: 423-427(1993)). For example, CD4 lymphocytes with CLA + vos memory are directed towards the skin (Berg, E.L., et al., Nature,174 (6): 1461-1466 (1991)), while CD4 lymphocytes with CLA_vo a4ß7 + vos memory are directed towards mucosal sites (Hamman, A., et al., J. Immunol., 152: 3282-3292 ( 1994)). It is believed that the adhesion of leukocytes to the endothelium involves several overlapping stages including rolling, activation and stopping. Rolling leukocytes are exposed to factors expressed at the site of adhesion, resulting in leukocyte activation and upregulation of integrin-mediated adhesion. As a consequence of such integrin-mediated interactions, leukocytes stand on the endothelium (Bargatze, RF and Butcher, EC, "Exp. Med., 178: 367-372 (1993); Bargatze, RF, et al., Immuni ty, 3: 99-108 (1995)). Activation of leukocytes and upregulation of integrin molecules takes place through a mechanism sensitive to pertussis toxin that is believed to involve chemokine receptors (Bargatze , RF and Butcher, EC, J. Exp. Med., 178: 367-372 (1993); Campbell, J.J., et al. , Science, 279: 381-383 (1998)). The CD4 + lymphocytes with memory can be grouped based on the expression of certain chemokine receptors. For example, CXCR3, CCR2 and CCR5 (Qin, S., et al., Eur. J. Iwmunol., 26: 640-647 (1996); Qin, S., et al., J. Clin. Invest., 101: 146-154 (1998); Liao, F., et al., J. I munol., 162: 186-194 (1999)) are all expressed in subsets of CD4 lymphocytes with memory, and certain chemokines selectively act in virgin T cells (Adema, GJ, et al., Nature, 387: 713-717 (1997)). In addition, it has been shown that several chemokines that are ligands for such receptors are expressed at sites of inflammation(Gonzalo, JA, et al., J., Clin.Invest., 98: 2332-2345 (1996)) and in some cases in the lymph nodes draining a challenged site (Tedia, N., et al., J I munol., 161: 5663-5672 (1998)) It has also been shown that the TH1 / T2 lymphocyte lines derived in vi tro express differentially chemokine receptors, Specifically, it has been shown that THI lymphocytes selectively express CXCR3 and CCR5, whereas TH2 lymphocytes selectively express CCR4, CCR8 andCCR3 (Bonecchi, R.G., et al., J ". Exp. Med., 187: 129-134(1998); Sallusto, F.D., et al., ". Exp. Med., 187: 875-883(1998); Sallusto, F.D., Science, 277: 2005-2007 (1997); Andrew,D.P., et al., J ". Immunol., 161: 5027-5038 (1998); Zingoni, A., et al.,". Iwmunol. , 161: 547-555 (1998)). Interestingly, in some cases the chemokines for these respective chemokine receptors, such as MDC for CCR4 and IP-10 for CXCR3, are induced by cytokines associated with a Tl / T2 environment (TAndrew, DP, et al., J .

Immunol. , 161: 5027-5038 (1998); Luster, A.D., et al., Nature, 315: 672-676 (1985)).

DETAILED DESCRIPTION OF THE INVENTION The abbreviations used herein include: ECV304, human umbilical vein endothelial cell line (Accession No. ATCC CRL-1998); ADEC, chemokine expressed by adenoids; IPÍO, 10 kDa protein inducible by IFN-gamma; IMDC, immature dendritic cell; I -TAC, alpha chemoattractant of T cells inducible by interferon; MCP-1, monocyte chemoattractant protein; SDF, a factor derived from stromal cells; MDC, chemokine of mature dendritic cells; MIG, monoquin induced by interferon-gamma; RANTES, regulated after activation, expressed by normal T cells; MIP3, inflammatory protein of macrophages 3; MIP4, inflammatory protein of macrophages 4; TECK, chemokine expressed in the thymus; SLC, chemokine of secondary lymphoid tissue; DC, dendritic cell. Chemokines and their receptors are an important component in the regulation of leukocyte directed migration. Chemokines are produced at sites of inflammation and attract several leukocytes carrying the corresponding receptors. Although the spectrum of chemokines expressed at the inflammatory site may differentially attract certain inflammatory cells, the selectivity and variation of chemokine receptor expression in lymphocytes provide additional regulation to ensure recruitment of the appropriate cells in response to inflammatory stimuli. particular. As the number of chemokine receptors identified and characterized continues to grow, it is increasingly clear that the cells selectively express several receptors that can identify, label or otherwise characterize functional subsets of leukocytes such as TH1 and TH2, activated and quiescent T cells. , virgins and with memory. Because several characterized and / or orphan chemokine receptors can be co-expressed in individual cells, it has been difficult to validate the role of specific receptors in the onset and progression of a disease or, for that reason, in normal immune function. As described in this, a study of the orphan chemokine receptor GPR-9-6 was carried out. During the course of the study an antibody was produced that binds to human GPR-9-6 (mAb 3C3) and was used to analyze the expression and function of the receptor in several types of leukocytes. The receptor was found to be predominantly expressed in thymocytes and in lymphocytes with elevated CD4 + a4ß7 memory that reach mucosal sites (eg, respiratory tract, urogenital tract, alimentary canal and associated tissues (pancreas, gallbladder)). As described herein, GPR-9-6 (CCR9) is a functional CC chemokine receptor that binds to, and is activated by, the chemokine CC known as chemokine expressed in the thymus (TECK). The invention relates to the chemokine receptor GPR-9-6 and to agents (eg, ligands, antibodies, antagonists, agonists) that bind to the receptor. In one aspect, the invention relates to an antibody that binds to mammalian GPR-9-6 or to a portion of GPR-9-6.

As described herein, an antibody termed "mAb GPR96-1" which binds to human GPR-9-6 has been produced. The GPR96-1 mAb can be produced by the murine hybridoma GPR96-1, also referred to as the murine hybridoma LS272 GPR96 1-5, which was deposited on March 9, 2000, on behalf of LeukoSite, Inc., 215 First Street, Cambridge , MA 02142, USA, at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA, under Accession No. PTA-1470. In another embodiment, the anti-GPR-9-6 antibody of the invention is the GPR96-1 mAb or an antigen-binding fragment thereof. In another embodiment, the binding of the antibody or the antigen-binding fragment to mammalian GPR-9-6 (eg, human) can be inhibited by the GPR96-1 mAb. Such inhibition may be the result of a competition for the same epitope or a similar epitope, of steric interference or due to a change in the conformation of GPR-9-6 that is induced after the binding of the antibody to the receptor. In yet another embodiment, the antibody or antigen-binding fragment of the invention has the same or similar epitope specificity as the mAb.

GPR96-1. Antibodies with an epitopic specificity that is the same or similar to that of mAb GPR96-1 can be identified by a variety of suitable methods, such as those described herein. The invention also relates to a bispecific antibody, or to a functional fragment thereof (eg, F (ab ') 2), which binds to a mammalian GPR-9-6 and at least one other antigen. In a particular embodiment, the bispecific antibody or functional fragment thereof has the same or similar epitopic specificity as the mAb.3C3 or that the GPR96-1 mAb and that at least one other antibody (see, for example, US Patent No. 5,141,736 (Iwasa et al.), US Patent No. 4,444,878, 5,292 .668, 5,523,210 (all for Paulus et al.) And U.S. Patent No. 5,496,549 (Yamazaki et al.)). In a preferred embodiment, the antibody or antigen-binding fragment of the invention binds specifically to a mammalian GPR-9-6. As used herein, the term "specific antibody" or "specific" when referring to an antibody-antigen interaction is used to indicate that the antibody can selectively bind to a mammalian GPR-9-6, rather than indicating that the antibody can bind only to one antigen. For example, an antibody can bind to one or more antigens with low affinity and bind to human GPR-9-6 with a high affinity. It is considered that such an antibody is specific for human GPR-9-6, because when it is used(for example, in a therapeutic or diagnostic application) at a suitable concentration, the antibody can selectively bind to human GPR-9-6. The concentration of antibody required to provide selectivity for a mammalian GPR-9-6 (e.g., a concentration that reduces or eliminates the low affinity binding) can be easily determined by suitable methods, e.g., titration. In another aspect, the invention relates to an isolated cell that produces an antibody or an antigen-binding fragment of an antibody that binds to a mammalian GPR-9-6. In a preferred embodiment, the isolated antibody producing cell of the invention is an immortalized cell, such as a hybridoma, a heterohybridoma, a lymphoblastoid cell or a recombinant cell. The antibody producing cells of the present invention have other uses than the production of antibodies.

For example, the cell of the present invention can be fused with other cells (such as human myeloma cells suitably labeled with a drug, mouse myeloma cells, human-mouse heteromyeloma cells or human lymphoblastoid cells) to produce, for example , additional hybridomas and thus provide for the transfer of the genes encoding the antibody. In addition, the cell can be used as a source of nucleic acids encoding the anti-GPR-9-6 immunoglobulin chains, which can be isolated and expressed (for example, after transfer to other cells using any suitable technique (see, for example, Cabilly et al., U.S. Patent No. 4,816,567.; Winter, U.S. Pat. No. 5,225,539)). For example, clones containing a sequence encoding a rearranged anti-GPR-9-6 light and / or heavy chain (eg, by PCR) can be isolated or 7DNA libraries can be prepared from mRNA isolated from the cell lines , and cDNA clones that encode a chain (s) of the anti-GPR-9-6 immunoglobulin can be isolated. Thus, nucleic acids encoding the heavy and / or light chains of the antibodies or portions thereof can be obtained and used for the production of the specific immunoglobulin, an immunoglobulin chain or variants thereof (e.g. humanized immunoglobulins) in a variety of host cells or in an in vi tro translation system. For example, nucleic acids, including cDNAs, or derivatives thereof encoding variants such as a humanized immunoglobulin or an immunoglobulin chain, can be placed in suitable prokaryotic or eukaryotic vectors (e.g., expression vectors) and introduced into a appropriate host cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is operably linked to one or more elements for the control of expression (e.g., in the vector or integrated in the genome of the host cell), to produce a cell that produces recombinant antibodies.

The antibody of the invention can be produced by any suitable method, for example, by collecting serum from an animal (eg, mouse, human, transgenic mouse) that has been immunized with a mammalian GPR-9-6. In another example, a suitable antibody producing cell (e.g., hybridoma, heterohybridoma, lymphoblastoid cell, recombinant cell) can be maintained, in vitro or in vivo, under conditions suitable for expression (e.g., in the presence of an inducing medium). adequate supplemented with salts, growth factors, antibiotics, appropriate nutritional supplements), whereby the antibody or antigen-binding fragment is produced. If desired, the antibody or antigen-binding fragment can be recovered and / or isolated (e.g., from host cells, from the culture medium) and purified to the desired degree. Recovery and purification of the antibody can be achieved using suitable methods, such as centrifugation, filtration, column chromatography (e.g., ion exchange, gel filtration, hydrophobic interaction, affinity), native preparative electrophoresis, precipitation and ultrafiltration. It will be understood that the production method encompasses expression in a host cell of a transgenic animal (see, for example, WO 92/03918, GenPharm International, published March 19, 1992). As described herein, the antibodies and functional fragments thereof of the present invention can inhibit (reduce or prevent) the binding of a ligand to a mammalian GPR-9-6 and / or inhibit one or more associated functions. with the binding of the ligand to GPR-9-6. As discussed below, various methods can be used to determine the inhibition of binding of a ligand to GPR-9-6 and / or a function associated with binding of the ligand to the receptor.

Binding Assays The invention also relates to methods for detecting or identifying an agent (ie, molecule or compound) that can bind to a mammalian GPR-9-6 or a variant thereof that binds to a ligand. As used herein, "mammalian GPR-9-6" refers to mammalian GPR-9-6 proteins that exist naturally or endogenously and to proteins that have an amino acid sequence that is the same as that of a mammalian protein. corresponding mammalian GPR-9-6 protein that exists naturally or endogenously (e.g., recombinant proteins, synthetic proteins (i.e., produced using synthetic inorganic chemistry methods)). Accordingly, as defined herein, the term includes mature receptor protein, polymorphic or allelic variants and other isoforms of a mammalian GPR-9-6 (eg, produced by alternative splicing or other cellular processes), and modified or unmodified forms of the above (for example, lipidated, glycosylated, non-glycosylated). Naturally or endogenously occurring mammalian GPR-9-6 proteins include wild-type proteins such as mature GPR-9-6, polymorphic or allelic variants and other isoforms that occur naturally in mammals (e.g., humans , non-human primates). Such proteins can be recovered or isolated from a source that naturally produces mammalian GPR-9-6, for example. Polymorphic, allelic, splice and other naturally occurring variants of mammalian GPR-9-6 can be expressed in particular organs, tissues or cells and have altered properties (e.g., an affinity for the altered ligand). (e.g., TECK)) and a specialized biological function (e.g., T cell development, T cell recruitment).

Naturally or endogenously occurring mammalian GPR-9-6 proteins and proteins having the same amino acid sequence as a corresponding mammalian GPR-9-6 that exists naturally or endogenously, are referred to by the name of the mammalian GPR-9-6. corresponding mammal. For example, when the corresponding mammal is a human, the protein is called human GPR-9-6 protein (eg, a recombinant human GPR-9-6 produced in a suitable host cell). "Functional variants" of mammalian GPR-9-6 proteins include functional fragments, functional mutant proteins and / or functional fusion proteins that can be produced using suitable methods (eg, mutagenesis (eg, chemical mutagenesis, mutagenesis by radiation), recombinant DNA techniques). A "functional variant" is a protein or polypeptide having at least one characteristic function of a mammalian GPR-9-6 protein as described herein, such as a binding activity, a signaling activity and / or ability to stimulate a cellular response. Preferred functional variants can be linked to a ligand (that is, to one or more ligands, such as TECK). Generally, fragments or portions of mammalian GPR-9-6 proteins include those that have a deletion (i.e., one or more deletions) of an amino acid (i.e., one or more amino acids) relative to the GPR protein. 9-6 mature mammal (such as N-terminal deletions, C-terminals or internal). Also provided are fragments or portions in which only contiguous amino acids have been removed or in which non-contiguous amino acids have been removed relative to the mature mammalian GPR-9-6 protein. Mutant mammalian GPR-9-6 proteins include natural or artificial variants of a mammalian GPR-9-6 protein that differ by the addition, deletion and / or substitution of one or more contiguous or non-contiguous amino acid residues (e.g. , chimeras of the receiver). Such mutations can take place at one or more sites of the protein, for example in a conserved region or in a non-conserved region (in comparison with other chemokine receptors or G-protein coupled receptors), in the extracellular region, in the region cytoplasmic or in the transmembrane region. The fusion proteins encompass polypeptides containing a mammalian GPR-9-6 (e.g., human GPR-9-6) or a variant thereof as a first moiety, linked by a covalent bond (e.g., a peptide bond). ) to a second residue that is not present in mammalian GPR-9-6 as found in nature. In this way, the second moiety can be an amino acid, an oligopeptide or a polypeptide. The second remainder can be attached to the first remainder in a suitable position, for example, to the N-end, to the C-end or internally. In one embodiment, the fusion protein comprises an affinity ligand (eg, an enzyme, an antigen, an epitope tag, a binding domain) as the first tag, and a second tag containing an adapter sequence and GPR-9- 6 human or a portion of it. Where appropriate, additional residues (eg, third, fourth) may be present. In one embodiment, a mammalian GPR-9-6 functional variant (e.g., a ligand-binding variant) shares at least about 80% amino acid sequence similarity with said mammalian GPR-9-6, preferably at least about 90% similarity of amino acid sequences, and more preferably at least about 95% similarity of amino acid sequences with said mammalian GPR-9-6. In another embodiment, a functional fusion protein comprises a first residue that shares at least about 85% sequence similarity with a mammalian GPR-9-6, preferably at least about 90% sequence similarity, and more preferably at least about 95% sequence similarity with a mammalian GPR-9-6 (e.g., a human GPR-9-6 (e.g., SEQ ID NO: 2)). In another embodiment, a functional mammalian GPR-9-6 protein or a functional variant of a mammalian GPR-9-6 protein shares at least about 80% similarity of amino acid sequences, preferably at least about 90% of similarity of amino acid sequences and more preferably at least about 95% similarity of amino acid sequences to a naturally occurring human GPR-9-6 (eg, SEQ ID NO: 2). The similarity of amino acid sequences can be determined using a suitable sequence alignment algorithm, such as the Lasergene system (DNASTAR, Inc., Madison, Wl), using the Clustal method with the PAM 250 waste weight table, a penalty by holes of 10, a penalty for the length of the holes of 10 and default parameters (parameters of alignment of pairs: ktuple = 1, penalty for holes = 3, window = 4 and diagonals conserved = 5). In another embodiment, a functional variant is encoded by a nucleic acid sequence that is different from the nucleic acid sequence that naturally exists, but which, due to the degeneracy of the genetic code, encodes mammalian GPR-9-6 or a portion of it. As used herein, "mammalian TECK" refers to mammalian TECK proteins that exist naturally or endogenously and to proteins that have an amino acid sequence that is the same as that of the corresponding mammalian TECK protein that exists in a manner natural or endogenous (for example, recombinant proteins, synthetic proteins (that is, produced using the methods of synthetic organic chemistry)). Accordingly, as defined herein, the term includes the mature receptor protein, polymorphic or allelic variants and other isoforms of a mammalian TECK (eg, produced by alternative splicing or by other cellular processes), and modified or modified forms. unmodified from the above (for example, lipidated, glycosylated, non-glycosylated). Naturally or endogenously occurring mammalian TECK proteins include wild type proteins such as mature TECK, polymorphic or allelic variants and other isoforms that exist naturally in mammals(for example, humans, non-human primates). Such proteins can be recovered or isolated from a source that naturally produces mammalian TECK, for example. Polymorphic, allelic, splice and other naturally occurring variants of mammalian TECK can be expressed in particular organs, tissues or cells and have altered properties (eg, altered affinity for the receptor (eg, GPR) -9-6)) and a specialized biological function (e.g., T cell development, T cell recruitment). For example, as described herein an alternately spliced form of human TECK, in which the amino acid at position 110 (Ala 110) has been removed, is more predominant in the small intestine than in the thymus. TECK mammalian proteins that exist naturally or endogenously and proteins that have the same amino acid sequence as a corresponding mammalian TECK that exists naturally or endogenously, are referred to by the name of the corresponding mammal. For example, when the corresponding mammal is a human, the protein is called human TECK protein (eg, a recombinant human TECK produced in a suitable host cell).

Functional Assays An agent that binds to a mammalian GPR-9-6 or a functional variant thereof can be further studied in one or more suitable assays to determine whether said agent can modulate (inhibit (reduce or prevent) or stimulate) a or more functions of GPR-9-6 as described herein. For example, an agent can be analyzed in an extracellular acidification assay, in a calcium flux assay, in a ligand binding assay, in a chemotaxis assay or in an assay that monitors the degranulation or release of inflammatory mediators ( see, for example, Hesselgesser, et al., J. Biol. Chem., 273 (25): 15687-15692 (1998) and WO 98/02151). For example, an agent that binds to a mammalian GPR-9-6 can be analyzed in a leukocyte chemotaxis assay using suitable cells. Suitable cells include, for example, cell lines, recombinant cells or isolated cells expressing a mammalian GPR-9-6 and undergoing chemotaxis induced by a GPR-9-6 ligand (eg, induced by TECK). In one example, recombinant Ll2 cells expressing GPR-9-6 (see Campbell, et al., J "Cell Biol., 134: 255-266 (1996) in relation to L1.2 cells), can to be used in a modification of a transendothelial migration assay (Carr, MW, et al, TA, Proc. Nati, Acad. Sci. USA, (91): 3652 (1994).) The endothelial cells used in this assay are preferably those of the endothelial cell line, ECV 304, which can be obtained in the American Type Culture Collection (Manassas, Va.) Endothelial cells can be cultured in 6.5 mm diameter Transwell culture inserts (Costar Corp., Cambridge , MA) with a pore size of 3.0 μm The culture medium for ECV 304 cells can consist of M199 + 10% FCS, L-glutamine and antibiotics The test medium can consist of equal parts of RPMI 1640 and M199 with 0.5% BSA Two hours before the assay, 2 x 10 5 ECV 304 cells can be plated on each insert of the chemo plate Transwell otaxis of 24 wells and incubated at 37 ° C. A chemotactic factor such as TECK (Peprotech, Rocky Hill, NJ) (diluted in assay medium) in a final volume of 600 μl can be added to the 24-well tissue culture plates. Transwells coated with endothelial cells can be inserted into each well and 106 cells of the leukocyte type being studied in a final volume of 100 μl of assay medium are added to the upper chamber. The plate can then be incubated at 37 ° C in 5% C02 / 95% air for 1-2 hours. Cells that migrate to the lower chamber during incubation can be counted using, for example, flow cytometry. To count the cells by flow cytometry, 500 μl of the cell suspension of the lower chamber can be placed in a tube and relative counts can be obtained for a set period of time, eg 30 seconds. This method of counting is highly reproducible and allows the selection of leukocytes and the exclusion of the analysis of cellular waste or other types of cells. Alternatively, the cells can be counted with a microscope. Assays can be made to evaluate agents that can inhibit or stimulate chemotaxis in the same manner as the control experiment described above, except that the solutions of the agents in the upper chamber and the lower chamber can be added before the addition of the cells. Test medium containing up to 1% co-solvent DMSO. The ability of an agent to inhibit or stimulate chemotaxis can be determined by comparing the number of cells migrating to the lower chamber in the wells containing the agent, with respect to the number of cells migrating to the lower chamber in the control wells. Control wells may contain equivalent amounts of DMSO, but not agent. An agent that binds to a mammalian GPR-9-6 can also be determined by monitoring the cellular responses induced by the active receptor., using suitable cells expressing a mammalian GPR-9-6 or a functional variant thereof. For example, exocytosis can be monitored by methods known in the art or by other suitable methods (eg, degranulation of cells leading to the release of one or more enzymes or other components of the granules, such as esterases (e.g. , serine esterases), perforin and / or granzymes), the release of inflammatory mediators (such as the release of bioactive lipids such as leukotrienes (eg, leukotriene C)) and respiratory burst (see, eg, Taub, DD , and col., J ".

Immunol. , 155: 3877-3888 (1995), regarding assays for releasing serine esterases derived from granules; Loetscher, et al., J ". Immunol., 156: 322-327 (1996), in relation to assays for the release of enzymes and granzymes, Rot, A., et al., J. Exp. Med., 176 : 1489-1495 (1992) regarding the respiratory outbreak, Bischoff, SC, et al., Eur. J. Im unol., 23: 161-161 (1993) and Baggliolini, M. and CA Dahinden, Immuno logy Today , 15: 127-133 (1994).) In one embodiment, an agent that can inhibit or stimulate a function of GPR-9-6 is identified by monitoring the release of an enzyme after degranulation or exocytosis. a cell capable of performing this function Cells expressing mammalian GPR-9-6 or a functional variant thereof can be maintained in a suitable medium under suitable conditions, and degranulation can be induced. an agent to be tested, and enzymatic release can be determined.The release of an enzyme to the medium can be detected or measured using a suitable assay, such as an immunological assay or a biochemical assay to determine the enzymatic activity. The medium can be analyzed directly, by introducing the test components (eg, substrate, cofactors, antibody) into the medium (eg, before, simultaneously, or after the cells and the agent have been combined). The assay can also be performed in medium that has been separated from the cells or further processed (eg, fractionated) before the assay. For example, useful assays for enzymes, such as serine esterases, are available (see, for example, Taub, DD, et al., J. Im unol., 155: 3877-3888 (1995) as regards the release of serine esterases derived from the granules). In another embodiment, cells expressing a mammalian GPR-9-6 or a functional variant thereof are combined with a GPR-9-6 ligand (e.g., TECK), an agent to be tested is added before, after or simultaneously with it and the flow of Ca2 + is determined. The inhibition of Ca2 + flux induced by the ligand is indicative that the agent is an inhibitor or an antagonist of mammalian GPR-9-6 function. The cell adhesion can be monitored by methods known in the art or by other suitable methods. The occupation of the chemokine receptors of a lymphocyte can cause the activation of integrins and the induction of adhesion to adhesion molecules expressed in the vasculature or in the perivascular space. In another embodiment, a ligand, an inhibitor and / or a GPR-9-6 function stimulator is identified by monitoring cell adhesion by a cell capable of adhering. For example, an agent to be tested can be combined with (a) cells expressing a mammalian GPR-9-6 or a functional variant thereof (preferably non-adherent cells that when transfected with the receptor acquire the adhesive capacity), ( b) a composition containing a suitable adhesion molecule (e.g., a substrate such as a culture well coated with an adhesion molecule, such as fibronectin) and (c) a ligand or stimulant (e.g., an agonist), and maintained under conditions suitable for adhesion induced by the ligand or by the stimulant. Labeling the cells with a fluorescent dye provides a useful means to detect adherent cells. Non-adherent cells can be removed (eg, by washing) and the number of adherent cells can be determined. The effect of the agent inhibiting or increasing the adhesion induced by the ligand or the stimulant may be indicative of an inhibitory or stimulatory activity, respectively. The active agents in the assay include inhibitors and stimulators of binding, signal transmission and / or cellular responses. In another embodiment, an agent to be tested can be combined with cells expressing a mammalian GPR-9-6 and a composition containing a suitable adhesion molecule, under conditions suitable for adhesion induced by the ligand or the stimulant, and the accession. Increased adhesion relative to adequate control is indicative of the presence of a ligand and / or a stimulant. An agent that binds to a mammalian TECK or to a functional variant thereof can be further studied in one or more suitable assays to determine whether said agent can modulate (inhibit (reduce or prevent) or stimulate) one or more functions mediated by the receiver (for example, GPR-9-6 (CCR9)) after the TECK junction. Suitable assays to determine whether a TECK-binding agent can modulate the function of the chemokine, include assays such as those described herein in which a cell expressing a TECK receptor is used (eg, GPR-9). -6 human (human CCR9)) and TECK (for example, human TECK). The binding assays and functional assays described above can be used, alone or in combination with each other or with other suitable methods, to detect or identify agents that bind to a mammalian GPR-9-6 protein (CCR9), agents that bind to a mammalian TECK protein and / or modulators (inhibitors, stimulants) of a function of the GPR-9-6 protein or TECK protein. The in vi tro methods of the present invention can be adapted for bulk and automated screening assays in which a large number of samples are processed (e.g., a 96-well format). Cells expressing a mammalian GPR-9-6 (eg, human GPR-9-6 (CCR9)) or a functional variant thereof at levels suitable for bulk and automated selection studies can be used and, therefore, are particularly valuable in the identification and / or isolation of agents that join GPR-9-6, that join TECK and modulators of the GPR-9-6 or TECK function. The expression of GPR-9-6 can be monitored in a variety of ways. For example, expression can be monitored using antibodies of the present invention that bind to the receptor or to a portion thereof. Commercially available antibodies can also be used to detect the expression of a fusion protein labeled with an antigen or with an epitope containing a receptor protein or polypeptide (e.g., FLAG-tagged receptors), and cells expressing GPR-9 can be selected. -6 to the desired level (for example, by flow cytometry).

Inflammation Models In vivo models of inflammation are available which can be used to determine the efficacy of the antibodies and the antigen-binding fragments of the invention, as well as of the agents identified by the methods described herein as therapeutic products in alive. For example, infiltration of leukocytes after intradermal injection of a chemokine and of an antibody or antigen-binding fragment of the same reagent with mammalian GPR-9-6 into a suitable animal, such as rabbit, mouse, can be monitored. rat, guinea pig or primate (for example, rhesus macaque) (see, for example, Van Damme, J., et al., J. "Exp. Med., 176: 59-65 (1992); Zacharie, COC; col., J. Exp. Med., 171: 2177-2182 (1990); José, PJ, et al., J. Exp. Med., 179: 881-887 (1994) .In one embodiment, they are histologically analyzed. skin biopsies to determine the infiltration of leukocytes (e.g., GPR-9-6 + T cells) In another embodiment, labeled cells are administered to the animal (e.g., stably transfected cells expressing a GPR-9-6) of mammal marked, for example, with L?: LIn) that have chemotaxis and extravasation capability, For example, an antibody or agent to be assayed that binds to a mammalian GPR-9-6, before, simultaneously, or after a GPR-9-6 ligand or agonist (e.g., TECK) has been administered to the test animal. A decrease in the degree of infiltration in the presence of the antibody or agent compared to the degree of infiltration in the absence of said antibody or agent is indicative of inhibition. As described herein, GPR-9-6 is selectively expressed in memory lymphocytes migrating to mucosal sites (e.g., in CD4 + CLA cells at ß7elevated). Therefore, animal models of inflammatory diseases of the mucosa (eg, of the respiratory tract, the urogenital tract, the alimentary canal and associated organs and tissues (eg, pancreas, liver, gallbladder)) can be used to determine the therapeutic efficacy of GPR-9-6 modulating agents. For example, the antibodies and antigen-binding fragments of the invention as well as the agents identified by the methods described herein, can be studied in the model of inflammatory bowel disease in cotton-topped tamarin (Podolsky, DK, et al., J. Clin Invest., 92: 312-380 (1993)). The CD45RB elevated / SCID model provides a mouse model with similarity to Crohn's disease and ulcerative colitis (Powrie, F., et al., Immunity, 1: 553-562 (1994)). The therapeutic efficacy in this model can be determined, for example, using parameters such as the inhibition of the recruitment to the colon of cells labeled with 1: L1In and the reduction of the number of CD4 + T lymphocytes in the lamina propria of the large intestine after administration (for example, intravenous (iv), intraperitoneal (ip) and oral (po)) of an agent. Knockout mice have also been described which develop intestinal lesions similar to those of human inflammatory bowel disease (Strober, W. and Ehrhardt, RO, Cell, 75: 203-205 (1993)), and NOD mice provide a model insulin-dependent diabetes mellitus animal. As described herein, GPR-9-6 is also expressed in cancer cells. Thus, animal models of cancer can be used to determine the in vivo anticancer activity of GPR-9-6 modulating agents. For example, the efficacy of the antibodies and antigen-binding fragments of the invention, as well as of the agents identified by the methods described herein, can be determined as therapeutic products for the treatment of leukemia (e.g., cell lymphoblastic leukemia Acute T) in rabbits (Simpson, RM, et al., Lab. Tnvest., 74: 696-710 (1996)) or in SCID or NOD mice (Stelle, JP, et al., Blood, 90: 2015-2019 (1997)).

Applications in Diagnosis The antibodies of the present invention have application in methods in which GPR-9-6 can be detected on the cell surface. The receptor provides a marker of the cell types of leukocytes in which it is expressed. For example, antibodies raised against a mammalian GPR-9-6 protein or peptide, such as the antibodies described herein (eg, mAb 3C3, mAb GPR96-1), can be used to detect and / or quantify cells that express a mammalian GPR-9-6. In one embodiment, the antibodies can be used to select cells expressing GPR-9-6 from a mixture of cells (e.g., to isolate leukocytes that migrate to the mucosa, such as T cells with CD4 + memory GPR-9-6 + CLA_vas a4ß7 + vs). For this purpose, suitable methods can be used to count and / or classify cells (eg, flow cytometry, fluorescence activated cell sorting). The cell count can be used in the diagnosis of diseases or conditions in which an increase or decrease in leukocyte cell types is observed (for example, leukocytes that migrate towards the mucosa, IEL, LPL). In addition, the antibodies can be used to detect or measure the expression of GPR-9-6. For example, antibodies of the present invention can be used to detect or measure a mammalian GPR-9-6 in a biological sample (e.g., in cells, tissues or bodily fluids of an individual such as blood, serum, leukocytes ( for example, activated T lymphocytes), bronchoalveolar lavage fluid, saliva, intestinal fluid, specimens of biopsies). For example, a sample (eg, tissue and / or fluid) can be obtained from an individual and a suitable assay can be used to determine the presence or amount of GPR-9-6 protein. Suitable assays include immunological and immunochemical methods such as flow cytometry(eg, FACS analysis) and enzyme-linked immunosorbent assays (ELISA), including chemiluminescence assays, radioimmunoassay, immunoblotting (e.g., Western blotting) and immunohistology. Generally, a sample and an antibody of the present invention are combined under conditions suitable for the formation of an antibody-GPR-9-6 complex, and the formation of the antibody-receptor complex is determined (directly or indirectly). The presence of an increased level of reactivity of GPR-9-6 in a sample (eg, a tissue sample) obtained from an individual, may be indicative of inflammation and / or infiltration and / or accumulation of leukocytes (e.g. activated T cells) associated with an inflammatory disease or condition, such as an inflammatory bowel disease, a graft rejection, a delayed-type hypersensitivity reaction or an infection such as a viral or bacterial infection. The presence of a decreased level of GPR-9-6 reactivity in the circulation (eg, on the surface of circulating lymphocytes) may also be indicative of an infiltration and / or accumulation of leukocytes at inflammatory sites. The level of expression of a mammalian GPR-9-6 protein or a variant can also be used to correlate the increased or decreased expression of a mammalian GPR-9-6 protein with a particular disease or condition, and in the diagnosis of a disease or condition in which increased or decreased expression of a mammalian GPR-9-6 protein occurs (eg, increased or decreased relative to an appropriate control, such as the level of expression in a normal individual) . Similarly, the course of a therapy can be monitored by determining the immunoreactivity of GPR-9-6 in a sample from a subject. For example, the antibodies of the present invention can be used to monitor the number of cells expressing GPR-9-6 in a sample (e.g., blood, tissue) of a subject being treated with an anti-inflammatory or immunosuppressive agent. Antibodies that bind to TECK can be used to detect or measure TECK expression. For example, antibodies of the present invention can be used to detect or measure a mammalian TECK in a biological sample (e.g., cells or bodily fluids of an individual such as blood, serum, leukocytes (e.g., activated T lymphocytes). , bronchoalveolar lavage fluid, saliva, intestinal fluid). For example, a sample (eg, serum) can be obtained from an individual and a suitable assay can be used to determine the presence or amount of TECK protein. Suitable assays include immunological and immunochemical methods such as flow cytometry (eg, FACS analysis, including intracellular staining) and immunosorbent assays with enzyme linked (ELISA), including chemiluminescence assays, radioimmunoassay, immunoblotting (e.g., Western blotting) ) and immunohistology. (See, for example, Kallas, EG, et al., J ".Infect. Dis., 179: 1124-1131 (1999), regarding the intracellular staining of cells to detect secreted proteins.) Generally, a sample and an antibody of the present invention are combined under conditions suitable for the formation of an antibody-TECK complex, and is detected(directly or indirectly) the formation of the antibody-TECK complex. The presence of an increased level of TECK reactivity in a sample (eg, a fluid sample) obtained from an individual may be indicative of inflammation and / or infiltration and / or accumulation of leukocytes (e.g., activated T cells) associated with an inflammatory disease or condition, such as an inflammatory bowel disease, a graft rejection, a delayed-type hypersensitivity reaction or an infection such as a viral or bacterial infection. The level of expression of a mammalian TECK protein or of a variant can also be used to correlate the increased or decreased expression of a mammalian TECK protein with a particular disease or condition, and in the diagnosis of a disease or condition in which increased or decreased expression of a mammalian TECK protein occurs (eg, increased or decreased relative to an appropriate control, such as the level of expression in a normal individual). Similarly, the course of a therapy can be monitored by determining TECK immunoreactivity in a sample from a subject. For example, the antibodies of the present invention can be used to monitor the amount of TECK in a sample (e.g., blood) of a subject being treated with an anti-inflammatory or immunosuppressant agent. Kits may also be prepared to be used in the detection of the presence of a mammalian GPR-9-6 protein or a mammalian TECK protein in a biological sample. Such kits include an antibody or a functional fragment thereof that binds to the target protein (i.e., a mammalian GPR-9-6 receptor or a portion of said receptor, a mammalian TECK protein or a portion thereof) as well as one or more auxiliary reagents suitable for detecting the presence of a complex between the antibody or the fragment and the target. The antibody compositions of the present invention may be provided in lyophilized form, alone or in combination with additional antibodies specific for other epitopes. Antibodies, which may be labeled or unlabelled, may be included in kits with accessory ingredients (for example, buffers such as Tris, phosphate and carbonate, stabilizers, excipients, biocides and / or inert proteins, for example bovine serum albumin). For example, the antibodies can be provided as a lyophilized mixture with the accessory ingredients, or the accessory ingredients can be provided separately to be combined by the user. Generally, these accessory materials will be present in less than about 5% by weight based on the amount of active antibody, and will normally be present in a total amount of at least about 0.001% by weight based on the concentration of antibody. When a second antibody capable of binding to the target protein (eg, a second anti-GPR-9-6 antibody or a second anti-TECK antibody) is employed, such an antibody can be provided in the kit, for example, in a vial or separate container. The second antibody, if present, is typically labeled and can be formulated analogously with the antibody formulations described above. The components of the kit (for example, the anti-GPR-9-6 antibody or the antigen-binding fragment thereof, the auxiliary reagent) may be packaged separately or together in a suitable containment medium (eg, a bottle, a box, an envelope, a tube). When the kit contains a plurality of individually packaged components, the individual packages may be contained in a single, larger containment means (eg, a bottle, a box, an envelope, a tube). Similarly, the present invention also relates to a method for detecting and / or quantifying the expression of a mammalian GPR-9-6 receptor or a portion of the receptor by a cell, in which a composition containing a cell or a fraction thereof (e.g., a membrane fraction) is contacted with an antibody or with a functional fragment thereof (e.g., mAb 3C3, mAb GPR96-1 ) binding to a mammalian GPR-9-6 (CCR9) or a portion of the receptor under conditions appropriate for antibody or fragment binding thereto, and binding is monitored. Detection of the antibody, indicative of the formation of a complex between the antibody and a mammalian GPR-9-6 (CCR9) or a portion thereof, indicates the presence of the receptor. The binding of the antibody to the cell can be determined using any suitable method. The method can be used to detect the expression of GPR-9-6 in cells of a subject (e.g., in a sample, such as a body fluid, such as blood, saliva or other suitable sample). The level of expression of GPR-9-6 on the surface of cells (e.g., leukocytes) can also be determined by, for example, flow cytometry and the level of expression (e.g., intensity of staining) can be correlated with susceptibility, progression or risk of disease.

Therapy Methods The modulation of mammalian GPR-9-6 function according to the present invention, through the inhibition or stimulation of at least one characteristic function of a mammalian GPR-9-6 protein, provides a form effective and selective to inhibit or stimulate the functions mediated by the receptor. Once lymphocytes have been recruited to a site, other types of leukocytes, such as monocytes, can be recruited by secondary signals. Therefore, agents that can modulate the function of GPR-9-6, including ligands, inhibitors and / or stimulants, such as those identified as described herein, can be used to modulate the function of leukocytes (e.g. , infiltration of leukocytes including recruitment and / or accumulation). In one aspect, the present invention provides a method for modulating (inhibiting or stimulating) an inflammatory response in a subject, comprising administering an effective amount of an agent that inhibits or stimulates mammalian GPR-9-6 function to a subject in need of such therapy. In one embodiment, an effective amount of an agent that inhibits one or more functions of a mammalian GPR-9-6 protein (eg, a human GPR-9-6) is administered to a subject to inhibit (i.e., reduce or prevent) inflammation. Preferred agents for modulating an inflammatory response in a subject are agents that inhibit (ie, reduce or prevent) the binding of a ligand (e.g., TECK) to GPR-9-6 (CCR9). For example, the antibodies of the present invention, including mAb 3C3, mAb GPR96-1, mAb 11.3.1 and mAb 16.3.1, can be used in the method. As a result, one or more inflammatory processes such as leukocyte migration, chemotaxis, exocytosis (for example, enzymes) or the release of inflammatory mediators are inhibited. For example, infiltration of leukocytes into inflammatory sites (e.g., in an inflamed mucosal membrane (e.g., colon, small intestine)) may be inhibited according to the present method. In another embodiment, a subject is administered an effective amount of an agent that inhibits one or more functions of a mammalian GPR-9-6 protein (eg, a human GPR-9-6) to inhibit (i.e., reduce or prevent) the arrival of leukocytes mediated by GPR-9-6. In particular embodiments, an effective amount of an agent that binds to a human GPR-9-6 is administered to a subject in need thereof.

Diseases or conditions of humans or other species that can be treated with stimulants (eg, an agonist) of the GPR-9-6 function include, but are not limited to: • diseases in which angiogenesis has a role or neovascularization, including neoplastic diseases, retinopathy (eg, diabetic retinopathy) and macular degeneration; • infectious diseases, such as bacterial infections and tuberculoid leprosy, and especially viral infections; • immunosuppression, such as that of individuals with immunodeficiency syndromes such as AIDS, individuals undergoing radiation therapy, chemotherapy, or other therapy that causes immunosuppression; immunosuppression due to a congenital deficiency of receptor function or other causes.

Modes of Administration According to the method, one or more agents can be administered to the subject through an appropriate route, alone or in combination with another drug. An effective amount of an agent (e.g., a molecule that inhibits the binding of a ligand, an anti-GPR-9-6 antibody or an antigen-binding fragment thereof, an anti-TECK antibody or a fragment of binding to the same antigen). An effective amount is an amount sufficient to achieve the desired therapeutic or prophylactic effect, under the conditions of administration, such as an amount effective to inhibit or stimulate the function of the GPR-9-6 receptor, and thereby, inhibit or stimulate, respectively, a process mediated by GPR-9-6 (e.g., an inflammatory response). The agents can be administered in a single dose or in multiple doses. The dosage can be determined by methods known in the art and depends, for example, on the particular agent chosen, the age, sensitivity and tolerance to drugs of the subject and the general well-being thereof. Suitable dosages for antibodies can be from about 0.01 mg / kg to about 100 mg / kg of body weight per treatment. A variety of routes of administration can be used including, for example, oral, dietary, topical, transdermal, rectal, parenteral routes of administration (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intrathecal, intradermal injection) and by inhalation (for example, intrabronchial, intranasal or oral inhalation, intranasal drops), depending on the agent and the disease or condition to be treated. Administration can be local or systemic as indicated. The preferred mode of administration may vary depending on the particular agent (e.g., GPR-9-6 antagonist, anti-TECK antibody) chosen, and the particular condition (e.g., disease) that is being treated, however, is it generally prefers oral or parenteral administration. The agent can be administered as a neutral compound or as a salt. Salts of the compounds containing an amine or other basic group can be obtained by, for example, the reaction of the compound with a suitable organic or inorganic acid such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like. Compounds with a quaternary ammonium group also contain a counter anion such as chloride, bromide, iodide, acetate, perchlorate and the like. Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reaction with a suitable base, for example a hydroxide base. The salts of the acid functional groups contain a countercation such as sodium, potassium and the like.

The agent (e.g., an agent that inhibits binding of TECK to GPR-9-6 (CCR9)) can be administered to the individual as part of a pharmaceutical or physiological composition. For example, the agent can be administered as part of a pharmaceutical composition for modulating the function of GPR-9-6 which contains an inhibitor or a GPR-9-6 function stimulant and a pharmaceutically acceptable carrier. The formulation will vary depending on the selected route of administration (e.g., solution, emulsion, capsule). Suitable pharmaceutical or physiological vehicles may contain inert ingredients that do not interact with the stimulant (agonist) or inhibitor (antagonist) of GPR-9-6 function. Standard techniques of pharmaceutical formulation can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Pharmaceutical carriers suitable for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing approximately 0.9% mg / ml of benzyl alcohol), phosphate buffered saline, Hank's solution. , Ringer's lactate and the like. Methods for encapsulating compositions (such as in a hard gelatin or cyclodextran coating) are known in the art (Baker, et al., "Controlled Relase of Biological Active Agents," John Wiley and Sons, 1986). For inhalation, the agent can be solubilized and loaded in a dispenser suitable for administration (eg, an atomizer, a nebulizer or a pressurized aerosol dispenser). In addition, when the agent is a protein or a peptide, the agent can be administered through in vivo expression of the recombinant protein. In vivo expression can be accomplished through expression in somatic cells according to suitable methods (see, for example, US Patent No. 5,399,346). In this embodiment, a nucleic acid encoding the protein can be incorporated into a retroviral, adenoviral, or other suitable vector (preferably, an infectious vector with poor replication) for delivery, or it can be introduced into a transfected host cell or transformed able to express the protein for its supply. In this latter embodiment, the cells can be implanted (alone or in a barrier device), injected or otherwise introduced in an amount effective to express the protein at a therapeutically effective level. The present invention will be illustrated below by the following Examples, which are not intended to be limiting in any way.

EXAMPLES Example 1 Purification of Cell Populations Human peripheral blood was collected in 10% (v / v) of 0.1 M EDTA, placed on a gradient of 1-Step Polymorphs (1113 ± 0.01 g / ml, Aecurate Chemical Co., Westbury, NY) and centrifuged at 400 xg for 30 minutes at room temperature. Neutrophil and mononuclear cells were harvested, resuspended in Duibecco's phosphate buffered saline (DPBS) without calcium or magnesium (Life Technologies, Grand Island, NY) and centrifuged for 15 minutes at -750 x g. Red blood cells were used in the neutrophil fraction by resuspension of the pellet in E-Lyse (5 ml / 107 cells)(Cardinal Associates, Santa Fe, NM) for 5 minutes on ice. Both cell fractions were washed twice with ice-cold DPBS. The mononuclear cells were allowed to adhere to a plastic coated with proteins for 2-3 hours and subsequently the non-adherent cells were removed from the plate by gentle washing. After an additional 12 hours the non-adherent dendritic cells were removed from the plate by washing and diminished in B lymphocytes and T lymphocytes with magnetic beads coated with anti-CD19 and anti-CD2 (Dynabeads;, Oslo, Norway) (5 spheres per cell). The remaining cells were cultured in 50 ng / ml of granulocyte and macrophage colony stimulating factor (GMCSF, R and D Systems, Minneapolis, MN) and 40 ng / ml of IL-4 (R and D Systems) in Eagle's medium. modified from Duibecco (DMEM, Gibco BRL, Grand Island, NY) with 10% fetal calf serum (FCS, HyClone, Logan, UT) plus additives: 50 U / ml penicillin, 50 μg / ml streptomycin, L- 2 mM glutamine, 10 mM HEPES, sodium pyruvate in 10 mM MEM, non-essential amino acids in 0.1 mM MEM and 2-mercaptoethanol 5.5 x 10 -5 M (all from Gibco BRL, Grand Island, NY) for 7 days (Sallusto, F. and Lanzabecchia, A., J. Exp. Med., 179: 1109-1118(1994)) to generate immature dendritic cells (IMDC), and in some cases an additional 24-hour culture was used in 10 ng / ml of LPS to mature the dendritic cells. The CD4 +, CD8 +, CD14 +, CD56 + and CD19 + populations were purified from the mononuclear cells with the relevant Miltenyi Spheres (Miltenyi Biotech, Bergisch, Gladbach, Germany) using 20 μl of spheres for 10 7 mononuclear cells in PBS / 1% BSA / 5 mM EDTA at 5 x 10 7 cells / ml for 30 minutes at 4 ° C. They were then sedimented by centrifugation, resuspended in PBS / 1% BSA / 5 mM EDTA at 5 x 10 7 cells / ml and passed over a VS column (Miltenyi Biotech, Auburn, CA 95603) in a magnetic field to separate unlabeled cells . The cells were extracted by forcing 20 ml of PBS / 1% BSA / 5 mM EDTA on the VS column, out of the magnetic field.

Antibodies and Reagents Marked antibodies that bind to CD4, CD8, CD14, CD19, CD49d, CD56, CD62L, CLA, CD45RA, CD45RO, CXCR5, CD80 and CD86 from PharMingen (San Diego, CA) were obtained and used for studies. of immunofluorescence, while anti-aE and anti-CD83 were obtained from Beckman Coulter(Fullerton, CA). 0KT3, a human anti-CD3 mAb, was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and the human anti-CD28 mAb was obtained from Becton Dickinson(Mountain View, CA). Some of the anti-chemokine receptor mAbs were produced at LeukoSite, Inc. (Cambridge,MA) and have the names of the anti-CCR3 (7B11), anti-CCR4 (2B10), anti-CCR6 (11A9) and anti-CXCR3 (1C6) clones. Several anti-chemokine receptor mAbs used in the analysis by FACS were obtained from commercial sources. Anti-CCR2, anti-CCR6 and anti-CXCR5 mAbs used for immunofluorescence studies were obtained from R and D Systems (Minneapolis, MN), while anti-CCR5 and anti-CXCR4 were obtained from PharMingen (San Diego, CA) . Recombinant human chemokines were obtained from Peprotech (Rocky Hill, NJ) and R &D Systems (Minneapolis, MN) and in some cases were synthesized using solid phase methods that were optimized and adapted to a fully automated peptide synthesizer (model 430A; Applied Biosystems, Foster City, CA) as described (Clark-Lewis, I., et al., Biochemistry, 30: 3128-3135 (1991)). The human endothelial cell line ECV304 was purchased from the ATCC. All cytokines were obtained from R &D Systems (Minneapolis, MN).

Production of Anti-GPR-9-6 mAbs A peptide consisting of the NH2-terminus of GPR-9-6 having the sequence MADDYGSESTSSMEDYVNFNFTDFYC (SEQ ID NO: 3) was produced. BALB / C i.p. mice were immunized. with 10 μg of GPR-9-6 / KLH peptide conjugate prepared in Freund's Complete Adjuvant (FCA, Sigma, St. Louis, MO) on day 1, 10 μg of GPR-9-6 / KLH peptide conjugate prepared in Incomplete Freund's adjuvant (IFA, Sigma, St. Louis, MO) on day 20 and 10 μg of conjugate of GPR-9-6 / KLH peptide prepared in PBS on day 40. On day 60, mice were boosted with 10 μg of GPR-9-6 / KLH peptide in PBS and after 4 days the spleens were extracted and fused to SP2 / 0 myeloma cells (ATCC) (Coligan, et al., Current Protocols in Immunology 2. 5. 1 (1992)). The fusions were subjected to selection by ELISA using plates coated with the GPR-9-6 peptide. Hybridomas producing anti-GPR-9-6 mAbs were analyzed for their reactivity with GPR-9-6 transfectants and subcloned for further characterization. The murine hybridoma 3C3, also referred to as hybridoma LS129-3C3-E3-1, can be cultured at 37 ° C in an atmosphere with 5% C02 in DMEM supplemented with FCS (10%), IL-6 (100 ng / ml), penicillin (50 U / ml), streptomycin (50 μg / ml), L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate in MEM (10 mM), non-essential amino acids in MEM (0 , 1 mM) and 2-mercaptoethanol (5.5 x 10 -5 M).

Preparation of Chronically Activated TH1 and T2 Lymphocytes As previously described (Murphy, E., et al., J. Exp. Med., 183: 901-913 (1997)), six-well Falcon plates were coated overnight with 10 μg / ml of anti-CD28 and 2 μg / ml of 0KT3, and then washed twice with PBS. CD4 + lymphocytes from umbilical cord blood(Poietic Systems, Germán Town, MD) were cultured at 105-106 cells / ml in DMEM with 10% FCS and IL-2 (4 ng / ml). IL-12 (5 ng / ml) and anti-IL-4 (1 μg / ml) were used to direct towards TH1, while IL-4 (5 ng / ml) and anti-IFN gamma (1 μg / ml) were used. ml) to direct towards TH2. After 4-5 days, the activated TH1 and TH2 lymphocytes were washed once in DMEM and cultured for 4-7 days in DMEM with 10% FCS and IL-2 (1 ng / ml). After this, activated T1 and T2 lymphocytes were restimulated for 5 days with anti-CD28 / OKT3 and cytokines as described above, but with the addition of anti-CD95L (1 μg / ml) to prevent apoptosis. After 4-5 days the TH1 and TH2 lymphocytes were washed and then cultured again with IL-2 for 4 days. The activated TH1 and TH2 lymphocytes were maintained in this way for a maximum of three cycles.

ECV304 Transmigration and Chemotaxis Assays Transwell tissue culture inserts of 3 micrometers pore diameter uncoated or coated with 2% gelatin for 2 hours were used. Subsequently, 0.45 ml of DMEM with 5% FCS were placed in the lower wells of the chambers and 2 x 10 5 ECV304 cells were added to each insert coated with gelatin in 0.2 ml of DMEM with 5% FCS . After two days, the wells and inserts were washed twice with RPMI-1640 (Gibco BRL, Grand Island, NY) containing 0.5% HSA (human serum albumin) and 10 mM HEPES, and subsequently the chemokine was added. to the lower well. The cells under study were washed once in RPMI and resuspended at 4 x 106 cells / ml for THL / TH2 lymphocytes, cell lines and transfectants, or at 107 cells / ml for the remaining CD4 lymphocytes in RPMI with 0.5% HSA and 10 mM HEPES. An aliquot of 200 μl of cell suspension (introduction of 8 x 10 5 cells and 2 x 10 6 cells, respectively) was added to each insert. After 2 to 4 hours the inserts were removed and the number of cells that had migrated through the monolayer of ECV304 to the lower well in a FACScan Becton Dickinson was counted for 30 seconds with the discriminators placed to acquire the cells of interest. Using this technique, 100% migration would be 25,000 cells for the THL / TH2 cells and 75,000 cells for the remaining CD4 lymphocytes, where this number represents the cells in the lower well counted in the FACScan for 1 minute. To study the phenotype of the migratory cells, identical experiments were performed with CD4 lymphocytes in 6-well plates using 24 mm diameter inserts. The chemotaxis assays were identical to the migration assays with ECV304 but inserts coated with Fibronectin (10 μg / ml) were used. In all cases, the data points were the result of duplicate wells, showing the mean value and the error bars representing the standard deviation of the samples.

Ca2 + Mobilization Assay (Ca2 + Flow). 107 cells / ml were labeled in DPBS for 30 minutes with the Fura 2 dye (Molecular Probes, Eugene, OR) at 2 mM, washed three times in DPBS and resuspended to 10 6 cells. / ml in DPBS containing 1 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES and 5.5 mM glucose. The cells were then analyzed in a fluorimeter (Hitachi fluorescence spectrophotometer model F2000, excitation 340 nm, emission 510 nm) using 10% NP-40 and 10 mM EDTA to establish the maximum and minimum Ca + mobilizations.

Recombinant DNA Methods Plasmid DNA was isolated using QIAGEN tips according to the manufacturer's recommendations (QIAGEN Inc., Chatsworth, CA). DNA ligatures, restriction endonuclease digestions and gel electrophoresis were performed as previously described (Sambrook, J., et al., Molecular Cloning: A Laboratory Handbook 2nd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY) (1989)). DNA purification by agarose gel extraction was performed using the QIAEXII Gel Extraction Kit according to the manufacturer's recommendations (QIAGEN Inc., Chatsworth, CA). Plasmid DNA was introduced into E. coli by chemical transformation (GIBCO, Inc.). Enzymes were purchased from New England Biolabs, Inc. (Beverly, MA), GIBCO Bethesda Research Laboratories, Inc. (Gaitherburg, MD) or Boehringer Mannheim, Inc. (Germany). RNA was isolated from tissues or frozen cells using the standard method of guanidinium isothiocyanate (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY) (1989). )) or the RNeasy kit as recommended (QIAGEN Inc., Chatsworth, CA). DNA sequencing was performed by Sequi-Net (Colorado State University) using the FS DyeDeoxy Terminator cyclic sequencing kit and a model 377 DNA sequencer (Perkin Elmer Applied Biosystems, Foster City, CA). The sequences were analyzed using SeqMan (DNASTAR, Inc., Madison, Wl).

PCR Primers were designed for use in PCR to amplify the entire coding region of GPR-9-6 on the basis of the nucleotide sequence deposited in GenBank (U45982) (SEQ ID NO: 1), which is incorporated into the present by reference. BairiHI and Xbal sites were incorporated into the pair of primers BAZ201 5 A. TCGAAGGGATCCCTAACATGGCTGATGACTATGGC .. 3 '(SEQ ID NO: 4) and BAZ202 5 A .AAGAAGTCTAGAACCCCTCAGAGGGAGAGTGCTCC .3' (SEQ ID NO: 5) for directional cloning (bold: coding sequence, italics: enzyme site). 5 μg of total human genomic DNA (Clontech, Palo Alto, CA) was used as template in PCR cycles with Pfu, with 60 mM Tris-HCl, pH 9.5, 1.5 mM MgCl2, 100 pmoles of primers, dNTP 200 μM and 5 units of Pful polymerase (Invitrogen, Carlsbad, CA) in a volume of 100 μl. The parameters of the cycles were an initial fusion at 95 ° C, 2 minutes, and then 35 cycles: 95 ° C, 30 seconds; 55 ° C, 30 seconds; 72 ° C, 2 minutes 15 seconds, followed by a final extension at 72 ° C, 7 minutes in a DNA thermal cycler (Perkin-Elmer Corp., Norwalk, NC). Primers were designed to amplify the entire coding region of TECK based on the published nucleotide sequence (access U86358), which is incorporated herein by reference. HindIII sites were incorporated andXbal in the pair of primers BAZ203 5 A. TCGAAGAAGCTTATGAACCTGTGGCTCCTG ..3 '(SEQ ID NO: 6) and BAZ204 5 A .AAGAAGTCTAGATCACAGTCCTGAATTAGC .3' (SEQ ID NO: 7) for directional cloning (bold: coding sequence, italics: enzyme site). 5 μg of human thymus RNA with oligo dT in a volume of 20 μl were reverse transcribed. The cDNA was mixed with 200 μM dNTP, 100 pmoles of primers, 60 mM Tris-HCl, pH 9.5, 1.5 mM MgCl2 and 10 units of AmpliTaq polymerase (Perkin-Elmer Roche Molecular Systems, Branchburg, NJ) in a volume of 50 μl. The parameters of the cycles were an initial fusion at 95 ° C, 2 minutes, and then 35 cycles: 95 ° C, 30 seconds; 55 ° C, 30 seconds; 72 ° C, 1 minute, followed by a final extension at 72 ° C, 7 minutes. The human thymus was obtained from Children's Hospital (Boston, MA). TECK amplification was performed by semiquantitative PCR using the primers BAZ203 (SEQ ID NO: 6) and BAZ204 (SEQ ID NO: 7), and GPR-9-6 using the primers BAZ201 (SEQ ID NO: 4) and BAZ202 (SEQ ID NO: 5), using equal amounts of cDNA (500 ng) thymus mold, small intestine, colon, brain, lymph node and spleen, as well as 500 ng of genomic DNA (ClonTech, Palo Alto, CA). The same conditions and the same PCR profile were used as in the PCR cycle with AmpliTaq described above, except that 30 cycles were performed. Amplification with glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primers (ClonTech, Palo Alto, CA, catalog number 5840-1) was used to demonstrate the equivalence of the template. After agarose gel electrophoresis, the PCR products were visualized in the presence of ethidium bromide with a UV light source. DNA fragments of the predicted size were isolated (-450 bp for TECK and ~1 kb for GPR-9-6) and cloned in pBluescript II KS + (Stratagene, Inc., La Jolla, CA) and in pcDNA3 (Stratagene, Inc.), respectively, for sequence analysis and subsequent manipulation.

Construction of Expression Vectors and Production of a Stable Cell Line Expressing GPR-9-6 The coding region of GPR-9-6 was amplified by PCR and directionally cloned into the Ba? L / Xbal sites of pcDNA3 (Invitrogen, San Diego, CA). Subsequently transfectants were generated in the L1.2 cell line of murine pre-lymphoma B, were maintained in RPMI-1640 supplemented with 10% fetal calf serum (HyClone, Logan UT), 2 mM L-glutamine, 50 units / ml pen / strep, 0.55 mM β-mercaptoethanol, 10 mM HEPES and 1 mM sodium pyruvate (Gibco BRL). 20 μg of linearized GPR-9-6 was used in pcDNA3 to transfect the cell line as follows. The L1.2 cells were washed twice in PBS and resuspended in 0, 8 mi of it. The plasmid DNA was mixed with the cells and incubated for 10 minutes at room temperature, transferred to a 0.4 cm electroporation cuvette, and then a single pulse was applied at 250 V, 960 μF. The electroporation was followed by a 10 minute incubation at room temperature. G418 (Geneticin, Gibco BRL) was added at a final concentration of 0.8 mg / ml 48 hours after transfection, and the cells were cultured in a mass culture under drug selection for 2-3 weeks. The transfectants were subsequently stained by mAbs with reactivity against the GPR-9-6 peptide (see below) and analyzed by FACScan (Becton Dickinson &; Co., Mountain View, CA) to confirm the expression of GPR-9-6 on the surface and cloned by limiting dilution. The transfected cells were treated with 5 mM n-butyric acid for 24 hours prior to experimentation (Palmero, D.P, et al., J. Biotech., 19: 35-47 (1991)).

/ Northern Blot Analysis Northern blots were purchased from ClonTech or prepared as follows. Total RNA was separated by electrophoresis in 1.2% formaldehyde agarose gels and transferred to nylon membranes (Hybond-N +, Amersham Corp., Arlington Heights, IL) by the capillary method as previously described (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY) (1989)) and cross-linked using a Stratalinker (Stratagene, Inc.). Hybridizations with radiolabeled probes were carried out with ExpressHyb Solution (Clonetech) using the protocol suggested by the manufacturer. The duration of the exposure of the autoradiographies is described in the legends of the appropriate figures. Hybridizations of gel-filled TECK and GPR-9-6 full-length DNA fragments were used in the hybridizations.

Results A mAb Produced Against GPR-9-6, the 3C3 mAb, Reacts Selectively with the GPR-9-6 Transfectants Due to its close phylogenetic association with other known leukocyte chemokine receptors (Figure 1), we cloned GPR-9- 6 by PCR using primers designed from the sequence deposited in the GenBank. GPR-9-6 / L1.2 transfectants were prepared and stained with mAbs produced against GPR-9-6 in fusions in which mice were immunized with the first 26 amino acids of the NH2-terminus of GPR-9-6 (SEQ. No.: 3) coupled to KLH. The mAb, designated mAb 3C3, reacted with the transfectants GPR-9-6 / L1.2 but not with the parental Ll2 cells. It was found that mAb 3C3 had an IgG2b isotype. In cross-reactivity studies, mAb 3C3 did not cross-react with CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 or CXCR1, CXCR2, CXCR3 and CXCR4 transfectants. The data for CCR6 is shown herein, as it is one of the chemokine receptors most closely related to GPR-9-6 (Figures 2A-2B). It was also found that the terminal NH2 peptide of GPR-9-6 (SEQ ID NO: 3) completely blocked the binding of mAb 3C3 to the transfectants of GPR-9-6 (data not shown), further validating the specificity of this mAb.

GPR-9-6 is expressed in all B lymphocytes, in subgroups of CD4 lymphocytes and in a small subset of peripheral blood CD8 lymphocytes, as well as in thymocytes. In initial studies of two peripheral blood colors, it was found that GPR- 9-6 was expressed in a small subgroup (2-4%) of CD4 lymphocytes as well as in a very small subset of CD8 lymphocytes (Figures 3A-3B), while B lymphocytes expressed low and heterogeneous levels of GPR-9- 6 Monocytes, basophils, eosinophils, neutrophils and NK cells did not express GPR-9-6 under the conditions used (Figures 3C-3I). GPR-9-6 was expressed in a large subgroup of thymocytes expressing all levels of TcR, although a small subset of TcReleva thymocytes or GPR-9-6_vos was evident. In three-color experiments, GPR-9-6 was found in the majority of CD4, CD8 and CD4 + CD8 + thymocytes and CD8 + vos and in approximately 50% of immature CD4 ~ CD8_vo thymocytes (data not shown). No expression of GPR-9-6 was seen in immature or mature dendritic cells (Figure 4D). However, as expected, the immature dendritic cells expressed CCR5, which was down-regulated after activation with LPS, while CD83 and CD86 were up-regulated (Figures 4A-4C). When examining a large panel of cell lines, GPR-9-6 was found in several T-cell lines (Table 1). The umbilical CD4 + lymphocytes did not express GPR-9-6 (Figure 4E) and the chronic activation of these cells in the presence of IL-12 or IL-14 to produce TH1 or T2 lymphocytes was not able to induce the expression of GPR-9 -6 (Figure 4H). However, as expected, CXCR3 was clearly upregulated in TH1 lymphocytes (Figure 4F), whereas a4β7, an integrin used in the movement of lymphocytes to mucosal sites, was upregulated in TH1 and TH2 lymphocytes (Figure 4G). The expression of GPR-9-6 in CD4 lymphocytes and in B lymphocytes was measured over time, and was found to be relatively constant (Figure 5A). However, activation of T lymphocytes with an anti-CD3 mAb resulted in a transient down-regulation of GPR-9-6 for 2 days, recovering expression after 10 days of culture in IL-2 (Figure 5B) . The chemokine receptors CCR6 and CCR5 show similar changes in expression after T lymphocyte activation (Figure 5C).

The Subgroup of CD4 Lymphocytes Expressing GPR-9-6 are Predominantly of the Memory Phenotype and Express Elevated Levels of the Mucosal Lymphoid Receptor Mucosal a4ß7 but not the Cutaneous Migration Receptor CLA The small subset of CD4 lymphocytes expressing GPR-9-6 was examined in more detail by staining with three colors (Figures 6A-6F). The CD4 lymphocytes expressing GPR-9-6 were mostly memory phenotype, and the cells expressing the highest levels of GPR-9-6 were all of memory phenotype. Interestingly, CD4 lymphocytes with CLA + vos memory, which migrate to the skin, did not express GPR-9-6. In contrast, a subset of CD4 lymphocytes with elevated ß7 memory, which are directed towards mucosal sites, clearly expressed GPR-9-6. The subset of CD4 lymphocytes with memory defined by the expression of aEß7 was also clearly subdivided into positive and negative GPR-9-6 subgroups. High CD4 + GPR-9-6 lymphocytes did not express CD62L, a migration receptor that is involved in trafficking to peripheral lymph nodes, although a small subset of GPR-9-6debllCD62L + vos lymphocytes was evident. The CD4 + GPR-9-6 + lymphocytes were also examined to determine the coexpression of other chemokine receptors that were known to be expressed on CD4 lymphocytes (Figures 7A-7F). Although GPR-9-6 was clearly found in the positive and negative subgroups of CCR5, CCR6, CXCR3 and CXCR5, the expression of CCR2 and GPR-9-6 in CD4 lymphocytes was mutually exclusive.

The Chemokine Receptor GPR-9-6 binds specifically to TECK Of all the published chemokines tested, only TECK proved capable of inducing chemotaxis of the transfectants GPR-9-6 / L1.2 (Figure 8A). MCP-1-4, MlP-la, MlP-lβ, eotaxin-1, eotaxin-2, RANTES, 1-309, TARC, MDC, MIP4, SLC, HCC1, fractalkine, lymphotactin, MIG, IP-10, ITAC, ADEC, IL-8, gro-a, gro-ß, gro- ?, leucotactin, SDF-la, SDF-lβ, MIP3 and MIP4 all demonstrated that they were unable to induce chemotaxis of GPR-9-6 / L1 transfectants. 2. The chemotaxis of transfectants L1.2 / GPR-9-6 induced by TECK was inhibited by mAb 3C3, but not by mAb 7B11 anti-CCR3 (Figure 8B). TECK did not act on any of the other transfectants tested (CCR1, CCR2, CCR4, CCR5, CCR6, CCR7 and CXCR1, CXCR2, CXCR3, CXCR4, data not shown). Interestingly, it was also found that TECK acted on the T cell lines MOLT-4 (Figure 8D) and MOLT-13 (Figure 8F), which express GPR-9-6 (Table 1). TECK was not chemotactic for other cell lines such as SKW3 (Figure 8E), which do not express GPR-9-6. Using the MOLT-4 T cell line, it was shown that TECK-induced chemotaxis was blocked by the pertussis toxin (Figure 8C). Additionally, anti-GPR-9-6 3C3 mAb blocked the chemotaxis of MOLT-13 cells towards TECK, but had no effect on the chemotaxis of these cells induced by SDFla (Figure 8F). In the calcium mobilization experiments, TECK was also found to induce a flow of Ca2 + in GPR-9-6 + cell lines such as MOLT-4 (Figures 9A-9C), while chemokines such as MDC for which these are cells do not express the relevant receptor, they had no effect.

Table 1 Expression of GPR-9-6 or Cell LinesWe also analyzed leukocyte subgroups (Figures 10A-10F) to determine if they were chemoattracted towards TECK. As observed in the mouse, neutrophils, monocytes, eosinophils, CD8 and NK cells were not chemoattracted towards TECK, but they were chemoattracted towards other chemokines. However, TECK was chemotactic for a small subset of CD4 lymphocytes. As the murine TECK induces thymocyte chemotaxis, the chemotaxis of human thymocytes towards TECK and SDFla, both of which mediate thymocyte chemotaxis (data not shown), was examined. Anti-GPR-9-6 3C3 mAb blocked the chemotaxis of thymocytes and CD4 lymphocytes towards TECK. The anti-GPR-9-6 3C3 mAb had no effect on the CD4 lymphocyte chemotaxis induced by TARC, indicating that the effect is specific (Figures 11A-11C). These results indicate that GPR-9-6 is the main physiological receptor for TECK.

Tissue Distribution of Transcripts of TECK and GPR-9-6 Due to the expression of GPR-9-6 in lymphocytes with mucosal migration, the distribution of transcripts of TECK and GPR-9-6 in lymphoid and mucosal tissue was examined (Figures 12A -12B). TECK was expressed selectively in thymus and small intestine (Figure 12A), while GPR-9-6 was expressed at high levels in thymus and weakly in spleen and in peripheral blood leukocytes (Figure 12B). Although no GPR-9-6 transcripts were detected by Northern blot analysis, GPR-9-6 messenger was detected in the small intestine, thymus, lymph node and spleen using the most sensitive RT-PCR technique (Fig. 12C). No messengers of TECK and GPR-9-6 were detected in brain or colon. In other Northern blots, TECK or GPR-9-6 were not detected in TH1, TH2, Trl (Groux, et al., Nature, 389: 737-742 (1997)), in LAK cells, in monocytes, in cells dendrites derived from CD34, in dendritic cells derived from monocytes, in astrocytes, in human umbilical vein endothelial cells (HUVEC) or in pulmonary vein endothelial cells (PUVEC) (data not shown). Finally, it was shown that the GPR-9-6 transcript was present only in cell lines that had previously been shown to be GPR-9-6 + by staining with mAb 3C3, further validating the specificity of the mAb (Figure 12B).

Only CD4 and CD8 Lymphocytes raised to 4ß7 Migrate Towards TECKSince GPR-9-6 is expressed mainly in CD4 a4ß7 lymphocytes with memory, CD45RA_vo CD4 and CD8 lymphocytes with memory were isolated that did not express, or that expressed intermediate or high levels of a4ß7. Only the CD8 lymphocytes with a4β7 elevated memory and the CD4 lymphocytes with a4β7 + vos CLA_vos memory underwent chemotaxis towards TECK (Figures 13A-13B).

Discussion Several different adhesion molecules are involved in the trafficking of lymphocyte subgroups to distinct physiological positions, such as peripheral lymph nodes (Gallitin, WM, et al., Nature, 304: 30-34 (1983)), Peyer's Plates (Hamman, A., et al., J. Immunol., 152: 3282-3292 (1994); Andrew, DP, et al., Eur. J. Immunol., 26: 897-905 (1996)) and places Inflammatory (Frenette, PS, et al., Cell, 84: 563-574 (1996); Tietz, WY, et al., J ". Immunol., 161 (2): 963-970 (1998); Picker, LJ , et al., J ". Immunol., 145: 3247-3255 (1990)). It is believed that the specific chemokine receptors expressed in these lymphocyte subgroups can interact with chemokines expressed in the areas that mediate the activation, arrest, and transendothelial migration of leukocytes. Therefore, CD4 subgroups defined by the expression of certain adhesion molecules can also express known chemokine receptors, orphaned or not yet discovered, which are important for the movement of lymphocytes to these sites. The work described herein refers to any of such chemokine receptors that may be involved in the selective trafficking of subsets of CD4 and CD8 lymphocytes with memory to mucosal sites. GPR-9-6 was originally chosen as a potentially interesting orphan chemokine receptor, due to its strong phylogenetic binding to other known chemokine receptors, including CCR6 and CCR7. In Northern blot analysis, GPR-9-6 was found in the thymus, which is indicative of some role in the development of T cells. Weak expression in spleen and blood may reflect the expression of GPR-9-6 in lymphocytes T with memory and in B lymphocytes. As GPR-9-6 is expressed by most thymocytes, and these GPR-9-6 + thymocytes express all levels of TcR, GPR-9-6 is apparently expressed in all stages of the development of the T cells. When leaving the thymus, the GPR-9-6 must be regulated downwards, since in the periphery only a small subset of CD4 lymphocytes and an even smaller subgroup of CD8 lymphocytes express GPR- 9-6. In three color experiments, GPR-9-6 is found predominantly in CD4 lymphocytes with memory. Of greater interest, although CD4 lymphocytes with CLA + vos memory (Picker, LJ, et al., J. Immunol., 145: 3247-3255 (1990)) do not express GPR-9-6, a subset of CD4 lymphocytes with elevated memory (Andrew, DP, et al., Eur. J. Immunol., 26: 897-905 (1996)) express this chemokine receptor. This may reflect a role of GPR-9-6 in the trafficking of lymphocytes to mucosal sites, or their effector action when there. Although GPR-9-6 was clearly expressed on CD4 lymphocytes migrating towards the mucosa, no GPR-9-6 transcripts were detected in the small intestine by Northern blot analysis. This may reflect the low number of CD4 + and / or CD8 + GPR-9-6 + ves lymphocytes in small intestine tissue compared to the thymus, in which the majority of cells are thymocytes GPR-9-6 + vos that they are actively divided. However, using the most sensitive RT-PCR technique, GPR-9-6 transcripts were detected in the small intestine but not in the brain. Interestingly, although transcripts of GPR-9-6 and TECK were expressed in the small intestine, neither GPR-9-6 nor TECK transcripts were detected in the colon by Northern blot analysis or by RT-PCR. Factors that are present in the mucosal environment can induce GPR-9-6 expression in T lymphocytes as well as TECK expression. Cytokines present in TH1 / TH2 environments induce the expression of certain chemokine receptors, such as CCR4 on TH2 and CXCR3 lymphocytes on TH1 lymphocytes, as well as the production of chemokines that bind to these receptors (Bonecchi, RG, et al. ., J ". Exp. Med., 187: 129-134 (1998); Sallusto, F.D., et al., J. Exp. Med., 187: 875-883 (1998); Sallusto, F., Science, 277: 2005-2007 (1997); / Andrew, D.P., et al., (1998); Zingoni, A., et al., J ". Immunol., 161: 547-555 (1998)). However, these conditions do not upregulate the expression of GPR-9-6 in T lymphocytes. induce the expression of GPR-9-6 in umbilical CD4 lymphocytes activated with the cytokines IL-1-18 or with TGF-β, which had previously been shown to induce aE in T lymphocytes (Kilshaw, PJ and Murant, SJ, Eur. J. Immunol., 21: 2591-2597 (1991)), could not identify a cytokine that upregulated the expression of GPR-9-6, therefore, the mechanism by which the expression of GPR-9- is controlled. 6 on CD4 lymphocytes is unclear.After activation through cross-linking to TcR, the expression of GPR-9-6 is regulated downward, as is the expression of the chemokine receptor CXCR4 (Bermejo, M., et al., J ". Immunol., 28: 3192-3204 (1998)). As cross-linking with TcR mimics the presentation of the antigen, we conclude that upon entering a lymph node and encountering APCs that express the antigenic peptide + MHC-II, those T lymphocytes will down-regulate chemokine receptors such as GPR-9 -6. This will keep the T lymphocytes in the lymph node, where the T lymphocytes can mediate other immune functions such as the change of B cell class through related T: B interactions. Of all the chemokines tested only TECK (Vicari, AP, et al., Immuni ty, 7 (2) -.291-301 (1991)) acted as a chemoattractant for GPR-9-6 / L1.2 transfectants, resulting in a optimal chemotaxis with 150 nM. This falls in the range of 1 nM-1 μM for which other leukocyte chemokines are active. However, as we are using a TECK that was produced by peptide synthesis, we can not be sure if post-translational modifications or a subsequent cut of TECK by factors outside the cell in vivo do not produce more active fragments, as is the case from CKB8 (Macphee, CH, et al., J. Immunol., 161: 6273-6279 (1998)). TECK did not act as a chemoattractant for transfectants CCR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR9 or for transfectants CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 L1.2. However, some weak TECK activity was detected in CCR3 / L1.2 transfectants which was approximately 20% of the chemotactic activity observed with eotaxin-1. This activity was blocked by anti-CCR3 mAbs, although TECK did not act as a chemoattractant for eosinophils. Therefore, TECK is probably not a physiological chemokine for the CCR3 receptor. This result has precedents, since previous studies have shown that MlP-la acts as a chemoattractant for transfectants CCR4 / HEK293 (Power, CA, et al., J. Biol. Chem., 270: 19495-19500 (1995)) , but not for CCR4 / L1.2 transfectants (Imai, TM, et al., J ". Biol. Chem., 272: 15036-15042 (1997).) In subsequent experiments, it was found that only T cell lines who expressed GPR-9-6 experienced chemotaxis towards TECK, whereas among TECK primary cells it was chemotactic only for a small subset of CD4 lymphocytes, presumably these cells represent the small subset of CD4 lymphocytes expressing GPR-9-6, and that the chemotaxis was blocked by the anti-GPR-9-6 mAb 3C3 Additionally, only the CD4 and CD8 lymphocytes with a4ß7 + memory had chemotaxis towards TECK, which would be the subgroup that was predicted to express GPR-9-6 The TECK was originally described as a chemokine produced This is caused by thymic dendritic cells, whose expression is restricted to the thymus and small intestine (Vicari, A.P., et al.,? mmunity, 7C2A291-301 (1997)). Our Northern data confirm this observation and show that the receptor for TECK, GPR-9-6, is also expressed in these sites. The expression of the chemokine receptor GPR-9-6 and its TECK ligand in the small intestine and in the thymus predicts a role for GPR-9-6 and for TECK in the development of T cells and in mucosal immunology. In summary, it was demonstrated that the orphan chemokine receptor GPR-9-6 was expressed in most thymocytes and in a subgroup of memory CD4 lymphocytes that move towards mucosal sites. The selective expression of TECK and GPR-9-6 in the thymus and in the small intestine implies a double role for GPR-9-6, in the development of T cells and in the mucosal immune response.

Example 2 Functional GPR-9-6 is Expressed in Leukemia Cell LinesAcute Lymphoblastic T Cells As described herein, expression of GPR-9-6 was detected in MOLT-4 and MOLT-13 cells (Table 1) using mAb 3C3. MOLT cell lines are human T cell lines that were derived from a patient diagnosed with acute T-cell lymphoblastic leukemia(ATL). Other T-cell leukemia cell lines including CEM, PEER, HUT78, PM1, SKW3 and JURKAT did not express GPR-9-6. In subsequent studies the ability of T cell lines to undergo TECK-induced chemotaxis in in vitro chemotaxis assays was determined. The cells ofATL, MOLT-4 and MOLT-13, experienced chemotaxis induced byTECK but other T cell lines (CEM, PEER) did not.

Example 3 Intraepithelial Lymphocytes (IEL) and Blade LymphocytesPropria (LPL) Express GPR-9-6 (CCR9) and Experience TECK-Induced Chemotaxis Isolation of Lymphocytes Lymphocytes from the epithelium and lamina propria of human intestines were isolated as previously described(Zabal, B.A., et al., J. Exp. Med., 190: 1241-1256 (1999)). Briefly, pieces of intestine were cut and opened, left flat and washed with ice-cold HBSS. The serosa was separated from the mucosa with scissors and discarded. The mucosa was cut into strips and incubated with cold 0.15% (w / v) dithiothreitol in HBSS (DTT / HBSS) for 30 minutes. The mucosa was then washed with cold HBSS to remove the mucus. The mucosal strips were then incubated in cold 1 mM EDTA in HBSS with shaking for 90 minutes to extract the epithelium and intraepithelial lymphocytes(IEL). Incubation in 1 mM EDTA in HBSS with shaking was repeated several times until no more epithelial cells were stripped from the strips. The remaining mucosal strips were crushed through a 50 mesh screen(Sigma, St. Louis, MO) to isolate lymphocytes from the lamina propria (LPL).

Analysis by FACS The isolated lymphocytes were resuspended in FACS buffer at a concentration of < 1 x 106 / ml. Non-specific antibody binding was blocked using horse IgG (Sigma, St. Louis, MO). Anti-GPR-9-6 (CCR9) unconjugated antibody (mAb 3C3) was detected using a biotinylated horse anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA) and PerCP streptavidin (PharMingen, San Diego, CA) .

Chemotaxis of Intestinal Lymphocytes Chemotaxis assays were performed using 24-well Transwell plates (Corning Costar, Cambridge, MA) with polycarbonate membranes having pores of 5 μm in diameter. Briefly, 600 μl of TECK diluted in RPMI 1640 with 0.5% BSA were placed in the lower chamber of the Transwell plates and 100 μl of cells (1 x 106 for IEL, 5 x 105 for LPL) were placed in each Insert For the inhibition experiments with antibodies, IEL or LPL were incubated with 40 μg / ml of mAb 3C3, with murine IgG2b control (clone 49.2, PharMingen, San Diego, CA) or with medium alone for 10 minutes at 4 ° C, before to add the insert to the wells. The plates were then incubated at 37 ° C in 5% C02 for 3 hours. The number of cells that migrated to the lower chamber during the assay was determined by FACS analysis, counting the number of events that passed through the detector with a characteristic light scattering profile of small lymphocytes during a 40 second interval. The number of events equivalent to 100% cell migration was equal to one sixth of the number of events recorded when the initial cell suspension was counted by FACS for 40 seconds.

Results GPR-9-6 expression (CCR9) was detected in only a small subset of peripheral blood leukocytes by flow cytometry. In contrast, essentially all the IELs and LPLs expressed high levels of GPR-9-6 (Figures 16A-16C). further, the in vitro chemotaxis assays revealed that IEL and LPL underwent TECK-induced chemotaxis that could be inhibited by an anti-GPR-9-6 antibody (mAb 3C3) but not by an isotype of control antibody (IgG2b) (Figures 17A and 17B). Therefore, GPR-9-6 (CCR9) is the main physiological receptor for TECK expressed by IEL and LPL. The data show that the total leukocyte trafficking in the intestinal epithelium is mediated through the interaction of TECK and GPR-9-6 (CCR9).

EXAMPLE 4 Additional Anti-GPR-9-6 mAbs C57 / Black mice were immunized with 10 million transfected L1.2 cells stably expressing GPR-9-6 (GPR-9-6 / L1.2) (see Example 1). Prior to immunization, transfected Ll2 cells were treated with mitomycin C (50 μg / ml) in PBS (Sigma). Three weeks later, the mice were again immunized with GPR-9-6 / L1.2 transfectants treated with mitomycin C. Subsequently, the mice were immunized with 10 million GPR-9-6 / L1.2 transfectants every three weeks . Mice were immunized with GPR-9-6 / L1.2 transfectants a minimum of 4 times. For the formation of hybridomas, the spleens were extracted from the immunized mice 3-4 days after the last immunization and the splenocytes were fused to SP2 / 0 myeloma cells. Hybridomas that produced antibodies that specifically bound to GPR-9-6 (CCR9) (ie, stained transfectants GPR-9-6 / L1.2 but did not stain transfectants from Ll.2 cells expressing other receptors of chemokines) were identified by analysis by FACS. The murine hybridoma GPR96-1 which produces the GPR96-1 mAb was isolated and the ability of the GPR96-1 mAb to inhibit the chemotaxis of GPR-9-6 / L1.2 induced by TECK in an in vitro chemotaxis assay was determined. . For this inhibition assay with antibodies, GPR-9-6 / L1.2 transfectants were incubated with various concentrations of mAb GPR96-1 or mAb 3C3 for 10 minutes on ice before exposure to TECK. The chemotaxis assay was performed essentially as described above, except that no ECV304 cells were used. The results, which are presented graphically in Figure 18, revealed that mAb GPR96-1 is more effective in inhibiting chemotaxis induced by TECK than mAb 3C3 under the conditions of the assay. The murine hybridoma GPR96-1, also referred to as hybridoma LS272 GPR96 1-5, can be cultured at 37 ° C in an atmosphere with 5% C02 in DMEM supplemented with FCS (10%), IL-6 (100 ng / ml), penicillin (50 U / ml), streptomycin (50 μg / ml), L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate in MEM (10 mM), essential amino acids in MEM (0, 1 mM) and 2-mercaptoethanol (5.5 x 10 -4 M).

Example 5 Anti-TECK mAbs Balb / c mice were immunized intraperitoneally first with 10 μg of human TECK (Peprotech, Rocky Hill, NJ 330-45) in Freund's Complete Adjuvant (Sigma, F 5881). Three weeks later the mice were immunized intraperitoneally with 10 μg with human TECK (Peprotech,Rocky Hill, NJ 330-45) in Freund's Incomplete Adjuvant(Sigma, F 5506). Subsequently, the mice were immunized intraperitoneally every three weeks with 10 μg of TECK in PBS. Each mouse was immunized a minimum of four times. For the formation of hybridomas, the spleens were extracted from the immunized mice 3-4 days after the last immunization and the splenocytes were fused to SP2 / 0 myeloma cells. Hybridomas that produced antibodies that specifically bound to TECK were identified by ELISA using plates coated with 2 μg / ml TECK. The anti-TECK antibodies were subsequently analyzed for their ability to inhibit the chemotaxis of transfectants GPR-9-6 / L1.2 induced by TECK (150 nM) in an in vi tro assay. The murine hybridomas 11.2, 11.3.1, 16.2 and 16.3.1 were isolated (hybridomas 11.3.1 and 16.3.1 are subclones of hybridomas 11.2 and 16.2, respectively) and the ability of the mAbs they produced to inhibit the GPR-9-6 / L1.2 cell chemotaxis induced by TECK in an in vitro chemotaxis assay. The TECK was diluted(final concentration 150 nM approximately) in culture medium containing a control IgGl mAb (20 mg / ml) or diluted 1: 4 in conditioned culture medium of hybridomas that produce mAbs that bind to TECK. The TECK solutions were placed on the bottom of a Transwell plate and incubated at room temperature for 10 minutes. The transfectants GPR-9-6 / L1.2 were then suspended in culture medium and placed in the inserts, which were arranged in the wells of the plate. The transfectants were allowed to migrate for 2-3 hours and subsequently the cells that accumulated in the lower well were counted in a FACScan. The results of the tests, which are presented graphically in Figure 19, revealed that mAbs 11.2, 11.3.1, 16.2 and 16.3.1 each inhibited TECK-induced chemotaxis, while mAb 20.2, which also binds to TECK, and non-specific IgG, did not. The murine hybridomas 11.3.1, also referred to as hybridoma LS250 11.3.1, and 16.3.1, also referred to as hybridoma LS250 16.3.1, can be cultured at 37 ° C in an atmosphere with 5% C02 in DMEM supplemented with FCS (10%), IL-6 (100 ng / ml), penicillin (50 U / ml), streptomycin (50 μg / ml), L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate in MEM (10 mM), essential amino acids in MEM (0.1 mM) and 2-mercaptoethanol (5.5 x 10-5 M).

EXAMPLE 6 Variants of TECK Existing Naturally RNA was prepared from samples of human thymus and of inflamed or non-inflamed small intestine using Qiagen Mini Kits. The RNA was reverse transcribed and the coding region of TECK was amplified by PCR using BAZ203 (SEQ ID NO 6) and BAZ204 (SEQ ID NO: 7) as described above (see Example 1). The PCR product was cut with the enzymes BamHI and Xbal and ligated into pBluescript IIKS +. The inserts were sequenced using primers that hybridized to pBluescript II sequences (M13 and T3). The sequencing data revealed that different forms of TECK are expressed in these tissues. A polymorphism at amino acid 104 was found with a threonine (T) or a methionine (M) (SEQ ID NO: 9). Spindle variants were also found that had a deletion of bases 326-328 by displacement of the framework, which causes the amino acid 109 (alanine) to be removed. In addition to this, the examination of the sequence of the inserts generated by PCR from separated samples of RNA, revealed the differential expression of the two forms of TECK resulting from the mutation by displacement of the frame, being more predominant in the small intestine than in the thymus the form with the alanine eliminated.

Example 7 TECK is Highly Expressed by Small Intestinal Epithelial Cells Hybridization In Si Tu The TECK probe was initially amplified from a group of murine cDNA prepared by RT-PCR from thymus, using a 5-prime synthetic oligonucleotide primer ( t aag gat ccg ca ggt gcc ttt gaa gac tgc t; SEQ ID NO: 12) and a synthetic oligonucleotide primer 3 prima (caa gaa ttc tta att gtt ctt tet ggg cat; SEQ ID NO: 13) and subcloned to through BapiHI and EcoRI. A second PCR amplification step was performed to introduce RNA polymerase sites, using the synthetic oligonucleotide primers m_TECK T3 (aat taa ccc tea cta aag gga act gtg gct ttt tgc ctg c; SEQ ID NO: 14) and m_TECK T7 (taa tac gac tea cta tag ggt gtt ggt ctt tet ggg cat c; SEQ ID N °: 15). Sense and antisense probes labeled with digoxigenin were synthesized using the DIG RNA Labeling Kit / Genius 4 Kit (Roche Molecular Biochemicals). Frozen sections of five micras of mouse small intestine were cut and thawed on Superfrost Plus slides (VWR), air-dried at room temperature for 1-2 hours and used the same day for hybridization. The sections were pretreated as described (Breitschopf, et al., Detection of mRNA on paraffin e bedded material of the central nervous system wi th DIG-labeled RNA probes., In"Nonradioactive In Situ Hybridization Application Manual" 2 edition Copyright 1996 Boehringer Mannheim GmbH, Biochemica. ), omitting the initial stage of xylene and including a digestion with Proteinase K (0.1 μg / ml) for 5 minutes at room temperature. The sections were hybridized for 16-18 hours at 60 ° C in a hybridization buffer containing 200 ng / ml of digoxigenin-labeled probe, 50% formamide (Gibco BRL) in 5x SSC, 5x Denhardt's solution (Sigma), 0 , 5 mg / ml of salmon sperm DNA (Gibco BRL) and 25 μg / ml of yeast RNA (Sigma). After hybridization, the sections were washed in 0.2x SSC for 1 hour at 60 ° C and then in 0.2x SSC for 5 minutes at room temperature. The probe labeled with digoxigenin was detected using the DIG Nucleic Acid Detection Kit / Genius 3 kit (Roche Molecular Biochemicals) as described, except that incubation with antibody (1: 100) was carried out at 4 ° C overnight and 10% polyvinyl alcohol 70-100 kD (Sigma) was added to the alkaline phosphatase reaction buffer.

Results Hybridization in si tu was used to directly determine the cellular sites of TECK expression in murine intestine. The expression of TECK was located in the epithelium on the villi and the Lieberkuhn crypts of the small intestine. The expression in the villi was maximum at the base, with lower levels of TECK hybridization detected toward the upper end of the villi. No expression of TECK was detected in the Peyer's (PP) plates associated with the small intestine (Figures 24A-24C). The data demonstrate that TECK is selectively expressed at high levels by epithelial cells of the small intestine. This pattern of expression also supports a highly selective role of TECK in the regulation of the recruitment of circulating lymphocytes "that migrate to the small intestine" as well as the local recruitment of IEL and LPL. Although this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention. invention as defined by the appended claims.

LIST OF SEQUENCES < 110 > LeukoSite, Inc. < 120 > ANTI-GPR-9-6 ANTIBODIES AND METHODS TO IDENTIFY MODULAR FUNCTIONS OF GPR-9-6 < 130 > 1855.1064-002 < 150 > US 09 / 266,464 < 151 > 1999-03-11 < 160 > 1: 5 < 170 > FastSEC for Windows Version 4.0< 210 > 1 < 211 > 2577 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (58) ... (1131) < 400 > 1 aatattttcc ttgacctaat gccatcttgt gtccccttgc agagccctat tcctaac atg 60Met1 gct gat gac tat ggc tet gaa tech here tet tec atg gaa gac tac gtt 108 Wing Asp Asp Tyr Gly Ser Glu Ser Thr Ser Ser Met Glu Asp Tyr Val 5 10 15 aac ttc aac ttc act gac ttc tac tgt gag aaa aac aat gtc agg cag 156 Asn Phe Asn Phe Thr Asp Phe Tyr Cys Glu Lys Asn Asn Val Arg Gln 20 25 30 ttt gcg age cat ttc etc cea ccc ttg tac tgg etc gtg ttc ate gtg 204 Phe Ala Ser His Phe Leu Pro Pro Leu Tyr Trp Leu Val Phe He Val 35 40 45 ggt gcc gtc gtc aac agt ctt gtt ate ctt gtc tac tgg tac tgc here 252 Gly Ala Leu Gly Asn Ser Leu Val He Leu Val Tyr Trp Tyr Cys Thr 50 55 6065 aga gtg aag acc atg acc gac atg ttc ctt ttg aat ttg gca att gct 300 Arg Val Lys Thr Met Thr Asp Met Phe Leu Leu Asn LeuAla He Ala 70 7580 gac etc etc ttt ctt gtc act ctt ccc ttc tgg gcc att gct gct gct 348 Asp Leu Leu Phe Leu Val Thr Leu Pro Phe Trp Wing He Wing Wing Wing 85 9095 gac cag tgg aag ttc cag acc ttc atg tgc aag gtc aac age atg 396 Asp Gln Trp Lys Phe Gln Thr Phe Met Cys Lys Val Val Asn Ser Met 100 105 110 tac aag atg aac ttc tac age tgt gtg ttg ctg ate atg tgc ate age 444 Tyr Lys Met Asn Phe Tyr Ser Cys Val Leu Leu Met Met Cys He Ser 115 120 125 gtg gac agg tac att gcc att gcc cag gcc atg aga gca cat act tgg 492 Val Asp Arg Tyr He Wing Wing Wing Gln Ala Met Arg Ala His Thr Trp 130 135 140145 agg gag aaa agg ctt ttg tac age aaa atg gtt tgc ttt acc ate tgg 540 Arg Glu Lys Arg Leu Leu Tyr Ser Lys Met Val Cys Phe Thr He Trp 150 155160 gta ttg gca gct gct etc tgc ate cea gaa ate tta tac age caa ate 588 Val Leu Ala Ala Ala Leu Cys He Pro Glu He Leu TyrSer Gln He 165 170175 aag gag gaa tec ggc att gct ate tgc acc atg gtt tac ect age gat 636 Lys Glu Glu Ser Gly He Wing He Cys Thr Met Val Tyr Pro Ser Asp 180 185 190 gag age acc aaa ctg aag tea gct gtc ttg acc ctg aag gtc att ctg 684 Glu Ser Thr Lys Leu Lys Ser Wing Val Leu Thr Leu Lys Val He Leu 195 200 205 ggg ttc ttc ctt ccc ttc gtg gtc atg gct tgc tgc tat acc ate ate 732 Gly Phe Phe Leu Pro Phe Val Val Met Wing Cys Cys Tyr Thr He He 210 215 220225 att falls acc ctg ata ca gcc aag aag tet tec aag falls aaa gcc cta 780 He His Thr Leu He Gln Ala Lys Lys Ser Ser Lys His Lys Ala Leu 230 235240 aaa gtg acc ate act gtc ctg acc gtc ttt gtc ttg tet cag ttt ccc 828 Lys Val Thr He Thr Val Leu Thr Val Phe Val Leu Ser Gln Phe Pro 245 250255 tac aac tgc att ttg ttg gtg cag acc att gac gcc tat gcc atg ttc 876 Tyr Asn Cys He Leu Leu Val Gln Thr He Asp Ala TyrWing Met Phe 260 265 270 ate tec aac tgt gcc gtt tec acc aac att gac ate tgc ttc cag gtc 924 He Ser Asn Cys Ala Val Ser Thr Asn He Asp He CysPhe Gln Val 275 280 285 acc cag acc ate gcc ttc ttc falls agt tgc ctg aac ect gtt etc tat 972 Thr Gln Thr He Ala Phe Phe His Ser Cys Leu Asn ProVal Leu Tyr 290 295 300305 gtt ttt gtg ggt gag aga ttc cgc cgg cg gat etc gtg aaa acc ctg aag 1020 Val Phe Val Gly Glu Arg Phe Arg Arg Asp Leu Val Lys Thr Leu Lys 310 315320 aac ttg ggt tgc ate age cag gcc cag tgg gtt tea ttt here agg aga 1068 Asn Leu Gly Cys He Ser Gln Ala Gln Trp Val Ser Phe Thr Arg Arg 325 330335 gag gga age ttg aag ctg teg tet atg ttg ctg gag here gga action 1116 Glu Gly Ser Leu Lys Leu Ser Being Met Leu Leu Glu Thr Ser Gly 340 345 350 gca etc tec tga ggggtcttct ctgaggtgca tggttctttt ggaagaaatg 1171 Wing Leu Ser Leu * 355 agaaatacat gaaacagttt ccccactgat gggaccagag agagtgaaag agaaaagaaa 1231 actcagaaag ggatgaatct gaactatatg attacttgta gtcagaattt gccaaagcaa 1291 atatttcaaa atcaactgac tagtgcagga ggctgttgat tggctcttga ctgtgatgcc 1351 cgcaattctc aaaggaggac taaggaccgg cactgtggag caccctggct ttgccactcg 1411 ccggagcatc aatgccgctg cctctggagg agcccttgga ttttctccat gcactgtgaa 1471 cttctgtggc ttcagttctc atgctgcctc ttccaaaagg ggacacagaa gcactggctg 1531 ctgetacaga ccgcaaaagc agaaagtttc gtgaaaatgt ccatctttgg gaaattttct 1591 accctgctct tgagcctgat aacccatgcc aggtcttata gattcctgat etagaacett 1651 tccaggcaat ctcagaccta atttccttct gttctccttg ttctgttctg ggccagtgaa 1711 ggtccttgtt ctgattttga aacgatctgc aggtcttgcc agtgaacccc tggacaactg 1771 accacaccca caaggcatcc aaagtctgtt ggcttccaat ccatttctgt gtcctgctgg 1831 aggttttaac ctagacaagg attccgctta ttccttggta tggtgacagt gtctctccat 1891 ggcctgagca gggagattat aacagctggg ttcgcaggag ccagccttgg ccctgttgta 1951 ggcttgttct gttgagtggc acttgctttg ggtccaccgt ctgtctgctc cctagaaaat 2011 gggctggttc ttttggccct cttctttctg aggcccactt tattctgagg aatacagtga 2071 gcagcagcca gcagatatgg ggtagggcaa aggggtgaag cgcaggcctt ta gctggaaggc 2131 tttacttc catgcttctc cttttcttac tctatagtgg caacatttta aaagctttta 2191 acttagagat taggctgaaa atggaattca aaaataagta cctttgcatc ttttgtgtct 2251 ttcttatcat gatttggcaa aatgcatcac ctttgaaaat atttcacata ttggaaaagt 2311 gctttttaat gtgtatatga agcattaatt acttgtcact ttctttaccc tgtctcaata 2371 gtgcaattaa ttttaagtgt agatcaaata gatacattaa gagtgtgaag gctggtctga 2431 aggtagtgag ctatctcaat cggattgttc acactcagtt acagattgaa ctccttgttc 2491 cttctctcta tacttccctg ctgcaattga ctagtcttta tgaagagtaa aaaaaaagtg 2551 gcaataggga taaggaaata agatct 2577< 210 > 2 < 211 > 357 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Wing Asp Asp Tyr Gly Ser Glu Ser Thr Ser Met Met Glu Asp Tyr 1 5 10 15 Val Asn Phe Asn Phe Thr Asp Phe Tyr Cys Glu Lys Asn Asn Val Arg 20 25Gln Phe Ala Ser His Phe Leu Pro Pro Leu Tyr Trp LeuVal Phe He 35 40 45Val Gly Ala Leu Gly Asn Ser Leu Val He Leu Val Tyr Trp Tyr Cys 50 55 60 Thr Arg Val Lys Thr Met Thr Asp Met Phe Leu Leu Asn Leu Ala He 65 70 7580 Wing Asp Leu Leu Phe Leu Val Thr Leu Pro Phe Trp WingHe Ala Ala 85 9095 Wing Asp Gln Trp Lys Phe Gln Thr Phe Met Cys Lys Val Val Asn Ser 100 105110 Met Tyr Lys Met Asn Phe Tyr Ser Cys Val Leu Leu He Met Cys He 115 120 125Ser Val Asp Arg Tyr He Wing Wing Wing Gln Wing Met Arg Wing His Thr 130 135 140 Trp Arg Glu Lys Arg Leu Leu Tyr Ser Lys Met Val Cys Phe Thr He 145 150 155160 Trp Val Leu Ala Ala Ala Leu Cys He Pro Glu He Leu Tyr Ser Gln 165 170175 He Lys Glu Glu Ser Gly He Wing He Cys Thr Met Val Tyr Pro Ser 180 185190 Asp Glu Ser Thr Lys Leu Lys Ser Wing Val Leu Thr Leu Lys Val He 195 200 205 Leu Gly Phe Phe Leu Pro Phe Val Val Met Wing Cys CysTyr Thr He 210 215 220 He He His Thr Leu He Gln Wing Lys Lys Ser Ser Lys His Lys Wing 225 230 235240 Leu Lys Val Thr He Thr Val Leu Thr Val Phe Val Leu Ser Gln Phe 245 250 255 Pro Tyr Asn Cys He Leu Leu Val Gln Thr He Asp Ala Tyr Ala Met 260 265270 Phe He Ser Asn Cys Ala Val Ser Thr Asn He Asp HeCys Phe Gln 275 280 285Val Thr Gln Thr He Wing Phe Phe His Ser Cys Leu Asn Pro Val Leu 290 295 300 Tyr Val Phe Val Gly Arlu Arg Pg Arg Arp Asp Leu Val Lys Thr Leu 305 310 315 320 Lys Asn Leu Gly Cys He Ser Gln Wing Gln Trp Val Ser Phe Thr Arg 325 330335 Arg Glu Gly Ser Leu Lys Leu Ser Ser Met Leu Leu GluThr Thr Ser 340 345350 Gly Ala Leu Ser Leu 355< 210 > 3 < 211 > 26 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Peptide NH2-Terminal of GPR-9-6 Humana < 400 > 3 Met Wing Asp Asp Tyr Gly Ser Glu Ser Thr Ser Ser Met Glu Asp Tyr 1 5 10Val Asn Phe Asn Phe Thr Asp Phe Tyr Cys 20 25< 210 > 4 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 4 tcgaagggat ccctaacatg gctgatgact atggc 35< 210 > 5 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 5 aagaagtcta gaacccctca gagggagagt gctcc 35< 210 > 6 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 6 tcgaagaagc ttatgaacct gtggctcctg 30< 210 > 7 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 7 aagaagtcta gatcacagtc ctgaattagc 30< 210 > 8 < 211 > 879 < 212 > DNA < 213 > Homo sapiens < 400 > 8 atgaacctgt ggctcctggc ctgcctggtg gccggcttcc tgggagcctg ggcccccgct 60 gtccacaccc aaggtgtctt tgaggactgc tgcctggcct accactaccc cattgggtgg 120 gctgtgctcc ggcgcgcctg gacttaccgg atccaggagg tgagcgggag ctgcaatctg 180 cctgctgcga tattetaect ccccaagaga cacaggaagg tgtgtgggaa ccccaaaagc 240 agggaggtgc agagagecat gaagctcctg gatgetegaa ataaggtttt tgcaaagctc 300 caccacaaca ygcagacctt ccaagcaggc cctcatgctg taaagaagtt gagttctgga 360 aactccaagt tatcatcatc caagtttagc aatcccatca gcagcagcaa gaggaatgtc 420 tccctcctga tatcagetaa ttcaggactg tgagccggct catttctggg ctccatcggc 480 acaggagggg ccggatcttt etcegataaa accgtcgccc tacagaccca gctgtcccca 540 ttttgggtca cgcctctgtc agtettaate cctgcacctg agttggtcct ccctctgcac 600 ccccaccacc tcctgcccgt ctggcaactg gaaagaagga gttggcctga ttttaacett 660 ttgccgctcc ggggaacagc acaatcctgg gcagccagtg gctcttgtag agaaaactta 720 ggatacctct ctcactttct gtttcttgcc gtccaccccg ggccatgcca gtgtgtcctc 780 tgggtcccct ccaaaaatct ggtcattcaa ggatcccctc ccaaggctat gcttttctat 840 aac ttttaaa taaaccttgg ggggtgaatg gaataaaaa 879< 210 > 9 < 211 > 150 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > VARIANT < 222 > (104) ... (104) < 223 > Xaa = Met or Thr < 400 > 9 Met Asn Leu Trp Leu Leu Wing Cys Leu Val Wing Gly Phe Leu Gly Wing 1 5 10Trp Wing Pro Wing Val His Thr Gln Gly Val Phe Glu Asp Cys Cys Leu 20 25Ala Tyr His Tyr Pro He Gly Trp Ala Val Leu Arg Arg Ala Trp Thr 35 40 45 Tyr Arg He Gln Glu Val Ser Gly Ser Cys Asn Leu ProAla Ala He 50 55 60 Phe Tyr Leu Pro Lys Arg His Arg Lys Val Cys Gly Asn Pro Lys Ser 65 70 7580 Arg Glu Val Gln Arg Wing Met Lys Leu Leu Asp Wing Arg Asn Lys Val 85 90 95 Phe Wing Lys Leu His His Asn Xaa Gln Thr Phe Gln Wing Gly Pro His 100 105110 Wing Val Lys Lys Leu Ser Ser Gly Asn Ser Lys Leu Ser Ser Ser Lys 115 120 125Phe Ser Asn Pro Be Ser Ser Lys Arg Asn Val Ser Leu Leu He 130 135 140 Ser Ala Asn Ser Gly Leu 145 150< 210 > 10 < 211 > 876 < 212 > DNA < 213 > Homo sapiens < 400 > 10 atgaacctgt ggctcctggc ctgcctggtg gccggcttcc tgggagcctg ggcccccgct 60 gtccacaccc aaggtgtctt tgaggactgc tgcctggcct accactaccc cattgggtgg 120 gctgtgctcc ggcgcgcctg gacttaccgg atccaggagg tgagcgggag ctgcaatctg 180 cctgctgcga tattctacct ccccaagaga cacaggaagg tgtgtgggaa ccccaaaagc 240 agggaggtgc agagagccat gaagctcctg gatgctcgaa ataaggtttt tgcaaagctc 300 caccacaaca ygcagacctt ccaaggccct catgctgtaa agaagttgag ttctggaaac 360 tccaagttat catcatccaa gtttagcaat cccatcagca gcagcaagag gaatgtctcc 420 ctcctgatat cagctaattc aggactgtga gccggctcat ttctgggctc catcggcaca 480 ggaggggccg gatctttctc cgataaaacc gtcgccctac agacccagct gtccccacgc 540 ctctgtcttt tgggtcaagt cttaatccct gcacctgagt tggtcctccc tctgcacccc 600 caccacctcc tgcccgtctg gcaactggaa agaaggagtt ggcctgattt taaccttttg 660 gaacagcaca ccgctccggg atcctgggca gccagtggct cttgtagaga aaacttagga 720 tacctctctc actttctgtt tcttgccgtc caccccgggc catgccagtg tgtcctctgg 780 aaaatctggt gtcccctcca tcccctccca cattcaagga aggctatgct tttctataac 840 tt ttaaataa accttggggg gtgaatggaa taaaaa 876< 210 > 11 < 211 > 149 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > VARIANT < 222 > (104) ... (104) < 223 > Xaa = Met or Thr < 400 > 11 Met Asn Leu Trp Leu Leu Wing Cys Leu Val Wing Gly Phe Leu Gly Wing 1 5 10 15 Trp Wing Pro Wing Val His Thr Gln Gly Val Phe Glu Asp Cys Cys Leu 20 25Ala Tyr His Tyr Pro He Gly Trp Ala Val Leu Arg Arg Ala Trp Thr 35 40 45Tyr Arg He Gln Glu Val Ser Gly Ser Cys Asn Leu Pro Wing Ala He 50 55 60 Phe Tyr Leu Pro Lys Arg His Arg Lys Val Cys Gly Asn Pro Lys Ser 65 70 75 80 Arg Glu Val Gln Arg Wing Met Lys Leu Leu Asp Ala Arg Asn Lys Val 85 9095 Phe Ala Lys Leu His His Asn Xaa Gln Thr Phe Gln GlyPro His Ala 100 105110 Val Lys Lys Leu Ser Ser Gly Asn Ser Lys Leu Ser Ser Ser Lys Phe 115 120 125Being Asn Pro Being Being Lys Arg Asn Val Being Leu Leu Being 130 135 140 Wing Asn Being Gly Leu 145< 210 > 12 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Synthetic oligonucleotide primer < 400 > 12 taaggatccg caaggtgcct ttgaagactg ct 32< 210 > 13 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Synthetic oligonucleotide primer < 400 > 13 caagaattct taattgttct ttctgggcat 30< 210 > 14 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Synthetic oligonucleotide primer < 400 > 14 aattaaccct cactaaaggg aactgtggct ttttgcctgc 40 < 210 > 15 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Synthetic oligonucleotide primer < 400 > 15 taatacgact cactataggg tgttggtctt tctgggcatc 40

Claims

CLAIMS 1. An antibody or an antigen-binding fragment thereof, which binds to a mammalian GPR-9-6 and inhibits the binding of a ligand to said GPR-9-6.
2. The antibody or antigen-binding fragment of Claim 1, wherein said mammalian GPR-9-6 is human GPR-9-6.
3. The antibody or antigen binding fragment of Claim 1, wherein said ligand is TECK.
4. The antibody or antigen-binding fragment of claim 1, wherein the binding of said antibody or said antigen-binding fragment to GPR-9-6 can be inhibited by peptide consisting of the amino acid sequence of SEQ ID NO 3.
5. - The antibody or antigen binding fragment of Claim 1, wherein the binding of said antibody or said antigen-binding fragment to GPR-9-6 can be inhibited by the mAb 3C3.
6. The antibody or antigen-binding fragment of Claim 5, wherein said antibody or said antigen-binding fragment binds to the same epitope or to a similar epitope as mAb 3C3.
7. The antibody or antigen-binding fragment of claim 1, wherein the binding of said antibody or said antigen-binding fragment to GPR-9-6 can be inhibited by mAb GPR96-1.
8. The antibody or antigen-binding fragment of claim 7, wherein said antibody or said antigen-binding fragment binds to the same epitope or to a similar epitope as mAb GPR96-1.
9. An antibody produced by the murine hybridoma 3C3 or an antigen-binding fragment thereof.
10. An isolated cell that produces an antibody or an antigen-binding fragment thereof that binds to a mammalian GPR-9-6 and inhibits the binding of a ligand to said GPR-9-6.
11. The isolated cell of Claim 10, wherein said mammalian GPR-9-6 is human GPR-9-6.
12. - The isolated cell of Claim 11, wherein said ligand is TECK.
13. The isolated cell of Claim 12, wherein said isolated cell is selected from the group consisting of an immortalized B cell, a hybridoma and a recombinant cell that contains one or more exogenous nucleic acid molecules encoding said antibody or said antigen-binding fragment thereof.
14. - The murine hybridoma 3C3.
15. A method for detecting a mammalian GPR-9-6 or a portion thereof in a biological sample, comprising: a) contacting a biological sample with an antibody or with an antigen-binding fragment of the same that binds to a mammalian GPR-9-6 or a portion of said receptor and inhibits the binding of a ligand to the receptor, under conditions appropriate for the binding of said antibody or said antigen-binding fragment thereof to a GPR-9-6 of mammal or a portion thereof; and b) detecting the binding of said antibody or said antigen-binding fragment thereof; wherein the binding of said antibody or said antigen binding fragment thereof indicates the presence of said receptor or a portion of said receptor.
16. The method according to claim 15, wherein the biological sample is of human origin.
17. The method according to claim 16, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: a) mAb 3C3; b) an antibody that can compete with mAb 3C3 for binding to a mammalian GPR-9-6; c) antigen binding fragments of (a) or (b) that bind to a mammalian GPR-9-6 or a portion thereof; and d) combinations of the above.
18. The method according to claim 16, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: a) mAb GPR96-1; b) an antibody that can compete with mAb GPR96-1 for binding to a mammalian GPR-9-6; c) antigen binding fragments of (a) or (b) that bind to a mammalian GPR-9-6 or a portion thereof; and d) combinations of the above.
19. A method for detecting and identifying an agent that binds to a mammalian GPR-9-6 or a variant thereof that binds to a ligand, comprising the combination of: a) a reference agent; b) a test agent; and c) a composition containing a functional mammalian GPR-9-6 or a variant thereof that binds to a ligand under conditions suitable for the binding of said reference agent to said GPR-9-6 or said variant thereof. binds to a ligand; and detecting or measuring the formation of a complex between said reference agent and said GPR-9-6 or a variant thereof that binds to a ligand, wherein, a decrease in the formation of said complex relative to suitable control indicates that said test agent binds to said GPR-9-6 or a variant thereof that binds to a ligand.
20. The method of Claim 19, wherein said reference agent is labeled with a tag selected from the group consisting of a radioisotope, an epitope, an affinity tag, an enzyme, a fluorescent group, and a chemiluminescent group.
21. The method of Claim 19, wherein said reference agent is TECK.
22. The method of Claim 19, wherein said reference agent is an antibody that binds said GPR-9-6 or an antigen-binding fragment thereof.
23. The method of Claim 19, wherein said composition containing a functional mammalian GPR-9-6 or a variant thereof that binds to a ligand is a cell expressing a mammalian GPR-9-6. .
24. The method of Claim 23, wherein said cell is a recombinant cell.
25. The method of Claim 23, wherein said cell is a cell line.
26. The method of Claim 25, wherein said cell is selected from the group consisting of MOLT-4 and MOLT-13.
27. The method of claim 19, wherein said composition containing a functional mammalian GPR-9-6 or a variant thereof that binds to a ligand is a membrane preparation of a cell expressing a GPR- 9-6 of mammal or a variant thereof that binds to a ligand.
28. A method for detecting or identifying an inhibitor of a mammalian GPR-9-6 receptor comprising: a) the combination of an agent to be tested, a ligand or stimulant of said GPR-9-6 and a cell expressing said GPR-9-6 under conditions suitable for detecting a response induced by the ligand or stimulant; and b) determining the ability of the test compound to inhibit said response, wherein the inhibition by the agent of a response induced by a ligand or stimulant is indicative that the agent is an inhibitor.
29. The method of Claim 28, wherein said cell is a recombinant cell expressing a human GPR-9-6.
30. The method of claim 29, wherein said ligand or stimulant is TECK.
31. The method of claim 28, wherein said response is chemotaxis or Ca2 + flux.
32. A method for treating a subject having an inflammatory disease, comprising administering to said subject an effective amount of an antagonist of a mammalian GPR-9-6 function.
33. The method of Claim 32, wherein said inflammatory disease is Crohn's disease or colitis.
34. The method of Claim 32, wherein said antagonist inhibits the binding of a ligand to a mammalian GPR-9-6.
35.- The method of Claim 34, wherein said ligand is TECK.
36. The method of Claim 34, wherein said antagonist is an antibody that binds to a mammalian GPR-9-6 or an antigen-binding fragment thereof.
37. A method for inhibiting the arrival of leukocytes mediated by GPR-9-6 in a subject, comprising the administration to said subject of an effective amount of an antagonist of the function of GPR-9-6.
38.- The method of Claim 37, wherein said antagonist inhibits the binding of a ligand to GPR-9-6.
39.- The method of Claim 38, wherein said ligand is TECK.
40. The method of Claim 39, wherein said antagonist is an antibody that binds to GPR-9-6 or an antigen-binding fragment thereof.
41. A method for inhibiting the arrival of leukocytes to mucosal tissue mediated by GPR-9-6 in a subject, comprising the administration to said subject of an effective amount of an antagonist of the function of GPR-9-6.
42. A method for treating a subject having an inflammatory bowel disease, comprising administering to said subject an effective amount of an antagonist of the function of GPR-9-6.
43.- The method of Claim 42, wherein said antagonist inhibits the binding of a ligand to GPR-9-6.
44. The method of Claim 43, wherein said ligand is TECK.
45. The method of Claim 44, wherein said antagonist is an antibody that binds to GPR-9-6 or an antigen-binding fragment thereof.
46.- A method for modulating a function of GPR-9-6 comprising the contacting of a cell expressing GPR-9-6 with an agent that binds to it, thereby modulating the function of said GPR-9 -6.
47. The method of Claim 46, wherein said agent can inhibit a function of GPR-9-6.
48. The method of claim 47, wherein said agent is an antibody or an antigen-binding fragment thereof.
49. The method of Claim 48, wherein said function is selected from the group consisting of binding to a ligand, chemotaxis induced by a ligand and Ca2 + flux induced by a ligand.
50.- The method of Claim 49, wherein said ligand is TECK.
51.- A test kit to be used in the detection of the presence of a mammalian GPR-9-6 or a portion thereof in a biological sample, which contains: a) at least one antibody or a binding fragment the antigen thereof which binds to a mammalian GPR-9-6 or a portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor; and b) one or more auxiliary reagents suitable for detecting the presence of a complex between said antibody or antigen-binding fragment thereof and said mammalian GPR-9-6 or a portion thereof.
52. - An assay kit according to claim 51, wherein the antibody is selected from the group consisting of: a) mAb 3C3; b) an antibody that can compete with mAb 3C3 for binding to a mammalian GPR-9-6; c) antigen binding fragments of (a) or (b) that bind to a mammalian GPR-9-6 or a portion thereof; and d) combinations of the above.
53. - An assay kit according to claim 51, wherein the antibody is selected from the group consisting of: a) mAb GPR96-1; b) an antibody that can compete with mAb GPR96-1 for binding to a mammalian GPR-9-6; c) antigen binding fragments of (a) or (b) that bind to a mammalian GPR-9-6 or a portion thereof; and d) combinations of the above.
54. - An antibody or antigen-binding fragment thereof, which binds to a mammalian TECK and inhibits the binding of said TECK to a receptor.
55.- The antibody or antigen-binding fragment of Claim 54, wherein said mammalian TECK is human TECK.
56.- The antibody or antigen-binding fragment of Claim 54, wherein said receptor is GPR-9-6.
57. The antibody or antigen binding fragment of Claim 54, wherein the binding of said antibody or said antigen-binding fragment to TECK can be inhibited by mAb 11.3.1 and / or by mAb 16.3.1 .
58. The antibody or antigen-binding fragment of Claim 57, wherein said antibody or antigen-binding fragment binds to the same epitope or to a similar epitope as mAb 11.3.1.
59. The antibody or antigen-binding fragment of Claim 57, wherein said antibody or antigen-binding fragment binds to the same epitope or a similar epitope as mAb 16.3.1.
60.- An antibody produced by the murine hybridoma GPR96-1 or an antigen-binding fragment thereof.
61.- The murine hybridoma GPR96-1.
62. An antibody produced by murine hybridoma 11.3.1 or an antigen-binding fragment thereof.
63.- The murine hybridoma 11.3.1.
64. An antibody produced by murine hybridoma 16.3.1 or an antigen-binding fragment thereof.
65.- The murine hybridoma 16.3.1.
66.- An isolated cell that produces an antibody or an antigen-binding fragment thereof that binds to a mammalian TECK and inhibits the binding of said TECK to a receptor.
67.- The isolated cell of Claim 66, wherein said mammal TECK is human TECK.
68.- The isolated cell of Claim 66, wherein the receiver is GPR-9-6.
69.- A method for detecting a mammalian TECK or a portion thereof in a biological sample, comprising: a) contacting a biological sample with an antibody or with an antigen-binding fragment thereof that is binds to a mammalian TECK or a portion thereof and inhibits the binding of a TECK to a receptor, under conditions appropriate for the binding of said antibody or said antigen binding fragment thereof to a mammalian TECK or a portion thereof Of the same; and b) detecting the binding of said antibody or said antigen-binding fragment thereof; wherein the binding of said antibody or said antigen binding fragment thereof indicates the presence of said receptor or a portion of said receptor.
70.- A test kit to be used in the detection of the presence of a mammalian TECK or a portion thereof in a biological sample, comprising: a) at least one antibody or an antigen-binding fragment of the same that binds to a mammalian TECK or a portion thereof, wherein said antibody or antigen-binding fragment thereof inhibits the binding of TECK to a receptor; and b) one or more auxiliary reagents suitable for detecting the presence of a complex between said antibody or antigen-binding fragment thereof and said mammalian TECK or a portion thereof.
71. A method for treating a subject having cancer comprising the administration to said subject of an effective amount of an antagonist of the function of GPR-9-6.
72. The method of Claim 62, wherein said antagonist is an antibody or an antigen-binding fragment thereof that binds to GPR-9-6.
73. A method for treating a subject having cancer comprising administering to said subject an effective amount of an antibody that binds to GPR-9-6 or an antigen-binding fragment thereof, wherein said antibody or fragment can activate the complement.
74. A method for treating a subject having cancer comprising administering to said subject an effective amount of an immunoconjugate or a fusion protein that binds to an antigen, wherein said immunoconjugate or fusion protein that binds an antigen contains at least one antigen-binding portion of an antibody that binds to GPR-9-6 that is directly or indirectly linked to an additional therapeutic agent. The method of Claim 74, wherein said additional therapeutic agent is a cytotoxic agent. 76.- An immunoconjugate containing at least one antigen-binding portion of an antibody that binds to GPR-9-6 that is directly or indirectly linked to an additional therapeutic agent. 77.- A fusion protein that binds to an antigen that contains at least one antigen-binding portion of an antibody that binds to GPR-9-6 that is directly or indirectly bound to an additional therapeutic agent, wherein said antigen binding portion of an antibody and said additional therapeutic agent are part of a contiguous polypeptide.