MXPA01007199A - Anti-ccr4 antibodies and methods of use therefor - Google Patents

Abstract

The present invention relates to an antibody or functional fragment thereof which binds to a mammalian (e.g., human) CC-chemokine receptor 4 (CCR4) or a portion of the receptor and blocks binding of a ligand to the receptor. The invention further relates to a method of inhibiting the interaction of a cell bearing mammalian CCR4 with a ligand thereof, and to use of the antibodies and fragments in research, therapeutic, prophylactic and diagnostic methods.

Description

ANTI-CCR4 ANTIBODIES AND METHODS OF UTILIZATION FOR THEMSELVES BACKGROUND OF THE INVENTION Over the past decade, chemokines have emerged as key mediators of inflammation as a result of their numerous proinflammatory activities, which affect virtually every type of leukocyte. More recently, chemokines have been recognized as a critical component of basal leukocyte trafficking, essential for normal surveillance and immune response, as well as for several other functions in hematopoiesis, angiogenesis, viral infection control and differentiation of T-cells (Bag-giolini et al., Ann. Rev. Immuno 1: 15: 675 (1997); Zou et al., Nature 393: 595 (1998); Tachibana et al., Nature 393: 591 (1998)) . This diverse series of biological activities, including the mediation of a range of proinflammatory effects on leukocytes, such as the triggering of chemotaxis, degranulation, the synthesis of lipid mediators and the activation of integrins, together with their critical role in the initiation and maintenance of inflammatory diseases and the recent identification of certain chemokine receptors as co-receptors for the entry of HIV-1, have made chemokines and chemokine receptors a new and attractive group of therapeutic targets. Members of the chemokine family are produced and secreted by many cell types in response to early inflammatory mediators, such as IL-1β or TNFα. The chemokine superfamily comprises two main branches: the a-chemokines (or CXC chemokines), which are characterized by a single amino acid that separates the first 2 cysteines, and the β-chemokines (CC chemokines), which contain two adjacent cysteines. The a-chemokine arm includes proteins such as IL-8, neutrophil activating peptide-2 (NAP-2), melanoma growth stimulating activity (MGSA / gro or GROa) and ENA-78, each of which have attractive and activating effects predominantly on neutrophils. Members of the β-chemokine arm affect other cell types, such as monocytes, lymphocytes, basophils and eosinophils (Oppenheim, JJ et al., Annu, Rev. Immunol., 9: 617-648 (1991); Baggiolini; , M. et al., Adv. Immunol., 55: 97-179 (1994), Miller and Krangel, Cri., Rev. Immunol., 12: 17-46 (1992), José, PJ et al., J. Exp. Med., 179: 881-118 (1994); Ponath, PD et al., J. Clin. Invest. 97: 604-612 (1996)), and include proteins such as monocyte chemotactic proteins 1-4 ( MCP-1, MCP-2, MCP-3 and MCP-4), RANTES, macrophage inflammatory proteins (MlP-la, MlP-lβ), chemokine of the thymus and regulated by activation (TARC, Imai et al., J " Biol. Chem. 271: 21514-21521 (1996)) and chemokine derived from macrophages (MDC, Godiska et al., J. Exp. Med. 185: 1595-1604 (1997).) Chemokines bind to 7 receptors coupled to the transmembrane expansion G protein (Murphy, PM, Annu, Rev. Immunol.12: 593-633 (1994)). a number of β-chemokine receptors (CCR1-CCR10) have been identified to date and the search for additional chemokine receptors is the subject of active research (Baggiolini, Nature 392: 565-568 (1998)). The chemokine receptor CCR4 was identified by Power et al. (J ". Biol. Chem. 270: 19495-19500 (1995); accession number of Genbank X85740) and Meyer et al (J. Biol. Chem. 271 (24): 14445-14451 (1996); Genbank accession X94151) A murine homolog of human CCR4 was also identified (Youn et al., Blood 89 (12): 4448-4460 (1997)). Originally, it was found that CCR4 emitted signal in response to MCP-1, MlP-la and RANTES, but more recently it has been shown to be specific for TARC and MDC chemokines (Imai et al., J. "Biol. Chem. 272 ( 23): 15036-15042 (1997), Imai et al., J. Biol. Chem. 278: 1764-1768 (1998).) Selective recruitment of leukocyte subgroups to sites of inflammation and ordered leukocyte trafficking to through circulation, the tissues, the lymphatic system and the secondary lymphoid organs are controlled in part by the differential expression of chemokine receptors in subgroups of cells. Such expression patterns would seem to ensure that a group of functionally related leukocytes can respond in a coordinated fashion to a specific group of chemokines induced by a given stimulus. For T cells, PCR or Northern blotting indicates that the known receptors for CC chemokines are expressed on subgroups of T cells. Exact delineation of which subgroups express particular receptors is an area of intense study, since the expression of Chemokine receptors can explain the localization or migration of several cell types, such as TH1 or TH2 cells or subgroups targeted to tissues. It can also determine which T cells are infected with different strains of HIV-1. However, most leukocytes express several chemokine receptors, many of them with complex and promiscuous ligand interactions. This makes it difficult to elucidate the normal immune function for a specific receptor on a given cell type and determine the relevance for the initiation and progression of the disease, especially because specific antibodies are not available for many chemokine receptors.

SUMMARY OF THE INVENTION The present invention relates to an antibody (immunoglobulin) or functional fragment thereof (e.g., an antigen binding fragment) that binds to a mammalian CC 4 chemokine receptor (also referred to such as CCR4, CKR-4, TARC receptor and MDC receptor) or portion of the receptor (anti-CCR4). In one embodiment, the antibody of the present invention or fragment thereof has specificity for human CCR4 or a portion thereof. In another embodiment, the antibody or fragment of the invention blocks the binding of a ligand (eg, TARC, MDC, MCP-1, MlP-la, RANTES) to the receptor and inhibits the function associated with the binding of the ligand to the receptor ( for example, leukocyte trafficking). In a preferred embodiment, the ligand is TARC and / or MDC. For example, as described herein, antibodies and fragments thereof of the present invention that bind to human CCR4 or a portion thereof can block the binding of a chemokine (e.g., TARC, MDC, MCP-1, MlP -la, RANTES) to the receptor and inhibit the function associated with the binding of chemokine to the receptor. In a preferred embodiment, the chemokine is TARC and / or MDC. In one embodiment, the antibody is monoclonal antibody (mAb) LS141-1G1 (1G1) or an antibody that can compete with 1G1 for binding to human CCR4 or a portion of human CCR4. In another embodiment, the antibody is the monoclonal antibody (mAb) LS185-2B10 (2B10) or an antibody that can compete with 2B10 for binding to human CCR4 or a portion of human CCR4. In another embodiment, the antibody is the monoclonal antibody (mAb) LS257-10E4 (10E4) or an antibody that can compete with 10E4 for binding to human CCR4 or a portion of human CCR4. Functional fragments of the above antibodies are also contemplated. The present invention also relates to an antibody or functional fragment thereof (eg, an antigen-binding fragment) that binds to a mammalian CCR4 or receptor portion and provides a higher fluorescent staining intensity of CCR4 or compositions that they contain CCR4 in relation to other anti-CCR4 antibodies. In one embodiment, the antibody is monoclonal antibody 1G1, 2B10 or 10E4 or an antibody that can compete with 1G1, 2B10 or 10E4 for binding to human CCR4 or a portion of human CCR4.

The present invention further relates to a method of inhibiting the interaction of a mammalian CCR4 carrying cell (eg, human, non-human or murine primate) with a ligand thereof, consisting of contacting the cell with an amount effective of an antibody or functional fragment thereof that binds to a mammalian CCR4 or a portion of CCR. Suitable cells include granulocytes, leukocytes, such as monocytes, macrophages, basophils and eosinophils, mast cells and lymphocytes, including T cells (e.g., CD8 + cells, CD4 + cells, CD25 + cells, CD45RO + cells), as Thl and Th2 cells, and other cells expressing CCR4, such as a recombinant cell expressing CCR4 or a portion thereof (eg, transfected cells). In a particular embodiment, the antibody is 1G1, 2B10 or 10E4 or an antibody that can compete with 1G1, 2B10 or 10E4 for binding to human CCR4 or a portion of human CCR4. Another embodiment of the invention relates to a method of inhibiting the interaction of a mammalian CCR4 carrier cell with a chemokine, comprising contacting said cell with an effective amount of an antibody or functional fragment thereof that binds to CCR4 or a portion of said receiver. In one embodiment of the method, the antibody or functional fragment thereof is any one or more than 1G1, 2B10, 10E4, an antigen-binding fragment of 1G1, 2B10 or 10E4, or an antibody or fragment thereof having a epitopic specificity that is equal to or similar to that of 1G1, 2B10 or 10E4. Moreover, the invention relates to a method of inhibiting a function associated with the binding of a chemokine to CCR4, comprising administering an effective amount of an antibody or a functional fragment thereof that binds to mammalian CCR4 or a portion thereof. of said receiver. In one aspect of the method, the antibody or functional fragment thereof is any one or more of 1G1, 2B10 or 10E4, an antigen-binding fragment of 1G1, 2B10 or 10E4, or an antibody or fragment thereof having an equal epitopic specificity or similar to 1G1, 2B10 or 10E4. Another aspect of the invention is a method of identifying the expression of a mammalian CCR4 or a portion of the receptor by a cell. According to the method, a composition containing a cell or fraction thereof (e.g., a membrane fraction) is contacted with an antibody or functional fragment thereof (e.g., 1G1, 2B10 or 10E4) that binds to a mammalian CCR4 protein or a portion of the receptor under conditions suitable for antibody binding thereto and the formation of a complex between said antibody or fragment and said protein or portion thereof is detected. The detection of the complex, directly or indirectly, indicates the presence of the receptor or portion thereof on the cell or fraction thereof. The present invention also relates to a kit for use in detecting the presence of CCR4 or a portion thereof in a biological sample, which contains an antibody or functional fragment thereof that binds to a mammalian CCR4 or a portion thereof. said receptor and one or more auxiliary reagents suitable for detecting the presence of a complex between said antibody or fragment and said protein or portion thereof. The present invention also includes methods of identifying additional ligands or other substances that bind to a mammalian CCR4 protein, including inhibitors and / or promoters of mammalian CCR4 function. For example, agents that have the same or similar binding specificity to that of the antibody of the present invention or functional fragment thereof can be identified by a competitive assay with said antibody or fragment. Thus, the present invention also encompasses methods of identifying li-gands or other substances that bind to the CCR receptor, including inhibitors (eg, antagonists) or promoters (eg, agonists) of receptor function. In one embodiment, cells that naturally express the CCR4 receptor protein or suitable host cells that have been engineered to express a CCR4 receptor or variant encoded by a nucleic acid introduced into said cells, are used in an assay to identify and assess the effectiveness of ligands, inhibitors or promoters of receptor function. Said cells are also useful in the assessment of the function of the protein or polypeptide of the expressed receptor. Thus, the invention also relates to a method of detecting or identifying an agent that binds to a mammalian CCR4 or a ligand-binding variant thereof, consisting of combining an agent to be studied, an antibody or fragment. of antigen binding of the present invention (for example, monoclonal antibody 1G1, monoclonal antibody 2B10, monoclonal antibody 10E4, an antibody having an epitopic specificity equal to or similar to that of 1G1, 2B10 or 10E4, antigen-binding fragments of 1G1 , 2B10 or 10E4) and a composition containing a mammalian CCR4 protein or ligand binding variant thereof. The above components can be combined under conditions suitable for the binding of the antibody or antigen-binding fragment to the mammalian CCR4 protein or a ligand-binding variant thereof and the binding of the antibody or fragment to the protein is detected or measured. mammalian CCR4 protein or ligand binding variant, directly or indirectly, according to the methods described here or other suitable methods. A reduction in the amount of complex formed relative to an adequate control (eg, in the absence of the agent to be studied) is indicative that the agent binds to said receptor or variant. The composition containing a mammalian CCR4 protein or a ligand-binding variant thereof can be a membrane fraction of a recombinant CCR4 protein carrier cell or ligand-binding variant thereof. The antibody or fragment thereof can be labeled with a label such as a radioisotope, spin labeling, antigenic labeling, enzyme labeling, fluorescent group and chemiluminescent group. These and other similar assays can be used to detect agents, including ligands (e.g., chemokines that interact with CCR4) or other substances, including inhibitors or promoters of receptor function, which can bind to CCR4 and compete with the antibodies herein. described by receptor binding. According to the present invention, li-gandos, inhibitors or promoters of receptor function can be identified in a suitable assay and their therapeutic effect assessed. Inhibitors of receptor function can be used to inhibit (reduce or prevent) the activity of the receptors and ligands and / or promoters can be used to induce (trigger or increase) the normal function of the receptors, when indicated. The present invention also provides a method of treating inflammatory diseases, autoimmune diseases, atherosclerosis and graft rejection or HIV infection, for example, by administering an inhibitor of receptor function (e.g. , the binding of chemokines or the binding of HIV) to an individual (e.g., a mammal, such as a human). The present invention further provides a method of stimulating the function of the receptors by administering a new ligand or promoter to an individual, providing a new approach to the selective stimulation of the function of leukocytes, which is useful, for example. , in the treatment of infectious diseases and cancer.

The present invention also encompasses a method of inhibiting leukocyte trafficking in a patient, comprising administering to the patient an effective amount of an antibody or functional fragment thereof that binds to a mammalian CCR4 or portion of said receptor and inhibits function. associated with the binding of a ligand to the receptor. The present invention also relates to a method of inhibiting or treating CCR4-mediated disorders, such as inflammatory disorders, comprising administering to a patient an effective amount of an antibody or functional fragment thereof that binds to a CCR4. of mammal or portion of said receptor and inhibits the function mediated by CCR4. The present invention is further related to an anti-body or fragment thereof as described herein (e.g., monoclonal antibody 1G1, monoclonal antibody 2B10, monoclonal antibody 10E4, an antigen-binding fragment of 1G1, 2B10 or 10E4 , an antibody having an epitopic specificity equal to or similar to that of 1G1, 2B10 or 10E4) for use in therapy (including prophylaxis) or diagnosis, and with the use of said antibody or fragment for the manufacture of a medicament for the treatment of a disorder mediated by CCR4 or another inflammatory disease or condition, as described herein. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a FACScan® profile showing the staining with 1G1 mAb of several transfectants Ll .2. Stable Ll2 transfectants that expressed different chemokine receptors, CCR1-CCR8, were stained with anti-CCR4 1G1 mAb. The negative control staining for all L1.2 transfectants resembled that of CCR1 / L1.2 cells. Figure 2 is a dot plot of FACScan® showing the expression of CCR4 in various populations of peripheral blood lymphocytes. A two-color staining protocol was used to assess the expression of CCR4 using mAb 1G1 (x-axis in all graphs) and cell markers (y-axis and in all graphs) CD14 (monocytes), CD4 and CD8 (T cells), CD16 and CD56 (NK cells) and CD19 and CD20 (B cells). The subgroup marker is indicated for each graph. The quadrants were fixed according to the staining of control mAbs. The staining was representative of multiple donors analyzed. Figure 3 is a dot plot of FACScan® showing the expression of CCR4 in CD4 + T cells. A three-color staining protocol was used to assess the expression of CCR4 (x-axis on all graphs) and markers of T-cell subgroups (y-axis and all graphs) on CD4 + T cells. In subgroup marker is indicated for each graph. The quadrants were fixed according to the staining of control mAbs. The staining was representative of multiple donors analyzed. The cells were analyzed by opening the gate on CD4 + lymphocytes and representing CCR4 against several cell markers. The quadrants were fixed according to the control mAbs staining. Memory / simple markers: CD45RO, CD45RA; ligands / selectin: CLA, selectin P ligand, selectin E ligand, selectin L; Integrins: ACT-1 (a4ß7), CD49d (a4), integrin ß7, CD29 (ßl), CD104 (ß4) and CD103 (ae). Staining of CCR4 by mAb 2B10 shows an identical pattern of staining. Figure 4 is a FACScan® profile showing the expression of CCR4 in Th2 cells. Thl (line) and Th2 (profile filled) chronically activated lymphocytes generated by two cycles of activation from umbilical CD4 lymphocytes were stained with mAb anti-a4ß7 Act 1 as a positive control, since this integrin is expressed in both Thl and Th2 lymphocytes . Staining with anti-CCR2 mAb 1D9 showed that both Thl and Th2 mAbs express CCR2, while staining with anti-CXCR3 mAb (R and D, Minneapolis, MN) showed that CXCR3 was selectively expressed in Thl cells derived in vi tro. Using 1G1, it was found that CCR4 was selectively expressed on Th2 lymphocytes. Figure 5 is a graph showing that Abs 1G1 and 2B10 inhibit the binding of 125I-TARC to transfectants CCR4 / L1.2. CCR4 / L1.2 cells were incubated with 125 I-TARC 0, 1 nM in the absence (total binding) or presence (competitive binding) of a dose range of mAb 1G1, mAb 2B10 or MOPC21, an isotype control IgG1. After 60 minutes, the excess of antibody and chemokine was washed and the reactions counted. The data shown are% inhibition of total binding. Figures 6A-6D are graphs showing the inhibition of CCR4 / L.12 chemotaxis towards TARC and MDC by mAb 1G1. The CCR4 / L1.2 cells were allowed to undergo chemotaxis towards TARC or MDC in the presence or absence of mAb 1G1 or mAb MOPC21 (the isotype control) in a transpocillo chemotaxis assay. In Figures 6A and 6B, a range of chemokine concentrations was used, with 20 ug / ml of mAb 1G1 and M0PC21. In Figures 6C and 6D, 5 nM chemokine was used with a range of concentrations of 1G1 and M0PC21. The number of migrated cells was counted by flow cytometry using forward and lateral scattering. Figures 7A-7B are graphs showing the inhibition of Peer cell chemotaxis (a line of human delta-gamma T cell receptors) towards TARC (Figure 7A) and MDC (Figure 7B) by mAb 1G1. The Peer cells were allowed to undergo chemotaxis towards TARC or MDC in the presence or absence of 1G1 mAb in a transpocillo chemotaxis assay. A range of concentrations of TARC or MDC was used, with 50 ug / ml of anti-CCR4 mAb 1G1 and mAb MOPC21 (isotype control). The number of migrated cells was counted by flow cytometry using forward and lateral scattering. Figure 8 shows the effect of anti-CCR4 mAbs 1G1 and 2B10 on Th2 migration. Chronically activated Thl / Th2 were generated by two activation cycles from umbilical CD4 lymphocytes and preincubated with 50 μg / ml of an IgGl control mAb, anti-CCR4 mAb 1G1 or anti-CCR4 mAb 2B10. After 10 minutes on ice, the Th2 cells were allowed to migrate for two hours at 100 ng / ml MDC, TARC or RANTES. In one case, the MDC was preincubated with a rabbit polyclonal for MDC for 10 minutes before use (MDC / -MDC). To establish background migration, chemokine was not used in the lower well (-). After this time, the cells accumulated in the lower well were counted using a FACSCAN. Figure 9 shows the effect of anti-CCR4 mAbs 1G1 and 2B10 on migration of CD4 lymphocytes of 24 hours of age. 24-hour-old CD4 lymphocytes were pre-incubated with 50 μg / ml of a control mAb, anti-CCR4 mAb 1G1 or anti-CCR4 mAb 2B10. At 10 minutes on ice, the CD4 lymphocytes were allowed to migrate for two hours at 100 ng / ml MDC, TARC or RANTES. In one case, the MDC was preincubated with a rabbit polyclonal for MDC for 10 minutes before use (MDC / -MDC). To establish background migration, chemokine was not used in the lower well (-). After this time, the cells accumulated in the lower well were counted using a FACSCAN. Figures 10A and 10B are graphs showing that monoclonal antibodies 10E4, 2B10 and 1G1 block the migration of transfectants L1.2 / CCR4 to MDC and TARC. Transfectants L1.2 / CCR4 (at 2 x 106 cells per ml in RPMI, 0.5% bovine serum albumin, 10 mM Hepes) were pretreated with various concentrations of purified anti-CCR4 monoclonal antibodies 10E4 (IgGl), 2B10 (IgG2a ) and 1G1 (IgGl) for ten minutes on ice and then aliquots of 200 μl of cells used in the chemotaxis assays were used with 3.0 μm Costar Transpore Filters (Costar, Cambridge, MA) and allowed to undergo chemotaxis to 50 ng / ml of MDC (Figure 10A) or 100 ng / ml of TARC (Figure 10B). The cells were then counted in a FACSCAN Becton Dickinson. 10E4 was shown to be the best anti-CCR4 blocking monoclonal antibody, followed by 2B10 and then 1G1. An IgG1 control monoclonal antibody had no effect on the migration of L1.2 / CCR4 transfectants to MDC (315 ± 30) or TARC (382111). DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antibody (anti-CCR4) or functional fragment thereof that binds to mammalian CC chemokine re-receptor 4 (CCR4)., CKR-4, TARC receptor or MDC receptor) or a portion of CCR4. In one embodiment, the antibody has specificity for human CCR4 or a portion thereof. In one embodiment, the antibodies (immunoglobulins) are produced against an isolated and / or recombinant mammalian CCR4 or portion thereof (eg, peptide) or against a host cell expressing mammalian CCR4. In a preferred embodiment, the antibodies specifically bind to the human CCR4 receptor or a portion thereof and, in a particularly preferred embodiment, the antibodies have specificity for a natural or endogenous human CCR4. Antibodies or functional fragments thereof that can inhibit one or more characteristic functions of a mammalian CCR4, such as a binding activity (eg, ligand binding, inhibitor and / or promoter), a signaling activity (eg. , activation of a mammalian G protein, induction of a rapid and transient increase in the concentration of cytosolic free calcium [Ca2 +] i) and / or stimulation of a cellular response (for example, stimulation of chemotaxis, exocytosis or release of mediators inflammatories by leukocytes, integrin activation) are also protected by the present invention, such as an antibody that can inhibit the binding of a ligand (i.e., one or more ligands) to CCR4 and / or one or more functions mediated by CCR4. in response to a ligand. For example, in one aspect, the antibodies or functional fragments thereof can inhibit (reduce or prevent) the interaction of the receptor with a natural ligand, such as MDC, TARC, MCP-1, MlP-la or RANTES. In one embodiment, the ligand is TARC and / or MDC. In another aspect, an antibody or functional fragment thereof that binds to CCR4 can inhibit the binding of TARC, MDC, MCP-1, MlP-la and / or RANTES to mammalian CCR4 (eg, human CCR4, primate CCR4). non-human, murine CCR4, CCR4 of guinea pig origin). The antibodies or functional fragments thereof of the present invention can inhibit functions mediated by human CCR4, including trafficking of leukocytes, activation of T cells, release of inflammatory mediators and / or degranulation of leukocytes. In a particular embodiment, the antibodies or functional fragments thereof demonstrate inhibition of chemotaxis of cells induced by chemokines (eg, induced by TARC or by MDC), preferably to less than about 0.50 μg / ml, preferably less than about 0.30 μg / ml and, more preferably, less than about 0.27 μg / ml. In another embodiment of the invention, the antibodies or functional fragments thereof of the invention can inhibit the binding of a CCR4 ligand (eg, a chemokine) to CCR4, preferably with an IC50 of less than about 1.5 μg. / ml. In another embodiment, the antibodies or functional fragments thereof of the invention can inhibit the binding of a CCR4 ligand (eg, a chemokine) to CCR4 with an IC50 of less than about 1.5 ng / ml. The murine monoclonal antibodies specific for CCR4, designated 1G1, 2B10 and 10E4, were produced as described herein. In a preferred embodiment, the antibodies of the present invention bind to human CCR4 and have an epitope specificity equal to or similar to that of the murine antibody 1G1, 2B10 or 10E4 described herein. Antibodies with epitopic specificity equal to or similar to that of the murine 1G1 monoclonal antibody can be identified by their ability to compete with the monoclonal antibody 1G1 u-kid for binding to human CCR4 (eg, to human CCR4 carrier cells, as transfectants carrying CCR4, CD8 + cells, CD4 + cells, CDR45RO + cells, CD25 + cells, monocytes, dendritic cells, macrophages and basophils). Similarly, antibodies with epitopic specificity similar or similar to that of murine monoclonal antibodies 2B10 or 10E4 can be identified by their ability to compete with murine antibodies 2B10 or 10E4., respectively, by binding to human CCR4. Using receptor chimeras (e.g., as described by Ruc-ker et al., Cell 87: 437-446 (1996)), one can map the CCR4 binding site (i.e., the epitope specificity) of 1G1 mAbs , 2B10 and 10E4. Alternatively, the epitope specificity of the antibodies can be determined using peptide blocking techniques wherein a peptide having a particular CCR4 amino acid sequence is used in competitive assays to identify peptides that can compete with CCR4 for binding to a given antibody . Peptides that can compete with CCR4 can be further studied to more specifically define the CCR4 epitope to which a given antibody binds. Using these and other suitable techniques, antibodies having epitopic specificity equal or similar to that of the antibody of the present invention can be identified. The invention also relates to a bi-specific antibody or functional fragment thereof (eg, F (ab ') 2) having the same or similar epitopic specificity as at least two of the antibodies described herein (see, for example, U.S. Patent No. 5,141,736 (Iwasa et al.), U.S. Patent Nos. 4,444,878, 5,292,668, 5,523,210 (all of Paulus et al.) and U.S. Pat. U.S. Patent No. 5,496,549 (Yamazaki et al.) For example, a bispecific antibody of the present invention may have the same or similar epitope specificity as mAbs 1G1 and 2B10, than mAbs 1G1 and 10E4 or mAbs 2B10. and 10E4 The murine hybridoma cell lines producing antibodies according to the present invention were deposited on January 6, 1999, in the name of LeukoSite, Inc., 215 First Street, Cambridge, MA 02142, USA, in American Type Cul-ture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA, under Accession No. HB-12624 (LS141-1G1-6 5-15-1 (1G1)) and HB-12625 (LS185-2B10-4-1 (2B10)). A line of murine hybridoma cells producing additional antibodies according to the present invention was deposited on January 14, 2000 in the name of LeukoSite, Inc., 215 First Street, Cambridge, MA 02142, USA, in the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA, under Accession No. PTA-1203 (LS257-10E4.1.1 (10E4)). The present invention also relates to the hybridoma cell lines deposited under ATCC Accession No. IIB-12624, Accession No. ATCC HB-12625 and Accession No. ATCC PTA-1203, as well as antibodies monoclonal antibodies produced by the hybridoma cell lines deposited under ATCC Accession No. HB-12624, HB-12625 and ATCC Accession No. PTA-1203. The antibodies of the present invention can be polyclonal or monoclonal and the term "antibody" is intended to include both polyclonal and monoclonal antibodies. Moreover, it is understood that the methods described herein using 1G1 may also utilize functional fragments (e.g., antigen-binding fragments) of 1G1, antibodies having the same or similar epitope specificity as 1G1 and its combinations, optionally in combination with antibodies or fragments that have an epitopic specificity that is not equal to or similar to 1G1; similarly, the methods described as 2B10 users may also utilize functional fragments of 2B10, antibodies having the same or similar epitope specificity as 2B10 and its combinations, optionally in combination with antibodies or fragments having an epitope specificity that does not is the same or similar to 2B10. The methods described as 10E4 users can also employ functional fragments of 10E4, antibodies having the same or similar epitope specificity as 10E4 and combinations thereof, optionally in combination with antibodies or fragments having an epitope specificity that is not the same or similar to 10E4. The antibodies of the present invention can be produced against an appropriate immunogen, such as isolated mammalian and / or recombinant CCR4 protein or a portion thereof, or synthetic molecules, such as synthetic peptides. In a preferred embodiment, cells that express receptor, such as transfected cells, can be used as immunogens or in a selection of antibodies that bind to the receptor. The antibodies of the present invention and their fragments are useful in therapeutic, diagnostic and research applications, as described herein. The present invention includes an antibody or functional portion thereof of the present invention (e.g., mAb 1G1, 2B10 or 10E4, or antigen-binding fragments thereof) for use in therapy (including prophylaxis) or diagnostics (e.g. of diseases or particular conditions, as described herein) and the use of said antibodies or functional portions thereof for the manufacture of a medicament for use in the treatment of diseases or conditions as described herein. The preparation of immunizing antigen and the production of polyclonal and monoclonal antibodies can be carried out as described herein or using other suitable techniques. A variety of methods have been described (see, for example, Kohler et al., Nature 256: 495-497 (1975), and Eur. J. Immunol., 6: 511-519 (1976); Milstein et al., Nature. 266: 550-552 (1977); Koprowski et al., US Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antijbodies: A Laboratory Manual (Cold Spring Harbor Laboratory: Cold Spring Harbor , NY), Current Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer 94), Ausubel, FM et al., Eds. (John Wiley &Sons: New York, NY), Chapter 11 (1991)) . In general, a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2 / 0) with antibody producing cells. The antibody-producing cells, preferably from the spleen or lymph nodes, are obtained from animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions and cloned by limiting dilution. Cells that produce antibodies with the desired binding properties can be selected by means of a suitable assay (e.g., ELISA). Other suitable methods of producing or isolating antibodies that bind to CCR4, including human or artificial antibodies, can be employed, including, for example, methods that select recombinant antibody (e.g., Fv or single-chain Fab) from a library, or based on the immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human or artificial antibodies (see, for example, Jakobo-vits et al., Proc. Nati. Acad. Sci. USA 90: 2551-2555 (1993), Jakobovits et al., Nature 362: 255-258 (1993), Lon-berg et al., US Patent No. 5,545,806; Surani et al., Patent EE US No. 5,545,807). Also included in the present invention and in the term "antibody" are single chain antibodies and chimeric, humanized or primatized antibodies (grafted with CDR), as well as chimeric or single chain antibodies grafted with CDR., and the like, which contain portions derived from different species. The various portions of these antibodies can be chemically linked together by conventional techniques, or they can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, for example, Cabilly et al., US Pat. No. 4,816,567; Cabilly et al., European Patent No. 0125.023 Bl; Boss et al., US Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 Bl; Neuberger, M.S. et al., WO 86/01533; Neuberger, M.S. et al., European Patent No. 0,194,276 Bl; Winter, US Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl, and Queen et al., Patents US Pat. Nos. 5,585,089, 5,698,761 and 5,698,762. See also Newman, R. et al., BioTechnology 10: 1455-1460 (1992) for primatized antibodies, and Ladner et al., US Pat. No. 4,946,778, and Bird, R.E. et al., Science 242: 423-426 (1988)) for single chain antibodies. In addition, functional fragments of antibodies can also be produced, including fragments of chimeric, humanized, primatized or single chain antibodies. Functional fragments of the above antibodies retain at least one binding function and / or modulating function of the full-length antibody from which they are derived. Preferred functional fragments retain an antigen-binding function of a corresponding full-length antibody (e.g., they retain the ability to bind to a mammalian CCR4). Particularly preferred functional fragments retain the ability to inhibit one or more functions characteristic of a mammalian CCR4, such as a binding activity, a signaling activity and / or stimulation of a cellular response. For example, in one embodiment, a functional fragment can inhibit the interaction of CCR4 with one or more of its ligands (eg, TARC, MDC, MCP-1, MlP-la and / or RANTES) and / or can inhibit one or more mediated receptor functions, such as leukocyte trafficking, HIV entry into cells, activation of T cells, release of inflammatory mediators and / or degranulation of leukocytes. For example, fragments of antibodies capable of binding to a mammalian CCR4 receptor or portion thereof, including, but not limited to, Fv, Fab, Fab 'and F (ab') 2 fragments, are included by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques, for example. For example, cleavage with papain or pepsin can generate Fab or F (ab ') 2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons needles have been introduced above the natural stop site. For example, a chimeric gene encoding a heavy chain portion of F (ab ') 2 can be designed to include DNA sequences encoding the CHi domain and the hinge region of the heavy chain. The term "humanized immunoglobulin", as used herein, refers to an immunoglobulin consisting of portions of immunoglobulins of different origin, where at least a portion is of human origin. Accordingly, the present invention relates to a human immunoglobulin that binds to mammalian CCR4 (eg, human CCR4, murine CCR4, guinea pig CCR4), whose immunoglobulin contains an antigen-binding region of non-human origin ( for example, rodent) and at least a portion of an immunoglobulin of human origin (eg, a human tag region, a human constant region or portion thereof). For example, the humanized antibody may contain portions derived from an immunoglobulin of non-human origin with the necessary specificity, such as from mouse, and from immunoglobulin sequences of human origin (eg, a chimeric immunoglobulin), chemically linked together by techniques conventional (eg, synthetic) or be prepared as a contiguous polypeptide using genetic engineering techniques (eg, DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized immunoglobulin of the present invention is an immunoglobulin containing one or more immunoglobulin chains containing a CDR of non-human origin (e.g., one or more CDRs derived from an antibody of non-human origin) and a framework region derived from a light and / or heavy chain of human origin (eg, CDR-grafted antibodies with or without frame changes). In one embodiment, the humanized immunoglobulin can compete with the murine monoclonal antibody 1G1, 2B10 or 10E4 for binding to human CCR4. In a preferred embodiment, the antigen-binding region of humanized immunoglobulin (a) is derived from monoclonal antibody 1G1, 2B10 or 10E4 (for example, as in a humanized immunoglobulin containing CDR1, CDR2 and CDR3 of the light chain of 1G1, 2B10 or 10E4 and CDR1, CDR2 and CDR3 of the heavy chain of 1G1, 2B10 or 10E4). Chimeric or single chain antibodies grafted with CDR are also included in the term humanized immunoglobulin. Said humanized immunoglobulins can be produced using synthetic and / or recombinant nucleic acids to prepare genes (e.g., cDNAs) encoding the desired humanized chain. For example, nucleic acid (eg, DNA) sequences encoding humanized variable regions can be constructed using PCR mutagenesis methods to alter the DNA sequences encoding a human or humanized chain, such as a DNA template from a region. previously humanized variable (see, for example, Kamman, M. et al., Nucí Acids Res. 17: 5404 (1989); Sato, K. et al., Cancer Research 53: 851-856 (1993); Dau-gherty, B.L. et al., Nucleic Acids Res. 19 (9): 2471-2476 (1991), and Lewis, A.P. and J.S. Crowe, Gene 101: 297-302 (1991)). Using these and other suitable methods, variants can also easily be produced. In one embodiment, cloned variable regions can be mutagenized and sequences coding for variants with the desired specificity can be selected (eg, from a phage library, see, for example, Krebber et al., US 5,514. 548; Hoogenboom et al., WO 93/06213, published April 1, 1993; Knappik et al., WO 97/08320, published March 6, 1997)). Anti-idiotypic antibodies are also provided. Anti-idiotypic antibodies recognize antigenic determinants associated with the antigen-binding site of another antibody. Anti-idiotypic antibodies can be prepared against a second antibody by immunizing an animal of the same species and, preferably, from the same strain, as the animal used to produce the second antibody. See, for example, US Pat. No. 4,699,880. The present invention also relates to the hybridoma cell lines deposited under ATCC Accession No. HB-12624, HB-12625 and PTA-1203, as well as the monoclonal antibodies produced by the hybridoma cell lines deposited under No. Access ATCC HB-125624, HB-12625 and PTA-1203. The cell lines of the present invention have uses other than the production of monoclonal antibodies. For example, the cell lines of the present invention can be fused to other cells (such as human myeloma, murine myeloma, human-murine heteromyeloma or human lymphoblastoid cells suitably labeled with drugs) to produce additional hybridomas and thus enable the transfer of the genes coding for monoclonal antibodies. In addition, the cell lines can be used as a source of nucleic acids encoding the anti-CCR4 immunoglobulin chains, which can be isolated and expressed (eg, by transfer to other cells using any suitable technique (see, for example, Cabilly et al., U.S. Patent No. 4,816,567; Winter, U.S. Patent No. 5,225,539.) For example, clones containing a redistributed anti-CCR4 heavy or light chain can be isolated (eg. example, by PCR) or cDNA libraries can be prepared from mRNA isolated from the cell lines and coding cDNA clones isolated from an anti-CCR4 immunoglobulin chain can be obtained. heavy and / or light chains of the antibodies or portions thereof and using them according to recombinant DNA techniques for the production of the specific immunoglobulin, immunoglobulin chain or variants thereof (eg, immunoglobulins) humanized) in a variety of host cells or in a translation system in vi tro. For example, nucleic acids, including cDNAs, or their variants-encoding derivatives, such as an immunoglobulin or humanized immunoglobulin chain, can be put into suitable prokaryotic or eukaryotic vectors (e.g., expression vectors) and introduced into a suitable host cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is operably linked to one or more expression control elements (e.g., in the vector or integrated into the genome of the host cell). For production, the host cells can be maintained under conditions suitable for expression (eg, in the presence of inducer, suitable means supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.), by which the encoded polypeptide is produced. If desired, the encoded protein can be recovered and / or isolated (e.g., from host, medium, milk cells). It will be appreciated that the production method includes 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 block (inhibit) the binding of a ligand to CCR4 and / or inhibit the function associated with the ligand to CCR4. As discussed below, various methods can be employed to assess the inhibition of a ligand to CCR4 and / or the function associated with binding of the ligand to the receptor. Binding assays As used herein, the term "mammalian CCR4" refers to natural or endogenous mammalian CCR4 proteins and to proteins having an amino acid sequence that is the same as that of a corresponding natural or endogenous mammalian CCR4 protein. (for example, recombinant proteins). Accordingly, as defined herein, the term includes protein from mature receptors, polymorphic or allelic variants and other isoforms of a mammalian CCR4 (eg, produced by adjustment or other cellular processes) and modified or unmodified forms of the previous ones (for example, glycosylated, non-glycosylated). The mammalian CCR4 proteins can be isolated and / or recombinant proteins (including synthetically produced proteins). Natural or endogenous mammalian CCR4 proteins include wild-type proteins, such as mature CCR4, polymorphic or allelic variants and other isoforms that occur naturally in mammals (e.g., humans, non-human primates). Said proteins can be recovered or isolated from a source that naturally produces mammalian CCR4, for example. Reference is made to these mammalian CCR4 proteins and proteins having the same amino acid sequence as a corresponding natural or endogenous mammalian CCR4 by the name of the corresponding mammal. For example, when the corresponding one is a human, the protein is designated as a human CCR4 protein (eg, a recombinant human CCR4 produced in an appropriate host cell). "Functional variants" of mammalian CCR4 proteins include functional fragments, functional mutant proteins and / or functional fusion proteins (e.g., produced by mutagenesis and / or recombinant techniques). In general, fragments or portions of mammalian CCR4 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 mature mammalian CCR4 protein ( such as N-terminal, C-terminal or inter-ñane deletions). Also contemplated are fragments or portions in which only contiguous amino acids have been deleted or where non-contiguous amino acids have been deleted relative to the mature mammalian CCR4 protein. In general, mutants of mammalian CCR4 proteins include natural or artificial variants of a mammalian CCR4 protein that differ only in the addition, deletion and / or substitution of one or more contiguous or non-contiguous amino acid residues (e.g. , chimeras of receivers). Such mutations may be in a conserved region or an unconserved region (as compared to other chemokine receptors CXC (a) and / or CC (ß)), or in an extracellular, cytoplasmic or transmembrane region, for example. In general, the fusion proteins include polypeptides containing a mammalian CCR4 (eg, human CCR4) or a variant thereof as the first residue, linked via a peptide bond to a second residue that does not appear in mammalian CCR4. as it is found in nature. Thus, the second moiety can be an amino acid, oligopeptide or polypeptide. The first residue may be at an N-terminal location, a C-terminal location or internal to the fusion protein. In one embodiment, the fusion protein comprises an affinity ligand (eg, an enzyme, an antigen, an epitope tag) as the first residue and a second residue consisting of a binding sequence and human CCR4 or a portion thereof. A "functional fragment or portion", "functional mutant" and / or "functional fusion protein" of a mammalian CCR4 protein refers to an isolated and / or recombinant protein or polypeptide having at least one characteristic function of a mammalian CCR4 protein as described herein, such as a binding activity, a signaling activity and / or the ability to stimulate a cellular response. Preferred functional variants may be linked to a ligand (ie, one or more ligands such as TARC, MDC, MCP-1, MlP-la and / or RANTES) and are referred to herein as "binding variants". ligand ". In one embodiment, a functional variant of mammalian CCR4 shares at least about 85% sequence identity with said mammalian CCR4, preferably at least about 90% sequence identity and, more preferably, at least about 95% sequence identity with said mammalian CCR4. In another embodiment, a functional fusion protein contains a first residue that shares at least about 85% sequence identity with a mammalian CCR4, preferably at least about 90% sequence identity and, more preferably, at least about 95% sequence identity with a mammalian CCR4. The sequence identity can be determined using a suitable program, such as the Blastx program (Version 1.4), using appropriate parameters, such as defect parameters. In one embodiment, the parameters for the Blastx search are the scoring matrix BLOSUM62, W = 3. In another embodiment, a functional variant includes a nucleic acid sequence that is different from the natural nucleic acid molecule, but which, due to the degeneracy of the genetic code, encodes mammalian CCR4 or a portion thereof. A composition containing an isolated and / or recombinant mammalian CCR4 or a functional variant thereof can be maintained under suitable binding conditions, the mammalian CCR4 or variant is contacted with an antibody or fragment to be studied and detects or measures the union directly or indirectly. In one embodiment, cells that naturally express CCR4 or cells that contain a recombinant nucleic acid sequence encoding a mammalian CCR4 or variant thereof are used. The cells are maintained under appropriate conditions for the ex-pressure of the receptor. The cells are contacted with an antibody or fragment under appropriate binding conditions (e.g., in a suitable binding buffer) and binding is detected by standard techniques. To determine the binding, the degree of binding can be determined in relation to a suitable control (for example, by comparing with a given background in the absence of antibody, comparing with the binding of a second antibody (ie, a standard), comparing with antibody binding to non-transfected cells). A cellular fraction, such as a membrane fraction, containing receptor or liposomes containing receptor, can be used in place of whole cells. In one embodiment, the antibody is labeled with a suitable labeling (e.g., fluorescent labeling, isotopic labeling, antigenic or epitope labeling) and binding is determined by label detection. In another embodiment, the labeled antibody can be detected by means of a labeled second antibody. The binding specificity can be assessed by competition or displacement, for example, using unlabelled anti-body or a ligand as a competitor. The inhibition of binding assays can also be used to identify antibodies or fragments thereof that bind to CCR4 and inhibit the binding of another compound, such as a ligand (TARC, MDC, MCP-1, MlP-la and / or RANTES) to CCR4 or a functional variant. For example, a binding assay can be carried out in which a reduction in the binding of a ligand to CCR4 (in the presence of an antibody) is detected or measured compared to the binding of the ligand in the absence of the antibody. A composition containing an isolated and / or recombinant mammalian CCR4 or functional variant thereof with the ligand and antibody simultaneously can be contacted., or one after the other, in any order. A reduction in the degree of binding of the ligand in the presence of the antibody is indicative of inhibition of binding by the anti-body. For example, ligand binding could be reduced or abolished. In one embodiment, the direct inhibition of the binding of a ligand (eg, a chemokine such as TARC or MDC) to a mammalian CCR4 or variant thereof by an antibody or fragment is monitored. For example, the ability of an antibody to inhibit the binding of labeled 125 I TARC, 125 I-labeled MDC, 125 I-labeled MCP-1, 125 I-labeled MlP or 125 I-labeled RANTES to mammalian CCR4 can be monitored. . Said assay can be conducted using suitable cells carrying CCR4 or a functional variant thereof, such as isolated blood cells (e.g., T cells) or a suitable cell line expressing CCR4 naturally, or a cell line containing nucleic acid encoding a mammalian CCR4, or a mere-brane fraction of said cells, for example. Other methods of identifying the presence of an antibody that binds to CCR4 are available, such as other suitable binding assays, or methods that monitor events that are triggered by binding to receptors, including the signaling function and / or the stimulation of a cellular response (for example, leukocyte trafficking). It will be understood that the inhibitory effect of the antibodies of the present invention can be determined in an assay of inhibition of binding. The competition between antibodies for binding to receptors can also be assessed in the method. Antibodies that are identified in this way can also be studied to determine whether, following the binding, they act by inhibiting other functions of CCR4 and / or to assess their therapeutic utility. Signaling assays The binding of a ligand or promoter, such as an agonist, to CCR4, can result in signaling by this G protein-coupled receptor and stimulates the activity of the G proteins, as well as other G protein molecules. intracellular signaling. The induction of the signaling function by a compound (e.g., an antibody or fragment thereof) can be monitored using any suitable method. Said assay can be used to identify CCR4 antibody agonists. The inhibitory activity of an antibody or functional fragment thereof can be determined by using a ligand or promoter in the assay and evaluating the ability of the antibody to inhibit the activity induced by the ligand or promoter. One can study the activity of the G protein, such as the hydrolysis of GTP to GDP, or the subsequent signaling events triggered by binding to receptors, such as the induction of a rapid and transient increase in intracellular free calcium concentration ( cytosolic) [Ca2 +] ?, by methods known in the art or by other suitable methods (see, e.g., Neote, K. et al., Cell 72: 415-425 (1993); Van Riper et al., J. Exp. Med. 177: 851-856 (1993); Dahinden, C.A. et al., J. "Exp. Med. 179: 751-756 (1994).) For example, the functional assay of Sledziewski et al. using hybrid G-protein coupled receptors can be used to monitor the capacity of a ligand or promoter to bind to the receptor and activate a G protein (Sled-ziewski et al., U.S. Patent No. 5,284,746, the teachings of which are incorporated herein by reference.) Such assays can be performed in the presence of the antibody or fragment of the The antibody or fragment is capable of inhibiting the activity induced by the ligand or promoter using known methods and / or the methods described herein.Chemotaxis and cell stimulation assays Chemotaxis assays can also be used. to study the ability of an antibody or functional fragment thereof to block the binding of a ligand to a mammalian CCR4 or functional variant thereof and / or to inhibit the function associated with the binding of the Going to the receiver. These assays are based on the functional migration of cells in vi tro or in vivo induced by a compound. Chemotaxis can be assessed as described in the Examples, for example in an assay using a 96-well chemotaxis plate or using other methods recognized in the art to study chemotaxis. For example, the use of a transendothelial chemotaxis assay in vi tro is described by Springer et al. (Springer et al., WO 94/20142, published Sep. 15, 1994, the teachings of which are incorporated herein by reference, see also Berman et al., Immunol Invest. 17: 625-627 (1988)). Migration through the endothelium to collagen gels has also been described (Kavanaugh et al., J. Immunol., 146: 4149-4156 (1991).) Stable transfectants of pre-B cells L1.2 can be used. of mouse or other suitable host cells capable of undergoing chemotaxis in chemotaxis assays, for example In general, chemotactic assays monitor the directional movement or migration of a suitable cell (such as a leukocyte (e.g., lymphocyte, eosinophil, basophil) ) to or through a barrier (eg, endothelium, a filter), to higher levels of a composite, from a first surface of the barrier to a second, opposite surface.Membranes or filters provide convenient barriers, so that It monitors the directional movement or migration of a suitable cell to or through a filter, towards higher levels of a compound, from a first surface of the filter to a second surface. opposite surface of the filter. In some assays, the membrane is coated with a substance to facilitate adhesion, such as ICAM-1, fibronectin or collagen. These trials provide an approximation of the "arrival" of leukocytes. For example, inhibition of migration of the cells in a suitable container (a containment medium) from a first chamber, to or through a microporous membrane, can be detected or measured to a second chamber containing an antibody under study and which is separated from the first chamber by a membrane. A suitable membrane having a suitable pore size is selected to monitor the specific migration in response to a compound, including, for example, nitrocellulose or polycarbonate. For example, pore sizes of about 3-8 microns and, preferably, about 5-8 microns can be used. The pore size can be uniform in a filter or be within a range of suitable pore sizes. To assess the migration and the inhibition of migration, the distance of migration to the filter, the number of cells crossing the filter that remain adherent to the second surface of the filter and / or the number of cells that accumulate can be determined. in the second chamber using standard techniques (for example, microscopy). In one embodiment, the cells are labeled with a detectable labeling (eg, radioisotope, fluorescent labeling, antigenic or epitope labeling) and migration can be assessed in the presence and absence of the antibody or fragment by determining the presence of the adherent labeling on the membrane and / or present in the second chamber using an appropriate method (e.g., detecting radioactivity, fluorescence, immunoassay). The degree of migration induced by an antibody agonist relative to an appropriate control can be determined (for example, compared to background migration determined in the absence of the antibody, as compared to the degree of migration induced by a second compound (ie. say, a pattern), compared to the migration of untransfected cells induced by the antibody). In one embodiment, particularly for T cells, monocytes or cells expressing a mammalian CCR4, transendothelial migration can be monitored. In this embodiment, transmigration is assessed through a layer of endothelial cells. To prepare the cell layer, the endothelial cells can be cultured on a microporous filter or membrane, optionally coated with a substance such as collagen, fibronectin or other proteins of the extracellular matrix, to facilitate binding of the endothelial cells. Preferably, the endothelial cells are cultured to form a confluent monolayer. A variety of mammalian endothelial cells are available for the formation of the monolayer, including, for example, veins, arteries or microvascular endothelial cells, such as human umbilical vein endothelial cells (Clonetics Corp., San Diego, CA ). To study chemotaxis in response to a particular mammalian receptor, endothelial cells of the same mammal are preferred.; however, endothelial cells of a heterologous mammalian species or genus can also be used. In general, the test is performed by detecting the directional migration of cells to or through a membrane or filter, in a direction toward higher levels of a compound, from a first filter surface to a second opposite surface of the filter, where the filter contains a layer of endothelial cells on a first surface. Directional migration occurs from an area adjacent to the first surface, to or through the membrane, to a compound located on the opposite side of the filter. The concentration of the compound present in the area adjacent to the second surface is greater than that of the area adjacent to the first surface. In an embodiment used to study an antibody inhibitor, a composition containing cells capable of migrating and expressing a mammalian CCR4 receptor can be placed in the first chamber. A composition containing one or more ligands or promoters capable of inducing the chemotaxis of the cells in the first chamber (which has a chemoattractant function) is placed in the second chamber. Preferably, sooner the cells are placed in the first chamber or, simultaneously with the cells, a composition containing the antibody to be studied is placed, preferably, in the first chamber. Antibodies or functional fragments thereof that can bind to the receptor and inhibit the induction of chemotaxis by a ligand or promoter of cells expressing a CCR4 in this assay are inhibitors of receptor function (eg, inhibitors of the function stimulant). A reduction in the extent of migration induced by the ligand or promoter in the presence of the antibody or fragment is indicative of inhibitory activity. Separate binding studies (see above) could be performed to determine whether the inhibition is a result of antibody binding to the receptor or occurs through a different mechanism. In the following, in vivo assays are described which monitor the leukocyte infiltration of a tissue in response to the injection of a compound (eg, chemokine or antibody) into the tissue (see Patterns of Inflammation). These in vivo delivery models measure the ability of cells to respond to a ligand or promoter by migration and chemotaxis to a site of inflammation and assess the ability of an antibody or fragment thereof to block this migration. In addition to the methods described, the effects of an antibody or fragment on the stimulating function of CCR4 can be studied by monitoring the cellular responses induced by the active receptor, using suitable host cells containing the receptor. Identification of additional ligands, inhibitors and / or promoters of mammalian CCR4 function The assays described above, which can be used to study the binding and function of the antibodies and fragments of the present invention, can be adapted to identify additional ligands or other substrates that bind to a mammalian CCR4 or functional variant thereof, as well as inhibitors and / or promoters of mammalian CCR4 function. For example, agents having the same or similar binding specificity as that of an antibody of the present invention or functional portion thereof can be identified by means of a competitive assay with said antibody or portion thereof. Thus, the present invention also includes methods of identifying ligands of the receptor or other substances that bind to a mammalian CCR4 protein, as well as inhibitors (eg, antagonists) or promoters (eg, agonists) of the function of the receivers. In one embodiment, cells carrying a mammalian CCR4 protein or functional variant thereof (eg, leukocytes, cell lines, or suitable host cells that have been engineered to express a mammalian CCR4 protein or functional variant encoded by a nucleic acid introduced into said cells) in an assay to identify and assess the efficacy of ligands or other substances that bind to the receptor, including inhibitors or promoters of receptor function. Said cells are also useful in the study of the function of the expressed protein or polypeptide of the receptor. According to the present invention, li-gands and other substances that bind to the receptor, inhibitors and promoters of receptor function can be identified in a suitable assay and further studied for their therapeutic effect. Inhibitors of receptor function can be used to inhibit (reduce or prevent) the activity of the receptors and the ligands and / or promoters can be used to induce (trigger or increase) the normal function of the receptors, when indicated . Thus, the present invention provides a method of treating inflammatory diseases, including autoimmune diseases and rejection of grafts, comprising administering an inhibitor of receptor function to an individual (e.g., a mammal). The present invention also provides a method of stimulating the function of the receptors by administering a new ligand or promoter of receptor function to an individual, offering a new approach to the selective stimulation of the leukocyte function, which is useful, for example , in the treatment of infectious diseases and cancer. As used herein, a "ligand" of a mammalian CCR4 protein refers to a particular class of substances that bind to a mammalian CCR4 protein, including natural ligands and synthetic and / or recombinant forms of natural ligands. Infectious agents that have a tropism for mammalian CCR4 positive cells (e.g., viruses such as HIV) can also bind to a mammalian CCR4 protein. A natural ligand of a selected mammalian receptor has a mammalian origin that is the same as that of the mammalian CCR4 protein (eg, a chemokine such as TARC, MDC, MCP-1, MlP-la and / or RANTES) . In a preferred embodiment, ligand binding of a mammalian CCR4 protein occurs with high affinity. As used herein, an "inhibitor" is a substance that inhibits (reduces or prevents) at least one characteristic function of a mammalian CCR4 protein (e.g., a human CCR4), such as a binding activity (eg. example, ligand binding, promoter binding, antibody binding), a signaling activity (eg, activation of a mammalian G protein, induction of a rapid and transient increase in cytosolic free calcium concentration [Ca2 +]?) and / or a cellular response function (eg, stimulation of chemotaxis, exocytosis or release of inflammatory mediators by leukocytes). An inhibitor is also a substance that inhibits the entry of HIV into a cell. The term "inhibitor" refers to substances, including agonists, that bind to the receptor (e.g., an antibody, a mutant of a natural ligand, organic molecules of small molecular weight, other competitive inhibitors of ligand binding) and substances that inhibit the function of receptors without binding to them (for example, an anti-idiotypic antibody). As used herein, a "promoter" is a substance that promotes (induces, causes, reinforces or enhances) at least one characteristic function of a mammalian CCR4 protein (e.g., a human CCR4), such as a binding activity (eg, ligand binding, inhibitor and / or promoter), a signaling function (eg, activation of a mammalian G protein, induction of a rapid and transient increase in the concentration of cytosolic free calcium [Ca2 +] i) and / or a cellular response function (eg, stimulation of chemotaxis, exocytosis or release of inflammatory mediators by leukocytes). The term "promoter" refers to substances, including agonists, that bind to the receptor (e.g., an antibody, a homolog of a natural ligand of another species) and substances that promote receptor function without binding to them (e.g., activating an associated protein). In a preferred embodiment, the agonist is different from a homolog of a natural li-gand. Thus, the invention also relates to a method of detecting or identifying an agent that binds to a mammalian CC chemokine receptor 4 or ligand-binding variant thereof, including ligands, inhibitors, promoters and other substances that they bind to a mammalian CCR4 receptor or functional variant. According to the method, a study agent, an antibody or antigen-binding fragment of the present invention can be combined (for example, 1G1, 2B10, 10E4, an antibody having an epitopic specificity equal to or similar to that of 1G1). , 2B10 or 10E4 and antigen-binding fragments thereof) and a composition containing a mammalian CC chemokine receptor 4 or a ligand-binding variant thereof. The above components are combined under suitable conditions to bind the antibody or antigen-binding fragment to mammalian CC chemokine receptor 4 or a ligand-binding variant thereof and the binding of the antibody or fragment to receptor 4 is detected or measured. mammalian CC chemokines or ligand-binding variant directly or indirectly according to the methods described herein or other suitable methods. A reduction in the amount of complex formed relative to an adequate control (eg, in the absence of the agent to be studied) is indicative that the agent binds to said receptor or variant. The composition containing a mammalian CC chemokine receptor 4 or ligand-binding variant thereof can be a membrane fraction of a recombinant chemokine receptor 4 protein carrier cell or ligand-binding variant thereof. The antibody or fragment thereof can be labeled with a label such as a radioisotope, a spin tag, an antigenic or epitope tag, an enzymatic tag, a fluorescent group and a qui-miumuminescent group. In one embodiment, the invention relates to a method of detecting or identifying an agent that binds to a mammalian CC chemokine receptor 4 or ligand binding variant thereof, consisting of combining a study agent, a antibody or antigen-binding fragment of the present invention (for example, (for example, 1G1, 2B10, 10E4, an antibody having an epitope specificity equal to or similar to that of 1G1, 2B10 or 10E4 or antigen-binding fragments of these) and a carrier cell of a mammalian CC chemokine receptor 4 or a ligand-binding variant thereof The above components are combined under conditions suitable to bind the antibody or antigen binding fragment to the CCR4 protein or ligand-binding variant thereof and the binding of the antibody or fragment to the mammalian or variant CC chemokine receptor 4 is detected or measured directly or indirectly according to the methods described herein or other methods. Suitable methods A reduction in the amount of complex formed relative to an adequate control is indicative that the agent binds to the receptor or variant. The antibody or fragment thereof can be labeled with a label such as a radioisotope, a spin label, an antigenic or epitope label, an enzymatic label, a fluorescent group and a chemiluminescent group. These and similar assays can be used to detect agents, including ligands (e.g., chemokines or HIV strains that interact with CCR4) or other substances, including inhibitors or promoters of receptor function, which can bind to CCR4 and compete with the antibodies described herein for binding to the receptor. The assays described herein may be used, alone or in combination with each other or with other suitable methods, to identify ligands or other substances that bind to a mammalian CCR4 protein and inhibitors or promoters of a mammalian or variant CCR4 protein. The in vi tro methods of the present invention can be adapted for a high-throughput, selective study in which large numbers of samples are processed (e.g., a 96-well format). Cells expressing mammalian CCR4 (eg, human CCR4) can be used at levels suitable for high throughput study and, therefore, are particularly valuable in the identification and / or isolation of ligands or other substances that bind to the receptor and inhibitors or promoters of mammalian CCR4 proteins. The expression of the receptor 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 a portion thereof. In addition, commercial antibodies can be used to detect the expression of an antigen or epitope-labeled fusion protein containing a receptor protein or polypeptide (eg, FLAG-tagged receptors) and cells expressing the desired level can be selected. Nucleic acid encoding a mammalian CCR4 protein or functional variant thereof can be incorporated into an expression system to produce a receptor protein or polypeptide. An isolated and / or recombinant mammalian CCR4 protein or variant, such as a receptor expressed in cells transiently or stably transfected with a construct containing a recombinant nucleic acid encoding a mammalian or variant CCR4 protein, or in a fraction can be used. cellular receptor-containing (e.g., a membrane fraction of transfected cells, liposomes that incorporate receptor) in assays for receptor function. The receiver can still be purified if desired. Tests of the function of the receptors can be carried out in vi tro or in vivo. An isolated and / or recombinant mammalian CCR4 protein or functional variant thereof, such as a human CCR4, can be used in the present method, where the effect of a compound is evaluated by monitoring the function of the receptors as described herein. or using other appropriate techniques. For example, stable or transient transfectants (e.g., Sf9 cells infected with crosier-virus, stable transfectants of pre-B Ll. 2 ra-ton cells) can be used in binding assays. Stable transfectants from Jurkat cells or other suitable cells capable of chemotaxis (eg, pre-B cells Ll.sub.2 from mice) can be used in chemotaxis assays, for example. According to the method of the present invention, the compounds can be studied individually or one or more compounds can be studied simultaneously according to the methods described herein. When studying a mixture of compounds, they can be separated (as appropriate) and the compounds selected by the methods described identified by suitable methods (eg, PCR, sequencing, chromatography, mass spectroscopy). The presence of one or more compounds (e.g., a ligand, inhibitor, promoter) in a test sample can also be determined according to these methods.

Large combinatorial libraries of compounds (eg, organic compounds, re-combining or synthetic peptides, "peptoids", nucleic acids) produced by combinatorial chemical synthesis or other methods can be studied (see, for example, Zuckerman, RN et al., J. Med. Chem. 37: 2678-2685 (1994) and the references cited herein, see also Ohlmeyer, MHJ et al., Proc. Nati, Acad. Sci. USA 90: 10922-10926 (1993), and DeWitt, SH et al., Proc. Nati, Acad. Sci. USA 90: 6909-6913 (1993), in relation to labeled compounds, Rutter, WJ et al., US Patent No. 5,010,175; Huebner , VD et al., U.S. Patent No. 5,182,366, and Geysen, HM, U.S. Patent No. 4,833,092). When the compounds selected from a combinatorial library by the present method carry unique labels, it is possible to identify individual compounds by chromatographic methods. In one embodiment, phage display methodology is used. For example, a mammalian or functional variant CCR4 protein, an antibody or functional portion thereof of the present invention and a phage (e.g., a phage or phage collection, such as a library) that exhibits a polypeptide under appropriate conditions for binding of the antibody or portion thereof to the mammalian CCR4 protein or variant (eg, in a suitable binding buffer). Phages that compete with the antibody or portion thereof and bind to the mammalian CCR4 protein or variant can be detected or selected using standard techniques or other suitable methods. The bound phages can be separated from the receptor using a suitable elution buffer. For example, a change in ionic strength or pH can result in phage release. Alternatively, the elution buffer can contain a release component or components designed to alter the binding of the compounds (e.g., one or more compounds that can alter the binding of the displayed peptide to the receptor, such as a ligand, inhibitor and / or promoter that competitively inhibits the binding). Eventually, the selection procedure may be repeated or another selection step may be used to further enrich for phages that bind to the recipient. The polypeptide displayed can be characterized (for example, by sequencing the phage DNA). The identified polypeptides can be produced and studied further in terms of binding and in terms of inhibitory or promoter function. Analogs of said peptides having greater stability or other desirable properties can be produced. In one embodiment, fusion proteins can be produced that express and display phages, consisting of a shell protein with an N-terminal peptide encoded by random sequence nucleic acids. Suitable host cells expressing a mammalian or variant CCR4 protein and an anti-CCR4 antibody or functional portion thereof are combined with the phage, the bound phages are selected, recovered and characterized. (See, for example, Doorbar and Win-ter, J. Mol. Biol. 244: 361 (1994), which discusses a method of displaying phage used with a G-protein coupled receptor, and WO 97/08320 (Morphosys) , published on March 6, 1997). Other sources of potential ligands or other substances that can bind to, or inhibitors and / or promoters of, mammalian CCR4 proteins include, but are not limited to, CCR4 ligand variants, including natural, synthetic or recombinant variants of TARC, MDC, MCP-1, MlP-la and / or RANTES, substances such as other chemoattractants or chi-miokines, variants thereof, low molecular weight organic molecules, other inhibitors and / or promoters (eg, anti-CCR4 antibodies, antagonists , agonists), other ligands, inhibitors and / or promoters of G protein-coupled receptors (eg, antagonists or agonists) and soluble portions of a mammalian CCR4 receptor, such as a suitable receptor or analog peptide that can inhibit the function of the receptors (see, for example, Murphy, R.B., WO 94/05695). Inflammation models In vivo models of inflammation are available that can be used to assess the effects of antibodies and fragments of the invention in vivo as therapeutic agents. For example, leukocyte infiltration can be monitored after intradermal injection of a chemokine and an antibody or fragment thereof reactive with mammalian CCR4 in a suitable animal, such as rabbit, mouse, rat, guinea pig or rhesus macaque (see, for example, example, Van Damme, J. et al., J ". Exp. Med. 176: 59-65 (1992); Zachariae, C.O.C. et al., J. Exp. Med. 171: 2177-2182 (1990); José, P.J. et al., J. Exp. Med. 179: 881-887 (1994)). In one embodiment, skin biopsies are histologically evaluated for leukocyte infiltration (e.g., eosinophils, granulocytes). In another embodiment, labeled animals (eg, stably transfected cells expressing a mammalian CCR4, labeled with 111 In, for example) capable of chemotaxis and extravasation are administered to the animal. For example, an antibody or fragment to be studied may be administered, either before, simultaneously or after administration of the ligand or agonist to the test animal. A reduction in the degree of infiltration in the presence of the antibody compared to the degree of infiltration in the absence of an inhibitor is indicative of inhibition. Diagnosis and therapeutic applications The antibodies and fragments of the present invention are useful in a variety of applications, including research, diagnostic and therapeutic applications. In one embodiment, the antibodies are labeled with suitable labeling (e.g., fluorescent labeling, chemiluminescent labeling, isotopic labeling, antigenic or epitope labeling, or enzyme labeling). For example, they can be used to isolate and / or purify receptors or portions thereof and to study the structure (e.g., conformation) and function of the receptors. In addition, the various antibodies of the present invention can be used to detect CCR4 or to measure expression of the receptor, for example, on T cells (e.g., CD8 + cells, CD45RO + cells), monocytes and / or on cells transfected with a receptor gene. Thus, they are also useful in applications such as cell sorting (eg, flow cytometry, fluorescence activated cell sorting), for diagnostic or research purposes. The anti-CCR4 antibodies of the present invention have value in diagnostic applications. An anti-CCR4 antibody or fragment thereof can be used to monitor the expression of this receptor in individuals, similar to how the anti-CDR has been used as a diagnostic indicator of the HIV phase. Typically, diagnostic assays involve detection of the formation of a complex resulting from the binding of an antibody or fragment thereof to CCR4. For diagnostic purposes, the antibodies or antigen binding fragments may not be labeled or untagged. The antibodies or fragments can be directly labeled. A variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands-two (eg, biotin, haptens). Numerous suitable immunoassays are known to the person skilled in the art (see, for example, US Patents 3,817,827, 3,850,752, 3,901,654 and 4,098,876). When they are not labeled, the antibodies or fragments can be detected using suitable means, as in agglutination tests, for example. Antibodies or unlabeled fragments may also be used in combination with another (i.e., one or more) suitable reagent that can be used to detect antibodies, such as a labeled anti-body (e.g., a second antibody) reactive with the first antibody (e.g., anti-idiotype antibodies or other antibodies that are specific for unlabeled immunoglobulin) or other suitable reagent (e.g., labeled A protein). In one embodiment, the antibodies or fragments of the present invention can be used in enzyme immunoassays, where the antibody or fragment in question, or the second antibodies, are conjugated to an enzyme. When a biological sample containing a mammalian CCR4 protein is combined with the antibodies in question, the binding between the antibodies and the CCR4 protein occurs. In an embodiment, a sample containing cells expressing a mammalian CCR4 protein, such as human blood, is combined with the antibodies in question and binding occurs between the antibodies and the cells carrying a human CCR4 protein containing a recognized epitope. by the antibody. These bound cells can be separated from the unbound reagents and the presence of the antibody-enzyme conjugate specifically linked to the cells can be determined, for example, by contacting the sample with a substrate of the enzyme that produces a color or other detectable change. when the enzyme acts on it. In another embodiment, the subject antibodies may be unlabelled and a labeled second antibody recognizing the target antibody may be added. Kits may also be prepared for use in detecting the presence of a mammalian CCR4 protein in a biological sample. Such kits will include an antibody or functional fragment thereof that binds to a mammalian CC chemokine receptor 4 or portion of said receptor, as well as one or more auxiliary reagents suitable for the detection of the presence of a complex between the antibody or fragment and CCR4 or portion thereof. The antibody compositions of the present invention may be provided in lyophilized form, either alone or in combination with additional antibodies specific for other epitopes. Antibodies, which may be labeled or unlabelled, may be included in the kits with accessory ingredients (e.g., buffers, such as Tris, phosphate and carbonate, stabilizers, excipients, biocides and / or inert proteins, e.g. seroal-butanol bovine). 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. In general, these accessory materials will be present in less than about 5% by weight, based on the amount of the 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 monoclonal antibody is employed, said antibody can be provided in the kit, for example in a separate vial or container. The second antibody, if present, is typically labeled and can be formulated in a manner analogous to the antibody formulations described above. Similarly, the present invention also relates to a method of detecting and / or quantifying the expression of a mammalian CCR4 or a portion of the receptor by a cell, wherein a composition containing a cell or fraction is contacted. of the same (e.g., membrane fraction) with an antibody or functional fragment thereof (e.g., 1G1 and / or 2B10 and / or 10E4) that binds to a mammalian CCR4 or portion of the receptor under conditions appropriate for binding of the antibody or fragment thereto and the binding is monitored. Detection of the antibody, indicative of the formation of a complex between the antibody and CCR4 or portion thereof, indicates the presence of the receptor. The binding of antibody to the cell can be determined as described above under the heading "Binding assays", for example. The method can be used to detect the expression of CCR4 in cells of an individual (e.g., in a sample, such as a body fluid, such as blood, saliva or other suitable sample). The level of expression of CCR4 on the surface of T cells or monocytes can also be determined, for example, by flow cytometry and the level of expression (e.g., binding intensity) can be correlated with susceptibility to the disease, its progression or its risk. The chemokine receptors function in the migration of leukocytes throughout the body, particularly to inflammatory sites. The emigration of inflammatory cells from the vasculature is regulated by a three-step process involving interactions of leukocytes and adhesion proteins of endothelial cells and cell-specific chemoattractants and activation factors.

(Springer, T.A., Cell 76: 301-314 (1994); Butcher, E.C., Cell 67: 1033-1036 (1991); Butcher, E.C. and Picker, L.J., Science (Wash. DC) 272: 60-66 (1996)). These are: (a) a low affinity interaction between leukocyte selectins and the carbohydrates of endothelial cells, (b) a high affinity interaction between chemoattractant leukocyte receptors and chemoattractant / activating factors and (c) a narrow binding between the integrins of the leukocytes and the adhesion proteins of the endothelial cells of the immunoglobulin superfamily. Different subgroups of leukocytes express different repertoires of selectins, chemoattractant receptors and integrins. Additionally, inflammation affects the expression of endothelial adhesion proteins and the expression of chemoattractant and leukocyte activating factors. As a consequence, there is great diversity to regulate the selectivity of the re-clumping of leukocytes to the extravascular sites. The second stage is crucial, in the sense that the activation of the chemoattractant receptors of the leukocytes is thought to cause the transition from selectin-mediated cell coiling to close binding mediated by integrists. This results in the leukocyte being ready to transmigrate to the perivascular sites. The chemoattractant / chemoattractant receptor interaction is also crucial for transendothelial migration and localization in a tissue (Campbell, JJ et al., J. Cell Biol. 134: 255-266 (1996); Carr, MW et al., Immuni ty 4: 179-187 (1996)). This migration is directed by a concentration gradient of chemoattractant that directs towards the inflammatory focus. The CCR4 has an important role in leu-cocitus traffic. It is likely that CCR4 is a key chemokine receptor for the migration of T cells or subsets of T cells or monocytes to certain inflammatory sites and, therefore, anti-CCR4 mAbs can be used to inhibit (reduce or prevent) migration of T cells or monocytes, particularly that associated with T cell dysfunction, such as autoimmune diseases or allergic reactions, or disorders mediated by monocytes, such as atherosclerosis. Accordingly, the antibodies and fragments thereof of the present invention can also be used to modulate the function of the receptors in research and therapeutic applications. For example, the antibodies and functional fragments described herein can act as inhibitors to inhibit (reduce or prevent) (a) the binding (eg, of a ligand, an inhibitor or a promoter) to the receptor, (b) a function signaling of the receiver and / or (c) a stimulating function. Antibodies that act as inhibitors of receptor function can block the binding of the ligand or the promoter directly or indirectly (for example, by causing a conformational change). For example, antibodies can inhibit the function of the receptors by inhibiting the binding of a ligand, or by desensitization (with or without inhibition of the binding of a ligand). Antibodies that bind to the receptor can also act as agonists of receptor function, triggering or stimulating a function of the receptors, such as a signaling function and / or stimulation of a receptor (eg, leukocyte trafficking) to join the receiver. Therefore, the present invention provides a method of inhibiting leukocyte trafficking in a mammal (e.g., a human patient), comprising administering to the mammal an effective amount of an antibody or functional fragment of the present invention. Administration of an antibody or fragment of the present invention may result in the amelioration or elimination of the disease state. The antibody of the present invention, or a functional fragment thereof, can also be used to treat disorders in which the activation of the CCR4 receptor is involved by chemokine binding. For example, antibodies or functional fragments thereof (eg, 1G1 and / or 2B10 and / or 10E4 or functional fragments thereof) can be used to treat allergy, atherogenesis, anaphylaxis, malignancies, chronic and acute inflammation, allergic reactions mediated by histamine and IgE, shock and rheumatoid arthritis, atherosclerosis, multiple sclerosis, allograft rejection, fibrotic disease, asthma and inflammatory glomerulopathies. Among the diseases or conditions of humans or other species that can be treated with inhibitors of the CCR4 receptor function (including antibodies or suitable fragments thereof), include, but are not limited to: • inflammatory or allergic diseases and conditions, including respiratory allergic diseases such as asthma, allergic rhinitis, pulmonary hypersensitivity diseases, hypersensitivity pneumonitis or interstitial lung diseases (EPI) (for example, idiopathic pulmonary fibrosis or PID associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, of Sjogren, polymyositis or dermatomyositis); anaphylaxis or hypersensitivity responses, drug allergies (for example, to penicillin or cephalosporins) or allergies to insect bites; inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis; spondyloarthropathies; scleroderma; inflammatory psoriasis and dermatoses, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis or urticaria; vasculitis (for example, necrotizing, cutaneous and hypersensitivity vasculitis); • autoimmune diseases, such as arthritis (for example, rheumatoid arthritis or psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, juvenile diabetes, nephritis such as glomerulonephritis, autoimmune thyroiditis or Behcet's disease; • rejection of grafts (for example, in a transplant), including allograft rejection or graft versus host disease; • atherosclerosis; • cancers with leukocyte infiltration of the skin or organs; • other diseases or conditions (including diseases or conditions mediated by CCR4) can be treated where undesirable inflammatory responses must be inhibited, including, but not limited to, reperfusion injury, certain hematologic malignancies, induced cytotoxicity by cytokines (for example, septic shock or endotoxic shock), polymyositis, dermatomyositis, and granulomatous diseases, including sarcoidosis. Diseases or conditions of humans or other species that can be treated with promoters of CCR4 receptor function (including antibodies or fragments thereof) include, but are not limited to: • immunosuppression, such as that which occurs in indivi-duos with immunodeficiency syndromes, such as AIDS, individuals undergoing radiation therapy, chemotherapy, therapy for autoimmune diseases or other drug therapy (eg, corticosteroid therapy) that causes immunosuppression, and immunosuppression due to a congenital deficiency in function of the recipients or other causes. The anti-CCR4 antibodies of the present invention can block the binding of one or more chemokines, thus blocking the downstream cascade of one or more events leading to the above disorders. Modes of administration One or more antibodies or fragments of the present invention can be administered to an individual by an appropriate route, either alone or in combination (before, simultaneously or after) with another drug or agent. For example, the anti-bodies of the present invention can be used in combination with other monoclonal or polyclonal antibodies (eg, in combination with antibodies that bind to other chemokine receptors, including, but not limited to, CCR3 and CCR5) or with existing blood plasma products, such as the commercialized products of gamma globulins and immunoglobulins used in prophylactic or therapeutic treatments. The antibodies or fragments of the present invention can be used as separately administered compositions given together with antibiotics and / or antimicrobial agents. An effective amount of an antibody or fragment (ie, one or more antibodies or fragments) is administered. An effective amount is an amount sufficient to achieve the desired therapeutic effect (including prophylactic) under the conditions of administration, such as an amount sufficient for the inhibition of a CCR4 function and, thus, for the inhibition of an inflammatory response or HIV infection, or an amount sufficient to promote a CCR4 function, as indicated. A variety of routes of administration are possible, including, but not necessarily limited to, oral, dietetic, topical, parenteral (e.g., intravenous, intraarterial, intramuscular or subcutaneous injection) and inhalation (e.g., intrabronchial, intraocular, intranasal or oral inhalation or intranasal drops), depending on the disease or condition that is to be treated. Other suitable methods of administration may also include rechargeable or biodegradable devices and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combination therapy with other agents. The formulation of an antibody or fragment to be administered will vary according to the route of administration and the formulation (eg, solution, emulsion, capsule) selected. An appropriate pharmaceutical composition containing an antibody or functional fragment thereof that is to be administered in a physiologically acceptable carrier or carrier can be prepared. A mixture of antibodies and / or fragments can also be used. For solutions or emulsions, suitable supports include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution.

Ringer dextrose, dextrose and sodium chloride, lactated Ringer or fixed oils. A variety of suitable aqueous supports are known to the person skilled in the art, including water, buffered water, buffered saline, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives or replenishers of fluids, nutrients or electrolytes (see, generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, Ed., 1980). The compositions may optionally contain pharmaceutically acceptable excipients as necessary to approximate physiological conditions, such as pH adjusting agents and buffers and tonicity adjusting agents, for example sodium acetate, sodium chloride, chloride potassium, calcium chloride and sodium lactate. The antibodies and fragments of this invention can be lyophilized for storage and reconstituted on a suitable support before use according to lyophilization and reconstitution techniques known in the art. The optimum concentration of the active component (s) in the chosen medium can be determined empirically according to procedures well known to the person skilled in the art and will depend on the desired final pharmaceutical formulation. For inhalation, the antibody or fragment can be solubilized and loaded in a dispenser suitable for administration (eg, an atomizer, nebulizer or pressurized aerosol dispenser). The invention will now be further illustrated by the following examples, which are not intended to limit the scope of the invention. The teachings of all the references cited here are here incorporated in full. EXAMPLES Material and methods Construction of suitable CCR4 transfectants CCR4 cDNA was obtained by PCR (polymerase chain reaction) using a 5'-oligonucleotide primer (5'-CCAACCAAGCTTATGAACCCCACGGATATAGCAG-3 '; SEQ ID NO: 1) and a 3' primer oligonucleotide (5'-CCAACCTCTAGATTAGAGCATCATGGAGATCATGATCC-3 '; SEQ ID NO: 2), which contained HindIII and Xbal flanking sites, respectively. The PCR fragment was subcloned into the HindIII and Xbal sites of pMRBIOl, where the inserted gene was driven by a CMV promoter. The DNA was stably transfected into a line of murine pre-B lymphoma cells (L1.2 or Ll / 2) as described (Ponath et al., J. Exp. Med. 183: 2437 (1996); Wu et al., J ". Biol. Chem. 271: 31202 (1996); Wu et al., Nature 384: 179 (1996).) Cells expressing high levels of CCR4 were selected by serial dilution / subcloning as soon as possible. to their ability to undergo chemotaxis to TARC and MDC For the production of monoclonal antibodies, the cells were treated with 5 mM butyric acid for 16-18 hours and used to immunize mice Cells and cell lines Venous blood was collected from voluntary donors and PBMCs (peripheral blood mononuclear cells) were isolated by ficoll-hypaque density gradient centrifugation as described (Ponath et al., J. "Exp. Med. 183: 2437 (1996)). Other cell lines used included transfectants from murine L-2 pre-B lymphoma cells expressing various chemokine receptors or orphan receptors coupled to protein G. Human peripheral blood was collected in 10% (v / v) EDTA 0, 1 M, was deposited on a gradient of 1-Step Poly-morphs (Accurate Chemical Co., NY) and centrifuged at 400xg for 30 minutes at room temperature. The neutrophil and mononuclear cells layers were harvested, resuspended in DPBS without calcium and magnesium (Life Technologies, Grand Island, NY) and centrifuged for 15 minutes at -750xg. The red blood cells were lysed in the neutrophil fraction by resuspending the pellet in E-Lyse (Cardinal Associates, Santa Fe, NM) for 5 minutes on ice. Both cell fractions were washed 2 times with ice-cold DPBS. CD14-positive monocytes were separated from peripheral mononuclear cells by incubation for 30 minutes at 4 ° C of 107 mononuclear cells in PBS with 1% BSA and 5 mM EDTA at 5 × 10 7 cells / ml with CD14 Miltenyi beads (Miltenyi Biotech Auburn, CA), where 20 μl of beads per 107 mononuclear cells were used. They were centrifuged, 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) in a magnetic field to remove unlabeled cells. The cells were separated by forcing 5 ml of PBS, 1% BSA, 5 mM EDTA, on the VS column, out of the magnetic field. The procedure was repeated using CD4 Miltenyi beads to isolate the CD4 lymphocytes, which were incubated overnight in DMEM, 2 nM glutamine, 50 U / ml penicillin, 50 ug / ml streptomycin, 1 nM sodium pyruvate MEM, 10 nM Hepes. all from Gibco BRL, Grand Island, NY 14072) with 10% FCS (Hyclone, Utah 84321) before use in chemotaxis assays. Preparation of chronically activated THl and TH2 lymphocytes Six-well tissue culture plates (Falcon 3046, Beckton Dickinson Labware, Franklin La-kes, NJ) were coated overnight with 10 μg / ml anti-CD28 (Beckton Dickinson) and 2 μg / ml of OKT3 (American Type Culture Collection, Manassas, VA) and then washed twice with PBS. CD4 lymphocytes from umbilical cord blood were cultured (Poietic Systems, Germán Town, MD) at 105-106 cells / ml in DMEM, 2 nM glutamine, 50 U / ml penicillin, 50 ug / ml streptomycin, MEM pyruvate sodium 1 nM , Hepes 10 nM (all from Gibco BRL, Grand Island, NY) with 10% FCS (Hyclone, Utah) and then used IL-2 (4 ng / ml), IL-12 (5 ng / ml) and anti-IL4 (1 mg / ml) to direct towards TH1, while IL-4 (5 ng / ml) and anti-IFN gamma (1 μg / ml) were used to direct towards TH2. After 4-5 days, activated THL and TH2 lymphocytes were washed once in DMEM and cultured for 4-7 days in DMEM with 10% FBS and IL-2 (1 ng / ml). After this, activated THL and TH2 lymphocytes were re-stimulated for 5 days with anti-CD28 / OKT3 and cytokines as described above, but with the addition of anti-CD95L (1 mg / 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 TH1 and TH2 lymphocytes were activated in this way for a maximum of three cycles. All cytokines were obtained from R and D Systems (Minneapolis, MN), while anti-IL4, anti-CD95L and anti-IFN gamma were obtained from Pharmingen (San Diego, CA). Generation of anti-CCR4 monoclonal antibodies, in-munofluorescent staining and FACS® analysis. Reactive mAbs with CCR4 were generated by immunizing mice with Ll2 cells expressing high levels of trans-fected CCR4. Six female C57BL6 mice were immunized with 10 7 cells intraperitoneally 8-12 times, at 2 week intervals, and six fusions were performed in an attempt to identify the specific mAbs of CCR4. Specifically, four days after an intravenous injection of CCR4 / L1.2 cells, the spleen was excised and the cells were fused with the SP2 / 0 cell line as described (Coligan et al., Current Protocols in Immunology, John Wiley and Sons, New York (1992)). In general, 3,000-5,000 hybrid-plus were studied per fusion. In one of the six mergers, an anti-CCR4 mAb was detected; this mAb was designated 1G1 (IgGl). In seven additional fusions, a second anti-CCR4 mAb was detected; this mAb was designated 2B10 (IgG2a). Another anti-CCR4 mAb was also identified and it was called 10E4 (IgGl). The 1G1, 2B10 and 10E4 hybridomas can be cultured in DMEM, 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate and 100 ng / ml IL-6, as well as penicillin (50 units / ml) and streptomycin. (50 ug / ml). 1G1, 2B10 and 10E4 were studied in terms of reactivity against numerous L1.2 transfectants that express chemokine receptors or orphan receptors coupled to protein G. Mabs conjugated to PE were obtained for CD4, CD8, CD14, CD20, CD25, CD26, CD69, CD45RO and CD45RA from Beckton Dickinson (San José, CA). Similar mAbs, as well as anti-CD95 PE, Cy-Chrome anti-CD3 and Cy-Chrome anti-CD4, were supplied by Pharmingen (La Jolla, CA). To assess the reactivity of the mAbs against transfected cells or leukocytes, indirect immunofluorescence and flow cytometry were used. The cells were washed once with PBS and resuspended in 100 μl of PBS containing 5% human serum and 0.1% sodium azide (staining buffer), 5 μg / ml purified antibody, 5 μg / ml. ml of MOPC21 control mAb with IgGl isotype mapping (Sigma Chemical Co., St. Louis, MO) or 50 ml of hybridoma culture supernatant. After 20 minutes at 4 ° C, the cells were washed twice with staining buffer and resuspended in 50 ml of affinity purified F (ab ') 2 goat anti-mouse IgG conjugated to FITC (Jackson ImmunoResearch Laboratories, West Grove, PA). After incubating for 20 minutes at 4 ° C, the cells were washed once in staining buffer and analyzed on the FACScan® to determine the level of surface expression. Tissues and Immunohistochemistry Normal human mediastinal lymph nodes were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA). Immunohistochemical analysis for CCR4 was performed on frozen tissue samples using previously described techniques (Silber et al., Lab. Invest. 70: 163 (1994)). Anti-CCR4 1G1 mAb (10 μg / ml in 0.3% Triton X 100 / 0.2% Tween 20/1% FCS / 5% human AB serum, 0.1% sodium azide) was applied to tissue sections , which were incubated overnight at 4 ° C. An irrelevant mAb with isotype correspondence (MOPC21; Sigma, St Louis, MO) was used at the same concentration as a negative control in serial sections of mediastinal nodule. Next, biotinylated goat anti-mouse IgG and avidin-biotin-alkaline phosphatase complexes were sequentially added (Biogenex, San Ramon, CA). Solid Red (Biogenex, San Ramón, CA) was used, which contained levamisole to block the activity of the endogenous alkaline phosphatase, as a chromogen, and Mayers' hematoxylin was used as contratinction. 125I-TARC binding Human TARC labeled with 125I was purchased from DuPont NEN (Bos-ton, MA) and unlabeled chemokines were from Peprotech (Rocky Hill, NJ) or R &D Systems (Minneapolis, MN). The binding of chemokines to the target cells was carried out using the following procedure: CCR4 / L1.2 cells were washed and resuspended in binding buffer (50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCl2 and 0.5% BSA) at 107 / ml. For each binding reaction (in a final volume of 100 μl), 25 μl of cell suspension (2.5 x 10 5 cells) was mixed with radiolabelled 0.1 nM chemokine with or without an appropriate amount of anti-CCR4 mAb , or a control mAb with isotypic correspondence. The total binding was terminated in the presence of only ra-di-labeled chemokines and the non-specific binding (background) was determined in the presence of 100 nM unlabeled chemokines. The reactions were incubated at room temperature for 45-60 minutes and stopped by transferring the mixture to GFB filter plates, which were then washed 2-3 times with binding buffer containing 0.5 M NaCl. The plates were dried and MicroScint scintillation fluid was added before counting. Each sample was studied in duplicate. The results are shown in Figure 5. The IC50 value for 2B10 was approximately 1 ng / ml and the IC50 for 1G1 was approximately 1 μg / ml (Kaleidagraph program). Chemotaxis assays Chemotaxis was performed with transfectant cells CCR4 / L1.2 and the Peer cell line as previously described (Wu et al., J. Biol. Chem. 271: 31202 (1996); Wu et al., J "Exp. Med. 186: 1373 (1997).) To explain it briefly, Transpocillo inserts of 3 μM pore diameter were used from Costar (Costar, MA). Chemokine was added at 100 ng / ml in 0.5 ml of RPMI, 0.5% BSA, 10 mM Hepes, to the bottom well Cells were washed once in RPMI and resuspended at 4 x 106 cells / ml in RPMI, 0.5% BSA and 10 mM Hepes In some cases, mAbs at 50 μg / ml were added to the cell suspension and allowed to bind for 10 minutes at 4 ° C. An aliquot of 200 μl of cell suspension was added (2 × 10 6 cells inlet). ) to each insert After 2 to 4 hours at 3 ° C in a 5% C0 incubator, the inserts were removed from the plates and the cells migrating to the bottom chamber of the Transpocillo were enumerated. using the FACScan®, counting the cells for 30 seconds. Dispersion gates of front and side angle were established to exclude remains or irrelevant cells. The IC50 value for inhibition by 1G1 of chemotaxis at MDC and TARC is approximately 0.25 μg / ml. Results CCR4 is a chemokine receptor expressed on T cells that can be specifically activated by TARC and MDC. To further investigate the expression and functions of CCR4, monoclonal antibodies (1G1, 2B10 and 10E4) were generated against the human CCR4 receptor. Monoclonal antibody 1G1 stains CCR4 / L1.2 transfectants, but not to a panel of more than 20 different L1.2 transfectants that express other chemokine receptors or orphan receptors coupled to G protein (Figure 1). The monoclonal antibody 2B10 reacts with the transfectants L1.2 CCR4, but not with two other transfectants of chemokine receptors studied so far. The monoclonal antibody (mAb) 1G1 stains at -15% of the peripheral blood CD4 + lymphocytes, but to very few CD8 + T lymphocytes (Figures 2 and 3). CCR4 is expressed in a subset of activated memory T cells; it is expressed in most of the CLA + / a4b7- cells, but not in the CLA- / a4b7 + cells. Most significantly, mAb 1G1 specifically stains Th2 cells derived in vi tro, but not Thl, whereas CXCR3 is expressed in most Thl cells, but not Th2 and CCR2 is expressed in both cell types (Figure 4 ). In addition, preliminary immunohistochemistry studies indicate that CCR4 can be detected in subgroups of T cells, macrophages and endothelium in human tonsils and several other tissues. 1G1 mAb inhibits the binding of 125I-labeled TARC to CCR4 / L1.2 transfectants (Figure 5), as well as the chemotaxis of these cells at TARC and MDC (Figures 6A-6D, 7A-7B, 8, 9 and 10). Monoclonal antibody 2B10 was found, which stained transfectants Ll.2 CCR4, but not parental line Ll.2 or two other transfectants of chemokine receptors (gpr-9-6 and V28). As seen with 1G1, 2B10 was found to stain a subset of approximately 15% of the peripheral CD4 lymphocytes, but very few CD8 lymphocytes. Lymphocytes CD19 and NK cells were not stained by 2B10. The CD4 lymphocytes stained by 2B10 were a subset of memory CD4 lymphocytes, as defined by the expression of CD45RO. As observed with 1G1, it was seen that 2B10 reacted selectively with Th2 lymphocytes derived in vitro, but not with Thl lymphocytes derived in vitro. In the chemotaxis assays, 2B10 significantly blocked the chemotaxis of peripheral blood CD4 lymphocytes and Th2 lymphocytes derived in vitro to MDC and TARC. Overall, 10E4 proved to be the best anti-CCR4 blocking monoclonal antibody, followed by 2B10 and then by 1G1 (Figures 10A and 10B). These combined results suggest that CCR4 and its ligands may play an important role in inflammatory responses mediated by Th2 cells and in the arrival of T lymphocytes to the skin, but not to mucous membranes. Although this invention has been particularly shown and described with reference 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 as defined by the invention. attached claims.

LIST OF SEQUENCES < 110 > LeukoSite, Inc. Wu, Lijun Ruffing, Nancy Andrew, David < 120 > ANTI-CCR4 ANTIBODIES AND METHODS OF USING THE SAME < 130 > 1855.1063002 PCT < 150 > 09 / 231.759 < 151 > 01-15-1999 < 160 > 2 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 34 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > oliqonucleot primer < 400 > 1 ccaaccaagc ttatgaaccc cacggatata gcag 34 < 210 > 2 < 211 > 38 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > oligonucleotide primer < 400 > 2 ccaacctcta gattagagca tcatggagat catgatcc 38

Claims (45)

Claims

1. An antibody or antigen-binding fragment thereof that binds to a mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor .

2. An antibody or antigen-binding fragment according to Claim 1, wherein said antibody or antigen-binding fragment thereof inhibits one or more functions associated with the binding of the ligand to the receptor.

3. An antibody or antigen-binding fragment thereof according to Claim 1, wherein the mammalian chemokine CC receptor (CCR4) 4 is a human chemokine CC (CCR4) receptor 4.

4. An antibody or antigen-binding fragment thereof according to Claim 1, wherein the antibody is selected from the group consisting of: a) monoclonal antibody 1G1, b) an antibody having an epitope specificity equal to or similar to that of 1G1, c ) an antibody that can compete with 1G1 for binding to chemokine CC receptor 4 (CCR4), d) monoclonal antibody 2B10, e) an antibody that has an epitope specificity equal to or similar to that of 2B10, f) an antibody that can compete with 2B10 for binding to chemokine CC receptor 4 (CCR4), g) monoclonal antibody 10E4, h) an antibody having an epitope specificity equal to or similar to that of 10E4, i) an antibody that can compete with 10E4 for binding to mammalian chemokine CC (CCR4) receptor 4 and j) antigen binding fragments of any of (a) to (i) that bind to mammalian chemokine CC receptor 4 (CCR4) or a portion of same.

5. An antibody or antigen-binding fragment thereof according to Claim 1, wherein the ligand is a chemokine.

6. An antibody or antigen-binding fragment thereof according to Claim 5, wherein the chemokine is any one or more of TARC, MDC, MCP-1, MlP-la and RANTES.

7. The 1G1 hybridoma cell line deposited under ATCC Accession No. HB-12624.

8. The hybridoma cell line 2B10 deposited under ATCC Accession No. HB-12625.

9. A monoclonal antibody or antigen-binding fragment thereof produced by the hybridoma cell line according to Claim 7.

10. A monoclonal antibody or antigen-binding fragment thereof produced by the hybridoma cell line according to Claim 8.

11. A test kit for use in detecting the presence of a mammalian chemokine CC (CCR4) receptor 4 or portion thereof in a biological sample, consisting of: a) at least one antibody or antigen-binding fragment thereof which binds to a mammalian chemokine CC (CCR4) receptor 4 or 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 suitable auxiliary reagents for detecting the presence of a complex between said antibody or antigen-binding fragment thereof and said mammalian chemokine CC (CCR4) receptor 4 or portion thereof.

12. A test kit according to Claim 11, the antibody is selected from the group consisting of: i) monoclonal antibody 1G1, ii) an antibody having an epitope specificity equal to or similar to that of 1G1, iii) an antibody that can compete with 1G1 for binding to mammalian chemokine CC 4 receptor (CCR4), iv) monoclonal antibody 2B10, v) an antibody that has an epitopic specificity equal to or similar to that of 2B10, vi) an antibody that can compete with 2B10 by binding to mammalian chemokine CC (CCR4) receptor 4, vii) monoclonal antibody 10E4, viii) an antibody having an epitope specificity equal to or similar to that of 10E4, ix) an antibody that can compete with 10E4 for binding to mammalian chemokine CC (CCR4) receptor 4 and x) antigen-binding fragments of any of (i) to (ix) that bind to mammalian CC chemokine receptor 4 (CCR4) or a portion thereof.

13. A method of inhibiting the interaction of a mammalian chemokine CC receptor 4 (CCR4) carrier cell with a ligand thereof, comprising contacting said cell with an effective amount of an antibody or antigen-binding fragment of the mammalian antigen. same that binds to the mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor and inhibits the binding of said ligand to the receptor.

14. A method according to Claim 13, wherein the cell is selected from the group consisting of lymphocytes, monocytes, granulocytes, T cells, basophils, and cells that contain a recombinant nucleic acid encoding CCR4 or a portion thereof.

15. A method according to Claim 14, wherein the T cells are selected from the group consisting of CD8 + cells, CD25 + cells, CD4 + cells and CD45R0 + cells.

16. A method according to Claim 13, wherein the li-gand is a chemokine.

17. A method according to Claim 16, wherein the chemokine is any one or more of TARC, MDC, MCP-1, MlP-la and RANTES.

18. A method according to Claim 13, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: a) monoclonal antibody 1G1, b) an antibody having an epitope specificity equal to or similar to that of 1G1, c ) an antibody that can compete with 1G1 for binding to chemokine CC receptor 4 (CCR4), d) monoclonal antibody 2B10, e) an antibody having an epitopic specificity equal to or similar to that of 2B10, f) an antibody which can compete with 2B10 for binding to mammalian chemokine CC (CCR4) receptor 4, g) monoclonal antibody 10E4, h) an antibody having an epitope specificity equal to or similar to that of 10E4, i) an antibody that can compete with 10E4 by binding to mammalian chemokine CC (CCR4) receptor 4 and j) antigen-binding fragments of any of (a) to (i) that bind to mammalian chemokine CC receptor 4 (CCR4) or a portion of the m ism.

19. A method of detecting the expression of mammalian chemokine CC (CCR4) receptor 4 or portion thereof by a cell or fraction of said cell, consisting of: a) contacting a composition containing a cell or fraction of said cell which is to be studied with an antibody or antigen-binding fragment thereof which binds to the mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor and inhibits binding of said ligand to the receptor, under conditions appropr to the binding of said antibody or antigen-binding fragment thereof to a mammalian CCR4 or portion thereof, and b) detecting the binding of said antibody or binding fragment thereof, wherein the binding of said antibody or fragment of binding to an Tigen thereof indicates the presence of said receptor or portion of said receptor on said cell.

20. A method according to Claim 19, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: i) monoclonal antibody 1G1, ii) an antibody having an epitope specificity equal to or similar to that of 1G1 , iii) an antibody that can compete with 1G1 for binding to chemokine receptor CC 4 (CCR4) of mammal, iv) monoclonal antibody 2B10, v) an antibody having an epitope specificity equal to or similar to that of 2B10, vi) an antibody that can compete with 2B10 for binding to mammalian chemokine CC (CCR4) receptor 4, vii) antigen-binding fragments of any of (i) to (vi) that can bind to chemokine receptor CC (CCR4) ) of mammal or a portion thereof, viii) monoclonal antibody 10E4, ix) an antibody having an epitope specificity equal to or similar to that of 10E4, x) an antibody that can compete with 10E4 for binding to chemokine receptor 4 CC (CCR4) of mammal and xi) combinations of the above.

21. The method according to Claim 20, wherein the composition is a sample containing human cells.

22. A method of detecting mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor, comprising: a) contacting a sample to be studied with an antibody or antigen binding fragment thereof that binds to mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor and inhibits the binding of said ligand to the receptor under conditions appropriate for the binding of said antibody or fragment thereof to said mammalian CCR4 or portion thereof, and b) detecting or measuring the binding of said antibody or antigen-binding fragment thereof, wherein the binding of said antibody or antigen-binding fragment thereof to the material of said sample is indicative of the presence of a chemokine CC receptor 4 (CCR4) ) of mammal or portion of said receptor in said sample.

23. A method according to Claim 22, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: i) monoclonal antibody 1G1, ii) an antibody having an epitope specificity equal to or similar to that of 1G1 , iii) an antibody that can compete with 1G1 for binding to chemokine receptor CC 4 (CCR4) of mammal, iv) monoclonal antibody 2B10, v) an antibody having an epitope specificity equal to or similar to that of 2B10, vi) an antibody that can compete with 2B10 for binding to mammalian chemokine CC (CCR4) receptor 4, vii) monoclonal antibody 10E4, viii) an antibody having an epitope specificity equal to or similar to that of 10E4, ix) an antibody that can compete with 10E4 for binding to mammalian chemokine CC (CCR4) receptor 4, x) antigen-binding fragments of any of (i) to (ix) that bind to mammalian chemokine CC receptor 4 (CCR4) or one for of the same and xi) combinations of the above.

24. A method according to Claim 22, wherein the sample is a cellular fraction which, in normal individuals, contains a mammalian chemokine CC receptor (CCR4) 4 or portion of said receptor.

25. A method of inhibiting a function associated with the binding of a chemokine to a chemokine receptor 4 CC (CCR4) or a functional portion of said receptor, comprising contacting a composition containing the receptor or portion with an effective amount of an antibody or antigen-binding fragment thereof that binds to a chemokine receptor. CC (CCR4) of mammalian or portion of said receptor, wherein said antibody or fragment inhibits the binding of said chemokine to mammalian chemokine receptor CC (CCR4) 4 and inhibits one or more functions associated with the binding of chemokine to the receptor.

26. A method according to Claim 25, wherein the chemokine is any one or more of TARC, MDC, MCP-1, MIP-1 and RANTES.

27. A method according to Claim 25, wherein the antibody or antigen-binding fragment is selected from the group consisting of: a) monoclonal antibody 1G1, b) an antibody having an epitope specificity equal to or similar to that of 1G1, c) a antibody that can compete with 1G1 for binding to mammalian chemokine CC receptor 4 (CCR4), d) monoclonal antibody 2B10, e) an antibody having an epitopic specificity equal to or similar to that of 2B10, f) an antibody that can compete with 2B10 for binding to mammalian chemokine CC (CCR4) receptor 4, g) monoclonal antibody 10E4, h) an antibody that has an epitope specificity equal to or similar to that of 10E4, i) an antibody that can compete with 10E4 by binding to mammalian chemokine CC (CCR4) receptor 4 and j) antigen binding fragments of any of (a) to (i) that bind to mammalian CC chemokine receptor 4 (CCR4) or a portion of the same .

28. A method of detecting or identifying an agent that binds a mammalian CC chemokine receptor 4 (CCR4) or ligand binding variant thereof, consisting of combining a) an agent to be studied, b) an antibody or antigen-binding fragment that binds to a mammalian CC chemokine receptor 4 (CCR4) or portion of said receptor, wherein said antibody or fragment antigen-binding thereof inhibits the binding of a ligand to the receptor and c) a composition containing a mammalian CC chemokine receptor 4 (CCR4) or a ligand-binding variant thereof, under conditions suitable for binding said antibody or antigen-binding fragment to said mammalian chemokine CC (CCR4) receptor 4 or ligand-binding variant thereof, and detecting or measuring the binding of said antibody or antigen-binding fragment to said chemokine receptor CC ( CCR4) of mammal or ligand-binding variant thereof.

29. A method according to Claim 28, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: a) monoclonal antibody 1G1, b) an antibody having an epitope specificity equal to or similar to that of 1G1, c ) an antibody that can compete with 1G1 for binding to chemokine CC receptor 4 (CCR4), d) monoclonal antibody 2B10, e) an antibody having an epitopic specificity equal to or similar to that of 2B10, f) an antibody which can compete with 2B10 for binding to mammalian chemokine CC (CCR4) receptor 4, g) monoclonal antibody 10E4, h) an antibody having an epitope specificity equal to or similar to that of 10E4, i) an antibody that can compete with 10E4 by binding to mammalian chemokine CC (CCR4) receptor 4, j) antigen-binding fragments of any of (a) to (i) that bind to mammalian chemokine CC receptor 4 (CCR4) or a portion of the same o and k) combinations of the above.

30. A method according to Claim 28, wherein the formation of a complex between said antibody or antigen binding fragment and said mammalian chemokine CC (CCR4) receptor 4 or ligand binding variant is monitized and where a reduction in the The amount of complex formed in relation to an adequate control is indicative that the agent binds to said receptor or ligand-binding variant thereof.

31. A method according to Claim 28, wherein the composition containing a mammalian chemokine CC (CCR4) receptor 4 or a ligand-binding variant thereof is a recombinant chemokine CC receptor (CCR4) carrier cell 4 or a variant of ligand binding thereof.

32. A method according to Claim 28, wherein the composition containing a mammalian chemokine CC (CCR4) receptor 4 or a ligand-binding variant thereof is a membrane fraction of said CC chemokine receptor 4 carrier cell ( CCR4) recombinant or ligand-binding variant thereof.

33. A method according to Claim 28, wherein the antibody or antigen-binding fragment thereof is labeled with a label selected from the group consisting of a radioisotope, a spin tag, an antigenic label, an enzyme label, a fluorescent group and a chemo-luminescent group.

34. A method according to Claim 28, wherein the agent is an antibody that has specificity for a receptor 4 of mammalian chemokine CC (CCR4) or antigen-binding fragment thereof.

35. A method of inhibiting leukocyte trafficking in a patient, comprising administering to the patient a composition containing an effective amount of an antibody or antigen-binding fragment thereof that binds to a mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor and inhibits the binding of a ligand to the receptor.

36. A method according to Claim 35, wherein the ligand is a chemokine.

37. A method according to Claim 36, wherein the qui-miokine is any one or more of TARC, MDC, MCP-1, MlP-la and RANTES.

38. A method according to Claim 35, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: a) monoclonal antibody 1G1, b) an antibody having an epitope specificity equal to or similar to that of 1G1 , c) an antibody that can compete with 1G1 for binding to chemokine receptor 4 CC (CCR4) of mammal, d) monoclonal antibody 2B10, e) an antibody having epitopic specificity equal to or similar to that of 2B10, f) an antibody that can compete with 2B10 for binding to chemokine CC receptor 4 (CCR4), g) monoclonal antibody 10E4, h) an antibody having an epitope specificity equal to or similar to that of 10E4, i) an antibody that can compete with 10E4 for binding to mammalian chemokine CC (CCR4) receptor 4 and j) antigen-binding fragments of any of (a) to (i) that bind to mammalian chemokine CC receptor 4 (CCR4) or a portion of my Saint.

39. A composition containing an antibody or antigen-binding fragment thereof that binds a mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor, and a physiologically acceptable eventual carrier.

40. An antibody or antigen-binding fragment thereof that binds to a mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor with an IC 50 of less than about 1.5 μg / ml.

41. An antibody or antigen-binding fragment thereof that binds to a mammalian chemokine CC (CCR4) receptor 4 or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor with an IC50 of less than about 1.5 ng / ml.

42. A method of treating a disorder mediated by chemokine CC receptor 4 (CCR4) in a patient, comprising administering to the patient an effective amount of an antibody or antigen binding fragment thereof that binds to chemokine receptor 4 CC (CCR4) of a mammal or a portion thereof.

43. A method of treating an inflammatory disorder in a patient, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof that binds to the mammalian CC chemokine receptor 4 (CCR4) or a portion thereof. of the same.

44. The 10E4 hybridoma cell line deposited under ATCC Accession No. PTA-1203.

45. A monoclonal antibody or antigen-binding fragment thereof produced by the hybridoma cell line according to Claim 44.