MXPA01007562A - Anti-ccr1 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 1 (CCR1) 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 CCR1 with a ligand thereof, and to use of the antibodies and fragments in research, therapeutic, prophylactic and diagnostic methods.

Description

ANTI-CCR1 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. Immunol., 15: 675 (1997); Zou et al., Nature 393: 591 (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 qui-myotaxis, degranulation, the synthesis of lipid mediators and the activation of integrins, together with its critical role in the initiation and maintenance of inflammatory diseases and the recent identification of certain chemokine receptors as co-receptors for HIV-1 entry, have made chemokines and chemokine receptors a new and attractive group of therapeutic objectives. 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 cysteines adjacent. The a-chemokine branch includes proteins such as IL-8, neutrophile-2 activating peptide (NAP-2), melanoma growth stimulating activity (MGSA / gro or GROa) and ENA-78, each of which have attractant 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-lß), chemokine of the thymus and regulated by activation (TARC) and chemokine derived from macrophages ( MDC) Chemokines bind to 7 receptors coupled to transmembrane expansion protein G (Murphy, PM, Annu, Rev. Immunol.12: 593-633 (1994)). A number of transmembrane receptors have been identified. β-chemokines (CCR1-CCR10) to date and the search for recep Additional chemokine targets are the subject of active research (Baggiolini, Nature 392: 565-568 (1998)). The chemokine receptor CCR1 was identified by Nomura et al. (Jnt Imol 5: 1239-1249 (1993); Neote et al (Cell 72: 415-425 (1993)) and Gao et al (J \ Exp. Med. 177: 1421-1427 (1993)). Originally, CCR1 was shown to emit signal in response to MlP-la and RANTES, but more recently it has been shown to also emit signal in response to additional chemokine ligands.Selective recruitment of leukocyte subgroups to sites of inflammation and Ordinary traffic of leukocytes through the circulation, tissues, lymphatic system and secondary lymphoid organs is controlled in part by the differential expression of chemokine receptors in cell subgroups.These expression patterns would seem to ensure that a group of leukocytes functionally related can respond in a coordinated manner to a specific group of chemokines induced by a given stimulus., most leukocytes express several chemokine receptors, many with complex and promiscuous ligand interactions. For T cells, PCR (polymerase chain reaction) 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 T cells or subgroups directed to tissues. It can also determine which T cells are infected with different strains of HIV-1. 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.

COMPENDIUM OF THE INVENTION CCR1 was the first chemokine receptor that was found to interact with chemokines C-C (β). Originally identified as a MIP-la / RANTES receptor, more recent studies have shown that CCR1 can have up to 6 or more β-chemokine ligands. The work described here characterizes the expression of CCR1 by flow cytometry and evaluates in vitro the relative functional contribution of this receptor on human leukocytes using a blocking monoclonal antibody. In peripheral blood, all monocytes express high levels of CCRl and monocyte responses to MlP-la and RANTES can be completely blocked by the anti-CCRl 2D mAb. CCR1 is expressed in a small percentage of CD45R0 + CD26 + T cells, suggesting that it marks a subset of memory T cells. Contrary to the chemokine receptors CCR5 and CXCR3, which are increasingly regulated in activated T cells, CCR1 expression decreases with the activation of T cells. Neutrophils express low levels of CCR1 and show a weak response to MlP -the in mobilization assays of [Ca2 +] i, which can be inhibited by the anti-CCR1 2D4 mAb. The expression of CCR1 in eosinophils is extremely variable between individuals, varying from > 90% positive to completely negative. The expression pattern of CCR1 suggests that it is involved in a wide range of immunological activities, having a greater role in the function of monocytes and eosinophils, as well as in the function of a subset of T lymphocytes. The present invention relates to an antibody (immunoglobulin) or functional fragment thereof (e.g., an antigen-binding fragment) that binds to a mammalian CC 1 chemokine receptor (also referred to as CCR1 or CKR-1) or portion thereof. receptor (anti-CCRl). In one embodiment, the antibody of the present invention or fragment thereof has specificity for human CCR1 or a portion thereof. In another embodiment, the antibody or fragment of the invention inhibits (reduces or prevents) the binding of a ligand (e.g., MlP-la, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF) to the receptor and inhibits one or more functions associated with the binding of the ligand to the receptor (eg, leukocyte trafficking). In a preferred embodiment, the ligand is MlP-la or RANTES. For example, as described herein, antibodies and fragments thereof of the present invention that bind to human CCRl or a portion thereof can block the binding of a chemokine (eg, MlP-la, RANTES, MCP-2). , MCP-3, leukotactin-1, HCC-1 or MPIF) to the receptor and inhibit the function associated with the binding of chemokine to the receptor. In a preferred embodiment, the antibody of the invention or fragment thereof has the same or similar epitope specificity as the monoclonal antibody (mAb) LS125-2D4 (2D4). For example, the antibody or fragment thereof may have epitope specificity by the second extracellular loop of CCR1, such as a portion of CCR1 from about amino acid 171 to about amino acid 205. In one embodiment, the antibody is the anti-body monoclonal 2D4 or an antibody that can compete with 2D4 for binding to human CCR1 or a portion of human CCR1. Functional fragments of the above antibodies are also contemplated. In another embodiment, the antibody or fragment thereof binds to CCR1 with an affinity greater than about 5 x 10"8 M and, more preferably, at least 5 x 10 ~ 9 M. In another embodiment, the antibody of the invention or fragment thereof inhibits the binding of chemokines to CCR1, preferably with an IC50 of less than about 10 μg / ml, more preferably less than about 5 μg / ml and, more preferably, less than about 1.0 μg. In one embodiment, the antibody of the invention or fragment thereof inhibits the binding of MlP-a to CCR1 with an IC50 of about 0., 5 μg / ml. In another embodiment, the antibody of the invention or fragment thereof inhibits the binding of RANTES to CCRl with an IC 50 of about 0.7 μg / ml. In another embodiment of the invention, the antibody or fragment thereof inhibits chemotaxis induced by chemokines (e.g., induced by MlP-la, RANTES or HCC-1) of cells (e.g., CCR1 carrier cells), preferably at less than about 50 μg / ml, more preferably less than about 20 μg / ml, and even more preferably less than about 10 μg / ml. It is contemplated that any of the antibodies or fragments described herein may be used in the methods described herein. The present invention also relates to an antibody or functional fragment thereof (eg, an antigen-binding fragment) that binds to a mammalian CCR1 or portion of the receptor and provides a higher fluorescent staining intensity of CCR1 or compositions that contain CCR1 relative to other anti-CCR1 antibodies. In one embodiment, the antibody is monoclonal antibody 2D4 or an antibody that can compete with 2D4 for binding to human CCR1 or a portion of human CCR1. The present invention further relates to a method of inhibiting the interaction of a mammalian CCR1-bearing 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 CCR1 or a portion of CCR1. 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, CD26 + cells, CD25 + cells, CD45RO + cells), such as Thl and Th2 cells, and other cells expressing CCR1, such as a recombinant cell expressing CCR1 or a portion thereof (eg, transfected cells). In a particular embodiment, the antibody is 2D4 or an antibody that can compete with 2D4 for binding to human CCR1 or a portion of human CCR1. Another embodiment of the invention relates to a method of inhibiting the interaction of a mammalian CCR1 carrier cell with a chemokine, comprising contacting said cell with an effective amount of an antibody or functional fragment thereof that binds to CCRl or a portion of said receiver. In one embodiment of the method, the antibody or functional fragment thereof is any one or more of 2D4, an antigen binding fragment of 2D4 or an antibody or fragment thereof having an epitope specificity that is the same or similar to that of 2D4. Moreover, the invention relates to a method of inhibiting a function associated with the binding of a chemokine to CCR1, consisting of administering an effective amount of an antibody or a functional fragment thereof that binds to mammalian CCR1 or a portion of said receiver. In one aspect of the method, the antibody or functional fragment thereof is any one or more of 2D4, an antigen binding fragment of 2D4 or an antibody or fragment thereof having an epitope specificity equal to or similar to that of 2D4. Another aspect of the invention is a method of identifying the expression of a mammalian CCR1 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., 2D4) that binds to a CCR1 protein. of mammal or a portion of the receptor under conditions appropriate 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 CCR1 or a portion thereof in a biological sample., which contains an antibody or functional fragment thereof that binds to a mammalian CCR1 or a portion of said receptor and one or more suitable auxiliary reagents to detect the presence of a complex between said antibody or fragment and said protein or portion thereof. same The present invention also includes methods of identifying additional ligands or other substances that bind to a mammalian CCR1 protein, including inhibitors and / or promoters of mammalian CCR1 function. For example, agents that have the same or similar binding specificity to the antibody of the pre-sent 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 ligands or other substances that bind to the CCR1 receptor, including inhibitors (eg, antagonists) or promoters (eg, agonists) of receptor function. In one embodiment, cells that naturally express the CCR1 receptor protein or suitable host cells that have been engineered to express a CCR1 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 CCR1 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 (e.g., monoclonal antibody 2D4, an antibody having an epitope specificity equal to or similar to that of 2D4, antigen binding fragments of 2D4) and a composition containing a CCR1 protein of mammal 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 CCR1 protein or a ligand-binding variant thereof and the binding of the antibody or fragment to the protein is detected or measured. mammalian CCR1 protein or li-gand 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 CCR1 protein or a ligand-binding variant thereof can be a membrane fraction of a recombinant CCR1 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 CCR1) or other substances, including inhibitors or promoters of receptor function, which can bind to CCR1 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. These ligands, inhibitors and promoters can be used to treat inflammatory diseases, autoimmune diseases, atherosclerosis and graft rejection or HIV infection, for example, in a me of 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). These ligands, inhibitors and promoters can also be used in a me 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 the leukocytes, which is useful, for example, in the treatment of infectious diseases and cancer. The present invention also encompasses a me 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 CCR1 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 me of inhibiting or treating CCR1-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 CCR1. of mammal or portion of said receptor and inhibits the function mediated by CCR1. The present invention is further related to an anti-body or fragment thereof as described herein (e.g., monoclonal antibody 2D4, an antigen-binding fragment of 2D4, an antibody having an epitope specificity equal to or similar to that of 2D4). ) 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 condition mediated by CCR1 or another inflammatory disease or condition, as described herein .

BRIEF DESCRIPTION OF THE DRAWINGS Figures IA and IB show the specific binding of monoclonal antibody 2D4 to CCR1. Figure 1A shows the results when the 2D4 monoclonal antibody was used to stain a variety of transfectants of CC chemokine receptors expressed in the parental cell line Ll-2. Only cells transfected with CCRl stained positively. Figure IB is a graph showing the results when cells transfected with CCR1 were used in ligand binding assays in the presence of increasing concentrations of 2D4 monoclonal antibody. The total union was 11,57411,355 cpm for MlP-la and 1,7341118 for RANTES. Figures 2A and 2B show that monocyte responses to MlP-la and RANTES are dependent on CCR1. Figure 2A is a flow cytometry histogram showing monocyte expression of CCR1. Normal human "PBMC" (peripheral blood mononuclear cells) were stained with an irrelevant mouse IgGl antibody (dotted line) and monoclonal antibody 2D4 (solid line). The monocytes were allowed to enter by forward and lateral dispersions. Figure 2B shows the mobilization of intracellular calcium from monocytes after stimulation with chi-kinas. Human PBMCs were loaded with Fluo-3 and stimulated with 20 nM of MlP-la, RANTES, IL-8 or 100 nM of HCC-1, as indicated by the arrows. When the 2D4 antibody was used, 50 μg / ml of 2D4 was added just before stimulation. Figures 3A and 3B show the expression of CCR1 in peripheral blood lymphocytes. Figure 3A shows the results when normal human PBMC were stained with 2D4 and monoclonal antibodies specific for T cells (CD3) or for B cells (CD19). Figure 3B shows the expression of CCR1 in subgroups of T cells. The graphics shown were first opened on CD3 + cells and then analyzed for CCR1 versus CD26., CD45RO, CXCR3 and CCR5. Figure 4 shows the expression of CCR1 on activated T cells. Human PBMC were stimulated with anti-CD3 and IL-2 in the absence or presence of IFNa. The cells were cultured for ten days and then stained with mAbs for CCR1, CCR5 and CXCR3 as indicated (solid line). Negative controls (dotted line) were stained with an irrelevant mouse IgGl. The data obtained from the staining on day 14 gave a similar profile. Figures 5A and 5B show the inhibition of chemo-taxis with anti-CCR1 mAb 2D4. Activated T cells were used in a chemotaxis assay for 50 nM of MlP-la, MlP-lβ or RANTES in the presence of 50 μg / ml of 2D4 (anti-CCR1, full bar), 2D7 (anti-CCR5, blank bar ) or both mAbs at 50 μg / ml each (striped bar). Figure 5A shows the results when the T cells were stimulated in the absence of INFa. Figure 5B shows the results when the T cells were cultured in the presence of INFa. The migrated cells were counted by flow cytometry. In this experiment, the counts of the total migration of CD3 blasts without IFNa were 3,155, 2,683 and 5,483 for MlP-la, MlP-lβ and RANTES, respectively, with a background of 246. The total migration of the cells treated with IFNa was 11,352, 6,542 and 12,136 for MlP-la, MlP-lß and RANTES, respectively, with a background of 612. The data are representative of three experiments that gave similar results. Figure 6 shows several levels of CCR1 expression in eosinophils. The eosinophils were purified from normal donors and stained with anti-CCRl 2D4 mAb. Spot plots were used to better demonstrate cells with positive staining. The data were the staining profiles of four different donors to represent the range of CCR1 expression in eosinophils from different individuals. Figures 7A and 7B show that the chemotaxis of eosinophils towards MlP-la can be completely inhibited by the anti-CCRl 2D monoclonal antibody. Eosinophils purified from a donor that showed >90% of CCRl positive cells were used in the chemotaxis assays (Forssmann et al., FEBS Lett 408: 211 (1997)) for increasing concentrations of MlP-la (Figure 7A) and eotaxin (Figure 7B), presence of anti-CCRl 2D4 mAb or anti-CCR3 7B11 mAb at 20 μg / ml. Chemotaxis was carried out in 96-well plates and the migrated cells were measured by means of a fluorescence plate reader. Figure 8 shows the staining of human blood with the anti-CCR1 2D4 mAb. Neutrophils were identified by the forward and lateral dispersions. Figures 9A-9F show the mobilization of intracellular Ca2 + in neutrophils. Fluo-3-charged neutrophils were stimulated, as indicated by the arrows, with 100 nM of MlP-la (Figures 9A and 9D), 100 nM of MPIF (Figures 9B and 9E) or 2 nM of IL-8 (Figures 9C). and 9F) in the absence of antibody (9A, 9B and 9C) or in the presence of 50 μg / ml of 2D4 (9D-9F). Figures 10A and 10B are schematic illustrations of epitope specificity determination of mAb 2D4 using CCR1 / CCR3 receptor chimeras. The upper row of the panels shows the junction of 2D4 (dotted contour) and a control antibody (blank contour) for several receptor chimeras constructed from portions of CCRl (punched) and CCR3 (shaded) as indicated. The lower row of panels shows the binding of the anti-CCR3 mAb 7B11 (dotted contour) and a control antibody (blank contour) for the indicated receptor chimeras. The 2D4 mAb binds to the second extracellular loop of CCR1. Figure 11 is a fluorescence graph illustrating the expression of CCR1 in human basophils. Human blood was stained with anti-CCRl and the basophils were identified by double staining with anti-IgE and opened by forward and lateral dispersions. Staining with anti-CCRl 2D4 is re-presented with shading. The IgGl staining of the control mouse is represented by solid lines. Figure 12 depicts the amino acid sequence of human CCR1. The predicted N-terminal, transmembrane (TM), extracellular (EC) and C-terminal portions are indicated.

DETAILED DESCRIPTION OF THE INVENTION The first C-C chemokine receptor identified, CCR1, was originally described as a MIP-la / RANTES receptor. From those publications, however, CCRl has been shown to interact with a series of β-chemokines, including MlP-la, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 and MPIF (Schall et al., Eur. J. Immunol., 22: 1477 (1992), Forssmann et al., FEBS Lett 408: 211 (1997), Gong et al., J. Biol. Chem. 272: 11682 (1997); Gong et al. , J. Biol. Chem. 272: 11682 (1997); Youn et al., J. Immunol., 159: 5201 (1997), and Youn et al., Blood 91: 3118 (1998)). Leukotactin-1 is also known as HCC-2 or MlP-ld, HCC-1 is also known as Ckßl or MlP-l? and MPIF is also known as CKβ8 or MIP-3. Most of these chemokines are also described as functional ligands for other chemokine receptors, such as CCR2 (Combadiere et al., J. "Biol. Chem. 270: 29671 (1995)), CCR3 (Ponath et al., J Exp. Med. 183: 2437 (1996)) and CCR5 (Samson et al., Biochemistry 35: 3362 (1996); Raport et al., J ". Biol. Chem. 271: 17161 (1996)), with patterns of expression that overlap with that of CCRl. CCR1 mRNA has been detected in T cells (Loetscher et al., J. "Exp. Med. 184: 569 (1996)), B cells (Gao et al., J. Exp. Med. 177: 1421 (1993)) , basophils (Uguccioni et al., J., Clin. Invest. 100: 1137 (1997)), eosinophils (Post et al., J. Immuno 1. 155: 5299 (1995)), monocytes and granulocytes (No-mura et al., Int. Immunol., 5: 1239 (1993)), as well as dendritic cells (Sozzani et al., J. Immunol., 161: 1083 (1998)), and all have been shown to functionally respond to CCR1 ligands.

The white disorganization of the CCR1 of mice has provided some insight into the role of this receptor in normal immune function and in the pathology of the disease. In one study (Gao et al., J. Exp. Med. 185: 1959 (1997)), CCRl - / - mice showed abnormal and induced steady state traffic of myeloid progenitors from bone marrow to blood and spleen , accelerated mortality when inoculated with Aspergillus fumigatus probably due to a malfunction of neutrophils, decreased granulomatous inflammatory responses and altered balance of Th1 / Th2 cytokines. An independent study (Lu et al., J. Exp. Med. 187: 601 (1998)) linked CCR1 with the syndrome of respiratory distress secondary to acute pancreatitis in mice, attributable to a lower intrapancreatic sequestration of neutrophils in the animals in those that CCRl had been removed. Several studies have demonstrated in vivo functions for MlP-la and RANTES, the two most extensively studied CCR1 ligands. The importance of MlP-la for virus removal both T-cell dependent and NK-dependent has been demonstrated in mice in which MlP-la had been removed (Cook et al., Science 269: 1583 (1995)). The use of neutralizing antisera against MlP-la and RANTES in rodent models of airway inflammation (Standiford et al., J., Immunol., 155: 1515 (1995)), experimental allergic encephalomyelitis (EAE) (Karpus. et al., J. Immunol., 155: 5003 (1995)) and arthritis (Barnes et al., J. Clin * Invest. 101: 2910 (1998)) suggests a role for these ligands in a range of inflammatory diseases. However, as MlP-la and RANTES are ligands for CCR1, CCR3 and CCR5 in the mouse, the role of these individual receptors in these models and their validation as targets for anti-inflammatory drugs is unclear.As a step towards understanding the CCRl function In humans, a monoclonal antibody (mAb) has been produced against this receptor as described herein and has been used to characterize the expression and functions of CCR1 on human leukocytes. in vi tro by blocking the receiver with the mAb supports and confirms the results of flow cytometry. These data demonstrate that the expression and regulation of CCR1 are distinct from other C-C chemokine receptors, such as CCR3 and CCR5, which share some common ligands, and provide further evidence for a complex and multi-faceted chemokine-receptor system. Chemokines and their receptors are an important component in the regulation of directed leucocyte migration. During an inflammatory response, chemokines are locally produced that attract several leukocytes to the corresponding receptors. While the spectrum of chemokines expressed in the lesion can differentially attract certain inflammatory cells, the selectivity and variation in the expression of chemokine receptors on leukocytes provides greater regulation to ensure appropriate cellular recruitment for a stimulus. dice. As the number of chemokine receptors identified and characterized continues to grow, it is increasingly clear that the cells selectively express several receptors that can identify, label or otherwise characterize functional subsets of leukocytes, such as Th1 and Th2. , activated and quiescent T cells, simple and memory. Since several chemokine receptors are co-expressed on individual cells, it has been difficult to validate the role of a specific receptor in the initiation and progression of the disease or, for that reason, in normal immune function. The expression pattern of CCR1 was assessed as described here and the functional contribution of this receptor to normal immune function was explored with anti-CCR1 2D4 mAb. T cells, especially activated T cells, demonstrate chemotactic responses to a number of CC chemokines, including MCP-1, RANTES, MlP-la, MlP-lβ and CXC chemokines such as IP-10, MIG and ITAC. An increase in the expression of CCR2 has been described (Qin et al., Eur. J. Immunol. 26: 640 (1996)), CCR5 and CXCR3 (Qin et al., J "Clin Invest. 101: 146 (1998)) on activated T cells, which correlates with an increase in chemotactic activities towards their ligands. Recent studies also described an increase in activated T-cell CCR1 mRNA (Sallusto et al., J. Exp. Med. 187: 875 (1998)), although a small population of resting T cells expressing themselves has been identified. In peripheral blood CCRl, a significant expression of CCR1 on the surface of T cells has not been identified by stimulation with anti-CD3 and IL-2, it is possible that there are post-transcription or post-translation mechanisms that control the surface expression of CCR1. Alternatively, CCR1 may be downregulated by other cytokines produced during T cell activation, as described in the TNFα-induced release of CXCR2 by neutrophils (Asagoe et al., J., Immunol., 160: 4518 (1998). )). This data was consistent with the previously published work on anti-CCR5 mAbs (Wu et al., J. Exp. Med. 186: 1373 (1997)), in that most of the effects of MlP-la and RANTES on activated T cells appears to be through CCR5. The fact that a population of CCR1-positive T cells could be expanded under particular activation conditions, such as the IFNa treatment described herein, suggests that CCR1 may be involved in the responses of T cells to a different set and perhaps more specific stimuli that CCR5 or CXCR3, for example. IFNa is an important cytokine in the defense of the host against viral infection and has been shown to induce the production of MIP-1 by human PBMC (Bug et al., Exp .. Hematol 26: 117 (1998)). Furthermore, studies with mice deficient in MlP-la show altered inflammatory responses of T cells associated with infection by various viruses, including resistance to myocarditis induced by Coxsackie virus and reduced pneumonitis associated with influenza virus. , as well as resistance to keratitis of the syndrome associated with herpes simplex virus type 1 (Tumpey et al., J. Virol. 72: 3705 (1998)). In addition to lymphocytes, monocytes also respond to C-C chemokines. It is shown here, by blocking receptors with mAb 2D4, that the responses of monocytes to MlP-la and RANTES, as well as to HCC-1, appear to be entirely dependent on CCR1. Until now, the only other major C-C chemokine receptor found in human monocytes is CCR2. Given the role that the lineage of monocytes / macrophages can have as antigen-presenting cells and the findings that monocyte-derived dendritic cells express CCR1 (Sozzani et al., J ". Immuno 1. 161: 1083 (1998) ), this receptor and its ligands may have important roles in specific immune responses by regulating the migration of antigen-presenting cells.A number of studies have demonstrated the importance of CCR1 in the migration of granulocytes in rodents. The expression and function of CCR1 on human granulocytes remain unclear With the specific mAb 2D4 described here, CCR1 was detected in most granulocytes On eosinophils, the expression of CCR1 was highly variable among donors, contrary to CCR3, which It was always expressed at uniformly high levels.Although CCR1 was seen to be expressed on neutrophils, no significant chemotactic response was observed. of these cells to the CCR1 MlP-la ligands, RANTES or MPIF. The inability of neutrophils to migrate to CCR1 ligands could be due to the low density of receptors on the cell surface, as indicated by weak immunostaining, the lack of a component of the signal transduction pathway or the lack of molecules of adhesion. Similarly, it was observed that, with resting T cells, approximately 30-40% of peripheral blood T cells express CXCR3, but fail to migrate in response to IP-10 or MIG unless activated with anti-CD3 and IL-2 (Qin et al., J. Clin Invest. 101: 146 (1998)). Alternatively, the results may suggest that chemotaxis is not the primary function of CCR1 on neutrophils. Certainly, evidence of differential function of chemokine receptors in neutrophils has been described, where the involvement with IL-8 of CXCR1, but not of CXCR2, resulted in activation of NADPH oxidase and phospholipase D, while both receptors they functioned in chemotaxis and elastase release (Jones et al., Proc. Natl. Acad. Sci. USA 93: 6682 (1996)). The lack of chemotactic activity of the neutrophils towards the CCR1 ligands is consistent with two previous publications, which also could not detect neutrophilic migration to MlP-la (McColl et al., J. Immunol. 150: 4550 (1993)) or MPIF (Forssmann et al., FEBS Lett 408: 211 (1997)), but disagrees with other studies that found that MPIF was chemotactic for neutrophils (Pa-tel et al., J ". Exp. Med. 185: 1163 (1997)). Approximately 10% of the Neutrophils responded to stimulation of MlP-la or MPIF with mobilization of intracellular Ca2 +, which was seen to be mediated by CCR1, since 2D4 could completely block the response.The observed flow of [Ca2 +] i was not deficient to eosinophils Tis because the eosinophils of these donors constitute less than 3% of the total granulocytes and is consistent with previous publications, which saw that both MlP-la and MPIF stimulated a flow of [Ca2 +] in neutrophils. Several leukocytes described herein using the anti-CCR1 2D4 mAb are in marked contrast to a previous publication using polyclonal antisera raised against an N-terminal peptide fusion protein of CCR1-GST (Su et al., J ". Leukoc. 60: 658 (1996)) . It is shown here that mAb 2D4 detected the expression of CCR1 in almost all monocytes and Su et al. they say that their polyclonal antisera detected the expression of CCR1 in monocytes. However, mAb 2D4 detected the expression of CCR1 only in a discrete subpopulation of T cells; on the contrary, Su et al. describe that the antisera dyed > 80% CD3 + peripheral blood. 2D4 also detected a discrete subpopulation of B cells (approximately 1-5%), whereas Su et al. describe that the antisera did not detect the expression of CCR1 in B cells, eosinophils or neutrophils. 2D4 not only detected texpression by flow cytometry, but was able to functionally block the chemotaxis mediated by MlP-la and the Ca2 + flux in these cells, confirming the authenticity of the staining and indicating that CCR1 is the predominant receptor , if not the only one, of MlP-la in granulocytes. The significant discrepancy in the staining profiles between mAb 2D4 and polyclonal rabbit antisera can be due to the different epitopes recognized by the reagents, since it is said that the rabbit polyclonal recognizes epitopes in the NH2-terminal domain and recognizes 2D4 the second extracellular loop. The poor sensitivity resulting from the low titer of antibodies that recognize native epitopes, as well as the low affinity antibodies in the antiserum, may explain the lack of detection of CCRl in B cells, eosinophils and neutrophils with rabbit polyclonal antisera. Su and col. describe that antisera inhibited less than 50% of the monocyte chemotaxis at 100 μg / ml, whereas 2D4 showed 100% inhibition at 50 μg / ml. However, the discrepancy in the identification of T cell expression between the two reagents probably occurs as a result of different accessibility to epitopes on different cell types. The CCR1 ligands include MlP-la, RaANTES and MCP-3, which are also shared by other receptors; for example, CCR3 interacts with MlP-a and RANTES, as well as with eo-taxin, and CCR5 interacts with MlP-la and RANTES, as well as with MlP-lβ. Tapparent redundancy may be necessary if the distribution of the receptors for these ligands is considered. CCR3 is predominantly expressed in eosinophils, basophils and subsets of T cells, but not in neutrophils or monocytes; CCR5 is expressed mostly in T cells, especially activated T cells. The work described here shows that CCR1 could be found in all monocytes, in most granulocytes, including basophils, and in a subpopulation of T cells. Moreover, the expression of CCR1 in eosinophils and T cells seemed to be actively regulated. Therefore, each receptor has its own distinct population of host cells, which may function differently during inflammatory responses. The present invention relates to an antibody (anti-CCR1) or functional fragment thereof that binds to mammalian CC chemokine receptor 1 (CCR1)., CKR-1) or portion of CCRl. In one embodiment, the antibody has specificity for human CCRl or portion thereof. In one embodiment, the antibody (immunoglobulin) is produced against an isolated and / or recombinant mammalian CCR1 or portion thereof (eg, peptide) or against a host cell expressing mammalian CCR1. In a preferred embodiment, the antibody binds specifically to the human CCR1 receptor or to a portion thereof and, in a particularly preferred embodiment, the antibody has specificity for a natural or endogenous human CCR1. Antibodies or functional fragments thereof that can inhibit one or more characteristic functions of a mammalian CCR1, 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 inflammatory mediators by leukocytes, activation of integrins) are also protected by the pre-sent invention, such as an antibody that can inhibit the binding of a ligand (ie, one or more ligands) to CCR1 and / or one or more functions mediated by CCRl in response to a ligand. For example, in one aspect, the antibodies or functional fragments thereof can inhibit (reduce or pre-come) the interaction of the receptor with a natural ligand, such as MlP-RANTES, MCP-2, MCP-3, leukotactin- 1, HCC-1 or MPIF. In one embodiment, the ligand is MlP-la, RANTES or HCC-1. In another aspect, an antibody or functional fragment thereof that binds to CCR1 can inhibit the binding of MlP-la, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF to mammalian CCR1 ( example, human CCRl, non-human primate CCRl, murine CCRl). The antibodies or functional fragments thereof of the present invention can inhibit functions mediated by human CCR1, including trafficking of leucocytes, activation of T cells, release of inflammatory mediators and / or degranulation of leukocytes. In a particular embodiment, antibodies or functional fragments thereof demonstrate inhibition of chemotaxis of cells (e.g., CCR1 carrier cells) induced by chemokines (e.g., induced by MlP-la, RANTES or HCC-1), preferably to less than about 50 μg / ml, preferably less than about 20 μg / ml and, more preferably, less than about 10 g / ml. In another embodiment of the invention, the antibodies or functional fragments thereof of the invention can inhibit the binding of a CCR1 ligand (eg, a chemokine) to CCR1, preferably with an IC50 of less than about 10 μg / ml, more preferably with an IC 50 of less than about 5 μg / ml, and even more preferably, with an IC 50 of less than about 1.0 μg / ml. In another embodiment, the antibodies or functional fragments thereof of the invention can inhibit the binding of one of MlP-la to CCRl with an IC50 of about 0.5 μg / ml. In yet another embodiment, the antibodies or fragments thereof of the invention can inhibit the binding of RANTES to CCR1 with an IC 50 of about 0.7 μg / ml. In another embodiment of the invention, the antibodies or fragments thereof bind to CCR1 with an affinity greater than about 5 x 10 ~ 8 M, and preferably at least about 5 x 10"9 M. A murine monoclonal antibody specific for CCR1, designated 2D4, was produced as described herein In a preferred embodiment, the antibodies of the present invention bind to human CCR1 and have an epitope specificity equal to or similar to that of the murine 2D4 antibody described herein. antibodies with epitopic specificity equal or similar to that of the murine 2D4 monoclonal antibody can be identified using recognized techniques in this field., antibodies having an epitope specificity equal to or similar to that of 2D4 can be identified by their ability to compete with the murine 2D4 monoclonal antibody for binding to human CCR1 (e.g., to human CCRl carrier cells, such as carrier translectants of CCR1, CD8 + cells, CD4 + cells, CDR45RO + cells, CD25 + cells, monocytes, dendritic cells, macrophages and basophils), for their ability to inhibit the binding of 2D4 to human CCR1 or through the use of receptor chimeras. Using receptor chimeras (Rucker et al., Cell 87: 437-446 (1996)), it has been shown that mAb 2D4 binds to the second extracellular loop of CCR1 (Figures 10A and 10B). For example, 2D4 can bind to a region consisting essentially of amino acids 171 to 205 of human CCRl (for example, from about amino acid 171 to about amino acid 205, Figure 12). Using these or other suitable techniques, antibodies having an epitope specificity equal to or similar to that of an antibody of the present invention can be identified. The invention also relates to a bispecific antibody or functional fragment thereof (eg, F (ab ') 2, which has the same or similar epitope specificity as 2D4 and at least one other antibody (see, for example, US Pat. 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. 5,496,549 (Yamazaki et al.) An antibody-producing hybridoma cell line according to the present invention was deposited on February 1, 1999 in the name of LeukoSite, Inc., 215 First Street, Cambridge, MA 02142, USA. US, at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA, under Accession No. HB-12644 (LS125-2D4-11-10-1 (2D4)). invention is also related to the hybridoma cell line deposited under Accession No. ATCC HB-12644, as well as to the monoclonal antibodies produced by the cell line of the invention. and hybridoma deposited under Accession No. ATCC HB-12644. 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 2D4 may also utilize functional fragments (e.g., antigen-binding fragments) of 2D4, antibodies that have the same or similar epitope specificity as 2D4 and its combinations, optionally in combination with antibodies or fragments that have an epitopic specificity that is not the same as or similar to 2D4. The antibodies of the present invention can be produced against an appropriate immunogen, such as isolated and / or recombinant mammalian CCR1 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 (eg, mAb 2D4, or antigen-binding fragments thereof) for use in therapy (including prophylaxis) or diagnosis (eg, of diseases or conditions particular, 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, Antibodies: A Labora-tory 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 to CCR1, 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. Natl. Acad. Sci. USA 90: 2551-2555 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Lon-berg et al., US Pat. No. 5,545,806; Surani et al., US Pat. No. 5,545,807). Also included in the present invention and in the term "antibody" are single chain antibodies and chimeric, humanized or primatized anti-bodies (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 Lad-ner 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 CCR1). Particularly preferred functional fragments retain the ability to inhibit one or more functions characteristic of a mammalian CCR1, 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 CCR1 with one or more of its ligands (e.g., MlP-RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF) and / or can inhibit one or more receptor-mediated 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 CCR1 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 pa-paine or pepsin can generate Fab or F (ab ') 2 / resctively fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of 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 humanized immunoglobulin that binds to mammalian CCR1 (eg, human CCR1, murine CCR1), whose immunoglobulin contains an antigen-binding region of non-human origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework 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 conventional techniques (e.g., synthetic) or be prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce an adjacent 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 (eg, one or more CDRs derived from an antibody of non-human origin) and a region of frame derived from a light and / or heavy chain of human origin (for example, antibodies grafted with CDR with or without frame changes). In one embodiment, the humanized immunoglobulin can compete with the murine 2D4 monoclonal antibody for binding to human CCR1. In a preferred embodiment, the antigen-binding region of the humanized immunoglobulin (a) is derived from the monoclonal antibody 2D4 (eg, as in a humanized immunoglobulin containing CDR1, CDR2 and CDR3 of the light chain of 2D4 and CDR1, CDR2 and CDR3 of the 2D4 heavy chain). Also included are chimeric or single chain antibodies grafted with CDR. 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); Daug-herty, 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 or other suitable methods, variants can also be easily 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; Khappik 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 line deposited under ATCC Accession No. HB-12644, as well as to the monoclonal antibody produced by the hybridoma 2D4 cell line deposited under Accession No. ATCC HB-12644 and fragments of antigen binding thereof. 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 make it possible to the transfer of the genes coding for the monoclonal antibodies. In addition, cell lines can be used as a source of nucleic acids encoding the anti-CCR1 immunoglobulin chains, which can be isolated and expressed (for example, by transfer to other cells using any suitable technique (see, for example, Cabilly et al. col., U.S. Patent No. 4,816,567; Winter, U.S. Patent No. 5,225,539.) For example, clones containing a redistributed anti-CCRl light or heavy chain (e.g. by PCR) or cDNA libraries can be prepared from mRNA isolated from the cell lines and cDNA clones encoding an anti-CCR1 immunoglobulin chain can be isolated, thus nucleic acids encoding the heavy chains can be obtained and / or light 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 of these (for example, immunoglobulins humanized) in a variety of host cells or in an in vi tro translation system. 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 host cell suitable by an appropriate method (eg, 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 media 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 the host cells, from the medium, from milk). 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 CCR1 and / or inhibit the function associated with the ligand to CCR1. As discussed below, various methods can be employed to assess the inhibition of a ligand to CCR1 and / or the function associated with binding of the ligand to the receptor. Binding Assays As used herein, the term "mammalian CCR1" refers to natural or endogenous mammalian CCR1 proteins and to proteins having an amino acid sequence that is the same as that of a corresponding natural or endogenous mammalian CCR1 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 CCR1 (eg, produced by alternative splicing or other cell processes) and modified or modified forms. unmodified from the above (eg, glycosylated, non-glycosylated). The mammalian CCR1 proteins can be isolated and / or recombinant proteins (including synthetically produced proteins). Natural or endogenous mammalian CCR1 proteins include wild-type proteins, such as mature CCR1, polymorphic or allelic variants and other isoforms that occur naturally in mammals (eg, humans, non-human primates). Said proteins can be recovered or isolated from a source that naturally produces mammalian CCR1, for example. Reference is made to these mammalian CCR1 proteins and proteins having the same amino acid sequence as a corresponding natural or endogenous mammalian CCR1 by the name of the corresponding mammal. For example, when the corresponding mammal is a human, the protein is designated as a human CCR1 protein (eg, a recombinant human CCR1 produced in a suitable host cell). "Functional variants" of mammalian CCR1 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 CCR1 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 CCR1 protein ( such as N-terminal, C-terminal or internal 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 CCR1 protein. In general, mutants of mammalian CCR1 proteins include natural or artificial variants of a mammalian CCR1 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). Said mutations may be in a conserved region or a non-conserved region (in comparison with other chemokine receptors CXC (a) and / or CC (ß)), or in an ex-tracelular, cytoplasmic or transmembrane region, for example. In general, fusion proteins include polypeptides containing a mammalian CCR1 (eg, human CCR1) or a variant thereof as the first moiety, linked through a peptide bond to a second moiety that does not appear in the CCR1. of mammal 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 an 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 CCR1 or a portion thereof. Additional remains (eg, third, fourth) may be present, as appropriate. A "functional fragment or portion", "functional mutant" and / or "functional fusion protein" of a mammalian CCR1 protein refers to an isolated and / or recombinant protein or polypeptide having at least one characteristic function of a CCR1 protein. of mammal as described herein, such as a binding activity, a signaling activity and / or the ability to stimulate a cellular response. Preferred functional variants can be linked to a ligand (ie, one or more ligands such as MlP-RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF) and reference is made herein to them as "ligand binding variants". In one embodiment, a functional variant of mammalian CCR1 shares at least about 85% sequence identity with said mammalian CCR1, preferably at least about 90% sequence identity and, more preferably, at least about 95% sequence identity with said mammalian CCR1. In another embodiment, a functional fusion protein contains a first residue that shares at least about 85% sequence identity with a mammalian CCR1, preferably at least about 90% sequence identity and, more preferably, at least about 95% sequence identity with a mammalian CCR1. 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 CCR1 or a portion thereof. A composition containing an isolated and / or recombinant mammalian CCR1 or a functional variant thereof can be maintained under suitable binding conditions, the mammalian CCR1 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 CCR1 or cells that contain a recombinant nucleic acid sequence encoding a mammalian CCR1 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, enzyme 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 unlabeled antibody or a ligand as a com-petitioner. Binding inhibition assays can also be used to identify antibodies or fragments thereof that bind to CCR1 and inhibit the binding of another compound, such as a ligand (e.g., MlP-la RANTES, MCP-2, MCP-3, leuko-tactin-1, HCC-1 or MPIF) to CCR1 or a functional variant. For example, a binding assay can be carried out in which a reduction in the binding of a ligand to CCR1 (in the presence of an antibody, such as 2D4) 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 CCR1 or functional variant thereof can be contacted with the ligand and antibody simultaneously, 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 antibody. For example, ligand binding could be reduced or abolished. In one embodiment, direct inhibition of the binding of a ligand (eg, a chemokine such as MlP-la RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF) is monitored at a CCR1 of mammal or variant thereof by an antibody or fragment. For example, the ability of an antibody to inhibit the binding of MlP-labeled with I, RANTES labeled with I, MCP-2 labeled with bI, MCP-3 labeled with 125I, leucotatin-1 labeled with 125I, HCC can be monitored. -1 labeled with 125I or MPIF labeled with 125I to mammalian CCRl. Said assay can be conducted using suitable cells carrying CCR1 or a functional variant thereof, such as isolated blood cells (e.g., T cells) or a suitable cell line expressing CCR1 naturally, or a cell line containing nucleic acid. encoding a mammalian CCR1, or a membrane fraction of said cells, for example. Other methods of identifying the presence of an antibody that binds to CCR1 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 a binding inhibition assay. The competition between antibodies for binding to receptors can also be assessed in the method. The antibodies that are identified in this way can also be studied to determine whether, following the binding, they act by inhibiting other CCR1 functions and / or to assess their therapeutic utility. Signaling assays The binding of a ligand or promoter, such as an agonist, to CCR1, can result in signaling by this G protein-coupled receptor and stimulates the activity of G proteins, as well as other intracellular signaling molecules. . 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 CCR1 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 +] i, by methods known in the art or by other suitable methods (see, for example, Neote, K. et al., Cell 72: 415-425 (1993); Van Riper et al., J. Exp. Med. 177: 851-856 (1993); Dahinden, CA et al., J ". Exp. Med. 179: 751-756 (1994).) For example, the functional trial of Sledziewski et al. using hybrid G-protein coupled receptors to monitor the ability of a ligand or pro-motor to bind to the receptor and activate a G protein (Sled-ziewski et al., US 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 thereof which has of being studied and the ability of the antibody or fragment to inhibit the activity induced by the ligand or is determined 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 CCR1 or functional variant thereof and / or to inhibit associated function to the binding of the ligand to the receptor. 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 September 15, 1994, the teachings of which are here incorporated by reference; see also Berman et al., Immunol. Invest. 17: 625-677 (1988)). Migration through the endothelium to collagen gels has also been described (Kavanaugh et al., J. Immunol., 146: 4149-4156 (1991)). Stable transfectants from mouse pre-B L1.2 cells or other suitable host cells capable of undergoing chemotaxis in chemotaxis assays, for example, can be used. In general, chemotactic tests monitor the directional movement or migration of a suitable cell (such as a leukocyte (e.g., lymphocyte, eosinophil, basophil)) to or through a barrier (e.g., d-endothelium, a filter), to higher levels of a compound, from a first surface of the barrier to a second opposite surface. The membranes or filters provide convenient barriers, in such a way that the directional movement or migration of a suitable cell to or through a filter is monitored towards higher levels of a compound, from a first surface of the filter to a second 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 that 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 migration and migration inhibition, one can determine the migration distance to the filter, the number of cells that cross the filter that remain adherent to the second filter surface and / or the number of cells that accumulate in the filter. second camera using standard techniques (for example, microscopy). In one embodiment, the cells are labeled with a detectable labeling (eg, radioisotope, fluorescent labeling, antigenic or epitope tagging) and migration can be assessed in the presence and absence of the antibody or fragment by determining the presence of adherent labeling to 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 the background migration determined in the absence of the antibody, as compared to the degree of migration induced by a second compound. (ie, 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 CCR1, transeridothelial migration can be monitored. In this realization, 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 towards higher levels of a compound, from a first surface of the filter 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, towards 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 CCR1 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, shortly before 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 CCR1 in this assay are inhibitors of receptor function (e.g., function inhibitors). 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 an-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 emi-gration. In addition to the methods described, the effects of an antibody or fragment on the stimulating function of CCR1 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 CCR1 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 substances that bind to a mammalian CCRl or functional variant thereof, as well as inhibitors and / or promoters of the mammalian CCR1 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 CCR1 protein, as well as inhibitors (eg, antagonists) or promoters (eg, agonists) of the function of the receivers. In one embodiment, cells carrying a mammalian CCR1 protein or functional variant thereof (eg, leukocytes, cell lines, or suitable host cells that have been engineered to express a mammalian CCR1 or functional variant encoded by a nucleic acid introduced into these 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, ligands 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 is indicated. These inhibitors can be used in methods of treating inflammatory diseases, including autoimmune diseases and rejection of grafts, consisting of administering an inhibitor of receptor function to an individual (eg, a mammal). The ligands and / or promoters identified as described herein can be used in 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 stimulation. selective leukocyte function, which is useful, for example, in the treatment of infectious diseases and cancer. As used herein, a "ligand" of a mammalian CCR1 protein refers to a particular class of substrates that bind to a mammalian CCR1 protein, including natural ligands and synthetic and / or recombinant forms of natural ligands. Infectious agents that have a tropism by mammalian CCR1-positive cells (e.g., viruses such as HIV) can also bind to a mammalian CCR1 protein. A natural ligand of a selected mammalian receptor has a mammalian origin that is the same as that of the mammalian CCR1 protein (eg, a chemokine such as MlP-RANTES, MCP-2, MCP-3, leuco-tactin -1, HCC-1 and / or MPIF). In a preferred embodiment, ligand binding of a mammalian CCR1 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 CCR1 protein (e.g., a human CCR1), 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 +] i) 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 increases) at least one characteristic function of a mammalian CCR1 protein (e.g., a human CCR1), 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, which bind to the receptor (e.g., an antibody, a homolog of a natural ligand of another species) and substances that promote the function of the receptors without binding to them (e.g., by 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 1 or ligand-binding variant thereof, including ligands, inhibitors, promoters and other substances that bind to a mammalian CCR1 receptor or functional variant. According to the method, a study agent, an antibody or antigen binding fragment of the present invention can be combined (eg, 2D4, an antibody having an epitope specificity equal to or similar to that of 2D4 and antigen-binding fragments). of these) and a composition containing a mammalian CC chemokine receptor 1 or a ligand binding variant thereof. The above components are combined under suitable conditions to bind the antibody or antigen-binding fragment to the mammalian CC chemokine receptor 1 or a ligand-binding variant thereof and the binding of the antibody or fragment to the receptor is detected or measured. 1 of 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 1 or ligand-binding receptor thereof can be a membrane fraction of a recombinant chemokine receptor 1 protein carrier or ligand-binding variant thereof. 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. In one embodiment, the invention relates to a method of detecting or identifying an agent that binds to a mammalian CC chemokine receptor 1 or ligand binding variant thereof, consisting of combining a study agent, an antibody or antigen-binding fragment of the present invention (for example, 2D4, an antibody having an epitope specificity equal to or similar to that of 2D4 or antigen-binding fragments thereof) and a carrier cell of a CC-chemokine receptor 1 of mammal or a ligand binding variant thereof. The above components are combined under suitable conditions to bind the antibody or antigen-binding fragment to the CCR1 protein or ligand-binding variant thereof and the binding of the antibody or fragment to the mammalian CC chemokine receptor 1 is detected or measured. variant directly or indirectly according to the methods described herein or other suitable methods. A reduction in the amount of complex formed relative to a suitable 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 CCR1) or other substances, including inhibitors or promoters of receptor function, which can bind to CCR1 and compete with the anti-bodies described here by receptor binding. 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 CCR1 protein and inhibitors or promoters of a mammalian CCR1 or variant protein. The in vi tro methods of the present invention can be adapted for a selective high-throughput study in which large numbers of samples are processed (e.g., a 96-well format). Cells expressing mammalian CCR1 (eg, human CCR1) 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 CCR1 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 level can be selected. wanted. Nucleic acid encoding a mammalian CCR1 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 CCR1 protein or variant can be used, such as a receptor expressed in cells stably or transiently transfected with a construct containing a recombinant nucleic acid encoding a mammalian CCR1 protein or variant, or a cellular fraction containing a receptor (for example, a membrane fraction of transfected cells, liposomes incorporating a receptor) in assays for the function of the receptors. 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 CCR1 protein or functional variant thereof, such as a human CCR1, can be used in the present method, where the effect of a compound is monitored by monitoring the function of the receptors as described herein. or using other appropriate techniques. For example, stable or transient transfectants (eg, Sf9 cells infected with baculoviruses, stable transfectants of mouse pre-B L1.2 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 references cited therein; see also Ohlmeyer, M.H.J. et al., Proc. Natl. Acad. Sci. USA 90: 10922-10926 (1993), and DeWitt, S.H. et al., Proc. Natl. Acad. Sci. USA 90: 6909-6913 (1993), in relation to labeled compounds; Rutter, W.J. et al., U.S. Pat. No. 5,010,175; Huebner, V.D. et al., U.S. Pat. No. 5,182,366, and Geysen, H.M., US Pat. 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 CCR1 protein, an antibody or functional portion thereof of the present invention and a phage (eg, a phage or phage collection, such as a library) that exhibits a suitable conditions for the binding of the antibody or portion thereof to the mammalian CCR1 protein or variant (e.g., in a suitable binding buffer). Phages that compete with the antibody or portion thereof and bind to the mammalian CCR1 or variant protein 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 that express and display phages can be produced, consisting of a shell protein with an N-terminal peptide encoded by random sequence nucleic acids. Suitable host cells expressing a mammalian CCR1 or variant protein and an anti-CCR1 antibody or functional portion thereof are combined with the phage, the bound phages are selected, recovered and characterized. (See, for example, Doorbar and Winter, 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 March 6, 1997). Other sources of potential ligands or other substances that can bind to, or inhibitors and / or promoters of, mammalian CCR1 proteins include, but are not limited to, variants of CCR1 ligands, including natural, synthetic or recombinant variants of MlP-la, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF, substances such as other chemoattractants or chemokines, variants thereof, low molecular weight organic molecules, other inhibitors and / or promoters (for example, anti-HIV antibodies). -CCR1, antagonists, agonists), other ligands, inhibitors and / or promoters of G protein-coupled receptors (eg, antagonists or agonists) and soluble portions of a mammalian CCR1 receptor, such as a suitable receptor or analog peptide that it can inhibit the function of the receptors (see, for example, Murphy, RB, 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 CCR1 in a suitable animal, such as rabbit, mouse, rat, guinea pig or rhesus macaque (see, for example, example, Van Dáñame, 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 CCR1, labeled with 11: LIn, 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 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, chemiluminesis 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 CCR1 or to measure the ex-pressure of the receptor, for example, on T cells (e.g., CD26 + cells, CD45RO + cells), neutrophils, eosinophils 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-CCR1 antibodies of the present invention have value in diagnostic applications. An anti-CCR1 antibody or fragment thereof can be used to monitor the expression of this receptor in individuals, similarly to how anti-CD4 has been used as a diagnostic indicator of the HIV phase. Typically, diagnostic assays involve the detection of complex formation resulting from the binding of an antibody or fragment thereof to CCR1. For diagnostic purposes, the antibodies or antigen binding fragments may be labeled or unlabeled. 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 noassays 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 not labeled, the antibodies or fragments can be detected using suitable means, as in agglutination assays, for example. Antibodies or unlabeled fragments can 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 antibody (e.g., a second antibody) reactive with the first antibody (eg, anti-idiotype antibodies or other antibodies that are specific for unlabeled noglobulin) or another suitable reagent (eg, labeled protein A). In one embodiment, the antibodies or fragments of the present invention can be used in enzyme noassays, where the antibody or fragment in question, or the second antibodies, are conjugated to an enzyme. When a biological sample containing a mammalian CCR1 protein is combined with the antibodies in question, binding occurs between the antibodies and the CCR1 protein. In a realization, a sample containing cells expressing a mammalian CCR1 protein, such as human blood, is combined with the antibodies in question and binding occurs between the antibodies and the cells carrying a human CCR1 protein containing an epitope recognized 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 subject antibody may be added. Kits may also be prepared for use in detecting the presence of a mammalian CCR1 protein in a biological sample. Such kits will include an antibody or functional fragment thereof which binds to a mammalian CC chemokine receptor 1 or portion of said receptor, as well as one or more suitable auxiliary reagents for the detection of the presence of a complex between the antibody or fragment and CCRl 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 pro-viewed 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 CCR1 or a portion of the receptor by a cell, wherein a composition containing a cell or fraction is contacted. thereof (e.g., membrane fraction) with an antibody or functional fragment thereof (e.g., 2D4) that binds to a mammalian CCR1 or portion of the receptor under conditions appropriate for antibody or fragment binding thereto and the union is monitored. The detection of the antibody, indicative of the formation of a complex between the antibody and CCR1 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 CCR1 in cells of an individual (for example, in a sample, such as a body fluid, such as blood, saliva or other suitable sample). The level of expression of CCR1 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 the 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 leukocyte recruitment to 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-binding to the tight integrin-mediated binding. 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, J.J. et al., J. Cell Biol. 134: 255-266. (nineteen ninety six); Carr, M.W. et al., I muni ty 4: 179-187 (1996)). This migration is directed by a concentration gradient of chemoattractant that directs towards the inflammatory focus. The CCRl has an important role in leukocyte trafficking. CCR1 is likely to be a key chemokine receptor for the migration of T cells or subsets of T cells or monocytes to certain inflammatory sites and, therefore, anti-CCR1 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 (for example, 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 a functional antibody or 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 activation of the CCR1 receptor is involved by chemokine binding. For example, antibodies or functional fragments thereof (eg, 2D4 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, rejection of allografts, fibrotic disease, asthma and inflammatory glomerulopathies. Among the diseases or conditions of humans or other species that can be treated with inhibitors of CCR1 receptor function (including antibodies or suitable fragments thereof), include, but are not limited to, those described in US Application Ser. No. 09 / 239,283, "Methods for Preventing Graft Rejection and Ischemia-Reperfusion Injury," by Wayne W. Hancock, filed January 29, 1999, and in the US Application. No. 09 / 240,253, "Methods of treatment of inflammatory demyelinating disease using CCR1 antagonists", by James B. Rottman and Way-ne W. Hancock, presented on January 29, 1999, the teachings of both of which are incorporated herein. its entirety, and the following: o Inflammatory or allergic diseases and conditions, including allergic respiratory diseases such as asthma, allergic rhinitis, pulmonary hypersensitivity diseases, hypersensitivity pneumonitis or interstitial lung diseases (EPI) (eg, idiopathic pulmonary fibrosis or EPI) associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome, 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; • chronic or acute rejection of grafts (for example, in a transplant), including allograft rejection or graft versus host disease and graft-associated arteriosclerosis; »Atherosclerosis; < »Cancers with leukocyte infiltration of the skin or organs; »Other diseases or conditions (including diseases or conditions mediated by CCRl) can be treated where undesirable inflammatory responses must be inhibited, including, but not limited to, ischemia / reperfusion injury, certain hematological malignancies, cytokines-induced cytotoxicity (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 CCR1 receptor function (including antibodies or fragments thereof) include, but are not limited to: • immunosuppression, such as that which occurs in individuals with syndromes of immunodeficiency, 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 receivers or other causes. The anti-CCR1 antibodies of the present invention can block the binding of one or more chemokines, thus blocking the downward 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 antibodies of the present invention can be used in combination with other monoclonal or polyclonal antibodies (e.g., in combination with antibodies that bind to other chemokine receptors, including, but not limited to, CCR2, CCR3, CCR4 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. The antibodies or fragments of the invention can also be administered in combination with antiviral agents, immunosuppressive agents (eg, calcineurin inhibitors, such as cyclosporin A, glucocorticoids, such as prednisone or methylpredni-solone, and inhibitors of the synthesis of nucleic acids, such as azathioprine or mycophenolic acid), cytokines, such as inferons and Th2-producing cytokines, and hormones, such as adrenocorticotropic hormone. 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 the prophylactic) under the conditions of administration, such as an amount sufficient for the inhibition of a CCR1 function and, thus, for the inhibition of an inflammatory response or infection by HIV, or an amount sufficient to promote a CCR1 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, infraocular, 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's dextrose, dextrose and sodium chloride, lactated Ringer's 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 in a. Suitable support before use according to lyophilization and reconstitution techniques known in this field. 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 following Examples are offered for the purpose of illustrating the present invention and are not to be considered as limiting the scope of the invention. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference. EXAMPLES Methods Cells, cell lines and tissue culture Leukocytes were isolated from normal human blood as described (Heath et al., J. Clin Invest 99: 178 (1997)). To generate CD3 blasts, 2 x 10e PBMC / ml in RPMI 1640 plus 10% FCS were added to tissue culture plates first coated with anti-CD3 antibody TR66. After 4-6 days, the blasts were removed to fresh medium and supplemented with IL-2 (kindly provided by Antonio Lanzavecchia, Basel Institute for Immunology, Basel, Switzerland) at 50 units / ml. In some cases, 10 ng / ml of IFNa (Peprotech, Rocky Hill, NJ) was added to the cultures 4 days after the stimulation. Human CCR1 was expressed in the murine pre-B cell line Ll-2 as previously described (Honda et al., J., Immunol., 152: 4026 (1994).) In summary, primer sequences were obtained for the coding and coding regions. 3'UTR of Genbank CCRl (accession number L09320) and the gene was amplified by rt-PCR from human peripheral blood.The amplified product was di-recionally cloned into the expression vector pMRB-101 and cells were transfected. -2 with linearized plasmid and were selected for growth in medium containing mycophenolic acid.The resistant cells were stained with anti-FLAG MI Ab (IBI-A Kodak Co., New Haven, CT) and sorted by flow cytometry. After several sorting operations, the cells were again selected by chemotaxis to 10 nM MlP-la. MAb production and flow cytometry mAbs reactive with CCR1 were generated by immunizing mice with 1 x 107 transfectants Ll-2 CCRl intraperi only at 2-week intervals. Four days after the last immunization, the mice were sacrificed, the spleen was excised and cell fusion was carried out using the SP2 / 0 cell line, as described (Coligan et al., Current Protocols in Immunology 2. 5.2 (1992)). Specific mAbs reactive with CCR1 were identified by flow cytometry using a FACScan (Becton Dickinson &; Co., Mountain View, CA), as described below. Antibodies were selected for immunofluorescent staining of CCR1 transfectants without staining of non-transfected cells or cells transfected with all other human chemokine receptors. The specific mAb of CCR1 described in this work, 2D4, was isotyped by ELISA (Southern Biotechnology, Birmingham, AL) and determined to be IgG1. The 2D4 hybridoma can be cultured in 90% DMEM, 10% fetal calf serum and 100 ng / ml IL-6. Mouse mAbs for human CCR3, CCR5 and CXCR3 have been described (Qin et al., J. "Clin. Invest. 101: 746 (1998); Health et al., J". Clin. Invest. 99: 178 ( 1997)). Mabs conjugated with fluorochrome for CD4, CD8, CD19, CD25, CD26, CD45RA, CD45RA and anti-biotinylated IgE 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 2% human serum and 0.1% sodium azide (staining buffer), 5 μg / ml purified antibody, μg / ml of control mAb with isotype correspondence (Sigma Chemical Co., St. Louis, MO) or 50 μl of hybridoma culture supernatant. After 20 minutes at 4 ° C, the cells were washed twice with staining buffer and re-suspended in 50 μl of affinity-purified goat anti-mouse IgG (ab ') 2 and conjugated to FITC (Jackson ImmunoRe -search Laboratories). After 20 minutes, the cells were washed twice in staining buffer and analyzed by FACScan to determine the level of surface expression. Propidium iodide was used to exclude dead cells. For multicolor analysis, the cells were first stained with anti-CCRl 2D4 mAb, followed by mouse anti-APC (Pharmingen). After blocking with mouse serum, mAbs conjugated to PE, FITC or Cy3 were used together to stain the cells. The results were analyzed by FACScan using gate and electronic compensation. Chemokines, chemotaxis assays and li-gand binding assays Recombinant human chemokines were obtained from Pe-protech (Rocky Hill, NJ) or from R &D Systems (Minneapolis, MN), except for IL-8, which was produced recombinantly in LeukoSite (Wu et al., J., Biol. Chem. 271: 31202 (1996)). The chemotaxis of human PMBC or CD3 blasts was assessed in transendothelial migration assays (Carr et al., Proc. Natl. Acad. Sci. USA 91: 3652 (1994)) using the ECV304 cell line, as described (Ponath et al., J. "Clin.Invest.97: 604 (1996)). The cells that migrated to the bottom chamber were collected and relative cell counts were obtained using the FACScan. Chemotaxis of human eosinophils and neutrophils was performed using 96-well chemotaxis plates (Neuro Probé, Gaithersburg, MD). 29 μl of appropriately diluted chemokine in RPMI-1640 plus 0.5% BSA was added to the wells, which were then covered with a 2 micron filter membrane. 5 x 104-1 x 10 5 cells were added in 25 μl on the filter above each well. The plate was incubated for 25-30 minutes at 37 ° C, the filter was removed and the plate was frozen at -80 ° C for 30 minutes. After thawing, 6 1 of CyQuant solution (Molecular Probes, Eugene, OR) was added to the cilia and the plate was read by a fluorescence plate reader. Migration is presented as the mean of Relative Fluorescence Units ("RFU") that is proportional to the number of migrated cells. Chemokines labeled with 125 I of NEN (Boston, MA) were obtained. The binding of chemokines to the target cells was carried out as previously described (Ponath et al., J ", Exp. Med. 183: 2437 (1996).) In summary, the cells were resuspended in binding buffer (HEPES 50 mM, 1 mM CaCl 2, 0, 5% BSA) and incubated with radiolabelled ligand in the presence or absence of competitors. After 60 minutes at 37 ° C, the cells were washed 3 times in binding buffer supplemented with 0.5 N NaCl and the pellets were counted. All the experiments were carried out in duplicate and repeated at least three times. The adjustment of the curve and the concentrations that inhibit 50% of the specific binding (IC50) were calculated by means of the KaleidaGraph program (Synergy Software, Reading, PA). Calcium flow assay Freshly isolated human PBMC were labeled with Fluo-3 and responses to various chemokines were measured in a FACScan as previously described (Ponath et al., J., Clin .. Invest. 97: 604 (1996) The gate was opened to different populations of leukocytes by lateral forward scatterings and a stable basal fluorescence of FL was first obtained.The cells were then stimulated with various chemokines and the change in fluorescence intensity was recorded and during the time course When anti-receptor antibodies were used, the cells were incubated with mAb for 5 minutes before the addition of chemokine Results Generation of a blocking mAb against CCRl A monoclonal antibody, called 2D4, was generated by immunizing mice with cells expressing high levels of transfected human CCR1 This mAb stained cells transiently transfected with cDNA CCRl and varied s cell lines transfected with stable CCR1 independently de-rivated, but not parental cells or cells transfected with other chemokine receptors (Figure 1A). When 2D4 was added to ra-di-labeled ligand binding assays, it completely inhibited the binding of 125 I-MIP-la with an IC50 of 3.4 nM (0.5 μg / ml) (Figure IB). In the same set of experiments, the binding of 125 I-MIP-la to CCR1 underwent competition from unlabeled MlP-la and RANTES, with an IC 50 of 1.1 nM and 2.5 nM, respectively. 2D4 inhibited the binding of 125 I-RANTES to CCRl transfectants with an IC 50 of 4.5 nM (0.7 μg / ml) (Figure IB). CCR1 is the predominant chemokine receptor for MIP-1 and RANTES on monocytes. All peripheral blood monocytes were brilliantly stained with anti-CCR1 2D4 mAb (Figure 2A). Mobilization of [Ca2 +] i in monocytes induced by 20 nM of MlP-la or 20 nM of RANTES was completely blocked by 50 μg / ml of 2D4, whereas the response to IL-8 (Figure 2B) or MCP-1 did not was affected by this antibody. HCC-1, a chemokine recently described as a ligand for CCR1, induced a weak calcium response at a concentration of 100 nM, which was completely inhibited by 2D4 as well. 2D4 only partially inhibited the activities of MCP-3, a chemokine that has been shown to mediate functions through several chemokine receptors, including CCR2 on monocytes (Franci et al., J. "Immunol. 154: 6511 (1995)) The data of the Ca2 + flow corresponded with the results of the chemotaxis assays, which showed that the migration of monocytes induced by MlP-la and RANTES could be completely blocked by 2D4, while there was no effect on the chemotaxis of monocytes. MCP-1, a specific ligand of CCR2 (Table 1) Only minimal monocyte chemotaxis was induced by MlP-lβ, a ligand for CCR5, but not for CCR1.

Table 1: Inhibition of monocyte chemotaxis by anti-CCRl mAb Freshly isolated human PBMCs were used in the chemotaxis assay with 10 nM of MCP-1, MlP-la, RANTES and MlP-lβ. Migrated monocytic cells were counted by flow cytometry using frontal and lateral dispersions. CCR1 is expressed in a subgroup of memory T lymphocytes In peripheral blood, 2D4 consistently stained a small percentage of CD3 + cells, varying between 1 and 5%, depending on the individual. Multicolour flow cytometric analysis showed that CCR1 + T cells were CD45R0 + CD26 +, a characteristic phenotype of post-activation memory T cells (Figure 3A and 3B). All CCR1 + T cells were contained in the CCR5 + and CXCR3 + T cell population, which constitutes 30-50% of the total peripheral blood T cells (Figure 3B). 2D4 also consistently stained a small percentage of B cells (0.5% -5%, n = 6). CD56 + NK cells were virtually negative when stained with 2D4. Expression of CCR1 in activated T cells Activated T cells respond much more vigorously to chemokines than resting T cells and also show increased expression of a series of chemokine receptors (Qin et al., J. Clin. Invest. : 146 (1998), Loetscher et al., J ". Exp. Med. 184: 569 (1996), Wu et al., J. Exp. Med. 186: 1373 (1997)), CCR5 and CXCR3 in particular. To determine if CCR1 also has an increasing regulation in a similar way, peripheral blood T cells were activated by anti-CD3 and IL-2 and the cells were analyzed at various times for the expression of chemokine receptors by cytometry. During the first three days of stimulation with anti-CDS, most of the chemokine receptors were modulated from the cell surface decreasing, a previously observed phenomenon (Qin et al., J. Clin. Invest. 101: 746 ( 1998), Loetscher et al., J. Exp. Med. 184: 569 (1996)). The expression of CXCR3 and CCR5 began to increase after day 4 and reached maximum levels on days 5-6 for CXCR3 and days 12-14 for CCR5. However, the expression of CCR1 remained undetectable by flow cytometry through this period of time (Table 2 and Figure 4). Chemotaxis assays confirmed that CCR1 did not have a significant role in migration induced by MlP-la or RANTES from activated T cells and demonstrated that the majority of migration was mediated by CCR5 (Figure 5A and 5B). The data in Figure 5A show that 2D4 failed to inhibit the chemotaxis of T cell blasts to MlP-la or RANTES, whereas the anti-CCR5 mAb 2D7 blocked the migration induced by both MlP-la and RANTES, as well as the chemotaxis induced by MlP-lβ in > 90% Recently, it was described that IFNa could increase CCRl mRNA levels when T cells were cultured under Thl or Th2 polarizing conditions (Sallusto et al., J.

Exp. Med. 187: 875 (1998)). In the present study, the ability of IFNa was determined to increasingly regulate the surface expression of CCRl in mass culture, where expression was usually down regulated. In all the donors studied, an increase in the expression of CCRl of the activated T cells was detected when they were cultured in the presence of IFNa. The data in Table 2 show that CCR1 started to appear in cells treated with IFNa by day 7 and gradually reached a maximum level between days 10-14, that is, 6-10 days after the addition of IFNa (Table 2 and Figure 4). The CCR1 expressed on T cells treated with IFNa made a significant functional contribution to the chemotactic potential of these cells, as demonstrated by the blocking of receptors with mAbs in migration assays to MlP-la and RANTES (Figure 5B). In the presence of IFNa, anti-CCR1 could inhibit cell migration to MlP-la by 39%, while anti-CCR5 inhibited migration to MlP-la by 58%. A combination of anti-CCR1 and anti-CCR5 inhibited chemotaxis by 89%. The anti-CCR1 mAb had no effect on the chemotaxis of MlP-la from CD3 blasts cultured only with IL-2, which could be completely inhibited by 50 μg / ml anti-CCR5 (Figure 5A). The anti-CCR1 mAb had no effect on migration to MlP-lß, whereas the anti-CCR1 mAb inhibited this migration in > 95%, regardless of IFNa treatment.

Table 2: Change of expression of chemokine receptors during activation of T cells Human PBMC were stimulated with anti-CD3 mAb on Day 0. On Day 4, the cells were divided and incubated with IL-2 alone or with IL-2 and IFNa. The cells were harvested on the indicated days and stained for the expression of the chemokine receptors. The data represent the results of one of eight donors; all gave a similar profile. Marked variation of CCR1 expression in eosinophils Human eosinophils respond vigorously to eo-taxin, RANTES, MCP-3 and MCP-4. Using a blocking mAb for CCR3, a previous study showed (Heath et al., J. Clin.Invest. 99: 178 (1997)) that these responses were predominantly mediated through CCR3. Although weak responses to MlP-la have been observed, the expression of CCR1 in human eosinophils has not been well characterized. With the anti-CCR1 mAb, a marked variation of donors was observed in the ex-pressure of CCR1 on human eosinophils. Figure 6 depicts the staining profile of purified eosinophils from four individuals. The expression of CCR1 varied between completely negative (Donor # 52) and more than 95% positive (Donor # 5). The expression of CCR3 in these cells was almost > 95% positive. Functionally, eosinophils that were completely negative for CCRl failed to respond to MlP-la in the chemotaxis assays, but those from donors that stained with 2D4 did respond to MlP-la, although the response was less effective than to eotaxin (Figure 7A and 7B). The limited number of donor samples analyzed to date and the weak chemotactic response of these donors to MlP-la does not allow a statistical analysis of the general pattern of CCRl expression in eosinophils among a population, nor does it allow the establishment of a strong correlation between the percentage of CCR1 + eosinophils and their ability to respond to MlP-la at this time. However, it should be noted that, among all the donors studied, eotaxin was significantly more potent than MlP-la in provoking an eosinophil chemotaxis, even when CCR1 and CCR3 showed a high expression of the humanBuferófilos human It is known that murine neutrophils produce a vigorous chemotactic response to MlP-la, presumably through CCRl (Gao et al., J., Exp. Med. 185: 1959 (1997).) On the contrary, human neutrophils migrate very poorly, if at all. to do, to MlP-la, although it has been seen that stimulation with MlP-la induces mobilization of Intracellular Ca (McColl and col J.) Immuno 1 150: 4550 (1993)). In the current study, the anti-CCRl 2D4 mAb stained 30-50% of the neutrophils of all the donors studied (Figure 8), but the ligands of CCRl MlP-la, RANTES and MPIF failed to induce any neutrophilic response in the chemotaxis, degranulation or adhesion assays at concentrations up to 200 nM. In the Ca2 + flux assays, approximately 6-8% of the neutrophils responded to stimulation with MlP-la or MPIF (Figure 9A, 9B), contrary to IL-8, which induced a response by essentially all neutrophils (Figure 9C). The anti-CCR1 2D4 mAb completely blocked the mobilization of [Ca2 +] i induced by MlP-la or MPIF, but not that induced by IL-8 (Figures 9D-9F). CCR1 is expressed in human basophils Basophils are potent inflammatory cells and have been shown to respond to a series of chemokines and express CCR3 on their surface (Uguccioni et al., J. Clin Invest 100: 1137 (1997)). By double staining of human blood with anti-IgE or CCR3, both markers for basophils given their different frontal and lateral dispersions, the expression of CCR1 was detected in virtually all basophils (Figure 11) with the anti-CCR1 mAb 2D4. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without deviating from the spirit and scope of the invention as it remains. defined by the appended claims.

LIST OF SEQUENCES < 110 > LeukoSite, Inc. Qin, Shixin Newman, Walter Kassam, Nasim < 120 > ANTI-CCR1 ANTIBODIES AND MET < DDC USE FOR THEM < 130 > LKS97-13A PCT < 140 > PCT / US99 / 04527 < 141 > 03-02-1999 < 150 > 09 / 239,938 < 151 > 01-29-1999 < 150 > PCT / US99 / 01915 < 151 > 01-29-1999 < 160 > 1 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 355 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > TRANSMEM < 222 > (38) ... (60) < 221 > TRANSMEM < 222 > (69) ... (90) < 221 > DOMAIN < 222 > (91) ... (107) < 223 > extracellular < 221 > TRANSMEM < 222 > (108) ... (129) < 221 > TRANSMEM < 222 > (150) ... (170) < 221 > DOMAIN < 222 > (171) ... (205) < 223 > extracellular < 221 > TRANSMEM < 222 > (206) ... (227) < 221 > TRANSMEM < 222 > (240) ... (260) < 221 > DOMAIN < 222 > (261) ... (280) < 223 > extracellular < 221 > TRANSMEM < 222 > (281) ... (304) < 400 > 1 Met Glu Thr Pro Asn Thr Thr Glu Asp Tyr Asp Thr Thr Thr Glu? Ne 1 5 10 15 Asp Tyr Gly Aso Wing Thr Pro Cys Gln Lys Val Asn Glu. Arg Wing Phe 20 25 30 Gly Wing Gln Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe Val He Gly 35 40 45 Leu Val Gly Asn He Leu Val Val Leu Val Leu Val Gln Tyr Lys Arg 50 55 60 Leu Lys Asn Met Thr Ser He Tyr Leu Leu Asn Leu Ala He Ser Asp 65 70 75 80 Leu Leu Phe Leu Phe Thr Leu Pro Phe Trp He Asp Tyr Lys Leu Lys 85 90 95 Asp Asp Trp Val Phe Gly Asp Wing Met Cys Lys He Leu Ser Gly Phe 100 105 110 Tyr Tyr Thr Gly Leu Tyr Ser Glu He Phe Phe He He Leu Leu Thr -115 120 125 He Asp Arg Tyr Leu Wing He Val His Wing Val Phe Wing Leu Arg Wing 130 135 140 Arg Thr Val Thr Phe Gly Val He Thr Ser He He He Trp Wing Leu 145 150 155 160 Ala He Leu -Ala Ser Met Pro Gly Leu Tyr Phe Ser Lys Thr Gln Trp 165 170 175 Glu Phe Thr Kis His Thr Cys Ser Leu His Phe Pro His Glu Ser Leu 180 185 190 Arg Glu Trp Lys Leu Phe Gln Ala Leu Lys Leu Asn Leu Phe Gly Leu 195 200 205 Val Leu Pro Leu Leu Val Met He He Cys Tyr Thr Gly He He Lys 210 215 220 He Leu Leu Arg Arg Pro Asn Glu Lys Lys Ser Lys Wing Val Arg Leu 225 230 235 240 He? H.e Val He Met He He Phe Phe Leu Phe Trp Thr Pro Tyr Asn 245 250 255 Leu Thr He Leu He Ser Val Phe Gln Asp Phe Leu Phe Thr His Glu 260 265 270 Cys Glu Gln Ser Arg His Leu -Asp Leu Ala Val Gln Val Thr Glu Val 275 280 285 He Ala Tyr Thr His Cys Cys Val Asn Pro Val He Tyr Ala Phe Val 290 295 300 Gly Glu - Arg Phe Arg Lys Tyr Leu Arg Gln Leu Phe His Arg Arg Val 305 310 315 320 Ala Val His Leu Val Lys Trp Leu Pro Phe Leu Ser Val Asp Arg Leu 325 330 335 Glu Arg Val Ser Ser Thr Ser Pro Ser Thr Gly Glu His Glu Leu Ser 340 345 350 Wing Gly Phe 355

Claims (49)

Claims

1. An antibody or antigen-binding fragment thereof that binds to a mammalian chemokine CC receptor (CCR1) 1 or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor and wherein said antibody or antigen-binding fragment thereof has an epitope specificity that is the same as or similar to that of monoclonal antibody 2D4.

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 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 (CCR1) 1 is a human chemokine CC receptor (CCR1).

4. An antibody or antigen-binding fragment thereof according to Claim 1, wherein the antibody or antigen-binding fragment thereof has specificity for the second extracellular loop of chemokine receptor CC (CCR1).

5. An antibody or antigen-binding fragment thereof according to Claim 4, wherein the antibody or antigen-binding fragment thereof has specificity for an epitope consisting essentially of amino acid 171 to amino acid 205 of chemokine receptor CC (CCR1). ) human.

6. An antibody or antigen-binding fragment thereof according to Claim 1, wherein the antibody is selected from the group consisting of: a) monoclonal antibody 2D4, b) an antibody that can compete with 2D4 for binding to chemokine receptor CC (CCR1) of mammal and c) antigen binding fragments of (a) or (b) that bind to mammalian chemokine CC receptor (CCR1) 1 or a portion thereof.

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

8. An antibody or antigen-binding fragment thereof according to Claim 7, wherein the chemokine is any one or more of MlP-RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF. .

9. The 2D4 hybridoma cell line deposited under ATCC Accession No. HB-12644.

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

11. A test kit for use in detecting the presence of a mammalian chemokine CC (CCR1) receptor 1 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 (CCR1) receptor 1 or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor and wherein said antibody or binding fragment The antigen thereof has an epitope specificity equal to or similar to that of monoclonal antibody 2D4, and b) one or more auxiliary reagents suitable for detecting the presence of a complex between said antibody or antigen-binding fragment thereof and said chemokine receptor 1. CC (CCR1) of mammal or portion thereof.

12. An assay kit according to Claim 11, wherein the antibody is selected from the group consisting of: a) monoclonal antibody 2D4, b) an antibody that can compete with 2D4 for binding to mammalian chemokine CC receptor (CCR1) 1, c) antigen-binding fragments of (a) or (b) that bind to mammalian chemokine CC receptor (CCR1) 1 or a portion thereof, and d) combinations of the foregoing.

13. A method of inhibiting the interaction of a carrier cell of a mammalian chemokine CC receptor (CCR1) with a ligand thereof, comprising contacting said cell with an effective amount of an antibody or binding fragment. antigen thereof which binds to the mammalian chemokine CC receptor (CCR1) 1 or portion of said receptor and inhibits the binding of said ligand to the receptor, wherein said antibody or antigen-binding fragment thereof has an epitope specificity equal or similar to that of monoclonal antibody 2D4.

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 CCR1 or a portion thereof.

15. A method according to Claim 14, wherein the T cells are selected from the group consisting of CD26 + cells and CD45RO + cells.

16. A method according to Claim 13, wherein the ligand is a chemokine.

17. A method according to Claim 16, wherein the chemokine is any one or more of MlP-RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF.

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 2D4, b) an antibody that can compete with 2D4 for binding to chemokine receptor CC (CCR1) of mammal, c) antigen-binding fragments of (a) or (b) that bind to mammalian chemokine CC receptor (CCR1) 1 or a portion thereof and d) combinations of the foregoing.

19. A method of detecting mammalian chemokine CC (CCR1) receptor 1 expression or portion thereof by a cell or fraction of said cell, comprising: a) contacting a composition containing a cell or fraction thereof; said cell to be studied with an antibody or antigen-binding fragment thereof that binds to the mammalian chemokine CC receptor (CCR1) or portion of said receptor and inhibits the binding of said ligand to the receptor, wherein said antibody or antigen-binding fragment thereof has an epitopic specificity equal or similar to that of monoclonal antibody 2D4, under conditions suitable for the binding of said antibody or antigen-binding fragment thereof to a mammalian CCR1 or portion thereof, and ) detecting the binding of said antibody or binding fragment thereof, wherein the binding of said antibody or antigen-binding fragment thereof indicates the presence of di receiver or portion of said receiver 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: a) monoclonal antibody 2D4, b) an antibody that can compete with 2D4 for receptor binding 1 of mammalian chemokine CC (CCR1), c) antigen binding fragments of (a) or (b) that can bind mammalian chemokine CC receptor (CCR1) 1 or a portion thereof and d) 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 receptor (CCR1) 1 or portion of said receptor, comprising: a) contacting a sample to be studied with an antibody or antigen-binding fragment thereof binds to the mammalian chemokine CC (CCR1) receptor 1 or portion of said receptor and inhibits the binding of said ligand to the receptor, and wherein said antibody or antigen-binding fragment thereof has an epitope specificity equal to or similar to that of the monoclonal antibody 2D4, under conditions appropriate for the binding of said antibody or fragment thereof to said mammalian CCR1 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 mammalian chemokine CC receptor (CCR1) 1 or portion of said receptor or 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: a) monoclonal antibody 2D4, b) an antibody that can compete with 2D4 for binding to chemokine receptor CC (CCR1) of mammal, c) antigen-binding fragments of (a) or (b) that bind to mammalian chemokine CC receptor (CCR1) 1 or a portion thereof and d) combinations of the foregoing.

24. A method according to Claim 22, wherein the sample is a cellular fraction which, in normal individuals, has a mammalian chemokine CC receptor (CCR1) 1 or portion of said receptor.

25. A method of inhibiting a function associated with the binding of a chemokine to a CC chemokine receptor 1 (CCR1) of a mammal 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 (CCR1) of mammal or portion of said receptor, wherein said antibody or fragment inhibits the binding of said chemokine to the mammalian chemokine CC receptor (CCR1) 1 and inhibits one or more functions associated with the binding of the chemokine to the receptor and wherein said antibody or antigen-binding fragment thereof. it has an epitopic specificity equal or similar to that of the mono-clonal antibody 2D4.

26. A method according to Claim 25, wherein the chemokine is any one or more of MIP-RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF.

27. A method according to Claim 25, wherein the antibody or antigen-binding fragment is selected from the group consisting of: a) monoclonal antibody 2D4, b) an antibody that can compete with 2D4 for binding to receptor 1 of mammalian chemokine CC (CCR1), c) antigen-binding fragments of (a) or (b) that bind to mammalian chemokine CC receptor (CCR1) 1 or a portion thereof and d) combinations of the foregoing.

28. A method of detecting or identifying an agent that binds to a mammalian receptor 1 chemokine CC (CCR1) or ligand-binding variant thereof, consisting of combining a) an agent to be studied, b) a antibody or antigen-binding fragment that binds to a mammalian chemokine CC receptor (CCR1) 1 or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor and wherein said antibody or antigen-binding fragment thereof has an epitope specificity equal to or similar to that of monoclonal antibody 2D4, and c) a composition containing a mammalian chemokine CC receptor (CCR1) or a ligand-binding variant of the same, under conditions suitable for the binding of said antibody or antigen-binding fragment to said mammalian chemokine CC receptor (CCR1) or ligand-binding variant thereof, and detecting or measuring the binding n of said antibody or antigen-binding fragment to said mammalian chemokine CC receptor (CCR1) 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 2D4, b) an antibody that can compete with 2D4 for binding to chemokine receptor CC (CCR1) of mammal, c) antigen-binding fragments of (a) or (b) that bind to mammalian chemokine CC receptor (CCR1) 1 or a portion thereof and d) combinations of the foregoing.

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 receptor (CCR1) or ligand-binding variant is moni- tored and where a reduction in the amount of complex formed relative 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 (CCR1) receptor 1 or a ligand-binding variant thereof is a recombinant chemokine CC receptor (CCR1) carrier cell or a ligand binding variant thereof.

32. A method according to Claim 31, wherein the composition containing a mammalian chemokine CC receptor (CCR1) 1 or a ligand binding variant thereof is a membrane fraction of said chemokine receptor 1 carrier cell. Recombinant CC (CCR1) 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 having specificity for a mammalian chemokine CC receptor (CCR1) 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 which binds to a mammalian chemokine CC reagent-1 (CCR1) or portion of said receptor and inhibits binding of a ligand to the receptor, wherein said antibody or antigen-binding fragment of the same has an epitopic specificity equal or similar to that of the 2D4 monoclonal antibody.

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

37. A method according to Claim 36, wherein the chemokine is any one or more of MlP-RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF.

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 2D4, b) an antibody that can compete with 2D4 for binding to receptor 1 of mammalian chemokine CC (CCR1), c) antigen-binding fragments of (a) or (b) that bind to mammalian chemokine CC receptor (CCR1) 1 or a portion thereof and d) combinations of the foregoing.

39. A composition containing an antibody or antigen-binding fragment thereof that binds to a receptor 1 of mammalian chemokine CC (CCR1) or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor, wherein said antibody or antigen-binding fragment thereof has a epitope specificity equal or similar to that of monoclonal antibody 2D4, and a physiologically acceptable eventual vehicle.

40. An antibody or antigen-binding fragment thereof that binds to a mammalian chemokine CC receptor (CCR1) 1 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 10 μg / ml.

41. An antibody or antigen-binding fragment thereof according to Claim 40, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor with an IC50 of less than about 5 μg / ml.

42. An antibody or antigen-binding fragment thereof according to Claim 41, wherein said antibody or antigen-binding fragment thereof inhibits the binding of a ligand to the receptor with an IC5o of less than about 1 μg / ml.

43. An antibody or antigen-binding fragment thereof that binds to a mammalian chemokine CC receptor (CCR1) 1 or portion of said receptor with an affinity greater than about 5 x 10 ~ 8 M.

44. An antibody or antigen-binding fragment thereof according to Claim 43, wherein the affinity is at least about 5 x 10 ~ 9 M.

45. An antibody or antigen-binding fragment thereof that binds to a chemokine CC receptor 1 ( CCR1) of mammal or portion of said receptor, wherein said antibody or antigen-binding fragment thereof inhibits chemo-taxis with an IC50 less than about 50 μg / ml.

46. An antibody or antigen-binding fragment thereof according to Claim 45, wherein the IC50 is less than about 20 μg / ml.

47. An antibody or antigen-binding fragment thereof according to Claim 46, wherein the IC50 is less than about IQ μg / ml.

48. A method of treating a disorder mediated by chemokine CC receptor 1 (CCR1) in a patient, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof that binds to receptor 1 of mammalian chemokine CC (CCR1) or a portion thereof and inhibits binding of a ligand to the receptor, wherein said antibody or antigen-binding fragment thereof has an epitope specificity equal or similar to that of monoclonal antibody 2D4.

49. 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 mammalian chemokine CC receptor (CCR1) 1 or a portion thereof. thereof and inhibits the binding of a ligand to the receptor, wherein said antibody or antigen-binding fragment thereof has an epitope specificity equal or similar to that of monoclonal antibody 2D4.