JP2003517810A - Anti-CCR1 antibodies and methods of use thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to a mammal (e.g., human) CC-chemokine receptor 1 (CCR1) or an antibody that binds to a part of the receptor and blocks binding of a ligand to the receptor, or a functional fragment thereof. Furthermore, the present invention relates to a method for inhibiting the interaction between a mammalian CCR1-producing cell and its ligand, and the use of said antibodies and fragments in research, therapy, prophylaxis and diagnostics.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Chemokines have gained attention as important inflammatory mediators during the last decade due to their numerous proinflammatory activities that act on almost all leukocyte types. Recently, chemokines have been shown to have normal immune surveillance and immune response as well as hematopoiesis,
It has become recognized as a key component of basal leukocyte trafficking, which is essential for angiogenesis, control of viral infection and several other functions in T cell differentiation (Baggiolini et al., Ann. Rev. Immu
nol. 15: 675 (1997); Zou et al., Nature 393: 59.
5 (1998); Tachibana et al., Nature 393: 591 (19).
98)). This diverse array of biological activities, including mediation of a range of proinflammatory effects on leukocytes, such as chemotaxis, degranulation, synthesis of lipid mediators and triggers for integrin activation, contributes to the initiation and maintenance of inflammatory diseases. Together with their important role and the recent identification of certain chemokine receptors as co-receptors for HIV-1 entry, chemokines and chemokine receptors have become an attractive new therapeutic target group.

Members of the chemokine family are produced and secreted by many cell types in response to early inflammatory mediators such as IL-1β and TNFα. The chemokine superfamily contains two major branches, an α-chemokine characterized by an insertion of one amino acid between the first two cysteines (ie the CXC chemokine) and two adjacent cysteines. Includes β-chemokines (CC chemokines). The branches of α-chemokines include proteins such as IL-8, neutrophil activating peptide-2 (NAP-2), melanoma growth stimulating activity (MGSA / gro or GROα) and ENA-78. Each of the proteins has primarily an attracting and activating effect on neutrophils. Members of the β-chemokine branch act on other cell types such as monocytes, lymphocytes, basophils and eosinophils (Openpenheim, JJ, et al., Annu. Rev.
． Immunol. , 9: 617-648 (1991); Baggiolini.
, M .; Et al., Adv. Immunol. 55: 97-179 (1994); Mil.
Ler and Krangel, Crit. Rev. Immunol. , 12: 1
7-46 (1992); Jose, P .; J. Et al., J. Exp. Med. , 179
: 881-118 (1994); Ponath, P .; D. Et al., J. Clin. I
nvest. , 97: 604-612 (1996)), and monocyte chemotactic proteins 1-4 (MCP-1, MCP-2, MCP-3 and MCP-4),
RANTES, macrophage inflammatory protein (MIP-1α, MIP-1β
), Thymus and activation-regulated chemokines (thymus and acti).
It includes proteins such as vation-regulated chemokines (TARCs) and macrophage-derived chemokines (MDCs).

Chemokines bind to the 7-transmembrane G protein-coupled receptor (Murp
hy, P.H. M. , Annu. Rev. Immunol. , 12: 593-633
(1994)). To date, many β-chemokine receptors (CCR1 to CCR
10) has been identified, and the search for further chemokine receptors has been the subject of active research (Baggiolini, Nature 392: 565-5).
68 (1998)). The chemokine receptor CCR1 is described by Nomura et al. (Int
． Immunol. 5: 1239-1249 (1993), Neote et al. (Ce
ll 72: 415-425 (1993)) and Gao et al. (J. Exp. Me.
d. 177: 1421-1427 (1993)). Initially CC
Although R1 was found to signal in response to MIP-1α and RANTES, it was also recently revealed to signal in response to other chemokine ligands.

Selective recruitment of leukocyte subsets to sites of inflammation and the regular transport of leukocytes through the circulatory system, tissues, lymphoid and secondary lymphoid organs is, in part, a difference in chemokine receptors in subsets of cells. It is regulated by subsequent expression. Such expression patterns appear to ensure that a group of functionally related leukocytes can respond cooperatively to a particular group of chemokines induced by a given stimulus. However, most leukocytes express several types of chemokine receptors, many with complex and intricate ligand interactions. In the case of T cells, PCR or Northern blotting shows that the known receptor for CC chemokines is expressed on a subset of T cells. The task of accurately describing which subset expresses a particular receptor has been an area of intense research. This is because chemokine receptor expression may account for the localization or migration of various cell types such as TH1 or TH2 T cells or tissue homing subsets. It may also determine which T cells are infected by different strains of HIV-1. This makes it difficult to elucidate normal immune function for specific receptors on a given cell type and to determine its association with disease initiation and progression.
This is because, in particular, many chemokine receptors do not have specific antibodies available.

SUMMARY OF THE INVENTION CCR1 is the first chemokine receptor demonstrated to interact with C—C (β) chemokines. Initially identified as the MIP-1α / RANTES receptor, more recent studies have shown that CCR1 can carry as many as six β-chemokine ligands. The studies described herein characterize the expression of CCR1 by flow cytometry and evaluate the relative functional contribution of this receptor to human leukocytes in vitro using blocking monoclonal antibodies. In peripheral blood, all monocytes express high levels of CCR1
Both monocyte responses to -1α and RANTES are anti-CCR1 mAb 2
Can be completely blocked by D4. CCR1 is CD45RO + CD26 + T
It is expressed in a small fraction of cells, suggesting that it characterizes a subset of memory T cells. In contrast to the chemokine receptors CCR5 and CXCR3, which are upregulated on activated T cells, CCR1 expression is reduced upon T cell activation. Neutrophils express low levels of CCR1 and show a weak response to MIP-1α in the [Ca ²⁺ ] _i mobilization assay, which is anti-CCR1.
It can be inhibited by mAb 2D4. Expression of CCR1 on eosinophils can vary extremely between individuals, from> 90% positive to completely negative. The expression pattern of CCR1 suggests that it is responsible for a wide range of immunological activities, including monocyte and eosinophil function and T
It is suggested to play an important role in the function of a subset of lymphocytes.

The present invention relates to mammalian CC chemokine receptor 1 (CCR1 or CKR-
1) or a part of its receptor (anti-CCR1) and an antibody (immunoglobulin) or a functional fragment thereof (eg, antigen-binding fragment). In one aspect, the antibody or fragment thereof of the invention has specificity for human CCR1 or a portion thereof. In another aspect, the antibody or fragment of the present invention comprises a ligand for the above receptor (eg, MIP-1α, RANTES, MCP-2, MCP-).
3, leukotactin-1, HCC-1 or MPI
It inhibits (reduces or prevents) the binding of F) and inhibits one or more functions (eg leukocyte trafficking) associated with the binding of the ligand to the receptor. In a preferred aspect, the ligand is MIP-1α or RANTES. For example, as described herein, antibodies of the invention and fragments thereof that bind human CCR1 or a portion thereof may be associated with chemokines (eg, MIP-1α, RAN) for this receptor.
TES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MP
IF) binding can be blocked to inhibit the functions associated with chemokine binding to the receptor.

In a preferred embodiment, the antibody or fragment thereof of the present invention has the same or similar epitope specificity as the monoclonal antibody (mAb) LS125-2D4 (2D4). For example, the antibody or fragment thereof may be the second extracellular loop of CCR1 (eg CC
(A portion of R1 from around amino acid 171 to around amino acid 205) can have epitope specificity. In one aspect, the antibody is monoclonal antibody 2D4 or an antibody capable of competing with 2D4 for binding to human CCR1 or a portion of human CCR1. Functional fragments of the antibodies described above are also contemplated.

In another aspect, the antibody or fragment thereof binds CCR1 with an affinity greater than about 5 × 10 ⁻⁸ M, more preferably at least about 5 × 10 ⁻⁹ M. In a further embodiment, the antibody or fragment thereof of the invention has a chemokine binding to CCR1 preferably less than about 10 μg / ml, more preferably about 5 μg / m 2.
It inhibits with an IC ₅₀ of less than 1, more preferably less than about 1.0 μg / ml. In one embodiment, the antibody or fragment thereof of the present invention is MIP-1α for CCR1.
Binding is inhibited with an IC ₅₀ of approximately 0.5 μg / ml. In another embodiment, the antibody or fragment thereof of the invention has about 0.7 μg RANTES binding to CCR1.
Inhibition with an IC ₅₀ of / ml. In a further embodiment of the invention the antibody or fragment thereof is induced by chemokines (eg MIP-1α, RANTES.
Or HCC-1 induced) cells (eg, CCR1 producing cells) chemotaxis is preferably inhibited by less than about 50 μg / ml, more preferably less than about 20 μg / ml, even more preferably less than about 10 μg / ml. . It is contemplated that any of the antibodies or fragments described herein can be used in the methods described herein.

The present invention also provides an antibody or a functional thereof that binds to a portion of mammalian CCR1 or its receptor and increases the fluorescence staining intensity of CCR1 or a CCR1-containing composition as compared with other anti-CCR1 antibodies. Fragments (eg antigen binding fragments). In one embodiment, the antibody is monoclonal antibody 2D4 or an antibody capable of competing with 2D4 for binding to human CCR1 or a portion of human CCR1.

The invention further relates to a method of inhibiting the interaction of a cell-carrying mammalian (eg, human, non-human primate or mouse) CCR1 with its ligand. The method comprises contacting the cells with an effective amount of an antibody or functional fragment thereof that binds to mammalian CCR1 or a portion of CCR1. Suitable cells include granulocytes, leukocytes (such as monocytes, macrophages, basophils and eosinophils), mast cells, lymphocytes containing T cells such as Th1 cells and Th2 cells (eg CD8 + cells, CD4 + cells, CD26 +). Cells, CD25 + cells, CD45R
O + cells) as well as other cells expressing CCR1 or a portion thereof, such as recombinant cells expressing CCR1 (eg, transfected cells).
In certain embodiments, the antibody is 2D4, or human CCR1 or human CC.
An antibody that can compete with 2D4 for binding to a portion of R1.

Another aspect of the invention relates to a method of inhibiting the interaction of mammalian CCR1-producing cells with chemokines. The method involves contacting the cells with an effective amount of an antibody or functional fragment thereof that binds to CCR1 or a portion of the receptor. In an embodiment of the method, the antibody or functional fragment thereof is any one or more of 2D4, 2D4 antigen-binding fragments, or an antibody or fragment thereof having the same or similar epitope specificity as 2D4. . Furthermore, the present invention provides a CCR
1. A method of inhibiting a function associated with the binding of a chemokine to 1. The method comprises the step of administering an effective amount of an antibody or functional fragment thereof that binds to mammalian CCR1 or a portion of the above receptors. In one aspect of this method, the antibody or functional fragment thereof is any one or more of 2D4, 2D4 antigen-binding fragments, or an antibody having the same or similar epitope specificity as that of 2D4 or a fragment thereof. It is a fragment.

Another aspect of the invention is a method of identifying expression of mammalian CCR1 or a portion of this receptor by cells. In this method, an antibody or a functional fragment (eg, 2D4) thereof that binds to a composition containing a cell or a fraction thereof (eg, a membrane fraction) to a mammalian CCR1 protein or a part of this receptor, and an antibody thereto Are contacted under conditions suitable for binding and the formation of a complex between the antibody or fragment and the protein or part thereof is detected. Detection of the complex indicates, either directly or indirectly, the presence of the receptor or part thereof on the cells or fractions thereof. The invention also relates to kits for use in detecting the presence of CCR1 or a portion thereof in a biological sample. This kit comprises an antibody or a functional fragment thereof that binds to mammalian CCR1 or a part of the above receptor,
It comprises one or more auxiliary reagents suitable for detecting the presence of a complex of said antibody or fragment with said protein or part thereof.

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, a reagent having the same or similar binding specificity as that of the antibody of the present invention or a functional fragment thereof can be identified by a competition assay using the above antibody or fragment. Accordingly, the present invention provides CC containing inhibitors (eg, antagonists) or enhancers (eg, agonists) of receptor function.
Also included are methods of identifying a ligand or other substrate that binds to the R1 receptor.
In one embodiment, cells that naturally express the CCR1 receptor protein, or suitable host cells that have been engineered to express the CCR1 receptor or variant encoded by a nucleic acid introduced into the cell, are treated with ligand, receptor function. For use in assays to identify and evaluate their potency. Such cells are also useful in assessing the function of the expressed receptor protein or polypeptide.

Accordingly, the present invention relates to a method of detecting or identifying a reagent that binds to mammalian CCR1 or a ligand binding variant thereof. This method can be performed by testing the reagents to be tested, the antibodies or antigen-binding fragments of the invention (eg, monoclonal antibody 2D4).
, An antibody with the same or similar epitopic specificity as 2D4, an antigen binding fragment of 2D4) and a composition containing a mammalian CCR1 protein or a ligand binding variant thereof. The above components are mammalian CCR1
The antibody or antigen-binding fragment can be mixed under conditions suitable for binding the antibody or antigen-binding fragment thereof to a protein or a ligand-binding variant thereof, and binding of the antibody or fragment to a mammalian CCR1 protein or a ligand-binding variant is herein described. It is detected or measured directly or indirectly according to the method described or other suitable method. A decrease in the amount of complex formed as compared to a suitable control (eg in the absence of the reagent being tested) is an indication that the reagent binds to the receptor or variant. The composition containing the mammalian CCR1 protein or ligand binding variant thereof may be a membrane fraction of cells producing the recombinant CCR1 protein or ligand binding variant thereof. The antibody or fragment thereof can be labeled with a label such as a radioisotope, a spin label, an antigen label, an enzyme label, a fluorescent group and a chemiluminescent group. These and similar assays demonstrate that substances that bind CCR1 and can compete with the antibodies described herein for binding to the receptor,
It can be used, for example, to detect ligands (eg chemokines that interact with CCR1) or other substrates including inhibitors or promoters of receptor function.

According to the present invention, ligands, inhibitors or promoters of receptor function can be identified in appropriate assays and evaluated for therapeutic efficacy. Inhibitors of receptor function can be used to inhibit (reduce or prevent) receptor activity, and ligands and / or promoters can be used to induce (induce or enhance) normal receptor function as needed. . These ligands, inhibitors and promoters inhibit inflammatory diseases, autoimmune diseases, atherosclerosis and graft rejection or HIV infection, eg, receptor function (eg chemokine binding or HIV binding). The substance may be used for treatment in a manner that includes administering the substance to an individual (eg, a mammal such as a human). Also, these ligands, inhibitors and promoters can be used in methods of stimulating receptor function by administering new ligands or promoters to an individual. This provides a new clue for selectively stimulating the function of white blood cells, which is useful, for example, in the treatment of infectious diseases and cancer.

The invention also includes a method of inhibiting leukocyte trafficking in a patient. This method
Administering to a patient an effective amount of an antibody or functional fragment thereof that binds to mammalian CCR1 or a portion of the receptor and inhibits the function associated with the binding of a ligand for that receptor.

The invention also relates to methods of inhibiting or treating CCR1-mediated disorders such as inflammatory disorders. The method comprises the step of administering to a patient an effective amount of an antibody or functional fragment thereof that binds to mammalian CCR1 or a portion of the receptor and inhibits CCR1-mediated function.

The present invention further provides the antibody or fragment thereof described herein for use in therapy (including prophylaxis) or diagnosis (eg, the same or similar to that of an antigen-binding fragment of monoclonal antibody 2D4, 2D4, 2D4). Antibody with epitopic specificity)
, As well as the use of the above antibodies or fragments for the manufacture of a medicament for treating CCR1-mediated disorders or other diseases or inflammatory conditions described herein.

DETAILED DESCRIPTION OF THE INVENTION The first identified CC chemokine receptor, CCR1, was originally MIP-
It was described as the 1α / RANTES receptor. But since those reports, C
CR1 has been shown to interact with many β chemokines including MIP-1α, 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. Am. Biol. Chem. 272: 11682 (19
97); Gong et al. Biol. Chem. 272: 11682 (199
7); Youn et al. Immunol. 159: 5201 (1997) and Youn et al., Blood 91: 3118 (1998)). Leukotactin-
1 is also known as HCC-2 or MIP-1δ, HCC-1 is also known as Ckβ1 or MIP-1γ, and MPIF is also known as CKβ8 or MIP-3. Most of these chemokines are found in other chemokine receptors, such as CCR2 (Combadiere), which have expression patterns that overlap those of CCR1.
Et al., J. Biol. Chem. 270: 29671 (1995)), CCR3 (
Ponath et al. Exp. Med. 183: 2437 (1996)) and CCR5 (Samson et al., Biochemistry 35: 3362 (19).
96); Raport et al., J. Am. Biol. Chem. 271: 17161 (19
96)) and the like have also been reported as functional ligands. CCR1 mR
NA is a T cell (Loetcher et al., J. Exp. Med. 184: 569 (
1996)), B cells (Gao et al., J. Exp. Med. 177: 1421 (1).
993)), basophils (Uguccioni et al., J. Clin. Invest.
100: 1137 (1997)), eosinophils (Post et al., J. Immunol.
155: 5299 (1995)), monocytes and granulocytes (Nomura et al., Int.
． Immunol. 5: 1239 (1993)) and dendritic cells (Sozza).
ni et al. Immunol. 161: 1083 (1998)), all of which have been shown to functionally respond to CCR1 ligands.

Targeted disruption of CCR1 in mice has provided some insight into the role of this receptor in normal immune function and disease pathology. A study (G
Ao et al. Exp. Med. 185: 1959 (1997)), CCR1
− / − Mice exhibit abnormal steady state and induced transport of myeloid progenitor cells from the bone marrow to blood and spleen, possibly due to impaired neutrophil function.
It promoted mortality, decreased granulomatous inflammatory response, and altered Th1 / Th2 cytokine balance when testing the immunity of Aspergillus fumigatus. Another report (Lu et al., J. Exp. Med. 187:
601 (1998)) linked CCR1 to respiratory distress syndrome associated with acute pancreatitis in mice that is believed to result from reduced intrapancreatic sequestration of neutrophils in CCR1 knockout animals.

[0021] Several studies have demonstrated in vivo function for the two most extensively studied CCR1 ligands, MIP-1α and RANTES. The importance of MIP-1α for both T cell-dependent and NK-dependent viral clearance has been demonstrated in MIP-1α knockout mice (Cook et al.,
Science 269: 1583 (1995)). Airway inflammation (Standif
ord et al. Immunol. 155: 1515 (1995)), experimental allergic encephalomyelitis (EAE) (Karpus et al., J. Immunol. 155:
5003 (1995)) and arthritis (Barnes et al., J. Clin. Inv.
est. 101: 2910 (1998)) in a rodent model with MIP-1α and R
The use of neutralizing antisera against ANTES suggests a role for these ligands in a range of inflammatory diseases. However, since MIP-1α and RANTES are ligands for CCR1, CCR3 and CCR5 in the mouse, the role of these individual receptors in these models and their relevance as anti-inflammatory drug targets remains unclear. Is.

As a step towards understanding the function of CCR1 in humans, a monoclonal antibody (mAb) against this receptor was generated as described herein to characterize the expression and function of CCR1 on human leukocytes. used. Inhibition of CCR1 ligand activity in vitro by receptor blockade with this mAb supports and supports the results of flow cytometry. These data show that CCR1 expression and regulation share some common ligands
It has been shown to be distinct from other CC chemokine receptors such as 3 and CCR5, providing further evidence for a complex and multi-faceted chemokine receptor system.

Chemokines and their receptors constitute important components in the regulation of directional leukocyte migration. Chemokines are produced locally during the inflammatory response,
It attracts various white blood cells that produce the corresponding receptors. While the spectrum of chemokines expressed at the site of injury can differentially attract specific inflammatory cells, the selectivity and variability of chemokine receptor expression on leukocytes depends on appropriate cell recruitment to a given stimulus. To provide an additional adjustment mechanism that ensures
As the number of identified and characterized chemokine receptors continues to grow, cells will identify functional subsets of leukocytes such as Th1 and Th2, naive and memory T cells, activated and resting T cells. The selective expression of several receptors that can be labeled, or otherwise characterized, is becoming increasingly apparent. Since some chemokine receptors are co-expressed on individual cells, it was difficult to establish the role of particular receptors in disease initiation and progression (and, in turn, normal immune function). The expression pattern of CCR1 was evaluated as described herein and the functional contribution of this receptor to normal immune function was sought using anti-CCR1 mAb 2D4.

T cells, particularly activated T cells, are MCP-1, RANTES, MIP-1α, M
It shows chemotactic response to many CC chemokines including IP-1β and CXC chemokines such as IP-10, MIG and ITAC. CCR2 for activated T cells (Qin et al., Eur. J. Immunol. 26: 640 (
1996)), CCR5 and CXCR3 (Qin et al., J. Clin. In).
vest. 101: 746 (1998)).
This correlated with an increase in the amount of chemotactic activity of those receptors to the ligand.
Recent studies have also described an increase in CCR1 mRNA levels in activated T cells (Sallusto et al., J. Exp. Med. 187: 875 (19).
98)). Although only a small group of resting T cells express CCR1 in peripheral blood, no significant CCR1 expression on the surface of T cells upon anti-CD3 and IL-2 stimulation has been identified. There may be post-transcriptional or post-translational machinery that regulates surface expression of CCR1. Alternatively, as reported by the loss of CXCR2 from neutrophils induced by TNFα (Asagoe et al., J.I.
mmunol. 160: 4518 (1998)), CCR1 may be downregulated by other cytokines produced during T cell activation. This data suggests that most of the effects of MIP-1α and RANTES on activated T cells are likely to be CCR5-mediated (Wu et al., J. Exp. Med. 186: 1373 (
1997)).

CCR1 is distinct, eg, either CCR5 or CXCR3, due to the fact that it was able to expand the CCR1 positive T cell population under certain activation conditions (IFNα treatment as described herein). It is suggested that it may be involved in the T cell response to a more specific series of stimuli. IFNα is an important cytokine in host defense against viral infection, and human PBMC
Have been found to induce MIP-1α production (Bug et al., Exp.
Hematol. 26: 117 (1998)). Furthermore, studies using MIP-1α-deficient mice showed resistance to, for example, coxsackievirus-induced myocarditis, reduction of influenza virus-associated pneumonia, and resistance to herpes simplex virus type 1-associated substrate keratitis. Defects in T cell inflammatory response associated with infection with several viruses are found, including (Tumpey et al., J. Virol).
． 72: 3705 (1998)).

In addition to lymphocytes, monocytes also respond to C—C chemokines. In this specification mAb
By receptor blockade with 2D4, MIP-1α and RANTES and H
It is shown that the monocyte response to CC-1 appears to be completely CCR1 dependent. So far, the only other major CC chemokine receptor found on human monocytes is CCR2. The role of the monocyte / macrophage lineage as an antigen-presenting cell and the finding that monocyte-derived dendritic cells express CCR1 (Sozz
ani et al. Immunol. 161: 1083 (1998)), this receptor and its ligands may play an important role in specific immune responses by regulating the migration of antigen presenting cells.

A number of studies have demonstrated the importance of CCR1 in rodent granulocyte migration. However, the expression and function of CCR1 on human granulocytes remains unclear.
CCR1 was detected on most granulocytes by the specific mAb 2D4 described herein. In eosinophils, CCR1 expression was significantly variable between donors, in contrast to CCR3, which is always expressed at uniformly high levels.

CCR1 was also found to be expressed on neutrophils, but no significant chemotactic response of these cells to the CCR1 ligands MIP-1α, RANTES or MPIF was observed. The inability of neutrophils to migrate towards the CCR1 ligand may be due to low receptor density at the cell surface, as shown by weak immunostaining, or due to lack of signaling pathway components or lack of adhesion molecules. Similarly, in resting T cells, approximately 30-40% of peripheral blood T cells express CXCR3, which, unless activated by anti-CD3 and IL-2, are unable to migrate in response to IP-10 or MIG. Can't (Qin et al., J. Clin. Inve
st. 101: 746 (1998)). Alternatively, these results may suggest that chemotaxis is not a major function of CCR1 on neutrophils. In fact, in neutrophils, IL-8 binding of CXCR1 resulted in activation of NADPH oxidase and phospholipase D, whereas IL-8 binding of CXCR2 did not result in their activation, whereas neither receptor chemotactic. Evidence for the differential function of chemokine receptors has been described to function in sex and elastase release (Jones et al., Proc. Natl. Acad. Sci. USA, 9
3: 6682 (1996)). The lack of neutrophil chemotaxis activity towards CCR1 ligands has been demonstrated by MIP-1α (McColl et al., J. Immunol. 150: 455).
0 (1993)) or MPIF (Forssmann et al., FEBS Lett.
． 408: 211 (1997)), which was consistent with the previous two reports that also failed to detect migration of neutrophils to neutrophils, but found that MPIF was chemotactic for neutrophils. Different from the study (Patel et al., J. Exp. Med. 18).
5: 1163 (1997)). About 10% of neutrophils are MIP-1α or MPI
It was shown to be mediated by CCR1 as 2D4 was able to completely block this response, accompanied by intracellular Ca ²⁺ mobilization in response to F stimulation. Since the proportion of eosinophils obtained from these donors in total granulocytes is less than 3%, the observed [Ca ²⁺ ] _i flux was not due to contaminated eosinophils, and MIP-1α
And MPIF are consistent with previous reports found to stimulate [Ca ²⁺ ] _i flux in neutrophils.

CC on various leukocytes reported here using anti-CCR1 mAb 2D4
Expression of R1 was reported previously using a polyclonal antiserum raised against the N-terminal peptide-GST fusion protein of CCR1 (Su et al., J. Leukoc. B.
iol. 60: 658 (1996)). Where m
It was shown that Ab 2D4 detected CCR1 expression in almost all monocytes, Su
Et al. Reported that their polyclonal antisera detected CCR1 expression on monocytes. However, while the 2D4 mAb detected CCR1 expression only in a discrete subpopulation of T cells, Su et al. Reported that the antisera stained more than 80% of peripheral blood CD3 +. 2D4 also detected a discrete subpopulation of B cells (about 1-5%), but Su et al. Reported that the antisera did not detect CCR1 expression on B cells, eosinophils or neutrophils. There is. 2D4 not only detected this expression by flow cytometry, but was also able to functionally block MIP-1α-mediated chemotaxis and Ca ²⁺ flux in these cells, confirming the credibility of the staining. Have shown that CCR1 is the major (if not the only) MIP-1α receptor on granulocytes. Since rabbit polyclonal is reported to recognize an epitope in the NH ₂ -terminal domain and 2D4 recognizes the second extracellular loop, a significant discrepancy in the staining profile between the 2D4 mAb and the rabbit polyclonal antiserum resulted in these This may be because the epitopes recognized by the reagents are different. Low titers of antibodies recognizing native epitopes as well as low susceptibility due to low affinity antibodies in antiserum were associated with B polyclonal cells in rabbit polyclonal antiserum,
It may explain the lack of detection of CCR1 on eosinophils and neutrophils. Su et al. Report that the antiserum inhibited less than 50% monocyte chemotaxis at 100 μg / ml, while 2D4 showed 100% inhibition at 50 μg / ml.
However, the discrepancy in identifying T cell expression seen between these two reagents is probably due to the different accessibility of the epitope to different cell types.

CCR1 ligands include MIP-1α, RANTES and MCP-3, which are also shared by other receptors. For example, CCR3 interacts with MIP-α, RANTES and eotaxin, and CCR5 interacts with MIP.
It interacts with -1α, RANTES and MIP-1β. This apparent duplication may be necessary given the distribution of receptors for these ligands. CC
R3 is mainly expressed on eosinophils, basophils and a subset of T cells, but not on neutrophils and monocytes, CCR5 is mostly expressed on T cells, especially activated T cells. The studies described herein show that CCR1 could be found in all monocytes, the majority of granulocytes, including basophils, and a subpopulation of T cells.
Furthermore, CCR1 expression on eosinophils and T cells appeared to be actively regulated. Thus, each receptor has its own population of host cells that can serve different functions during the inflammatory response.

The present invention provides mammalian CC-chemokine receptor 1 (CCR1, CKR-1
Or an antibody (anti-CCR1) or a functional fragment thereof that binds to a part of CCR1. In one embodiment, the antibody has specificity for human CCR1 or a portion thereof. In one embodiment, the antibody (immunoglobulin) is directed against isolated and / or recombinant mammalian CCR1 or a portion thereof (eg, peptide) or against a host cell expressing mammalian CCR1. Produced.
In a preferred embodiment, the antibody specifically binds to the human CCR1 receptor or a portion thereof, and in a particularly preferred embodiment, the antibody is specific for naturally occurring or endogenous human CCR1. Have sex. Mammal CC
One or more functions specific to R1, such as binding activity (eg, binding of ligands, inhibitors and / or promoters), signaling activity (eg, activation of mammalian G proteins, cytosolic free calcium concentration). Induction of a rapid and transient increase in [Ca ²⁺ ] _i and / or stimulation of cellular responses (eg chemotaxis, exocytosis or leukocyte release of inflammatory mediators, integrin activation). The present invention also includes an antibody or a functional fragment thereof capable of inhibiting the above. This antibody is a ligand for CCR1 (ie, one or more ligands)
And / or can inhibit one or more functions mediated by CCR1 in response to a ligand. For example, in one aspect, the antibody or functional fragment thereof comprises the receptor, MIP-1α, RANTES, MCP-2, MCP-3,
It may inhibit (reduce or prevent) interaction with natural ligands such as leukotactin-1, HCC-1 or MPIF. In one aspect, the ligand is MIP-1.
α, RANTES or HCC-1. In another aspect, the antibody or functional fragment thereof that binds CCR1 is a mammalian CCR1 (eg, human CCR1).
1. MIP-1α for non-human primate CCR1, mouse CCR1),
It may inhibit the binding of RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF. The antibodies or functional fragments thereof of the present invention may inhibit human CCR1 mediated functions including, for example, leukocyte trafficking, T cell activation, inflammatory mediator release and / or leukocyte degranulation. In certain embodiments, cells in which the antibody or functional fragment thereof is induced by chemokines (eg, induced by MIP-1α, RANTES or HCC-1) (
Inhibition of chemotaxis (eg CCR1 producing cells) is preferably less than about 50 μg / ml, more preferably less than about 20 μg / ml, even more preferably about 10 μg / ml.
Shown in less than ml.

In yet another aspect of the invention, the antibody or functional fragment thereof of the invention has a binding of a CCR1 ligand (eg a chemokine) to CCR1 preferably with an IC _{50 of} less than about 10 μg / ml, more preferably about 5 μg. IC ₅₀ less than / ml,
Even more preferably, it can inhibit with an IC ₅₀ of less than about 1.0 μg / ml. In another aspect, the antibody of the present invention or a functional fragment thereof can inhibit the binding of MIP-1α to CCR1 with an IC ₅₀ of about 0.5 μg / ml. In yet another embodiment, the antibody or functional fragment thereof of the present invention is capable of inhibiting the binding of RANTES to CCR1 with an IC ₅₀ of about 0.7 μg / ml. In a further aspect of the invention the antibody or fragment thereof is stronger than about 5 × 10 ⁻⁸ M, preferably at least about 5 × 1.
Bind to the CCR1 at 0 ^-9 M of affinity.

A mouse monoclonal antibody specific for CCR1 (designated 2D4) was generated as described herein. In a preferred embodiment, the antibody of the present invention is human CCR1.
And has the same or similar epitope specificity as that of the mouse 2D4 antibody described herein. Antibodies with the same or similar epitopic specificity as that of the mouse 2D4 monoclonal antibody can be identified using techniques well known in the art. For example, an antibody with the same or similar epitope specificity as that of 2D4 is a human C
CR1 (eg CCR1-producing transfectants, CD8 + cells, CD
4D cells, CDR45RO + cells, CD25 + cells, monocytes, dendritic cells, human CCR1 producing cells such as macrophages and basophils) or the ability to compete with mouse 2D4 monoclonal antibody for binding, or to inhibit binding of 2D4 to human CCR1 Ability to perform or through the use of receptor chimeras. Receptor chimera (Rucker et al., Cell 87: 437)
-446 (1996)), mAb 2D4 was shown to bind to the second extracellular loop of CCR1 (FIGS. 10A and 10B). For example,
2D4 can bind to a region of human CCR1 consisting essentially of amino acids 171 to 205 (eg, from about amino acid 171 to about amino acid 205; FIG. 12). By using these techniques or other suitable techniques, antibodies with the same or similar epitopic specificity as those of the antibodies of the invention can be identified. The present invention also includes 2
Relates to bispecific antibodies or functional fragments thereof (eg F (ab) ₂ ) having the same or similar epitopic specificity as D4 and at least one other antibody (eg US Pat. No. 5,141,736 (Iwasa)). Et al., U.S. Pat. No. 4,444.
, 878, 5,292,668, 5,523,210 (above Pa.
ulus et al.) and US Pat. No. 5,496,549 (Yamazaki et al.)).

The hybridoma cell line producing the antibody of the present invention was transformed into Le on February 1, 1999.
ukoSite, Inc. , 215 First Street, Cambri
dge, MA 02142, U.S.A. S. A. On behalf of the American Type Culture Col (American Type Culture Col)
direction), 10801, University Boulevard,
Manassas, Virginia 20110, U.S.A. S. A. , Under accession number HB-12644 (LS125-2D4-11-10-1 (2D4)). The present invention is 2D4 deposited under ATCC Registration No. HB-12644.
It also relates to the hybridoma cell line, and the monoclonal antibody produced by the 2D4 hybridoma cell line deposited under ATCC Accession No. HB-12644.

The antibody of the present invention may be polyclonal or monoclonal.
The term "antibody" is meant to include both polyclonal and monoclonal antibodies. Furthermore, in the methods described herein using 2D4, functional fragments of 2D4 (eg, antigen-binding fragments), antibodies with the same or similar epitope specificity as 2D4, and combinations thereof, optionally also the same as 2D4. It is understood that it can be used in combination with an antibody or fragment having an epitopic specificity which is neither similar nor similar. The antibodies of the present invention can be raised against a suitable immunogen, eg, an isolated and / or recombinant mammalian CCR1 protein or synthetic molecule such as a fragment or synthetic peptide thereof. In a preferred embodiment, cells expressing the receptor (eg, transfected cells) can be used as an immunogen or as a screening tool for antibodies that bind to the receptor.

The antibodies of the invention and fragments thereof are useful in therapeutic, diagnostic and research applications as described herein. The invention is for use in treatment (including prophylaxis) or diagnosis (eg, diagnosis of a particular disease or condition as described herein) and for treating a disease or condition as described herein. The use of an antibody or a functional portion thereof as described above in the manufacture of a medicament for treating the antibody of the invention or a functional portion thereof (eg mAb 2D4 or an antigen binding fragment thereof).

Preparation of immunizing antigen and production of polyclonal and monoclonal antibodies can be performed as described herein or using other suitable techniques. Various methods have been described (eg, Kohler et al., Nature, 256: 495-.
497 (1975) and Eur. J. Immunol. 6: 511-519
(1976); Milstein et al., Nature 266: 550-552.
(1977); Korowski et al., U.S. Pat. No. 4,172,124; Ha.
rlow, E .; And D. Lane (1998) Antibodies: A
Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY); Ausubel, F .; M. Ed.,
Current Protocols In Molecular Biolo
gy Vol. 2 (Supplement 27, Summer 1994) (John
Chapter at Wiley & Sons, New York, NY
11 (1991) etc.). Generally, hybridomas can be produced by fusing a suitable immortalized cell line (eg, a myeloma cell line such as SP2 / 0) with antibody-producing cells. Antibody producing cells, preferably cells from the spleen or lymph nodes, are obtained from animals immunized with the antigen of interest. Fused cells (hybridomas) can be isolated using selective culture conditions and cloned by the limiting dilution method. Cells which produce antibodies with the desired binding characteristics are labeled with a suitable assay (eg, ELISA).
).

Other suitable methods of producing or isolating antibodies that bind CCR1 (including human or artificial antibodies) can be used. This is, for example, a recombinant antibody (eg,
Single-chain Fv or Fab) from a library, immunization of transgenic animals (eg mice) capable of producing a repertoire of human or artificial antibodies (eg Jakobovits et al., Proc.
． Natl. Acad. Sci. USA, 90: 2551-2555 (1993.
); Jakobovits et al., Nature, 362: 255-258 (199).
3); Lonberg et al., US Pat. No. 5,545,806; Surani et al.
See, eg, US Pat. No. 5,545,807).

Single-chain and chimeric antibodies, humanized antibodies or primatize
d) (CDR-grafted) antibodies as well as chimeric antibodies or CDR-grafted single-chain antibodies containing moieties from different species are also encompassed by the present invention and the term "antibody".
The various parts of these antibodies may be chemically linked to each other by conventional techniques,
It can be produced as a continuous protein using genetic engineering techniques. For example, a nucleic acid encoding a chimeric or humanized chain can be expressed to produce a continuous protein. For example, Cabilly et al., US Pat. No. 4,816,567,
Cabilly et al., EP 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., EP 0,120,694 B.
No. 1; Neuberger, M .; S. , WO 86/01533; Ne
Uberger, M .; S. Et al., European Patent No. 0,194,276 B1; Win
Ter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,23.
9,400 B1; Queen et al., US Pat. Nos. 5,585,089, 5,
See 698,761 and 5,698,762, and the like. For primatized antibodies, see Newman, R. et al. Et al., BioTechnology, 1
0: 1455-1460 (1992), for single chain antibodies Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. et al. E. Et al., Science,
242: 423-426 (1988)).

In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. The functional fragments of the above-mentioned antibodies retain at least one binding function and / or regulatory function of their original full-length antibody. Preferred functional fragments retain the antigen binding function of the corresponding full-length antibody (eg, retain the ability to bind mammalian CCR1). Particularly preferred functional fragments retain the ability to inhibit one or more functions unique to mammalian CCR1 such as binding activity, signaling activity and / or stimulation of cellular responses. For example, in one embodiment, the functional fragment inhibits the interaction of CCR1 with one or more ligands (eg, MIP-1α, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF). And /
Or functions mediated by one or more receptors (eg leukocyte trafficking, H to cells)
IV invasion, T cell activation, inflammatory mediator release and / or leukocyte degranulation) can be inhibited.

For example, antibody fragments (including but not limited to Fv, Fab, Fab ′ and F (ab ′) ₂ ) that are capable of binding to the mammalian CCR1 receptor or a portion thereof are encompassed by the present invention. Such fragments may be produced, for example, by enzymatic cleavage or recombinant technology. For example, cleavage with papain or pepsin can generate Fab fragments or F (ab ') ₂ fragments, respectively. Antibodies can also be produced in various 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 an F (ab ′) ₂ heavy chain portion can be designed to include DNA sequences encoding the CH ₁ domain and the hinge region of the heavy chain.

As used herein, the term “humanized immunoglobulin” refers to an immunoglobulin that comprises portions of immunoglobulins of different origin, at least one portion of which is of human origin. Accordingly, the present invention provides mammalian CCR1 (eg,
A humanized immunoglobulin that binds to human CCR1 and mouse CCR1), which is an antigen-binding region of non-human origin (eg, rodent) and an immunoglobulin of human origin (eg, human framework region, human) Constant region or a part thereof). For example, humanized antibodies are chemically linked to each other by conventional techniques (eg, synthetic) or prepared as contiguous polypeptides using genetic engineering techniques (eg, DNA encoding the protein portion of a chimeric antibody is A portion derived from a non-human origin immunoglobulin (eg, mouse) having the required specificity (which can be expressed to produce a continuous polypeptide chain) and a portion derived from an immunoglobulin sequence of human origin (eg, , Chimeric immunoglobulins). Another example of a humanized immunoglobulin of the present invention is a CDR of non-human origin (eg, one or more CDRs from an antibody of non-human origin) and a frame of light and / or heavy chain of human origin. An immunoglobulin containing one or more immunoglobulin chains containing a work region (eg, a CDR-grafted antibody with or without framework mutations). In one embodiment, the humanized immunoglobulin is mouse 2 for binding to human CCR1.
It can compete with the D4 monoclonal antibody. In a preferred embodiment, the antigen-binding region of humanized immunoglobulin (a) is (eg as in the case of a humanized immunoglobulin consisting of 2D4 light chain CDR1, CDR2 and CDR3 and 2D4 heavy chain CDR1, CDR2 and CDR3). 2) derived from the 2D4 monoclonal antibody. Chimeric or CDR-grafted single chain antibodies are also included in the term humanized immunoglobulin.

Such humanized immunoglobulins can be produced using synthetic and / or recombinant nucleic acids to produce the gene (eg, cDNA) encoding the desired humanized chain. For example, a nucleic acid (eg, DNA) sequence encoding a humanized variable region is
It can be constructed using PCR mutagenesis to modify DNA sequences encoding human or humanized chains (eg, DNA templates derived from previously humanized variable regions) (eg, Kamman, M. et al., Nucl). . Acids. Res., 1
7: 5404 (1989)); Sato, K .; Et al, Cancer Research
ch, 53: 851-856 (1993); Daugherty, B .; L. ,
Nucleic Acids Res. , 19 (9): 2471-2476 (1
991); Lewis, A .; P. And J. S. Crowe, Gene, 10
1: 297-302 (1991) and the like). Variants can be easily produced using these or other suitable methods. In one embodiment,
The cloned variable region can be mutated and a sequence encoding a variant with the desired specificity selected (eg, from a phage library, eg, K
Rebber et al., US Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993; March 1997.
See Knappik et al., Published on June 6, WO 97/08320, etc.).

Anti-idiotypic antibodies are also provided. Anti-idiotypic antibodies recognize antigenic determinants involved in the antigen binding site of another antibody. Anti-idiotypic antibodies can be raised against a secondary antibody by immunizing an animal of the same species, preferably the same strain, as the animal used to produce the secondary antibody. See, eg, US Pat. No. 4,699,880.

The present invention also relates to the 2D4 hybridoma cell line deposited under ATCC Accession No. HB-12644 as well as the monoclonal antibody produced by the 2D4 hybridoma cell line deposited under ATCC Accession No. HB-12644. The cell line of the present invention has uses other than the production of monoclonal antibodies. For example,
The cell lines of the present invention can be used to generate additional hybridomas using other cells (suitable drug-marked human myeloma, mouse myeloma, human-mouse heteromyeloma or human lymphoblasts). Fused to a sphere cell, etc.) to provide transfer of the gene encoding the monoclonal antibody. These cell lines can also be used as a source of nucleic acids encoding anti-CCR1 immunoglobulin chains. It can be isolated and expressed (eg, when transferred to other cells using any suitable technique (eg, Cabilly et al., US Pat. No. 4,816,567; Winter, US Pat. No. 5 , 225, 539))). For example, clones containing rearranged anti-CCR1 light or heavy chains can be isolated (eg, by PCR), or cDNA libraries can be generated from mRNA isolated from these cell lines to produce anti-CCR1. A cDNA clone encoding the immunoglobulin chain can be isolated. Therefore, in order to produce specific immunoglobulins, immunoglobulin chains or variants thereof (eg humanized immunoglobulins) in various host cells or in an in vitro translation system, heavy chains and / or light chains of antibodies or parts thereof are produced. The nucleic acid encoding the strand can be obtained and used according to recombinant DNA technology. For example, nucleic acids (including cDNA, or derivatives thereof that encode variants such as humanized immunoglobulins or immunoglobulin chains) can be placed into a suitable prokaryotic or eukaryotic vector (eg, an expression vector). It can be introduced into a suitable host cell by a suitable method (eg transformation, transfection, electroporation, infection etc.). As a result, the nucleic acid is operably linked to one or more expression control elements (eg, integrated in the vector or in the genome of the host cell). For production, maintaining host cells under conditions suitable for expression (eg, in the presence of a suitable medium supplemented with inducers, suitable salts, growth factors, antibiotics, nutrient supplements, etc.) Which results in the production of the encoded polypeptide. If desired, the encoded protein can be recovered and / or isolated (eg, from host cells, medium, milk). It will be appreciated that this method of production includes expression in host cells of transgenic animals (eg, WO 92/03918, GenPharm Interna, published Mar. 19, 1992).
(see topical).

As described herein, the antibodies of the invention and functional fragments thereof are CCR1.
Can block (inhibit) the binding of a ligand to and / or inhibit one or more functions associated with the binding of that ligand to CCR1. As described below, various methods can be used to assess the function associated with the inhibition of ligand binding to CCR1 and / or the binding of ligand to the receptor.

Binding Assay As used herein, “mammalian CCR1” refers to naturally occurring
ccuring) or an endogenous mammalian CCR1 protein, and a protein having an amino acid sequence identical to that of a naturally occurring or endogenous mammalian CCR1 protein (eg, a recombinant protein). Thus, as defined herein, the term refers to mature receptor proteins, polymorphisms or allelic variants, and isoforms of mammalian CCR1 (eg, produced by alternative splicing or other cellular processes). , And modified or unmodified forms of the foregoing (eg, glycosylated, non-glycosylated). The mammalian CCR1 protein can be an isolated and / or recombinant protein (such as a synthetically produced protein). Naturally occurring or endogenous mammal C
CR1 proteins include wild-type proteins such as mature CCR1, polymorphic or allelic variants and other isoforms that naturally occur in mammals (eg, human, non-human primates). Such proteins can be recovered or isolated from sources that naturally give rise to mammalian CCR1, for example. These proteins and the mammalian CCR1 proteins having the same amino acid sequence as the naturally occurring or endogenous corresponding mammalian CCR1 are designated by the name of the corresponding mammal. For example, if the corresponding mammal is a human, the protein is
Human CCR1 protein (eg, recombinant human CC produced in a suitable host cell)
R1 protein).

A “functional variant” of a mammalian CCR1 protein is a functional fragment,
Functional muteins and / or functional fusion proteins (eg, those produced via mutagenesis and / or recombinant techniques) are included. In general,
As a fragment or part of a mammalian CCR1 protein, a deletion of amino acids (ie one or more amino acids) with respect to the mature mammalian CCR1 protein (such as N-terminal, C-terminal or internal deletion) (ie one or more amino acids). Deletion). Fragments or proteins in which only contiguous amino acids have been deleted with respect to the mature mammalian CCR1 protein, or fragments or proteins in which non-contiguous amino acids have been deleted are also contemplated.

In general, variants of the mammalian CCR1 protein include mammalian CCRs that differ by the addition, deletion and / or substitution of one or more contiguous or non-contiguous amino acid residues.
Includes natural or artificial variants of one protein (eg, receptor chimera). Such mutations can be, for example, conserved or non-conserved regions (relative to other CXC (α) and / or CC (β) chemokine receptors), extracellular regions, cytoplasmic regions, or transmembrane regions.

Generally, the fusion protein is linked to a mammalian CCR1 (via a peptide bond) to a second moiety not found in naturally-occurring mammalian CCR1.
For example, a polypeptide containing human CCR1) or a variant thereof as the first part is included. Thus, the second portion can be an amino acid, oligopeptide or polypeptide. The first portion can be N-terminal, C-terminal or internal to the fusion protein. In one embodiment, the fusion protein has an affinity ligand (eg, enzyme, antigen, epitope tag) as a first site and a linker sequence and a second site containing human CCR1 or a portion thereof. . Additional (eg, third, fourth) sites may be present as appropriate.

“Functional fragment or portion”, “functional variant” and / or “functional fusion protein” of mammalian CCR1 protein means binding activity, signal transduction activity and / or cellular response. Refers to an isolated and / or recombinant protein or polypeptide having at least one functional characteristic of a mammalian CCR1 protein described herein, such as its ability to stimulate. Preferred functional variants are ligands (ie MIP-1α, RANTES, MCP-2,
One or more ligands such as MCP-3, leukotactin-1, HCC-1, or MPIF) and are referred to herein as "ligand binding variants".

In one embodiment, the functional variant of mammalian CCR1 is the mammalian CCR1.
It has at least about 85% sequence identity with CR1, preferably at least about 90% sequence identity with the mammalian CCR1 and more preferably at least about 95% sequence identity. In other embodiments, the functional fusion protein is a mammalian CCR
1 with at least about 85%, preferably at least about 90% sequence identity, and more preferably with at least about 95% sequence identity with mammalian CCR1. Sequence identity can be determined using an appropriate program, such as the Blastx program (version 1.4), with appropriate parameters, such as default parameters. In one aspect, the parameters for the Blastx search are the scoring matrix BLOSUM62, W = 3.
Is. In other embodiments, the functional variant contains a nucleic acid sequence that differs from a naturally occurring nucleic acid molecule but, due to the degeneracy of the genetic code, encodes mammalian CCR1 or a portion thereof.

A composition containing isolated and / or recombinant mammalian CCR1 or a functional variant thereof can be maintained under conditions suitable for binding, with mammalian CCR1 or variant, The antibody or fragment to be tested is contacted and binding is detected or measured 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 conditions suitable for receptor expression. The cells are contacted with the antibody or fragment under conditions suitable for binding (eg, in a suitable binding buffer) and binding detected by standard techniques. To measure binding, the degree of binding can be measured relative to an appropriate control (eg, compared to background measured in the absence of antibody and compared with binding of a second antibody (eg, standard)). Compare and compare the binding of the antibody to non-transfected cells). Cell fractions such as receptor-containing membrane fractions or receptor-containing liposome-containing membrane fractions can be used in place of whole cells.

In one embodiment, the antibody is labeled with a suitable label (eg, fluorescent label, isotope label, antigen or epitope label, enzyme label) and binding is measured by detection of the label. In other embodiments, bound antibody can be detected by a labeled second antibody. The specificity of binding can be assessed by competition or displacement, using, for example, unlabeled antibody or unlabeled antibody or ligand as a competitor.

A binding inhibition assay was also used to bind CCR1 and to ligand (eg, MIP-1α, RANTES, MCP-2, MCP-3, leukotactin-1).
, HCC-1, or MPIF), or antibodies or fragments thereof that inhibit the binding of other compounds, such as CCR1 or functional variants, to CCR1. For example, a binding assay can be performed that detects or measures a reduction in ligand binding of CCR1 (in the presence of an antibody such as 2D4) compared to ligand binding in the absence of the antibody. The composition containing isolated and / or recombinant mammalian CCR1 or a functional variant thereof may be contacted with the ligand and antibody either simultaneously, or after the other, in either order. A decrease in the extent of ligand binding in the absence of antibody is an indication of inhibition of binding by the antibody. For example, binding of the ligand can be reduced or abolished.

In one embodiment, the ligand (eg, MIP-1α, RANTES, MC
Direct inhibition by antibodies or fragments of the binding of mammalian CCR1 or a variant thereof to P-2, MCP-3, leukotactin-1, HCC-1, or chemokines such as MPIF) is monitored. For example, ¹²⁵ I-labeled MIP-α, ¹²⁵ I
-Labeled RANTES, ¹²⁵ I-labeled MCP-2, ¹²⁵ I-labeled MCP-3, ¹²⁵ I-labeled leukotactin, ¹²⁵ I-labeled HCC-1 or ¹²⁵ I-labeled MPIF
The ability of the antibody to inhibit the binding of Escherichia coli to mammalian CCR1 can be monitored. Such assays can be performed, for example, on isolated blood cells (eg, T cells, etc.)
Or a suitable cell line which naturally expresses CCR1, or a cell line containing a nucleic acid encoding a mammalian CCR1, or a membrane fraction from said cell, or a suitable cell line which produces CCR1 or a functional variant thereof. Can be done using.

Antibodies that bind CCR1, such as other suitable binding assays, or methods of monitoring events triggered by receptor binding, including stimulation of signal transduction function and / or cellular response (eg, leukocyte trafficking) Other methods of identifying the presence of

It will be appreciated that the inhibitory effect of the antibodies of the invention may be evaluated in a binding inhibition assay. Competition between antibodies for receptor binding can also be evaluated in the method.
The antibodies thus identified were further evaluated and, following binding, they were CC
It can be determined whether it acts to inhibit other functions of R1 and / or to assess their therapeutic utility.

Signaling Assay Binding of ligands or facilitators, such as agonists, to CCR1 can result in signal transduction by this G protein-coupled receptor, leading to the activation of G proteins as well as other intracellular signaling molecules. Be stimulated. Induction of signaling function by a compound (eg, antibody or fragment thereof) can be monitored using any suitable method. Such assays can be used to identify antibody agonists of CCR1. The inhibitory activity of an antibody or functional fragment thereof can be measured by using the ligand or enhancer in the assay and assessing the antibody's ability to inhibit the activity induced by the ligand or enhancer.

[0060]   Hydrolysis of GTP to GDP, or intracellular (cytoplasmic) free calcium [Ca ²⁺ ]_iCaused by receptor binding, such as inducing a rapid and transient increase in the concentration of
G protein activity, such as subsequent signaling events, is well known in the art.
Assay or any other suitable method (eg, Neot
e, K.E. Et al., Cell 72: 415-425 1993); Van R.
iper et al. Exp. Med. , 177: 851-856 (1993).
Dahinden, C .; A. Et al., J. Exp. Med. , 179: 75
1-756 (1994)).

For example, Sledziewsk using a hybrid G protein-coupled receptor
The functional assay of i. et al. can be used to monitor the ability of a ligand or promoter to bind si to the receptor and activate the G protein (Sledzie.
wski et al., US Pat. No. 5,284,746, the teachings of which are incorporated herein by reference).

Such an assay can be carried out in the presence of the antibody or fragment thereof to be evaluated, and the ability of the antibody or fragment to inhibit the activity induced by the ligand or enhancer can be determined by known methods and / or methods. It is measured using the method described in the specification.

Chemotactic and Cellular Stimulation Assays Chemotactic assays are also used to determine the ability of a ligand to block binding to mammalian CCR1 or a functional variant thereof, and / or to bind a ligand to a receptor. The ability of the antibody or functional fragment thereof to inhibit a related function can be assessed. These assays are based on the compound-induced functional migration of cells in vitro or in vivo. Chemotaxis can be assessed as described in the Examples, for example, in assays using 96-well chemotaxis plates, or in assays using other art-recognized methods for assessing chemotaxis. . For example, the use of an in vitro transendothelial cell chemotaxis assay is described in Springer.
(Springer et al., WO 94/20142).
No. Pamphlet, published September 15, 1994, the teachings of which are incorporated herein by reference; see also Berman et al., Immunol. Invest.
, 17: 625-677 (1988)). Migration to collagen gels through the endothelium has also been described (Kavanaugh et al., J. Immunol.,
146: 4149-4156 (1991)). For example, mouse L1.2 pre-
Stable transfectants of B cells and of suitable host cells capable of chemotaxis can be used in chemotaxis assays.

In general, a chemotaxis assay involves a second surface opposite the surface of the barrier, towards an increased level of compound, into or through the barrier (eg endothelium, filter). Towards (for lymphocytes, eosinophils, basophils, etc.)
Monitor proper cell directional movement or migration. The membrane or filter is directed toward or through the appropriate cells to or from the filter's second surface, from the filter's first surface to the filter's opposing second surface. It provides a convenient barrier so that is monitored. In some assays, the membrane is coated with an agent that facilitates adhesion, such as ICAM-1, fibronectin or collagen. Such an assay is called leukocyte'homing
(homing) "in vitro approximation.

For example, from the first chamber to or through the porous membrane into a second chamber which is contacted with the antibody to be tested and which is divided from the first chamber by the membrane (inclusion means) (a containig means)) Inhibition of cell migration in a suitable container can be detected or measured. Appropriate membranes with appropriate pore sizes to monitor specific migration in response to compounds are selected, including, for example, nitrocellulose, polycarbonate. For example, about 3-8 microns, preferably about 5-
A pore size of 8 microns can be used. The pore size is uniform on the filter or within the appropriate pore size range.

To assess migration and inhibition of migration, the distance of migration to the filter, the number of cells across the filter that remain adherent to the second surface of the filter, and / or accumulate in the second chamber. Count cells by standard techniques (eg, microscopy)
Can be detected using. In one embodiment, the cell detectable label (
(Eg, radioisotope, fluorescent label, antigen or epitope label) and migration is attached to the membrane and / or present in the second chamber or the presence of the label using an appropriate method. Can be assessed in the presence or absence of the antibody or fragment by measuring (eg, by radioactivity, fluorescence, immunoassay). The extent of migration induced by an antibody agonist can be measured relative to a suitable control (eg, by a second compound (ie, standard) as compared to background migration measured in the absence of antibody). Compared to the degree of migration induced, compared to the migration of non-transfected cells induced by the antibody).

In one embodiment, transendothelial cell migration can be monitored, particularly for T cells, monocytes, or mammalian CCR1 expressing cells. In this aspect,
Assess migration through the endothelial cell layer. To prepare the cell layer, the endothelial cells are placed on a porous filter or membrane, optionally coated with a substance such as collagen, fibronectin, or other intracellular matrix proteins to facilitate attachment of the endothelial cells. It can be cultured. Preferably, the endothelial cells are cultured until a confluent monolayer is formed. For example, human umbilical vein endothelial cells (Clon
A variety of mammalian endothelial cells, such as venous, arterial or microvascular endothelial cells, such as e.g. etics Corp, San Diego, CA) are available for monolayer formation. Endothelial cells from the same mammal are preferred for assessing chemotaxis in response to a particular mammalian receptor; however, endothelial cells from different mammalian species or genera can also be used.

In general, the assay is directed from a first surface of a filter comprising an endothelial cell layer on a first surface towards an opposing second surface of the filter towards increasing levels of compound. Of the cells to or through the membrane or filter is detected. Directional migration occurs from the region adjacent the first surface to the compound on or opposite the filter to or through the membrane. The concentration of the compound present in the area adjacent to the second surface is greater than that in the area adjacent to the first surface.

In one embodiment used to test for antibody inhibitors, a composition containing cells that are able to migrate and express the mammalian CCR1 receptor can be placed in the first chamber. Can induce chemotaxis of cells in the first chamber 1
A composition (having a chemo-attracting function) containing the above ligand or promoter is placed in the second chamber. Preferably, immediately before or at the same time as the cells are placed in the first chamber, the composition containing the antibody to be tested is preferably placed in the first chamber. In this assay, the receptor binds to the mammalian CC
An antibody or a functional fragment thereof that can inhibit the induction of chemotaxis of R1-expressing cells by a ligand or a promoter is an inhibitor of receptor function (eg, an inhibitor of stimulatory function). A reduction in the extent of migration induced by a ligand or enhancer in the presence of antibody or fragment is an indicator of inhibitory activity. Alternative binding test (see above)
Can be performed to determine whether inhibition is the result of antibody or receptor binding or occurs through a different mechanism.

An in vivo assay for monitoring leukocyte infiltration of tissues in response to injection of compounds (eg chemokines or antibodies) into tissues is described below (Models o.
f Inflammation). These models of in vivo homing measure the ability of cells to respond to ligands or enhancers by migration and chemotaxis to sites of inflammation and to assess the ability of antibodies or fragments thereof to block this migration. .

In the described method, the effect of the antibody or fragment on the stimulatory function of CCR1 can be assessed by monitoring the cellular response elicited by active receptors using a suitable host containing the receptor.

Identification of Additional Ligands, Inhibitors, and / or Promoters of Mammalian CCR1 Function The above assay, which can be used to assess the binding and function of the antibodies and fragments of the invention, is adjusted to provide mammalian CCR1 function. , Or another ligand or other substance that binds a functional variant thereof, and an inhibitor of CCR1 function and / or a promoter of mammalian CCR1 function can be identified. For example, a competitive assay method using the antibody of the present invention or a part thereof can identify a drug having the same or similar binding specificity as the function of the antibody of the present invention or a functional part thereof. Accordingly, the present invention also includes methods for identifying receptor ligands, or other substances that bind to mammalian CCR1 protein, and inhibitors (eg, antagonists) or enhancers (eg, agonists) of receptor function. In one embodiment, a cell producing a mammalian CCR1 protein, or a functional variant thereof (eg, a white blood cell modified to express a mammalian CCR1 protein or a functional variant encoded by a nucleic acid introduced into the cell, a cell Strains or suitable host cells) are used in assays to identify and determine the potency of other substances that bind to the receptor, such as ligands and inhibitors or promoters of receptor function. Such cells are useful in assessing the function of the expressed receptor protein or polypeptide.

According to the invention, ligands and other substances that bind to the receptor, ie inhibitors or promoters of receptor function, are identified by a suitable assay method,
Furthermore, the therapeutic effect can be evaluated. Inhibitors of receptor function can be used to inhibit (reduce or prevent) receptor activity, and ligands and / or enhancers can be used to induce (initiate or promote) the indicated normal receptor function. sell. These inhibitors can be used in methods of treating inflammatory diseases such as autoimmune diseases and graft rejection, which include administering inhibitors of receptor function to an individual (eg, a mammal). The ligand and / or enhancer identified as described herein further provides a method of stimulating receptor function by administering to the individual a novel ligand or enhancer of receptor function. It can be used to provide new clues for selectively stimulating white blood cell function, which is useful, for example, in the treatment of infections and cancer.

As used herein, a mammalian CCR1 “ligand” is a substance that binds to a mammalian CCR1 protein, including natural ligands, as well as synthetic and / or recombinant forms of natural ligands. Means a specific taxon. Mammal CC
Infectious agents that have an affinity for R1-positive cells (eg, viruses such as HIV) can also bind to mammalian CCR1 protein. The natural ligand of the receptor of choice is a ligand for the mammalian CCR1 protein (eg, M
IP-1α, RANTES, MCP-2, MCP-3, leukotactin-1, H
Chemokines such as CC-1 and / or MPIF) and the same mammal origin. In a preferred embodiment, ligand binding of the mammalian CCR1 protein occurs with high affinity.

As used herein, “inhibitor” refers to binding activity (eg, ligand binding, promoter binding, antibody binding), signal transduction activity (eg, mammalian G protein activation, cytoplasmic). free calcium such as [Ca ^2+] rapid and transient increase in the concentration of induction of _i), and / or cellular response function (e.g. in, leukocyte chemotaxis, exocytosis or inflammatory mediator release), etc., Mammal CC
It is a substance that inhibits (reduces or prevents) one or more functions characteristic of R1 protein (eg, human CCR1). Inhibitors are also substances that block the entry of HIV into cells. The term inhibitor refers to an antagonist that binds to the receptor (eg, an antibody, a variant of a natural ligand, a low molecular weight organic molecule, or other competitive inhibitor of ligand binding), and an inhibitor that inhibits receptor function without binding to the receptor. By substance (eg, anti-idiotypic antibody) is meant.

Here, the term “promoter” means binding activity (eg, ligand binding, promoter binding, antibody binding), signal transduction activity (eg, mammalian G protein activation, cytosolic free calcium [Ca]. ²⁺ ] Induction of rapid and transient concentration increase of _i )
And / or promotes (induces) one or more of the functions characteristic of mammalian CCR1 proteins (eg, human CCR1), such as cell response functions (eg, leukocyte chemotaxis, exocytosis, or inflammatory mediator release). , Induce, enhance, or increase) substances. The term enhancer refers to an agonist that binds to the receptor (eg, an antibody, a homologue of a natural ligand from another species), and a receptor function (eg, by activating associated proteins) without binding to the receptor. A substance that promotes. In a preferred embodiment,
Agonists are other than homologs of natural ligands.

Accordingly, the present invention provides ligands, inhibitors, enhancers, and other substances that bind to mammalian CCR1 protein or functional variants thereof, which bind to mammalian CC-chemokine receptor 1 or ligand binding variants thereof. It also relates to methods of detecting or identifying agents such as. According to the method, an agent to be tested, an antibody or an antigen-binding fragment of the present invention (for example, an antibody having the same or similar epitope specificity as that of 2D4, 2D4, and an antigen-binding fragment thereof), and Compositions containing mammalian CC-chemokine receptor 1 or ligand binding variants thereof can be combined. Antibodies to mammalian CC-chemokine receptor 1 or its ligand binding variants, combining the above components under conditions suitable for the antibody or antigen binding fragment to bind to mammalian CC-chemokine receptor 1 or its ligand binding variant. Alternatively, binding of fragments thereof is detected or measured directly or indirectly by the methods described herein or other suitable methods. A decrease in the amount of complex formed relative to a suitable control (eg, in the absence of the drug being tested) is indicative of binding of the drug to the receptor or variant thereof. The composition containing the mammalian CC-chemokine receptor 1 or ligand binding variant thereof may be a membrane fraction of cells producing the recombinant chemokine receptor 1 or ligand binding variant thereof. The antibody or fragment thereof can be labeled with a label such as a radioisotope, a spin label, an antigen or epitope label, an enzyme label, a fluorescent group and a chemiluminescent group.

In one embodiment, the invention provides for an agent to be tested, an antibody or antigen-binding fragment of the invention (eg, an antibody with an epitope specificity identical or similar to that of 2D4, 2D4, or Antigen-binding fragment), and mammalian CC
A method of detecting or identifying an agent that binds to mammalian CC-chemokine receptor 1 or a ligand binding variant thereof comprising combining cells that produce chemokine receptor 1 or a ligand binding variant thereof. The above components are combined under conditions suitable for the antibody or antigen binding fragment to bind to mammalian CC chemokine receptor 1 or a ligand binding variant thereof, and the antibody or fragment thereof binds to a CCR1 protein or a ligand binding variant thereof. Binding is detected or measured directly or indirectly by the methods described herein or other suitable methods. A decrease in the amount of complex formed relative to a suitable control is an indication that the agent binds to the receptor or variant thereof. The antibody or fragment thereof can be labeled with a label selected from the group consisting of radioisotopes, spin labels, antigen or epitope labels, enzyme labels, fluorescent groups and chemiluminescent groups. These assays, and similar assays, can be used to bind CCR1 with a ligand (eg, chemokine, HIV strain that interacts with CCR1) or other substance, such as an inhibitor or enhancer of receptor function, to receptor binding. For, agents such as substances that compete with the antibodies described herein can be detected.

The assays may, alone or in combination with each other, or in combination with suitable alternatives, inhibit ligands or other substances that bind to mammalian CCR1 protein and mammalian CCR1 protein or variants thereof. It can be used to identify substances or promoters. The in vitro methods of the invention can be tailored to perform high throughput screening (eg, 96-well format) that processes large numbers of samples. Since cells expressing a suitable amount of mammalian CCR1 (eg, human CCR1) can be used to perform high throughput screening, such cells can be used as a ligand or other agent that binds to the receptor, and to mammalian cells. Useful for identifying and / or isolating inhibitors or promoters of CCR1 protein. Receptor expression can be monitored in a variety of ways. For example, expression can be observed using an antibody of the invention that binds to the receptor or a portion thereof. In addition, a commercially available antibody can be used to detect the expression of a fusion protein containing a receptor protein or polypeptide (for example, a receptor tagged with FLAG) and tagged with an antigen or an epitope, and a cell expressing a desired amount. Can be selected.

Nucleic acid encoding a mammalian CCR1 protein or functional variant thereof may be incorporated into an expression system to produce a receptor protein or polypeptide.
Intracellularly or transiently transfected with a construct containing a recombinant nucleic acid encoding a mammalian CCR1 protein or variant thereof, or a cell fraction containing the receptor (eg, transfected cells, incorporating the receptor Isolated and / or recombinant mammalian CCR1 proteins or variants, such as those expressed in liposomes) can be used to determine receptor function. If desired, the receptor can be further purified. Testing for receptor function can be performed in vitro or in vivo.

Isolated and / or recombinant mammalian CCR1 protein or functional variants thereof, such as human CCR1, may be monitored for receptor function or other suitable techniques as described herein. When used, it can be used in the present method for determining the effect of a compound. For example, stable or transient transfectants (eg, baculovirus-infected Sf9 cells, mouse L1.2 pre-B cells, etc.) can be used in the binding assay. For example, Jurkat cells, or other suitable cells capable of chemotaxis (eg, mouse L1.2
Pre-B cells) stable transfectants can be used in chemotaxis assays.

According to the method of the present invention, compounds can be individually screened, or one or more compounds can be simultaneously examined by the method described herein. When testing a mixture of compounds, the compounds selected by the described process can be (appropriately) separated and identified by a suitable method (eg PCR, sequencing, chromatography, mass spectrometry). . The presence of one or more compounds (eg, ligands, inhibitors, promoters, etc.) in the sample to be tested can also be determined by these methods.

Compounds made by combinatorial chemical synthetic methods or otherwise (eg,
Large combinatorial libraries of organic compounds, recombinant or synthetic peptides, "peptoids," nucleic acids, etc. can be interrogated (eg, Zucke
rman, R.R. N. Et al., J. Med. Chem. , 37: 2678-2685 (
1994) and references cited therein; also Ohlmey.
er, M.E. H. J. Et al., Proc. Natl. Acad. Sci. USA 90
: 10922-10926 (1993), and for tagged compounds see DeWitt, S .; H. Et al., Proc. Natl. Acad. Sci. US
A 90: 6909-6913 (1993); Rutter, W .; J. Et al., U.S. Patent No. 5,010,175; Huebner, V .; D. Et al., U.S. Patent No. 5,182,366; and Geysen, H .; M. US Patent No. 4,8
33,092 specification). If the compound selected from the combinatorial library by this method has a unique tag, each compound can be identified by the chromatography method.

In one embodiment, the phage display method is used. For example, mammal C
The CR1 protein or a functional variant thereof, the antibody of the present invention or a functional site thereof, and a phage displaying a polypeptide (for example, a phage, or a collection of phage such as a library) can be a mammalian C
The CR1 proteins or variants can be combined under conditions suitable for binding (eg, in a suitable binding buffer). Phage that bind to the mammalian CCR1 protein or a functional variant thereof in competition with the antibody or portion thereof can be detected or selected using standard techniques or other suitable methods. Bound phage can be separated from the receptor using a suitable elution buffer. For example, ionic strength or pH can be altered to effect phage release. Alternatively, the elution buffer is a free component, or
A component designed to disrupt binding of a compound (eg, one or more compounds capable of disrupting binding of a presented peptide to a receptor, such as a ligand, inhibitor, and / or enhancer that competitively inhibits binding. ) Is included. Optionally, the selection process can be repeated or another selection step can be used to further enrich for phage that bind to the receptor. The characteristics of the displayed polypeptide can be examined (eg, by sequencing the phage DNA). The identified polypeptides can be produced and further tested for binding and the function of inhibitors or promoters. With such peptide analogs, it is possible to produce peptide analogs with increased stability or with other desired properties.

In one embodiment, a phage can be produced that contains a coat protein with an N-terminal peptide encoded by a nucleic acid of any sequence and that expresses and displays the fusion protein. Appropriate host cells expressing a mammalian CCR1 protein or variant and an anti-CCR1 antibody or functional site thereof are combined with the phage, bound to select for phage, recovered and characterized. (For example,
Doorbar and Winter, J. et al. Mol. Biol. 244: 361 (
1994) (considering a phage display method for use with G protein-coupled receptors), and WO 97/08320 (Morphosys) published March 6, 1997).

Other sources from which a ligand of the mammalian CCR1 protein, other substances that bind to it, or potential inhibitors and / or promoters thereof, may be obtained, including but not limited to, MIP-1α, RANTES. , MCP-2, MCP-
3, variants of CCR1 ligands such as natural, synthetic, or recombinant variants of leukotactin-1, HCC-1, or MPIF, other chemoattractants or substances such as chemokines, their variants, low molecular weight organic molecules, other Inhibitors and / or promoters (eg anti-CCR1 antibodies, antagonists, agonists, etc.), other G protein-coupled receptor ligands, inhibitors and / or promoters (eg antagonists, agonists, etc.), and suitable Soluble portions of the mammalian CCR1 receptor, including receptor peptides, or suitable receptor peptides or analogs capable of inhibiting receptor function (
For example, Murphy, R .; B. , International Publication No. 94/05695 pamphlet) and the like.

Models of Inflammation In vivo models of inflammation are available that can be used to determine the effectiveness of the antibodies and fragments of the invention as therapeutic agents in vivo. For example, leukocyte infiltration can be monitored upon intradermal injection of an appropriate animal, such as rabbit, mouse, rat, guinea pig, or rhesus monkey, with a chemokine and an antibody or fragment thereof that reacts with mammalian CCR1 (eg, Van Damme, J
． Et al., J. Exp. Med. , 176: 59-65 (1992); Zachar.
iae, C.I. O. C. J. et al .; Exp. Med. , 171: 2177-218
2 (1990); Jose, P .; J. Et al., J. Exp. Med. , 179: 88
1-887 (1994)). In one embodiment, a skin biopsy is histologically determined for leukocyte (eg, eosinophil, granulocyte, etc.) infiltration. In another embodiment, labeled cells with chemotaxis and extravasation capacity (eg, stably transfected cells expressing mammalian CCR1, ¹¹¹ In
(Labeled with) is administered to the animal. For example, the antibody or fragment to be determined is
The ligand or agonist can be administered before, after, or simultaneously with the administration of the test animal. The decrease in the degree of infiltration in the presence of the antibody, as compared with the degree of infiltration in the absence of the inhibitor, is an index of inhibition.

Diagnostic and Therapeutic Applications The antibodies and fragments of the present invention find utility in a variety of applications, including research, diagnostic, and therapeutic applications. In one embodiment, the antibody is labeled with a suitable label (eg, fluorescent label, chemiluminescent label, isotope label, antigen or epitope label, or enzyme label). For example, they can be used to isolate and / or purify the receptor or portions thereof, or to study receptor structure (eg, conformation) and function.

In addition, various antibodies of the invention can be used to detect CCR1 or, for example,
Receptor expression on T cells (eg, CD26 + cells, CD45RO + cells), neutrophils, eosinophils, and / or cells transfected with the receptor gene can be measured. Therefore, it can also be applied to cell sorting (for example, flow cytometry, fluorescence-marked cell sorting) for diagnostic or research purposes.

The anti-CCR1 antibody of the present invention has value in diagnostic applications. Anti-CCR1
The antibody or fragment thereof can monitor the expression of this receptor in each individual in the same manner as when anti-CD4 was used as a diagnostic indicator of the HIV stage.

In general, diagnostic assays need to detect the formation of complexes formed by the antibody or fragment thereof binding to CCR1. The antibody or antigen-binding fragment can be labeled or unlabeled for diagnostic purposes. The antibody or fragment can be directly labeled. A variety of labels can be used, including but not limited to radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (eg, biotin, haptens, etc.). Many suitable immunoassays are known to those of skill in the art (eg, US Pat. No. 3,817,827;
850,752; 3,901,654 and 4,098,876). When unlabeled, the antibody or fragment can be detected using any suitable means, eg, an agglutination assay. The unlabeled antibody or fragment may be another (ie, one or more) suitable reagent (eg, anti-idio) that can be used to detect the antibody, such as a labeled antibody that reacts with the first antibody (eg, a secondary antibody). It can also be used in combination with a type antibody, or another antibody specific for unlabeled immunoglobulin), or other suitable reagent (eg, labeled protein A).

In one embodiment, the antibody or fragment of the present invention can be used in an enzyme immunoassay, in which case the antibody or fragment of the present invention or a secondary antibody is linked to an enzyme. . When a biological sample containing mammalian CCR1 protein is combined with the antibody, binding occurs between the antibody and CCR1 protein. In one embodiment, a sample containing cells that express a mammalian CCR1 protein, such as human blood, is combined with the antibody so that the antibody and cells having the human CCR1 protein that contains an epitope recognized by the antibody are Bonding occurs between them. These bound cells are separated from the unbound reagents and, for example, the sample is contacted with an enzyme substrate that produces a color change or other detectable change when subjected to an enzymatic action, whereby an antibody that specifically binds to the cell- The presence of enzyme conjugate can be measured. In another embodiment, a labeled secondary antibody that recognizes the antibody of the invention can be added without labeling the antibody of the invention.

Kits for use in detecting the presence of mammalian CCR1 protein in a biological sample can also be prepared. Such kits include mammalian C
Antibody or functional fragment thereof that binds to C-chemokine receptor 1 or a portion of said receptor, and one or more suitable for detecting the presence of a complex between the antibody or fragment and CCR1 or a portion thereof Includes auxiliary reagents. The antibody composition of the invention can be provided alone or in combination with another antibody specific for another epitope in lyophilized form. The antibody may be labeled or unlabeled with additional components (eg, buffers such as Tris, phosphate and carbonic acid, stabilizers,
Included in the kit with excipients, biocides, and / or inactive proteins such as, for example, bovine serum albumin). For example, the antibody can be provided as a lyophilized mixture with additional components, or the additional components can be provided separately for the user to combine. Generally, these additives will be less than about 5% by weight, based on the amount of active antibody, and usually in total amounts about 0% of the antibody concentration.
． 001 wt% or more is present. When utilizing a secondary antibody that can bind to a monoclonal antibody, the antibody can be provided in a kit, for example, in a separate vial or container. When present, the secondary antibody is generally labeled and can be formulated in the same manner as the antibody formulations described above.

Similarly, the present invention provides a method of detecting and / or quantifying the cellular expression of mammalian CCR1 or a portion of this receptor under conditions suitable for the antibody or fragment thereof to bind to the cells or A method for monitoring binding by contacting a composition containing the fraction (eg, membrane fraction) with an antibody or a functional fragment thereof (eg, 2D4) that binds to mammalian CCR1 or a part of this receptor, Concerned. Detection of the antibody, which indicates that it has formed a complex between the antibody and CCR1 or a portion thereof, indicates the presence of the receptor. Antibody binding to cells can be measured, for example, as described above under the heading "Binding Assay". This method can be used to detect CCR1 expression on cells from an individual (eg, in a sample such as blood, body fluid such as saliva, or other suitable sample). Also,
The expression level of CCR1 on the surface of T cells or monocytes can be measured by, for example, flow cytometry, and the expression level (eg, staining intensity) is correlated with disease susceptibility, disease progression or risk. is there.

Chemokine receptors function when leukocytes migrate throughout the body, especially to sites of inflammation. Emigration of inflammatory cells from the vasculature is regulated by a three-step process involving interactions with leukocytes and endothelial cell adhesion proteins and cell-specific chemoattractants and activators (Springer, TA et al. , Cell, 7
6: 301-314 (1994); Butcher, E .; C. , Cell, 67
: 1033-1036 (1991); Butcher, E .; C. And Picker
L. J. , Science (Wash. DC), 272: 60-66 (199).
6)). These are: (a) low affinity interactions between leukocyte selectins and endothelial cell carbohydrates; (b) high affinity interactions between leukocyte chemoattractant receptors and chemoattractants / activators. And (c) intimate binding between leukocyte integrins and the immunoglobulin superfamily of endothelial cell adhesion proteins. Different subsets of white blood cells express different repertoires of selectins, chemoattractant receptors, and integrins. In addition, inflammation alters the expression of endothelial adhesion proteins and chemoattractants and leukocyte activating factors. As a result, there is great variability in the control of selectivity when recruiting leukocytes to extravascular sites. The second step is important in that activation of leukocyte chemoattractant receptors is believed to cause a change from selectin-mediated cell rolling to integrin-mediated tight binding. As a result, leukocytes are ready to migrate to the perivascular site. Chemoattractant / chemoattractant receptor interactions are also important for transendothelial migration and localization within tissues (Campbell, JJ, et al., J. Cell Biol., 134: 25.
5-266 (1996); Carr, M .; W. Et al., Immunity, 4:17.
9-187 (1996)). This migration is triggered by a concentration gradient of chemoattractant that leads to the center of inflammation.

CCR1 plays an important role in leukocyte trafficking. Since CCR1 is likely to be an important chemokine receptor for T cell, T cell subset, or monocyte migration to certain inflammatory sites, anti-CCR1 mAbs were used to specifically stimulate T cell or monocyte migration, particularly , T cell dysfunction such as autoimmune disease or allergic reaction, or migration associated with monocyte-mediated disorders such as atherosclerosis can be inhibited (reduced or prevented). Therefore, the antibodies and fragments thereof of the present invention can also be used to modulate receptor function in research and therapeutic applications. For example, the antibodies and functional fragments described herein include (a) binding to a receptor (eg, of a ligand, inhibitor, or promoter),
(B) Receptor signal transduction function and / or (c) Stimulatory function is inhibited (
Can act as an inhibitor to reduce or prevent). Antibodies that act as inhibitors of receptor function can block ligand or promoter binding either directly or indirectly (eg, by causing a conformational change). For example, the antibody can inhibit receptor function by inhibiting ligand binding, or by desensitizing (with or without inhibiting ligand binding). Antibodies that bind to the receptor can also act as agonists of receptor function and bind to the receptor to initiate or stimulate receptor functions, such as receptor signaling and / or stimulatory functions (eg, leukocyte trafficking). To do.

Thus, the present invention involves administering to a mammal (eg, a human patient) an effective amount of an antibody of the present invention or functional fragment thereof in a method of inhibiting leukocyte trafficking in the mammal. Provide a way. Administration of the antibody or fragment of the invention can result in amelioration or elimination of the disease state.

In addition, the antibody of the present invention or a functional fragment thereof has a CCR by binding of a chemokine.
It can be used for the treatment of diseases in which activation of one receptor is involved. For example, using an antibody or a functional fragment thereof (eg 2D4 or a functional fragment thereof),
Allergy, atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation, histamine and IgE mediated allergic reactions, shock, and rheumatoid arthritis, atherosclerosis, multiple sclerosis, graft rejection, ischemia / re Perfusion disorders, fibrosis, asthma, and inflammatory glomerulopathy can be treated.

Diseases or conditions in humans or other species treatable with inhibitors of CCR1 receptor function (such as antibodies or suitable fragments thereof) include January 2, 1999.
Wayne W. US Application 09/239 by Hancock
, 283, "Methods for preventing graft rejection and ischemia-reperfusion injury", 1999 1
James B. Rottman and Wayne W. H
Ancock, US Application Serial No. 09 / 240,253, "Methods of Treating Demyelinating Inflammatory Diseases Using CCR1 Antagonists," the entire teachings of both of which are incorporated herein in their entirety. , But are not limited to: Ie:

● Asthma, allergic rhinitis, hypersensitivity lung disease, hypersensitivity pneumonia, interstitial lung disease (I
LD) (eg, ILD associated with idiopathic pulmonary fibrosis, or rheumatoid arthritis,
Systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome,
Allergic diseases of the respiratory system such as polymyositis or dermatomyositis; anaphylactic or hypersensitivity reactions, drug allergies (eg to penicillin, cephalosporins), insect sting allergies; Crohn's disease and ulcerative colitis Inflammatory bowel disease; spondyloarthropathies; scleroderma; psoriasis and dermatitis, eczema, atopic dermatitis,
Allergic contact dermatitis, inflammatory dermatitis such as urticaria; inflammatory or allergic diseases and conditions, including vasculitis (eg necrotizing vasculitis, cutaneous vasculitis and irritable vasculitis);

● Arthritis (eg, rheumatoid arthritis, psoriatic arthritis), demyelinating disorder (eg, multiple sclerosis), systemic lupus erythematosus, myasthenia gravis, juvenile diabetes, glomerulonephritis, and other nephritis, autoimmunity Autoimmune diseases such as systemic thyroiditis and Behcet's disease;

• Chronic or acute graft rejection, including allograft rejection or graft-versus-host disease (eg in organ transplantation), and graft-related arteriosclerosis;

[0103]   ● Atherosclerosis;

[0104]   ● Cancer with infiltration of white blood cells in the skin or organs;

Other diseases or conditions that can be treated by inhibiting unwanted inflammatory reactions
(Including CCR1-mediated diseases or conditions) include ischemia / reperfusion injury, certain hematological malignancies, cytokine-induced toxicity (eg septic shock, endotoxic shock), polymyositis, Includes but is not limited to dermatomyositis, and granulomatous disorders including systemic granulomatosis

Diseases or conditions treatable with a promoter of CCR1 receptor function (including antibodies and fragments thereof) include, but are not limited to:

In individuals suffering from immunodeficiency syndromes such as AIDS, in individuals undergoing radiotherapy that results in immunosuppression, chemotherapy, treatment of autoimmune diseases or other drug treatments (eg corticosteroid treatment). Immunosuppression; and immunosuppression due to congenital failure of receptor function or other causes.

The anti-CCR1 antibodies of the invention may block the binding of one or more chemokines, and thus block the cascade of one or more events leading to the disorders described above.

Methods of Administration One or more antibodies or fragments of the invention will be administered to an individual by any suitable route, alone or in combination with another drug or agent (prior to, concurrently with, or after administration). can do. For example, the antibodies of the invention may be combined with other monoclonal or polyclonal antibodies (eg, CCR2, CCR3, CCR
4 and CCR5, etc., in combination with other chemokine receptor-binding antibodies), or existing blood such as commercially available gamma globulin and immunoglobulin preparations for prophylactic or therapeutic treatment. It can also be used in combination with plasma products. The antibody or fragment of the present invention can be used as a separately administered composition provided with an antibiotic and / or antibacterial agent. The antibody or fragment of the present invention is an antiviral agent, an immunosuppressant (for example, a calcineurin inhibitor such as cyclosporin A; a glucocorticoid such as prednisone or methylprednisolone; and a nucleic acid synthesis inhibitor such as azathioprine or mycophenolic acid), interferon. And in combination with cytokines such as Th2-producing cytokines and hormones such as adrenocorticotropic hormone.

An effective amount of antibody or fragment (ie, one or more antibody or fragment) is administered. An effective amount inhibits CCR1 function, thereby causing an inflammatory response or HIV.
Achieving the desired therapeutic effect (including prophylaxis) under dosing conditions, such as an amount sufficient to inhibit infection or, as described above, sufficient to promote CCR1 function That's enough to do.

Oral, feeding, local, parenteral (eg, intravenous, intraarterial, intramuscular, and subcutaneous injection), inhalation, (eg, intrabronchial, intraocular, intranasal or oral inhalation, nasal drop) Agents, etc., but not necessarily limited to these, various routes of administration are possible depending on the disease and condition to be treated. Other suitable administration methods include refillable,
Or biodegradable devices, slow release polymeric devices, etc. The pharmaceutical compositions of this invention can also be administered as part of a combination therapy with other agents.

The formulation of the antibody or fragment to be administered depends on the route and formulation of choice (eg,
Solutions, emulsions, capsules, etc.). A suitable pharmaceutical composition containing the antibody or functional fragment thereof to be administered is a physiologically acceptable vehicle (vehicl).
e) or a carrier. It is also possible to use mixtures of antibodies and / or fragments. In the case of solutions or emulsions, suitable carriers are, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, such as saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's or fixed oils. A variety of suitable aqueous carriers are known to those of skill in the art, such as water, buffered water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), dextrose solution, and glycine. ing. As an intravenous vehicle, various additives,
Preservatives, solutions, supplements for nutrients or electrolytes, etc. (Generally, Remington's Pharmaceutical Sciences, 16th Edition, Mac Edition, 1980 (Remington's
Pharmaceutical Science, 16th, Edition
, Mack, Ed. 1980)). The composition is a pharmaceutical agent required to approximate physiological conditions, such as pH adjusting agents such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, buffering agents, and toxicity adjusting agents. Can optionally contain auxiliary substances. Antibodies and fragments of the invention may be prepared by lyophilization and reconstitution techniques known in the art.
It can be lyophilized for storage and reconstituted in a suitable carrier before use. The optimum concentration of active ingredient in the chosen medium is empirically determined by procedures known to those skilled in the art depending on the final drug formulation desired. In the case of inhalation, the antibody or fragment is solubilized and a suitable dosing container (eg atomizer, nebulizer,
Alternatively, it can be placed in a pressurized aerosol dosage container).

The following examples are provided for the purpose of illustrating the invention, which is not intended to limit 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 Cultures Normal human blood leukocytes were isolated as described (Heath et al., J. Clin.
Invest. 99: 178 (1977)). To produce CD3 blast cells,
RPMI-1640 plus 2 × 10 ⁶ PBMC / ml in 10% FCS, anti-C
D3 antibody TR66 was added to tissue culture plates initially coated. After 4 to 6 days, the blast cells were taken out to a fresh medium, and 50 units / ml of IL-2 (Anton) was used.
io Lanzavechia, Basel Institute for
Immunology, Basel, kindly provided by Switzerland). In some cases 10 ng / ml IFN 4 days after stimulation
α (Peprotech, Rocky Hill, NJ) was added to the culture.

Human CCR1 was expressed as previously described in the mouse pre-B cell line L1-2 (Honda et al., J. Immunol. 152: 4026 (1994)).
. Briefly, r r from Genbank (registration number L09320) and human peripheral blood
From the gene amplified by t-PCR, 3'UT of coding region and CCR1
The primer sequence for R was obtained. The amplified product was directionally cloned into the expression vector pMRB-101 and L1-2 cells were transfected with the linear plasmid,
Selected by growth on medium containing mycophenolic acid. Anti-FLAG Ab M1
Resistant cells were stained with (IBI-A Kodak Co., New Haven., CT) and sorted by flow cytometry. 10 nM M after several classifications
Cells were further selected by chemotaxis against IP-1α.

MAb Production and Flow Cytometry CCR1-reactive mAbs were generated by immunizing mice intraperitoneally with 1 × 10 ⁷ CCR1 L1-2 transfectants at 2-week intervals. Four days after the last immunization, the mice were sacrificed and the spleens were removed and cell fusions were performed as described using the cell line SP2 / O (Coligan et al., Current Pr).
otocols in Immunology 2.5.2 (1992)). FA
CScan® (Becton Dickinson & Co., M
Specific mAbs reactive with CCR1 were identified by flow cytometry using the Outaintain View, CA) as described below. CCR1
Antibodies were screened against immunofluorescent stains that stain transfectants and not untransfected cells or cells transfected with all other human chemokine receptors. CCR1-specific mAbs described herein
2D4 is an isotype by ELISA (Southern Bi
technology, Birmingham, AL), IgG1. 2D4 hybridomas can be cultured in 90% DMEM, 10% fetal bovine serum and 100 ng / ml IL-6.

Mouse mAbs against human CCR3, CCR5 and CXCR3 have been described (Qin et al., J. Clin. Invest. 101: 746 (1998);
(Heath et al., J. Clin. Invest. 99: 178 (1997)).
CD4, CD8, CD19, CD25, CD26, CD45RO, CD45RA
And a fluorochrome-conjugated mAb to biotinylated anti-IgE was supplied by Pharmingen (La Jolla, CA). Indirect immunofluorescence and flow cytometry were used to assess mAb reactivity to transfected cells and leukocytes. Cells were washed once with PBS and washed with 2% human serum and 0.1% sodium azide (staining buffer), 5 μg / ml purified antibody, 5 μg / ml isotype matched control mAb (Si.
gma Chemical Co. , St. Louis, MO) including 100μ
Resuspended in PBS or 50 μl hybridoma culture supernatant. After 20 minutes at 4 ° C, cells were washed twice with staining buffer and purified with FITC-conjugated affinity F.
(Ab ') ₂ goat anti-mouse IgG (Jackson ImmunoResea
rch Laboratories) 50 μl. After 20 minutes, the cells were washed twice with staining buffer and analyzed by FACScan® to determine the level of surface expression. Dead cells were eliminated using propidium iodide. Cells were treated with anti-CCR1 mAb 2D for multicolor analysis.
4 was stained first and then with anti-mouse APC (Pharmingen). PE-, FITC- or Cy3-conjugated mA after blocking with mouse serum
b was used together to stain cells. Results were analyzed using electronic gating and correction by FACScan®.

Chemokines, Chemotactic Analysis, and Ligand Binding Assays IL-8 recombinantly produced on LeukoSite (Wu et al., J. Bio.
l. Chem. 271: 31202 (1996)) except Peprotech
(Rocky Hill, NJ) or R & D system (Minneapolis)
s, MN) to obtain a recombinant human chemokine. (Ponath et al., J. Clin.
． Invest. 97: 604 (1996)).
4 was used to determine the chemotaxis of human PMBC or CD3 blast cells by transendothelial transduction.
sendotherial) migration assay (Carr et al., Proc. Natl.
Acad. Sci. USA 91: 3652 (1994)). Cells that migrated to the bottom chamber were collected and relevant cell numbers were obtained using a FACScan®. Human eosinophil and neutrophil chemotaxis was performed using 96-well chemotaxis plates (Neuro Probe, Gaithersburg, MD). 29 μl of chemokine appropriately diluted in RPMI-1640 plus 0.5% SBA was added to the wells and then covered with a 2 micron filter membrane.
5 × 10 ⁴ to 1 × 10 ⁵ cells in 25 μl were added on the filter on each well. Plates were incubated at 37 ° C for 25-30 minutes, filters were removed and plates were frozen at -80 ° C for 30 minutes. After thawing, CyQuant (Mole
6 μl of a solution of (Curar Probes, Eugene, OR) was added to the wells and the plate was read by a fluorescence plate reader. Migration is expressed as the average of relative fluorescence units (RFU), which is proportional to the number of cells migrated.

¹²⁵ I-labeled chemokines were obtained from NEN (Boston, MA). Binding of chemokines to target cells was performed as previously described (Ponath et al., J.E.
xp. Med. 183: 2437 (1996)). Briefly, cells were resuspended in binding buffer (50 mM HEPES, 1 mM CaCl ₂ , 0.5% BSA) and incubated with radiolabeled ligand in the presence or absence of competitor.
After 60 minutes at 37 ° C., cells were washed 3 times with binding buffer supplemented with 0.5 N NaCl and pellets were counted. All experiments were done in duplicate and repeated at least 3 times. The curve fit and the concentration that inhibits specific binding by 50% (IC ₅₀ ) were calculated using KaleidaGraph software (Synergy So).
FW, Reading, PA).

Calcium flux assay Freshly isolated human PBMCs were labeled with Fluo-3 and the response to various chemokines was measured as previously described on the FACScan® (P
onath et al. Clin. Invest. 97: 604 (1996)). Different leukocyte populations were gated by forward scatter vs. side scatter to obtain FL1 stationary baseline fluorescence first. Cells were then stimulated with various chemokines and changes in fluorescence intensity were recorded and plotted over the time course. When anti-receptor antibody was used, cells were incubated with mAb for 5 minutes before addition of chemokines.

Results Production of blocking mAb against CCR1 Immunizing mice with cells expressing high levels of transfected human CCR1 produced a monoclonal antibody called 2D4. This mA
b is cells transiently transfected with CCR1 cDNA and stable C
Some stained independently derived from the CR1 transfected cell line, but did not stain parental cells or cells transfected with other chemokine receptors (FIG. 1A). When 2D4 was added to the radiolabeled ligand binding assay, it completely bound ¹²⁵ I-MIP-1α binding to 3.4 nM (0.5 μg / ml) I.
It was inhibited by C ₅₀ (FIG. 1B). ¹²⁵ I-MI to CCR-1 in the same set of experiments
P-1α binding was determined by unlabeled MIP-1α and RANTES respectively 1.
It was competed with an IC ₅₀ of 1 nM and 2.5 nM. 2D4 binds ¹²⁵ I-RANTES to CCR-1 transfectants at 4.5 nM (0.7 μg / ml)
It was inhibited by IC ₅₀ (FIG. 1B).

CCR1 is the predominant chemokine receptor for MIP-1α and RANTES on monocytes Anti-CCR1 mAb 2D4 vividly stained whole peripheral blood monocytes (FIG. 2A).
. [Ca ²⁺ ] _i mobilization in monocytes induced by 20 nM MIP-1α or 20 nM RANTES was completely blocked by 50 μg / ml 2D4, whereas IL-8 (FIG. 2B) or MCP-1 The response was not affected by this antibody. HCC-1, a chemokine recently reported to be a ligand for CCR1, induced a weak calcium response at a concentration of 100 nM, which was also completely inhibited by 2D4. MC, a Chemokine Shown to Mediate Function Through Several Chemokine Receptors Including CCR2 on Monocytes
2D4 only partially inhibited the activity of P-3 (Franci et al., J. Immu.
nol. 154: 6511 (1995)). A chemotaxis assay showing that MIP-1α and RANTES-induced monocyte migration can be completely blocked by 2D4, while having no effect on monocyte chemotaxis to the CCR2-specific ligand MCP-1. The Ca ²⁺ flux data corresponded to the results from (Table 1). Only a few monocyte chemotaxis were induced by MIP-1β, a ligand for CCR5 but not CCR1.

[0123]

[Table 1]

Freshly isolated human PBMC was treated with 10 nM MCP-1, MIP-1α, RANT.
Used in chemotaxis assay with ES and MIP-1β. Migrated monocyte cells were counted by flow cytometry using forward versus side scatter.

CCR-1 is expressed in a subset on memory T lymphocytes In peripheral blood, 2D4 consistently stained a small percentage of CD3 + cells in the range of 1-5% depending on the individual. Analysis by multicolor flow cytometry showed that CCR1 + T cells were a phenotype characterized by CD45RO + CD26 +, post-activation memory T cells (FIGS. 3A and 3B). Total CCR1 + T cells are contained within the CCR5 + and CXCR3 + T cell populations, which contain 30-50% of total peripheral blood T cells (FIG. 3B). 2D4 also consistently stained a small percentage of B cells (0.5%
~ 5%, n = 6). CD56 + NK cells were substantially negative when stained with 2D4.

CCR1 Expression on Activated T Cells Activated T cells respond much more actively to chemokines than do resting T cells,
Also, in particular, a large number of chemokine receptors (Qin et al., J. Clin.
t. 101: 746 (1998); Loetscher et al. Exp. Med
． 184: 569 (1996); Wu et al. Exp. Med. 186: 137
3 (1997)), showing increased expression of CCR5 and CXCR3. To determine whether CCR1 is also upregulated in a similar manner, peripheral blood T
Cells were activated with anti-CD3 and IL-2 and cells were analyzed by flow cytometry for chemokine receptor expression at various times. Anti-CDS
During the first 3 days of stimulation with, most of the chemokine receptors were down-modulated from the cell surface, a phenomenon previously observed (Qin et al., J. Clin. In.
vest. 101: 746 (1998); Loetscher et al. Exp.
Med. 184: 569 (1996)). Expression of CXCR3 and CCR5 is 4
It began to increase after day and reached maximum levels in 5-6 days for CXCR3 and 12-14 days for CCR5. However, CCR1 expression remained undetected by flow cytometry throughout this period (Table 2 and FIG. 4). A chemotaxis assay shows that CCR1 induces migration of activated T cells to MIP-1α or R
Confirming that it does not play an important role in ANTES, it was demonstrated that the majority of migration was mediated by CCR5 (FIGS. 5A and 5B). The data in FIG. 5A is T
While 2D4 was unable to inhibit chemotaxis of cytoblasts to MIP-1α or RANTES, chemotaxis induced by both MIP-1α and RANTES and MIP-1β-induced chemotaxis was increased by 90%. Anti-CCR5 mAb until exceeded
Indicates that 2D7 has blocked.

When T cells are cultured under Th1 or Th2 polarization conditions, IFNα becomes CCR
1 was recently reported to be able to increase mRNA levels (Sallusto et al.,
J. Exp. Med. 187: 875 (1998)). Recent studies have evaluated the ability of IFNα to upregulate CCR1 surface expression in large cultures where expression is normally downregulated. IFNa in all donors tested
An increase in CCR1 expression was detected in activated T cells when cultured in the presence of A. The data in Table 2 show that CCR1 begins to appear on cells treated with IFNα by day 7 and gradually reaches a maximum level on days 10-14, ie 6-10 days after addition of IFNα. Is shown (Table 2 and FIG. 4). MIP-1α and RANTES
CCR1 expressed on IFNα-treated T cells, as evidenced by receptor inhibition with mAb in a migration assay for C. cerevisiae, made important functional contributions to these potentially chemotactic cells. (FIG. 5B). In the presence of IFNα, anti-CCR1 can inhibit cell migration to MIP-1α by 39% while anti-CCR1.
CCR5 inhibited migration to MIP-1α by 58%. Anti-CCR1 and anti-C
The combination with CR5 inhibited chemotaxis by 89%. Anti-CCR1 mA
b did not affect chemotaxis of CD3 blasts cultured only with IL-2 to MIP-1α, which could be completely inhibited by 50 μg / ml anti-CCR5 (FIG. 5A).
). The anti-CCR1 mAb did not affect migration to MIP-1β, whereas the anti-CCR1 mAb inhibited this migration to> 95% regardless of IFNα treatment.

[0128]

[Table 2]

Human PBMCs were stimulated with anti-CD3 mAb on day 0. On day 4, cells were detached and incubated with IL-2 alone or with IL-2 and IFNα. Cells were removed on the day of incubation and stained for chemokine receptor expression. Data represent results from one of eight donors, all of which gave similar profiles.

Significant changes in CCR1 expression on eosinophils Human eosinophils are eotaxin, RANTES, MCP-3.
And actively respond to MCP-4. Previous studies have shown that using blocking mAbs for CCR3, these responses are mediated predominantly through CCR3 (Heath et al., J. Clin. Invest. 99: 178 (1997)).
Although a weak response to MIP-1α was observed, the expression of CCR1 on human eosinophils is less well characterized. A significant donor change in CCR1 expression on human eosinophils was observed with the anti-CCR1 mAb. FIG. 6 shows the staining profile of eosinophils purified from 4 individuals. Expression of CCR1 ranged from completely negative (donor # 52) to over 95% positive (donor # 5). Expression of CCR3 on these cells was almost> 95% positive. Functionally, C
Eosinophils that were completely negative for CR1 failed to respond to MIP-1α in the chemotaxis assay, whereas those from the 2D4-stained donor responded to MIP-1α. However, the response was less effective than eotaxin (Figures 7A and 7B). A limited number of donor samples were analyzed daily. The weak chemotactic response of those donors to MIP-1α did not allow statistical analysis of the general pattern of CCR1 expression on eosinophils between populations, at which time the proportion of CCR1 + eosinophils and MI.
It also does not allow the establishment of a strong correlation with its response to P-1α. However,
Among all tested donors, Eotaxin is significantly more effective than MIP-1α in eliciting eosinophil chemotaxis, even when CCR1 and CCR3 show equal amounts of surface expression. It should be noted.

CCR1 is expressed on human neutrophils Mouse neutrophils are known to increase the active chemotactic response to MIP-1α, presumably via CCR1 (Gao et al. , J. Exp. Med. 185.
: 1959 (1997)). In contrast, human neutrophils migrate little and MIP
In the case of -1α, there is no movement. However, MIP-1α stimulation has been shown to induce intracellular Ca ²⁺ mobilization (McColl et al., J. Im.
munol. 150: 4550 (1993)). In the latest study, anti-CCR1
mAb 2D4 stained 30-50% of neutrophils from all donors tested (FIG. 8), while the CCR1 ligands MIP-1α, RANTES and MPIF chemotaxis, degranulation or up to a concentration of 200 nM. We were unable to induce any neutrophil response in the adhesion assay. In the Ca ²⁺ flux assay, approximately 6-8% of neutrophils responded to MIP-1α or MPIF stimulation (FIGS. 9A, 9B), in contrast IL-.
8 induced essentially all neutrophil responses (FIG. 9C). Anti-CC
R1 mAb 2D4 completely blocked [Ca ²⁺ ] _i mobilization induced by either MIP-1α or MPIF, but not by IL-8 (FIGS. 9D-9F).

CCR1 is Expressed on Human Basophils Basophils have been shown to be potent inflammatory cells, responding to many chemokines and expressing CCR3 on their surface (Uguccioni et al. , J. Cli
n. Invest. 100: 1137 (1997)). Anti-IgE or CCR3, markers for basophils, which both give a clear forward and side scatter
CCR1 expression was detected on virtually all basophils with anti-CCR1 mAb 2D4 (FIG. 11) by double-staining human blood with.

While the present invention has been particularly shown and described in the references for the preferred embodiments thereof, there are various changes in form and detail without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will appreciate that changes can be made therein.

[Brief description of drawings]

1A and 1B show specific binding of monoclonal antibody 2D4 to CCR1. FIG. 1A shows that monoclonal antibody 2D4 was used to stain various CC chemokine receptor transfectants expressed on the L1-2 parental cell line.
The result when using is shown. Only CCR1 transfected cells stained positive. FIG. 1B is a graph showing the results when CCR1 transfected cells were used in a ligand binding assay in the presence of increasing concentrations of monoclonal antibody 2D4. Total binding was 11574 ± 1355 cpm for MIP-1α and 1 for RANTES
It was 734 ± 118.

2A and 2B show that monocyte responses to MIP-1α and RANTES are C.
It is a figure which shows CR1 dependence. FIG. 2A is a flow cytometry histogram showing monocyte expression of CCR1. Normal human PBMC were stained with an irrelevant mouse IgG1 antibody (dotted line) and monoclonal antibody 2D4 (solid line). Monocytes were gated by forward and side scatter. FIG. 2B shows intracellular calcium mobilization of monocytes after chemokine stimulation. Loading human PBMC with Fluo-3,
20 nM MIP-1α, RANTES, IL-8 or 10 as indicated by the arrow
It was stimulated with 0 nM HCC-1. If antibody 2D4 is used, 5
0 μg / ml 2D4 was added.

FIG. 3A and FIG. 3B are diagrams showing CCR1 expression in peripheral blood lymphocytes. FIG. 3A shows normal human PBMC with 2D4 and T cells (CD3) or B cells (CD1
The results when stained with a monoclonal antibody specific to 9) are shown. FIG. 3B shows CCR1 expression on T cell subsets. These plots first gated CD3 + cells, then CCR1 vs. CD26, CD45RO, CXCR3 and CC.
It analyzed about R5.

FIG. 4 is a diagram showing CCR1 expression in activated T cells. IF human PBMC
Stimulation with anti-CD3 and IL-2 was performed in the absence or presence of Na. Cells were cultured for 10 days, then mAc for CCR1, CCR5 and CCR3 as indicated.
Stained with b (solid line). Negative control (dotted line) was stained with irrelevant mouse IgG1. The data obtained on day 14 staining also showed a similar profile.

5A and 5B show inhibition of chemotaxis by anti-CCR1 mAb 2D4. 50 μg / ml 2D4 (anti-CCR1, solid bar), 2D7
(Anti-CCR5, hollow bars) or 50 nM MIP-1α, MIP-1β or RANTE in the presence of both mAbs (hatched bars) at 50 μg / ml each.
Activated T cells were used in the chemotaxis assay for S. Figure 5A shows T cells INF
The result when stimulated in the absence of α is shown. FIG. 5B shows the results when T cells were cultured in the presence of INFα. Migrated cells were counted by flow cytometry. The total migration number of CD3 blasts in this experiment was MIP-1α, MIP-1β and RA.
3155, 2683 and 5483 respectively for NTES and background was 246. The total migration number of IFNα-treated cells is MIP-1α, M
11352, 6542 and 1 for IP-1β and RANTES, respectively
2136 and the background was 612. This data represents 3 experiments with similar results.

FIG. 6 shows various expression levels of CCR1 in eosinophils. Eosinophils were purified from normal donors and stained with anti-CCR1 mAb 2D4. Dot plots were used to better demonstrate the positively stained cells. Data are staining profiles from 4 different donors, representing the extent of CCR1 expression on eosinophils from different individuals.

7A and 7B show that eosinophil chemotaxis towards MIP-1α can be completely inhibited by anti-CCR1 monoclonal antibody 2D4. 20μ
g / ml anti-CCR1 mAb 2D4 or anti-CCR3 mAb 7B1
Increasing concentrations of MIP-1α (FIG. 7A) and eotaxin (in the presence of 1
7B) chemotaxis assay (Forssmann et al., FEBS Let).
t. 408: 211 (1997)), purified eosinophils from donors showing> 90% CCR1-positive cells were used. Chemotaxis was performed in 96 well plates and migrated cells were measured with a fluorescent plate reader.

FIG. 8 shows staining of human blood with anti-CCR1 mAb 2D4.
Neutrophils were identified by forward and side scatter.

9A to 9F are diagrams showing Ca ²⁺ mobilization in neutrophil cells. Fluo-
3 loaded neutrophils in the absence of antibody (9A, 9B and 9C) or 50μ
In the presence of g / ml 2D4 (9D-9F), 100 nM MIP-1α (FIGS. 9A and 9D), 100 nM MPIF (FIGS. 9B and 9E) as indicated by the arrows.
) Or 2 nM IL-8 (FIGS. 9C and 9F).

10A and 10B show m using CCR1 / CCR3 receptor chimeras.
It is a schematic diagram showing the determination of the epitope specificity of Ab2D4. The top row shows the 2D4 (dotted outline) and control antibody against various receptor chimeras constructed as shown from CCR1 (dotted) and part of CCR3 (shaded). The (hollow outline) bond is shown. The bottom row of panels shows the binding of anti-CCR3 mAb 7B11 (dotted outline) to control antibody (hollow outline) for the indicated receptor chimeras. mAb 2D4 binds to the second extracellular loop of CCR1.

FIG. 11 is a fluorescence plot illustrating CCR1 expression on human basophils.
Human blood was stained with anti-CCR1 and basophils were identified by double staining with anti-IgE and gated by forward and side scatter. Anti-by adding a shadow
CCR1 2D4 staining is represented. Control mouse IgG1 staining is represented by the solid line.

FIG. 12 represents the amino acid sequence of human CCR1. The predicted N-terminal portion, transmembrane portion (TM), extracellular portion (EC) and C-terminal portion are shown.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 G01N 33/50 Z 4H045 15/02 33/566 G01N 33/15 C12P 21/08 33/50 C12N 15/00 ZNAA 33/566 C // C12P 21/08 5/00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH , GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Cassam, Nasime United States Massachusetts 02154 Waltham, Number One, Adams Street 58 F Term (Reference) 2G045 AA40 DA36 FB03 4B024 AA01 AA11 BA44 GA03 GA18 HA01 HA15 4B064 AG27 CA10 CA20 CC24 CE12 DA01 DA13 4B065 AA91X AA92X AB05 AC15 CA25 CA44 CA11 ABBCA4 CAB 4A0A A20 AA30 CA40 DA76 DA86 EA22 EA23 EA28 EA50 FA72 GA26

Claims (49)

[Claims] 1. Epitope specificity which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part of said receptor, inhibits the binding of a ligand to said receptor and is the same or similar to that of monoclonal antibody 2D4. An antibody or an antigen-binding fragment thereof, which has sex. 2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof inhibits one or more functions associated with binding of a ligand to a receptor. 3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the mammalian CC-chemokine receptor 1 (CCR1) is human CC-chemokine receptor 1 (CCR1). 4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has specificity for the secondary extracellular loop of CC-chemokine receptor 1 (CCR1). 5. The antibody or the antibody thereof according to claim 4, wherein the antibody or the antigen-binding fragment thereof has specificity for an epitope consisting essentially of amino acids 171 to 205 of human CC-chemokine receptor 1 (CCR1). Antigen-binding fragment. 6. The antibody is: a) monoclonal antibody 2D4; b) 2 for binding to mammalian CC-chemokine receptor 1 (CCR1).
An antibody capable of competing with D4; c) selected from the group consisting of antigen-binding fragments of (a) or (b), which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof. The antibody or the antigen-binding fragment thereof according to claim 1, which is one. 7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the ligand is a chemokine. 8. The chemokine is MIP-1α, RANTES, MCP-2,
The antibody or antigen-binding fragment thereof according to claim 7, which is any one or more of MCP-3, leukotactin-1, HCC-1 and MPIF. 9. 2D4 deposited under ATCC registration number HB-12644
Hybridoma cell line. 10. A monoclonal antibody or an antigen-binding fragment thereof produced by the hybridoma cell line according to claim 9. 11. A) mammalian CC-chemokine receptor 1 (CCR1)
) Or at least one antibody or antigen-binding fragment thereof which binds to a part of said receptor, inhibits the binding of a ligand to said receptor and has the same or similar epitopic specificity as that of monoclonal antibody 2D4, and b) one or more auxiliary reagents suitable for detecting the presence of a complex of said antibody or antigen-binding fragment thereof with said mammalian CC-chemokine receptor 1 (CCR1) or part thereof, Mammalian CC-chemokine receptor 1 (CC
A test kit for use in detecting the presence of R1) or a part thereof. 12. The antibody is a) monoclonal antibody 2D4; b) 2 for binding to mammalian CC-chemokine receptor 1 (CCR1).
An antibody capable of competing with D4; c) an antigen-binding fragment of (a) or (b), which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof, and d) above. The test kit according to claim 11, which is selected from the group consisting of combinations. 13. A mammalian CC-chemokine receptor 1 (CCR1) -producing cell binds to a mammalian CC-chemokine receptor 1 (CCR1) or a part of the receptor and inhibits binding of a ligand to the receptor. And producing a mammalian CC-chemokine receptor 1 (CCR1), the method comprising contacting the ligand with an effective amount of an antibody or an antigen-binding fragment thereof having the same or similar epitope specificity as that of the monoclonal antibody 2D4. Method of inhibiting interaction with cells. 14. The cell is selected from the group consisting of lymphocytes, monocytes, granulocytes, neutrophils, T cells, basophils, and cells containing a recombinant nucleic acid encoding CCR1 or a part thereof. 14. The method of claim 13, which is 15. The method of claim 14, wherein the T cells are selected from the group consisting of CD26 + cells and CD45RO + cells. 16. The method of claim 13, wherein the ligand is a chemokine. 17. The chemokine is MIP-1α, RANTES, MCP-2.
17. The method according to claim 16, which is one or more of MCP-3, leukotactin-1, HCC-1 and MPIF. 18. The antibody or antigen-binding fragment thereof is: a) monoclonal antibody 2D4; b) 2 for binding to mammalian CC-chemokine receptor 1 (CCR1).
An antibody capable of competing with D4; c) an antigen-binding fragment of (a) or (b), which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof, and d) above. 14. The method of claim 13, which is selected from the group consisting of combinations. 19. A composition comprising a) a cell or a fraction of a cell to be tested, which binds to a mammalian CC-chemokine receptor 1 (CCR1) or a portion of said receptor and which comprises a ligand for said receptor. An antibody or antigen-binding fragment thereof that inhibits binding and has the same or similar epitope specificity as that of monoclonal antibody 2D4, is suitable for binding said antibody or antigen-binding fragment thereof to mammalian CCR1 or a part thereof. Contacting under the following conditions; and b) detecting the binding of the antibody or the antigen-binding fragment thereof, wherein the binding of the antibody or the antigen-binding fragment is one of the receptor or the receptor on the cell. CC-Chemokine Receptor 1 (CCR1) by a cell or a fraction of said cell showing the presence of Some methods of detecting expression. 20. An antibody or antigen-binding fragment thereof a) monoclonal antibody 2D4; b) 2 for binding to mammalian CC-chemokine receptor 1 (CCR1).
An antibody capable of competing with D4; c) an antigen-binding fragment of (a) or (b), which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof, and d) above. 20. The method of claim 19, which is selected from the group consisting of combinations. 21. The method of claim 20, wherein the composition is a sample containing human cells. 22. a) binding to a sample to be tested and mammalian CC-chemokine receptor 1 (CCR1) or part of said receptor, inhibiting the binding of a ligand to said receptor, and of monoclonal antibody 2D4 Contacting an antibody or an antigen-binding fragment thereof with the same or similar epitopic specificity as those under conditions suitable for the binding of said antibody or fragment thereof to said mammalian CCR1 or part thereof; and b ) The step of detecting or measuring the binding of the antibody or the antigen-binding fragment thereof, wherein the binding of the antibody or the antigen-binding fragment thereof to the substance in the sample is the mammalian CC-chemokine receptor 1 ( CCR1) or mammalian CC-chemokine receptor 1 (CCR1) showing the presence of a portion of the receptor. 1) or a method for detecting a part of the receptor. 23. The antibody or antigen-binding fragment thereof is a) monoclonal antibody 2D4; b) 2 for binding to mammalian CC-chemokine receptor 1 (CCR1).
An antibody capable of competing with D4; c) an antigen-binding fragment of (a) or (b), which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof, and d) above. 23. The method of claim 22, which is selected from the group consisting of combinations. 24. The method according to claim 22, wherein the sample is a fraction of cells containing a mammalian CC-chemokine receptor 1 (CCR1) or a part of the receptor in a normal individual. 25. Contacting an effective amount of mammalian CC-chemokine receptor 1 (CCR1) or an antibody or an antigen-binding fragment thereof that binds to a part of the receptor with a composition containing the receptor or a part thereof. Wherein the antibody or fragment is a mammalian CC-chemokine receptor 1 (C
A mammalian CC-, which inhibits binding to CR1) and inhibits one or more functions associated with the binding of chemokines to said receptor and has the same or similar epitope specificity as that of monoclonal antibody 2D4. Chemokine receptor 1 (C
CR1) or a method of inhibiting the function associated with the binding of chemokines to a functional fragment of said receptor. 26. The chemokine is MIP-1α, RANTES, MCP-2.
26. The method according to claim 25, wherein the method is any one or more of, MCP-3, leukotactin-1, HCC-1 or MPIF. 27. The antibody or antigen-binding fragment is: a) monoclonal antibody 2D4; b) 2 for binding to mammalian CC-chemokine receptor 1 (CCR1).
An antibody capable of competing with D4; c) an antigen-binding fragment of (a) or (b), which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof, and d) above. 26. The method of claim 25, which is selected from the group consisting of combinations. 28. A) a reagent to be tested, b) a binding to mammalian CC-chemokine receptor 1 (CCR1) or a part of said receptor, inhibiting the binding of a ligand to said receptor, and monoclonal antibody 2D4. Antibody or antigen-binding fragment having the same or similar epitopic specificity as that of c), and c) a composition containing a mammalian CC-chemokine receptor 1 (CCR1) or a ligand-binding variant thereof, wherein Binding of said antibody or antigen binding fragment to chemokine receptor 1 (CCR1) or a ligand binding variant thereof, and of said antibody or antigen binding fragment to said mammalian CC-chemokine receptor 1 (CCR1) or a ligand binding variant thereof Conditions suitable for detecting or measuring binding Mixing (combining) a step, mammalian CC- chemokine receptor 1 (CCR1) or detection or identification methods of the reagent to bind its ligand binding variant. 29. The antibody or antigen-binding fragment thereof is: a) monoclonal antibody 2D4; b) 2 for binding to mammalian CC-chemokine receptor 1 (CCR1).
An antibody capable of competing with D4; c) an antigen-binding fragment of (a) or (b), which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof, and d) above. 29. The method of claim 28, which is selected from the group consisting of combinations. 30. A complex formed by monitoring the formation of a complex between said antibody or antigen binding fragment and said mammalian CC-chemokine receptor 1 (CCR1) or ligand binding variant and comparing to an appropriate control. 29. The method of claim 28, wherein the reduction in body mass is indicative of the agent binding to the receptor or a ligand binding variant thereof. 31. A composition comprising a mammalian CC-chemokine receptor 1 (CCR1) or a ligand binding variant thereof in a cell producing a recombinant CC-chemokine receptor 1 (CCR1) or a ligand binding variant thereof. 29. The method of claim 28, wherein: 32. A composition comprising mammalian CC-chemokine receptor 1 (CCR1) or a ligand binding variant thereof, wherein the recombinant CC-
32. The method of claim 31, which is a membrane fraction of cells that produce chemokine receptor 1 (CCR1) or a ligand binding variant thereof. 33. The antibody or antigen-binding fragment thereof is labeled with a label selected from the group consisting of radioisotopes, spin labels, antigen labels, enzyme labels, fluorescent groups and chemiluminescent groups. the method of. 34. The agent is a mammalian CC-chemokine receptor 1 (CC
29. An antibody having specificity for R1) or an antigen-binding fragment thereof.
The method described. 35. Epitope specificity identical to or similar to that of monoclonal antibody 2D4, which binds to mammalian CC-chemokine receptor 1 (CCR1) or a portion of said receptor and inhibits binding of ligands to the receptor. A method for inhibiting leukocyte trafficking in a patient, which comprises the step of administering to the patient a composition containing an effective amount of an antibody having the above or an antigen-binding fragment thereof. 36. The method of claim 35, wherein the ligand is a chemokine. 37. The chemokine is MIP-1α, RANTES, MCP-2.
37. The method of claim 36, wherein the method is any one or more of :, MCP-3, leukotactin-1, HCC-1 or MPIF. 38. The antibody or antigen-binding fragment thereof is: a) monoclonal antibody 2D4;
b) 2 for binding to mammalian CC-chemokine receptor 1 (CCR1)
An antibody capable of competing with D4; c) an antigen-binding fragment of (a) or (b), which binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof, and d) above. 36. The method of claim 35, wherein the method is selected from the group consisting of combinations. 39. Epitope specificity that binds to mammalian CC-chemokine receptor 1 (CCR1) or a portion of said receptor, inhibits binding of a ligand to said receptor and is the same as or similar to that of monoclonal antibody 2D4. A composition comprising an antibody or an antigen-binding fragment thereof, and optionally a pharmaceutically acceptable vehicle. 40. An antibody or antigen thereof which binds to mammalian CC-chemokine receptor 1 (CCR1) or a portion of said receptor and inhibits binding of a ligand to said receptor with an IC ₅₀ of less than about 10 μg / ml. Combined fragment. 41. The antibody or antigen-binding fragment thereof of claim 40, which inhibits binding of a ligand to the receptor with an IC ₅₀ of less than about 5 μg / ml. 42. The antibody or antigen-binding fragment thereof of claim 41, which inhibits binding of a ligand to the receptor with an IC ₅₀ of less than about 1 μg / ml. 43. A mammalian CC with an affinity of greater than about 5 × 10 ^-8 M.
-Binding to chemokine receptor 1 (CCR1) or a part of said receptor,
An antibody or an antigen-binding fragment thereof. 44. The affinity according to claim 43, wherein the affinity is about 5 × 10 ⁻⁹ M or higher.
The described antibody or antigen-binding fragment thereof. 45. An antibody or antigen-binding fragment thereof that binds to mammalian CC-chemokine receptor 1 (CCR1) or a portion of the receptor and inhibits chemotaxis with an IC ₅₀ of less than about 50 μg / ml. 46. The antibody or antigen-binding fragment thereof of claim 45, which has an IC ₅₀ of less than about 20 μg / ml. 47. The antibody or antigen-binding fragment thereof of claim 46, which has an IC ₅₀ of less than about 10 μg / ml. 48. It binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof and inhibits the binding of a ligand to the receptor, and has the same or similar epitope specificity as that of monoclonal antibody 2D4. A method for treating a CC-chemokine receptor 1 (CCR1) -mediated disorder in a patient, comprising the step of administering to the patient an effective amount of an antibody or antigen-binding fragment thereof. 49. It binds to mammalian CC-chemokine receptor 1 (CCR1) or a part thereof and inhibits the binding of a ligand to the receptor, and has the same or similar epitope specificity as that of the monoclonal antibody 2D4. A method for treating an inflammatory disorder in a patient, which comprises the step of administering to the patient an effective amount of an antibody or an antigen-binding fragment thereof.