Methods of use for anti-CCR1 antibodies

Details Methods of use for anti-CCR1 antibodies Methods of use for anti-CCR1 antibodies

2001-09-21

2004-04-20

Ulm, John (Department: 1646)

Chemistry: analytical and immunological testing

Biospecific ligand binding assay

C436S512000, C436S513000

Reexamination Certificate

active

06723570

ABSTRACT:

BACKGROUND OF THE INVENTION
Over the last decade, chemokines have emerged as key mediators of inflammation as a result of their numerous proinflammatory activities which affect virtually every leukocyte type. More recently, chemokines have been recognized as a critical component of basal leukocyte trafficking essential for normal immune surveillance and response, as well as for several other functions in hematopoiesis, angiogenesis, control of viral infection, and T cell differentiation (Baggiolini et al.,
Ann. Rev. Immunol.
15:675 (1997); Zou et al.,
Nature
393:595 (1998); Tachibana et al.,
Nature
393:591 (1998)). This diverse array of biological activities, including mediation of a range of pro-inflammatory effects on leukocytes, such as triggering of chemotaxis, degranulation, synthesis of lipid mediators, and integrin activation, together with their critical role in the initiation and maintenance inflammatory diseases, and the recent identification of certain chemokine receptors as co-receptors for HIV-1 entry, have made chemokines and chemokine receptors an attractive new set of therapeutic targets.
Members of the chemokine family are produced and secreted by many cell types in response to early inflammatory mediators such as IL-1&bgr; or TNF&agr;. The chemokine superfamily comprises two main branches: the &agr;-chemokines (or CXC chemokines) which are characterized by a single amino acid separating the first 2 cysteines, and the &bgr;-chemokines (CC chemokines), which contain two adjacent cysteines. The &agr;-chemokine branch includes proteins such as IL-8, neutrophil activating peptide-2 (NAP-2), melanoma growth stimulatory activity (MGSA/gro or GRO&agr;), and ENA-78, each of which have attracting and activating effects predominantly on neutrophils. The members of the &bgr;-chemokine branch affect other cell types such as monocytes, lymphocytes, basophils, and eosinophils (Oppenheim, J. J. et al.,
Annu. Rev. Immunol.,
9:617-648 (1991); Baggiolini, M., et al.,
Adv. Imunol.,
55:97-179 (1994); Miller and Krangel, Crit. Rev. Immunol, 12:17-46 (1992); Jose, P. J., et al.,
J. Exp. Med.,
179:881-118 (1994); Ponath, P. D., et al.,
J. Clin. Invest.,
97:604-612 (1996)), and include proteins such as monocyte chemotactic proteins 1-4 (MCP-1, MCP-2, MCP-3, and MCP-4), RANTES, macrophage inflammatory proteins (MIP-1&agr;, MIP-1&bgr;), thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC).
Chemokines bind to 7 transmembrane spanning G protein-coupled receptors (Murphy, P. M.,
Annu. Rev. Immunol.,
12:593-633 (1994)). A number of &bgr; chemokine receptors (CCR1-CCR10) have been identified to date, and the search for additional chemokine receptors is the subject of active research (Baggiolini,
Nature
392:565-568 (1998)). Chemokine receptor CCR1 was identified by Nomura et al., (
Int. Immunol.
5:1239-1249 (1993); Neote et al., (
Cell
72:415-425 (1993)) and Gao et al., (
J. Exp. Med.
177:1421-1427 (1993)). CCR1 was originally found to signal in response to MIP-1&agr; and RANTES, but more recently has also been shown to signal in response to additional chemokine ligands.
The selective recruitment of leukocyte subsets to sites of inflammation and the ordered trafficking of leukocytes through the circulation, tissues, lymphatic system and secondary lymphoid organs is controlled in part by the differential expression of chemokine receptors on subsets of cells. Such expression patterns would seem to ensure that a functionally related group of leukocytes can coordinately respond to a specific set of chemokines induced by a given stimulus. However, most leukocytes express several chemokine receptors, many with complex and promiscuous ligand interactions. For T cells, PCR or Northern blotting indicates that the known receptors for CC chemokines are expressed on subsets of T cells. Delineating exactly which subsets express particular receptors is an area of intense study, because chemokine receptor expression may explain 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 with different strains of HIV-1. This makes elucidating the normal immune function for a specific receptor on a given cell type and determining the relevance to initiation and progression of disease difficult, especially since specific antibodies are not available for many chemokine receptors.
SUMMARY OF THE INVENTION
CCR1 was the first chemokine receptor shown to interact with C—C (&bgr;) chemokines. Originally identified as a MIP-1&agr;/RANTES receptor, more recent studies have demonstrated that CCR1 may have as many as 6 or more &bgr; chemokine ligands. Work described herein characterizes the expression of CCR1 by flow cytometry and assesses in vitro the relative functional contribution of this receptor on human leukocytes using a blocking monoclonal antibody. In peripheral blood, all monocytes express high levels of CCR1, and monocyte responses to both MIP-1&agr; and RANTES can be completely blocked by the anti-CCR1 mAb 2D4. CCR1 is expressed on a small percentage of CD45RO+ CD26+ T cells, suggesting that it marks a subset of memory T cells. In contrast with chemokine receptors CCR5 and CXCR3, which are upregulated on activated T cells, CCR1 expression is diminished upon T cell activation. Neutrophils express low levels of CCR1 and show a weak response to MIP-1&agr; in [Ca
2+
]
i
mobilization assays, which can be inhibited by the anti-CCR1 mAb 2D4. The expression of CCR1 on eosinophils is extremely variable among individuals, ranging from >90% positive to completely negative. The expression pattern of CCR1 suggests that it is involved in a broad range of immunological activities, playing a major role in monocyte and eosinophil function, as well as in the function of a subset of T lymphocytes.
The present invention relates to an antibody (immunoglobulin) or functional fragment thereof (e.g., an antigen-binding fragment) which binds to a mammalian CC-chemokine receptor 1 (also referred to as CCR1 or CKR-1) or portion of the receptor (anti-CCR1). In one embodiment, the antibody of the present invention or fragment thereof has specificity for human CCR1 or a portion thereof. In another embodiment, the antibody or fragment of the invention inhibits (reduces or prevents) binding of a ligand (e.g., MIP-1&agr;, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF) to the receptor and inhibits one or more functions associated with binding of the ligand to the receptor (e.g., leukocyte trafficking). In a preferred embodiment, the ligand is MIP-1&agr; or RANTES. For example, as described herein, antibodies and fragments thereof of the present invention which bind human CCR1 or a portion thereof, can block binding of a chemokine (e.g., MIP-1&agr;, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1, or MPIF) to the receptor and inhibit function associated with binding of the chemokine to the receptor.
In a preferred embodiment, the antibody of the invention or fragment thereof has the same or similar epitopic specificity as monoclonal antibody (mAb) LS125-2D4 (2D4). For example, the antibody or fragment thereof can have epitopic specificity for the second extracellular loop of CCR1, such as a portion of CCR1 from about amino acid 171 to about amino acid 205. In one embodiment, the antibody is monoclonal antibody 2D4 or an antibody which can compete with 2D4 for binding to human CCR1 or a portion of human CCR1. Functional fragments of the foregoing antibodies are also envisioned.
In another embodiment, the antibody or fragment thereof binds CCR1 with an affinity of greater than about 5×10
−8
M, and more preferably at least about 5×10
−9
M. In a further embodiment, the antibody of the invention or fragment thereof inhibits chemokine binding to CCR1, preferably with an IC
50
of less than about 10 &mgr;g/ml, more preferably less than about 5 &mgr;g/ml, and more preferably less than about 1.0 &mgr;g/ml. In one embodiment, the antibody of the i

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