WO2000044365A1 - Methods for preventing graft rejection and ischemia-reperfusion injury - Google Patents

Abstract

A method for inhibiting the rejection of transplanted grafts is disclosed. The method comprising administering an effective amount of an antagonist of CCR1 function to a graft recipient. Also disclosed is a method of inhibiting ischemia/reperfusion injury comprising administering to a subject in need thereof an effective amount of an antagonist of CCR1 function. The disclosed methods can also comprise the co-administration of one or more additional therapeutic agents, for example, immunosuppressive agents and cell adhesion inhibitors.

Description

METHODS FOR PREVENTING GRAFT REJECTION AND ISCHEMIA-

REPERFUSION INJURY

RELATED APPLICATION

This application is a continuation-in-part of U. S. Serial No. 09/239,283, filed January 29, 1999, the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

In many instances, the best and only treatment available to patients suffering from certain end stage degenerative conditions or congenital genetic disorders is transplantation of a healthy graft (e.g., organs, tissues). Advances in surgical techniques and post-operative immunosuppressive therapy have mitigated some of the barriers to long-term survival of grafts and graft recipients, and ushered this once experimental therapy into wider clinical practice.

A major barrier to the long-term survival of transplanted grafts is rejection by the recipient's immune system. Graft rejection can be classified as hyper-acute rejection which is mediated by preformed antibodies that can bind to the graft and are present in the circulation of the recipient, acute rejection which is mediated by the recipient's cellular immune response or chronic rejection which occurs via a multi-factorial process that includes an immune component. The practice of matching the allelic variants of cellular antigens, most notably major histocompatability antigens (MHC), also referred to as tissue typing, as well as matching of the blood type of the donor and recipient has reduced the incidence of hyper-acute rejection. However, most grafts which are transplanted do not exactly match the tissue type of the recipient (e.g., allografts) and will not remain viable without therapeutic intervention.

The rejection of allografts can be inhibited by long-term (e.g., life-long) prophylactic immunosuppressive therapy, most notably with agents that inhibit calcineurin (e.g., cyclosporin A (CsA), FK-506). Immunosuppressive therapy not only inhibits rejection of the graft, but can render the recipient susceptible to infection with, for example, viruses, bacteria and fungi (e.g., yeasts, molds), and at higher risk for the development of certain malignancies. Additionally, therapeutic doses of immunosuppressive agents can produce adverse side effects, such as diabetes mellitus, neurotoxicity, nephrotoxicity, hyperlipidemia, hypertension, hirsutism and gingival hyperplasia (Spencer, CM., et al, Drugs 54(6):925-915 (1997)). Thus, the degree of immunosuppression must be carefully tailored to prevent rejection of the graft and to preserve the general health of the recipient. Despite such prophylactic immunosuppression, the acute and chronic rejection of grafts remains a clinical problem. Acute episodes of rejection are characterized by infiltration of the graft by the recipient's leukocytes (e.g., monocytes, macrophages, T cells) and cellular necrosis. These episodes usually occur during the days to months following transplantation. Acute rejection has been treated with high doses of certain immunosuppressive agents, such as, glucocorticoids (e.g., prednisone) and certain antibodies which bind to leukocytes (e.g., OKT3). However, these therapies do not always stop the rejection, are associated with systemic side effects and can lose efficacy in cases of recurrent rejection activity.

Chronic rejection becomes the major cause of graft failure and recipient death for those patients that survive past the first year. Evidence of chronic rejection can be found in about 40-50% of heart and/or lung allograft recipients who survive for five years, and most kidney grafts succumb to chronic rejection. The pathogenesis of chronic rejection is complex and involves accelerated arteriosclerosis (e.g., atherosclerosis) of the graft-associated vasculature and leukocyte infiltration. Unlike acute rejection episodes, chronic rejection is not generally responsive to further immunosuppressive therapy. Furthermore, the graft accelerated arteriosclerosis characteristic of chronic rejection is generally diffuse and not amenable to conventional therapeutic procedures (e.g., angioplasty, bypass grafting, endarterectomy). Thus, patients who chronically reject their grafts can require a second transplant. (Schroeder J.S. "Cardiac Transplantation", pp. 1298- 1300; Maurer, J.R. "Lung Transplantation", pp. 1491-1493; Carpenter, C.B. and Lazarus, J.M. "Dialysis and Transplantation in the Treatment of Renal Failure", pp. 1524-1529; Dienstag, J. "Liver Transplantation", pp. 1721-1725; all in Harrison 's Principles of Internal Medicine, 14^th ed., Fauci et al. Eds. McGraw Hill (1998)). The flow of oxygenated blood to all transplanted grafts is stopped during graft procurement and transplantation surgery. The lack of blood results in hypoxia and necrosis of some or all of the cells of the graft, depending on the amount of time that blood flow is stopped. The resulting damage or injury is apparent after the flow of blood is returned (reperfusion). This type of injury, referred to as ischemia reperfusion injury, can lead to the death of endothelial cells which line the graft associated blood vessels (e.g., arteries) (Gohra, H, et al., Transplantation, 60(l):96-l02 (1995)). Thus, ischemia/reperfusion injury can be a contributing factor to graft accelerated arteriosclerosis and graft rejection.

A need exists for therapeutic methods for preventing graft rejection and ischemia/reperfusion injury.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a Kaplan-Meier survival curve that illustrates the survival of

CCRl-/- mice (-0-) and wild type B6/129 mice (-Δ-) following renal ischemia/reperfusion. CCRl -/- mice survived significantly longer than B6/129 mice (p<0.001, log rank sum-test, survival limited to 100 days).

Figure 2 is a histogram illustrating the serum creatinine levels of CCR1-/- and B6/129 mice following renal ischemia/reperfusion or sham surgery (unilateral nephrectomy without ischemia/reperfusion). Creatinine levels were measured 2, 7 and 100 days following ischemia/reperfusion or sham surgery. Only B6/129 (control) mice had significantly elevated serum creatinine levels 2 and 7 days following ischemia/reperfusion (**p<0.005). All groups had elevated levels of serum creatinine 100 days after ischemia/reperfusion or sham surgery (*p<0.01). Figure 3 is a histogram illustrating serum alanine aminotransferase (SGPT) levels, an indicator of liver dysfunction, in CCRl-/- (KO) and B6/129 (WT) mice at predetermined times following hepatic ischemia reperfusion. Also shown are the SGPT levels in the serum of B6/129 mice that underwent surgery but did not experience hepatic ischemia/reperfusion (sham). CCRl-/- mice experienced significantly less liver dysfunction than B6/129 mice, as assessed by serum SGPT levels, over the period from 0.5 to 7 days following hepatic ischemia/reperfusion (*p<0.0001).

Figure 4A is a histogram illustrating concanavilin-A-induced proliferation of T cells in cultures of splenocytes isolated from CCRl-/- or B6/129 mice (CCR1+/+). Concanavilin-A-induced T cell proliferative responses in cultures of cells from CCRl-/- mice or cultures of B6/129 mice were about equivalent. Data are mean ± standard deviation of six cultures that were stimulated for 48 hours. The presented data are representative of four assays.

Figure 4B is a histogram illustrating mixed lymphocyte responses (MLR) of cells isolated from CCRl-/- mice (CCRl KO) or wild type B6/129 mice (WT) stimulated with allogeneic splenocytes (isolated from Balb/c mice). Cells from both CCRl-/- and wild type B6/129 mice displayed robust MLRs toward mitomycin-c treated Balb/c stimulator cells (**p<0.001 versus unstimulated CCRl-/- or unstimulated wild type B6/129 responder cells). CCRl-/- cells proliferated less than wild type cells in the assay (*p<0.01). Data are mean ± standard deviation of six 5- day cultures. The presented data are representative of four assays. BALB/c stim.: mitomycin-c treated splenocytes isolated from a Balb/c mouse; WT resp.: splenocytes isolated from B6/129 mouse; CCRl KO resp.: splenocytes isolated from CCRl-/- mouse; BALB/c- WT: splenocytes isolated from B6/129 mouse stimulated with mitomycin-c treated splenocytes isolated from a Balb/c mouse; BALB/c-* KO: splenocytes isolated from CCRl-/- mouse stimulated with mitomycin-c treated splenocytes isolated from a Balb/c mouse.

SUMMARY OF THE INVENTION

The invention relates to transplantation and to promoting the viability of transplanted grafts. In one aspect, the invention relates to a method for inhibiting (reducing or preventing) graft rejection (e.g., acute rejection, chronic rejection). In one embodiment, the method comprises administering to a graft recipient an effective amount of an antagonist of CCRl function. In another embodiment, the graft is an allograft. In a particular embodiment, the allograft is a heart. In a preferred embodiment, the method comprises administration of an antagonist of CCRl function and one or more immunosuppressive agents to a graft recipient. In another aspect, the invention relates to a method for inhibiting (reducing or preventing) ischemia reperfusion injury. In one embodiment, the method comprises administering to a subject in need there of an effective amount of an antagonist of CCRl function. In some embodiments, the ischemia/reperfusion injury can be a consequence of trauma or a medical procedure, for example, surgery. In other embodiments, the ischemia/reperfusion injury can be the result of a pathological condition, for example, arteriosclerosis, myocardial infarction, stroke or transient ischemic attack. In a particular embodiment, the ischemia/reperfusion injury is a consequence of graft transplantation. In another particular embodiment, the graft is a kidney. In another embodiment, the method comprises administration of an antagonist of CCRl function and one or more additional therapeutic agents, for example, thrombolytic agents, cell adhesion inhibitors, anti-coagulants, anti- thrombotic agents and activators or inhibitors of nitric oxide synthase.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to transplantation and the viability of transplanted grafts. Specifically, the invention relates to inhibiting graft rejection (e.g., acute graft rejection, chronic graft rejection) by administering to a graft recipient an effective amount of an antagonist of the mammalian (e.g., human) C-C chemokine receptor, CCRl . Chemokines are a family of proinflammatory mediators that promote recruitment and activation of multiple lineages of leukocytes (e.g., lymphocytes, macrophages). They can be released by many kinds of tissue cells after activation. Continuous release of chemokines at sites of inflammation can mediate the ongoing migration and recruitment of effector cells to sites of chronic inflammation. The chemokines are related in primary structure and share four conserved cysteines, which form disulfide bonds. Based upon this conserved cysteine motif, the family can be divided into distinct branches, including the C-X-C chemokines (α-chemokines), and the C-C chemokines (β-chemokines), in which the first two conserved cysteines are separated by an intervening residue, or are adjacent residues, respectively (Baggiolini, M. and Dahinden, C. A., Immunology Today, 15:121-133 (1994)). The C-X-C chemokines include a number of potent chemoattractants and activators of neutrophils, such as interleukin 8 (IL-8), PF4 and neutrophil-activating peptide-2 (NAP-2). The C-C chemokines include, for example, RANTES (Regulated on Activation, Normal T Expressed and Secreted), the macrophage inflammatory proteins 1 α and 1 β (MIP-1 and MIP-1 β), eotaxin and human monocyte chemotactic proteins 1-3 (MCP-1, MCP-2, MCP-3), which have been characterized as chemoattractants and activators of monocytes or lymphocytes. Chemokines, such as RANTES and MIP-1 α, have been implicated in human acute and chronic inflammatory diseases including respiratory diseases, such as asthma and allergic disorders.

The chemokine receptors are members of a superfamily of G protein-coupled receptors (GPCR) which share structural features that reflect a common mechanism of action of signal transduction (Gerard, C. and Gerard, N.P., Annu Rev. Immunol., 72.775-808 (1994); Gerard, C. and Gerard, N. P., Cwrr. Opin. Immunol, (5:140-145 (1994)). Conserved features include seven hydrophobic domains spanning the plasma membrane, which are connected by hydrophilic extracellular and intracellular loops. The majority of the primary sequence homology occurs in the hydrophobic transmembrane regions with the hydrophilic regions being more diverse. The receptors for the C-C chemokines include: CCRl which can bind, for example, MIP-1 , RANTES, MCP-2, MCP-3, MCP-4, CKbetaδ, CKbeta8-l, leukotactin-1, HCC-1 and MPIF-1 ; CCR2 which can bind, for example, MCP-1, MCP-2, MCP-3 and MCP-4; CCR3 which can bind, for example, eotaxin, eotaxin-2, RANTES, MCP-2, MCP-3 and MCP-4; CCR4 which can bind, for example, TARC, RANTES, MIP-1 α and MCP-1 ; CCR5 which can bind, for example, MIP-1 , RANTES, and MIP-1 β; CCR6 which can bind, for example, LARC/MIP- 3α/exodus; CCR7 which can bind, for example, ELC/MIP-3β and CCR8 which can bind, for example 1-309 (Baggiolini, M., Nature 392:565-568 (1998); Luster, A.D., New England Journal of Medicine, J35(7):436-445 (1998); Tsou, et al., J. Exp. Med., 188:603-608 (1998); Nardelli, et al., J Immunol, 7<52(l):435-444 (1999); Youn, et al, Blood, P7(9):3118-3126 (1998); Youn, et al, J Immunol, 759(11):5201-5201 (1997)). CCR1 as well as processes and cellular responses mediated by CCRl, are involved in rejection of transplanted grafts. As described herein, studies of allograft survival using a murine cardiac transplantation model were undertaken. Mice which lack functional chemokine receptor CCRl as a result of targeted disruption of the CCRl gene (CCRl KO; Gerard, C. et al, J. Clin. Invest. 100:2022-2021 (1997)) did not reject transplanted allografts, which were mismatched at MHC class I and MHC class II, as rapidly as control mice which comprise a functional CCRl gene and are otherwise genetically identical to CCRl KO mice (Example 1, Table 1, groups 1 and 2). As described herein, administration of low dose immunosuppressive therapy

(cyclosporin A) to CCRl +/+ control mice resulted in only a 2-3 day increase in graft viability as compared with untreated CCRl +/+ animals (Example 1, Table 1, group 3). Surprisingly, administration of the same low dose of CsA with inhibition of CCRl function led to permanent (>100 days) engraftment in CCRl KO mice which received CsA for a maximum period of only 21 days (Table 1, group 4).

In further studies, allografts that were mismatched only at MHC class II were transplanted into CCRl KO and CCR1+/+ control mice. As expected, the partial tissue match led to prolonged survival of the graft in CCR1+/+ control mice. However, all CCR1+/+ control mice still rejected the graft by the thirty-fifth day (Example 1, Table 1, group 5). Partial matching of the MHC antigens, like low dose immunosuppression, with inhibition of CCRl function led to permanent (>100 days) engraftment (Example 1, Table 1, group 6).

Histological examination of permanently engrafted hearts removed from group 4 and group 6 (see Table 1) mice at 100 days after transplantation revealed only minimal mononuclear cell infiltration and no evidence of transplant accelerated arteriosclerosis.

The survival of Class I and Class II mismatched allografts can be prolonged by the administration of anti-CD4 monoclonal antibody (mAb) (Mottram, et al, Transplantation, 59:559-565 (1995)). However, the long-term survival of these grafts is complicated by the development of chronic rejection with wide-spread arteriosclerosis in the vasculature of the graft (Hancock, et al , Nature Medicine, ^:1392-1396 (1998)). As described herein, Class I and Class II mismatched allografts survived for 60 days in CCRl KO and CCRl +/+ control mice that received anti-CD4 mAb therapy. Morphological examination of grafts removed from CCRl +/+ control recipients at sixty days revealed severe arteriosclerosis. In contrast, grafts removed from CCRl KO recipients showed no evidence of arteriosclerosis (Example 2, Table 2). Thus, disruption of CCRl function can provide the dual benefit of inhibiting both acute and chronic rejection of allografts.

Accordingly, a first aspect of the invention provides a method for inhibiting rejection (e.g., acute and/or chronic rejection) of a graft, comprising administering to a graft recipient an effective amount of an antagonist of CCRl function.

CCRl antagonists

As used herein, the term "antagonist of CCRl function" refers to an agent (e.g., a molecule, a compound) which can inhibit a (i.e., one or more) function of CCRl. For example, an antagonist of CCRl function can inhibit the binding of one or more ligands (e.g., MIP-1 cc, RANTES, MCP-2, MCP-3, MCP-4, CKbetaδ, CKbeta8-l, leukotactin-1, HCC-1, MPIF-1) to CCRl and/or inhibit signal transduction mediated through CCRl (e.g., GDP/GTP exchange by CCRl associated G proteins, intracellular calcium flux). Accordingly, CCRl -mediated processes and cellular responses (e.g., proliferation, migration, chemotactic responses, secretion or degranulation) can be inhibited with an antagonist of CCRl function.

Preferably, the antagonist of CCRl function is a compound which is, for example, a small organic molecule, natural product, protein (e.g., antibody, chemokine, cytokine), peptide or peptidomimetic. Several molecules that can antagonize one or more functions of chemokine receptors (e.g., CCRl) are known in the art, including the small organic molecules disclosed in, for example, international patent application WO 97/24325 by Takeda Chemical Industries, Ltd.; WO 98/38167 by Pfizer, Inc.; WO 97/44329 by Teijin Limited; WO 98/04554 by Banyu Pharmaceutical Co., Ltd.; WO 98/27815, WO 98/25604, WO 98/25605, WO 98/25617 and WO 98/31364 by Merck & Co., Inc.; WO 98/02151 and WO

99/37617, by LeukoSite, Inc.; WO 99/37651 and WO 99/37619 by LeukoSite, Inc., et al; United States Provisional Patent Application Number 60/021,716, filed July 12, 1996; United States Patent Application Numbers: 09/146,827 and 09/148,236, filed September 4, 1998; Hesselgesser et al, J. Biol. Chem. 275(25):15687-15692 (1998); and Howard et al, J. Medicinal Chem. 7(13):2184-2193 (1998); proteins, such as antibodies (e.g., polyclonal sera, monoclonal, chimeric, humanized) and antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')₂, Fv), for example, those disclosed in Su et al, J. Leukocyte Biol. (50:658-656 (1996); chemokine mutants and analogues, for example, those disclosed in U.S. Patent No. 5,739,103 issued to Rollins et al, WO 96/38559 by Dana Farber Cancer Institute and WO 98/06751 by Research Corporation Technologies, Inc.; peptides, for example, those disclosed in WO 98/09642 by The United States of America. The entire teachings of each of the above cited patent applications and references is incorporated herein by reference. Antagonists of CCRl function can be identified, for example, by screening libraries or collections of molecules, such as, the Chemical Repository of the National Cancer Institute (U.S.A.), as described herein or using other suitable methods.

Another source of antagonists of CCRl function are combinatorial libraries which can comprise many structurally distinct molecular species. Combinatorial libraries can be used to identify lead compounds or to optimize a previously identified lead. Such libraries can be manufactured by well-known methods of combinatorial chemistry and screened by suitable methods, such as the methods described herein.

The term "natural product", as used herein, refers to a compound which can be found in nature, for example, naturally occurring metabolites of marine organisms (e.g., tunicates, algae) and plants and which possess biological activity, e.g., can antagonize CCRl function. For example, lactacystin, paclitaxel and cyclosporin A are natural products which can be used as anti-proliferative or immunosuppressive agents.

Natural products can be isolated and identified by suitable means. For example, a suitable biological source (e.g., vegetation) can be homogenized (e.g., by grinding) in a suitable buffer and clarified by centrifugation, thereby producing an extract. The resulting extract can be assayed for the capacity to antagonize CCRl function, for example, by the assays described herein. Extracts which contain an activity that antagonizes CCRl function can be further processed to isolate the CCRl antagonist by suitable methods, such as, fractionation (e.g., column chromatography (e.g., ion exchange, reverse phase, affinity), phase partitioning, fractional crystallization) and assaying for biological activity (e.g., antagonism of CCRl activity). Once isolated the structure of a natural product can be determined (e.g., by nuclear magnetic resonance (NMR)) and those of skill in the art can devise a synthetic scheme for synthesizing the natural product. Thus, a natural product can be isolated (e.g., substantially purified) from nature or can be fully or partially synthetic. A natural product can be modified (e.g., derivatized) to optimize its therapeutic potential. Thus, the term "natural product", as used herein, includes those compounds which are produced using standard medicinal chemistry techniques to optimize the therapeutic potential of a compound which can be isolated from nature. The term "peptide", as used herein, refers to a compound consisting of from about two to about ninety amino acid residues wherein the amino group of one amino acid is linked to the carboxyl group of another amino acid by a peptide bond. A peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (see Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). A "peptide" can comprise any suitable L- and/or D-amino acid, for example, common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6- aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine). The amino, carboxyl and/or other functional groups on a peptide can be free (e.g., unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups, and means for adding or removing protecting groups are know in the art and are disclosed in, for example, Green and Wuts, "Protecting Groups in Organic Synthesis ", John Wiley and Sons, 1991. The functional groups of a peptide can also be derivatized (e.g., alkylated) using art-known methods.

Peptides can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry, and can be screened as described herein or using other suitable methods to determine if the library comprises peptides which can antagonize CCRl function. Such peptide antagonists can then be isolated by suitable methods.

The term "peptidomimetic", as used herein, refers to molecules which are not polypeptides, but which mimic aspects of their structures. For example, polysaccharides can be prepared that have the same functional groups as peptides which can antagonize CCRl . Peptidomimetics can be designed, for example, by establishing the three dimensional structure of a peptide agent in the environment in which it is bound or will bind to CCRl. The peptidomimetic comprises at least two components, the binding moiety or moieties and the backbone or supporting structure.

The binding moieties are the chemical atoms or groups which will react or form a complex (e.g., through hydrophobic or ionic interactions) with CCRl, for example, with the amino acid(s) at or near the ligand binding site. For example, the binding moieties in a peptidomimetic can be the same as those in a peptide antagonist of CCRl. The binding moieties can be an atom or chemical group which reacts with the receptor in the same or similar manner as the binding moiety in a peptide antagonist of CCRl . Examples of binding moieties suitable for use in designing a peptidomimetic for a basic amino acid in a peptide are nitrogen containing groups, such as amines, ammoniums, guanidines and amides or phosphoniums. Examples of binding moieties suitable for use in designing a peptidomimetic for an acidic amino acid can be, for example, carboxyl, lower alkyl carboxylic acid ester, sulfonic acid, a lower alkyl sulfonic acid ester or a phosphorous acid or ester thereof. The supporting structure is the chemical entity that, when bound to the binding moiety or moieties, provides the three dimensional configuration of the peptidomimetic. The supporting structure can be organic or inorganic. Examples of organic supporting structures include polysaccharides, polymers or oligomers of organic synthetic polymers (such as, polyvinyl alcohol or polylactide). It is preferred that the supporting structure possess substantially the same size and dimensions as the peptide backbone or supporting structure. This can be determined by calculating or measuring the size of the atoms and bonds of the peptide and peptidomimetic. In one embodiment, the nitrogen of the peptide bond can be substituted with oxygen or sulfur, thereby forming a polyester backbone. In another embodiment, the carbonyl can be substituted with a sulfonyl group or sulfmyl group, thereby forming a polyamide (e.g., a polysulfonamide). Reverse amides of the peptide can be made (e.g., substituting one or more -CONH- groups for a -NHCO- group). In yet another embodiment, the peptide backbone can be substituted with a polysilane backbone.

These compounds can be manufactured by known methods. For example, a polyester peptidomimetic can be prepared by substituting a hydroxyl group for the corresponding α-amino group on amino acids, thereby preparing a hydroxyacid and sequentially esterifying the hydroxyacids, optionally blocking the basic and acidic side chains to minimize side reactions. Determining an appropriate chemical synthesis route can generally be readily identified upon determining the chemical structure. Peptidomimetics can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using well- known methods of combinatorial chemistry, and can be screened as described herein to determine if the library comprises one or more peptidomimetics which antagonize CCRl function. Such peptidomimetic antagonists can then be isolated by suitable methods.

In one embodiment, the CCRl antagonist is an antibody or antigen-binding fragment thereof having specificity for CCRl. The antibody can be polyclonal or monoclonal, and the term "antibody" is intended to encompass both polyclonal and monoclonal antibodies. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production. The term "antibody" as used herein also encompasses functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies. Functional fragments include antigen-binding fragments which bind to CCRl . For example, antibody fragments capable of binding to CCRl or portions thereof, including, but not limited to Fv, Fab, Fab' and F(ab')₂ fragments are encompassed by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')₂ fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')₂ fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')₂ heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain. Single chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody". The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al, U.S. Patent No. 4,816,567; Cabilly et al, European Patent No. 0,125,023 Bl; Boss et al, U.S. Patent No. 4,816,397; Boss et al, European Patent No. 0,120,694 B 1 ; Neuberger, M.S. et al, WO 86/01533; Neuberger, M.S. et al, European Patent No. 0,194,276 Bl; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen et al, European Patent No. 0 451 216 Bl; and Padlan, E.A. et α/., EP 0 519 596 Al. See also, Newman, R. et al, BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al, U.S. Patent No. 4,946,778 and Bird, R.E. et al, Science, 242: 423-426 (1988)) regarding single chain antibodies.

Humanized antibodies can be produced using synthetic or recombinant DNA technology using standard methods or other suitable techniques. Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al, Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al, Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al, Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P. and J.S. Crowe, Gene, 101: 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213, published April 1, 1993). Antibodies which are specific for mammalian (e.g., human) CCRl can be raised against an appropriate immunogen, such as isolated and/or recombinant human CCRl or portions thereof (including synthetic molecules, such as synthetic peptides). Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with cells that express CCRl, such as activated T cells (see e.g., U.S. Pat. No. 5,440,020, the entire teachings of which are incorporated herein by reference). In addition, cells expressing recombinant CCRl such as transfected cells, can be used as immunogens or in a screen for antibody which binds receptor (See e.g., Chuntharapai et al, J. Immunol, 152: 1783-1789 (1994); Chuntharapai et al, U.S. Patent No. 5,440,021). Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique. A variety of methods have been described (see e.g., Kohler et al, Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al, Nature 266: 550-552 (1977); Koprowski et al, U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer ^*94), Ausubel, F.M. et al, Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). Generally, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0 or P3X63Ag8.653) with antibody producing cells. The antibody producing cells, preferably those obtained from the spleen or lymph nodes, can be obtained from animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select recombinant antibody from a library (e.g., a phage display library), or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see e.g., Jakobovits et al, Proc. Nat I. Acad. Sci. USA, 90: 2551- 2555 (1993); Jakobovits et al, Nature, 362: 255-258 (1993); Lonberg et al, U.S. Patent No. 5,545,806; Surani et al, U.S. Patent No. 5,545,807; Lonberg et al, WO97/13852).

In one embodiment, the antibody or antigen-binding fragment thereof has specificity for a mammalian CC chemokine receptor- 1 (CCRl), such as human CCRl . In a preferred embodiment, the antibody or antigen-binding fragment can inhibit binding of a ligand (i.e., one or more ligands) to CCRl and/or one or more functions mediated by CCRl in respond to ligand binding. Preferred antibody antagonists of CCRl function are disclosed in our co-pending United States Patent Application titled "Anti-CCRl Antibodies and Methods of Use Therefor", by Shixin Qin, Walter Newman and Nasim Kassam, Attorney's docket number LKS97-13, U.S. Serial No. 09/239,938, filed January 29, 1999, and in International Application No. PCT/US99/04527, the teachings of each of these applications are incorporated herein by reference in their entirety.

Assessment of Activity of Antagonists

The capacity of an agent (e.g., proteins, peptides, natural products, small organic molecules, peptidomimetics) to antagonize CCRl function can be determined using a suitable screen (e.g., high through-put assay). For example, an agent can be tested in an extracellular acidification assay, calcium flux assay, ligand binding assay or chemotaxis assay (see, for example, Hesselgesser et al, J. Biol. Chem. 273(25):15687-15692 (1998) and WO 98/02151).

In a particular assay, membranes can be prepared from cells which express CCRl, such as THP-1 cells (American Type Culture Collection, Manassas, VA; Accession No. TIB202). Cells can be harvested by centrifugation, washed twice with PBS (phosphate-buffered saline), and the resulting cell pellets frozen at -70 to -85°C. The frozen pellet can be thawed in ice-cold lysis buffer consisting of 5 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.5, 2 mM EDTA (ethylenediaminetetraacetic acid), 5 μg/ml each aprotinin, leupeptin, and chymostatin (protease inhibitors), and 100 μg/ml PMSF (phenyl methane sulfonyl fluoride - also a protease inhibitor), at a concentration of 1 to 5 x 10⁷ cells/ml, to achieve cell lysis. The resulting suspension can be mixed well to resuspend all of the frozen cell pellet. Nuclei and cell debris can be removed by centrifugation of 400 x g for 10 minutes at 4°C. The resulting supernatant can be transferred to a fresh tube and the membrane fragments can be collected by centrifugation at 25,000 x g for 30 minutes at 4°C. The resulting supernatant can be aspirated and the pellet can be resuspended in freezing buffer consisting of 10 mM HEPES pH 7.5, 300 mM sucrose, 1 μg/ml each aprotinin, leupeptin, and chymostatin, and 10 μg/ml PMSF (approximately 0.1 ml per each 10⁸ cells). All clumps can be resolved using a minihomogenizer, and the total protein concentration can be determined by suitable methods (e.g., Bradford assay, Lowery assay). The membrane solution can be divided into aliquots and frozen at -70 to -85°C until needed.

The membrane preparation described above can be used in a suitable binding assay. For example, membrane protein (2 to 20 μg total membrane protein) can be incubated with 0.1 to 0.2 nM ¹²⁵I-labeled RANTES or MIP-1 α with or without unlabeled competitor (RANTES or MIP-1 α) or various concentrations of compounds to be tested. ¹²⁵I-labeled RANTES and ¹²⁵I-labeled MIP-1 α can be prepared by suitable methods or purchased from commercial vendors (e.g., DuPont- NEN (Boston, MA)). The binding reactions can be performed in 60 to 100 μl of a binding buffer consisting of 10 mM HEPES pH 7.2, 1 mM CaCl₂, 5 mM MgCl₂, and 0.5% BSA (bovine serum albumin), for 60 min at room temperature. The binding reactions can be terminated by harvesting the membranes by rapid filtration through glass fiber filters (e.g., GF/B or GF/C, Packard) which can be presoaked in 0.3% polyethyleneimine. The filters can be rinsed with approximately 600 μl of binding buffer containing 0.5 M NaCl, dried, and the amount of bound radioactivity can be determined by scintillation counting. The CCRl antagonist activity of test agents (e.g., compounds) can be reported as the inhibitor concentration required for 50% inhibition (IC₅₀ values) of specific

125 125 binding in receptor binding assays (e.g., using I-RANTES or MIP-1 as ligand and THP-1 cell membranes). Specific binding is preferably defined as the total binding (e.g., total cpm on filters) minus the non-specific binding. Non-specific binding is defined as the amount of cpm still detected in the presence of excess unlabeled competitor (e.g., RANTES or MIP-1 ).

If desired, membranes prepared from cells which express recombinant CCRl can be used in the described assay. The capacity of compounds to antagonize CCRl function can also be determined in a leukocyte chemotaxis assay using suitable cells. Suitable cells include, for example, cell lines, recombinant cells or isolated cells which express CCRl and undergo CCRl ligand-induced (e.g., MIP-1 a, RANTES, MCP-2, MCP-3, MCP-4, HCC-1 or MPIF-1) chemotaxis. In one example, CCRl -expressing recombinant LI .2 cells (see Campbell, et al. J Cell Biol, 134:255-266 (1996)), peripheral blood mononuclear cells or HL60 cells differentiated with butyric acid, can be used in a modification of a transendothelial migration assay (Carr, M.W., et al T.A., Proc. Nad Acad Sci, USA, (91):3652 (1994)). Peripheral blood mononuclear cells can be isolated from whole blood by suitable methods, for example, density gradient centrifugation and positive or preferably negative selection with specific antibodies. The endothelial cells used in this assay are preferably the endothelial cell line, ECV 304, obtained from the European Collection of Animal Cell Cultures (Porton Down, Salisbury, U.K.). Endothelial cells can be cultured on 6.5 mm diameter Transwell culture inserts (Costar Corp., Cambridge, MA) with 3.0 μm pore size. Culture media for the ECV 304 cells can consist of M199+10% FCS, L-glutamine, and antibiotics. The assay media can consist of equal parts RPMI 1640 and M199 with 0.5% BSA. Two hours before the assay, 2x10 ECV 304 cells can be plated onto each insert of the 24 well Transwell chemotaxis plate and incubated at 37°C. Chemotactic factors such as RANTES or MIP-1 (Peprotech)(diluted in assay medium) can be added to the 24- well tissue culture plates in a final volume of 600 μL. Endothelial-coated Transwells can be inserted into each well and 10 cells of the leukocyte type being studied are added to the top chamber in a final volume of 100 μL of assay medium. The plate can then be incubated at 37°C in 5% CO₂/95% air for 1-2 hours. The cells that migrate to the bottom chamber during incubation can be counted, for example using flow cytometry. To count cells by flow cytometry, 500 μL of the cell suspension from the lower chamber can be placed in a tube and relative counts can be obtained for a set period of time, for example, 30 seconds. This counting method is highly reproducible and allows gating on the leukocytes and the exclusion of debris or other cell types from the analysis. Alternatively, cells can be counted with a microscope. Assays to evaluate chemotaxis inhibitors can be performed in the same way as control experiment described above, except that antagonist solutions, in assay media containing up to 1% of DMSO co-solvent, can be added to both the top and bottom chambers prior to addition of the cells. Antagonist potency can be determined by comparing the number of cell that migrate to the bottom chamber in wells which contain antagonist, to the number of cells which migrate to the bottom chamber in control wells. Control wells can contain equivalent amounts of DMSO, but no antagonist.

The activity of an antagonist of CCRl function can also be assessed by monitoring cellular responses induced by active receptor, using suitable cells expressing receptor. For instance, exocytosis (e.g., degranulation of cells leading to release of one or more enzymes or other granule components, such as esterases (e.g., serine esterases), perform, and/or granzymes), inflammatory mediator release (such as release of bioactive lipids such as leukotrienes (e.g., leukotriene C₄)), and respiratory burst, can be monitored by methods known in the art or other suitable methods (see e.g., Taub, D.D. et al, J. Immunol, 155: 3877-3888 (1995), regarding assays for release of granule-derived serine esterases; Loetscher et al, J. Immunol, 156: 322-327 (1996), regarding assays for enzyme and granzyme release; Rot, A. et al, J. Exp. Med., 176: 1489-1495 (1992) regarding respiratory burst; Bischoff, S.C. et al, Eur. J. Immunol, 23: 761-767 (1993) and Baggliolini, M. and CA. Dahinden, Immunology Today, 15: 127-133 (1994)). In one embodiment, an antagonist of CCRl is identified by monitoring the release of an enzyme upon degranulation or exocytosis by a cell capable of this function. Cells expressing CCRl can be maintained in a suitable medium under suitable conditions, and degranulation can be induced. The cells are contacted with an agent to be tested, and enzyme release can be assessed. The release of an enzyme into the medium can be detected or measured using a suitable assay, such as in an immunological assay, or biochemical assay for enzyme activity. The medium can be assayed directly, by introducing components of the assay

(e.g., substrate, co-factors, antibody) into the medium (e.g., before, simultaneous with or after the cells and agent are combined). The assay can also be performed on medium which has been separated from the cells or further processed (e.g., fractionated) prior to assay. For example, convenient assays are available for enzymes, such as serine esterases (see e.g., Taub, D.D. et al, J. Immunol, 155: 3877-3888 (1995) regarding release of granule-derived serine esterases).

In another embodiment, cells expressing CCRl are combined with a ligand of CCRl or promoter of CCRl function, an agent to be tested is added before, after or simultaneous therewith, and degranulation is assessed. Inhibition of ligand- or promoter-induced degranulation is indicative that the agent is an inhibitor of mammalian CCRl function.

In a preferred embodiment, the antagonist of CCRl function does not significantly inhibit the function of other chemokine receptors (e.g., CCR2, CXCR1, CCR3). Such CCRl -specific antagonists can be identified by suitable methods, such as by suitable modification of the methods described herein. For example, cells which do not express CCRl (CCRl^") but do express one or more other chemokine receptors (e.g., CCR2, CXCR1, CCR3) can be created or identified using suitable methods (e.g., transfection, antibody staining, western blot, RNAse protection). Such cells or cellular fractions (e.g., membranes) obtained from such cells can be used in a suitable binding assay. For example, when a cell which is CCRl^" and

CCR3" is chosen, the CCRl antagonist can be assayed for the capacity to inhibit the binding of a suitable CCR3 ligand (e.g., RANTES, MCP-3) to the cell or cellular fraction, as described herein.

In another preferred embodiment, the antagonist of CCRl function is an agent which binds to CCRl . Such CCRl -binding antagonists can be identified by suitable methods, for example, in binding assays employing a labeled (e.g., enzymatically labeled (e.g., alkaline phosphatase, horse radish peroxidase), biotinylated, radio- labeled (e.g., ³H, ¹⁴C, ¹²⁵I)) antagonist.

In another preferred embodiment, the antagonist of CCRl function is an agent which can inhibit the binding of a (i.e., one or more) CCRl ligand to CCRl (e.g., human CCRl).

In particularly preferred embodiment, the antagonist of CCRl function is an agent which can bind to CCRl and thereby inhibit the binding of a (i.e., one or more) CCRl ligand to CCRl (e.g., human CCRl).

Methods of Therapy The term "graft", as used herein, refers to organs and/or tissues which can be obtained from a first mammal or donor and transplanted into a second mammal, preferably a human. The term "graft" encompasses, for example, skin, eye or portions of the eye (e.g., cornea, retina, lens), muscle, bone marrow or cellular components of the bone marrow (e.g., stem cells, progenitor cells), heart, lung, heart- lung (e.g., heart and a single lung, heart and both lungs), liver, kidney, pancreas, parathyroid, bowel (e.g., colon, small intestine, duodenum), neuronal tissue, bone and vasculature (e.g., artery, vein). A graft can be obtain from a suitable mammal (e.g., human, pig, baboon, chimpanzee), or under certain circumstances a graft cah be produced in vitro by culturing cells, for example, embryonal cells, fetal cells, skin cells, blood cells and bone marrow cells which were obtained from a suitable mammal. The graft can be obtained from a genetically modified animal or can be modified (e.g., genetically, chemically, physically) by suitable means. A graft is preferably obtained from a human. An "allograft", as the term is used herein, refers to a graft comprising antigens which are allelic variants of the corresponding antigens found in the recipient. For example, a human graft comprising an MHC class II antigen encoded by the HLA-DRB 1*0401 allele is an allograft if transplanted into a human recipient whose genome does not comprise the HLA-DRB 1*0401 allele.

In one embodiment, the method of inhibiting (reducing or preventing) graft rejection comprises administering an effective amount of an (i.e., one or more) antagonist of CCRl function to a recipient of a graft. In another embodiment, the method of inhibiting graft rejection comprises administering an effective amount of an antagonist of CCRl function to a recipient of an allograft. In a preferred embodiment, the method comprises administering an effective amount of an antagonist of CCRl function to a recipient of a cardiac allograft. In another embodiment, the antagonist of CCRl function is selected from the group consisting of small organic molecules, natural products, peptides, peptidomimetics and proteins, wherein said proteins are not chemokines or mutants or analogues thereof.

In a preferred embodiment, the invention provides a method for inhibiting (reducing or preventing) graft rejection comprising administering to a graft recipient an effective amount of an antagonist of CCRl function and an effective amount of an (i.e., one or more) additional therapeutic agent, preferably, an immunosuppressive agent. Advantageously, the rejection- inhibiting effects of CCRl antagonists and immunosuppressive agents can be additive or synergistic, and can result in permanent engraftment.

A further benefit of co-administration of a CCRl antagonist and an immunosuppressive agent is that the dose of immunosuppressive agent required to inhibit graft rejection can be reduced to sub-therapeutic levels (e.g., a dose that does not inhibit graft rejection when administered as the sole therapeutic agent). The ability to reduce the dose of the immunosuppressive agent can greatly benefit the graft recipient as many immunosuppressive agents have severe and well-known side effects including, for example, increased incidence of infection, increased incidence of certain malignancies, diabetes mellitus, neurotoxicity, nephrotoxicity, hyperlipidemia, hypertension, hirsutism, gingival hyperplasia, impaired wound healing, lymphopenia, jaundice, anemia, alopecia and thrombocytopenia (Spencer, CM., et al, Drugs, 54(6):925-915 (1997); Physicians Desk Reference, 53^rd Edition, Medical Economics Co., pp. 2081-2082 (1999)).

The term "immunosuppressive agent", as used herein, refers to compounds which can inhibit an immune response. The immunosuppressive agent used in the invention can be a novel compound or can be selected from the compounds which are known in the art, for example, calcineurin inhibitors (e.g., cyclosporin A, FK-506), IL-2 signal transduction inhibitors (e.g., rapamycin), glucocorticoids (e.g., prednisone, dexamethosone, methylprednisolone), nucleic acid synthesis inhibitors (e.g., azathioprine, mercaptopurine, mycophenolic acid) and antibodies to lymphocytes or antigen-binding fragments thereof (e.g., OKT3, anti-IL2 receptor). Novel immunosuppressive agents can be identified by those of skill in the art by suitable methods, for example, screening compounds for the capacity to inhibit antigen-dependent T cell activation.

The immunosuppressive agent used for co-therapy (e.g., co-administration with an antagonist of CCRl function) is preferably a calcineurin inhibitor. More preferably the immunosuppressive agent used for co-therapy is cyclosporin A.

When the graft is bone marrow, cells (e.g., leukocytes) derived from the graft can mount an immune response directed at the recipient's organs and tissues. Such a condition is referred to in the art as graft versus host disease (GVHD). Administration of an antagonist of CCRl function with or without an additional therapeutic agent (e.g., immunosuppressive agent, hematopoietic growth factor) can inhibit GVHD. Accordingly, in another embodiment, the invention provides a method of inhibiting (reducing or preventing) GVHD in a bone marrow graft recipient comprising administering an effective amount of an antagonist of CCRl function. In an additional embodiment, the method of inhibiting GVHD comprises the administration of an antagonist of CCRl function and one or more additional therapeutic agents, for example, an immunosuppressive agent.

In another embodiment, the method of inhibiting GVHD comprises the administration of an antagonist of CCRl function, which is selected from the group consisting of small organic molecules, natural products, peptides, peptidomimetics and proteins, wherein said proteins are not chemokines or mutants or analogues thereof.

A further aspect of the invention related to inhibiting ischemia/reperfusion injury using CCRl antagonists. Ischemia/reperfusion injury refers to necrotic cell death that occurs when the flow of blood to an organ or tissue is restricted or stopped (ischemia), resulting in oxygen deprivation (hypoxia). The injury sustained by an organ or tissue under ischemic conditions is apparent after blood flow has been restored (reperfusion). Ischemia/reperfusion injury can be the result of a pathological condition in which ischemia can occur, for example, myocardial infarction, arteriosclerosis, stroke, transient ischemic attacks and the like. Ischemia/reperfusion injury can also result from trauma or medical procedures which stop, restrict, or redirect (e.g., shunt) the flow of blood. Examples of such trauma include, for example, frost bite, burns and pinch injuries which restrict the flow of blood to the limbs or extremities. Medical procedures which can result in ischemia/reperfusion injury include, for example, placement of a tourniquet, angioplasty (e.g., balloon angioplasty) and surgery (e.g., organ transplantation). In the context of organ transplantation, ischemia/reperfusion injury which occurs during the transplantation of all grafts, even isogenic grafts (e.g., when the donor and recipient are identical twins), can be a contributing factor to graft rejection (e.g., acute rejection, chronic rejection).

A murine model of cold renal ischemia reperfusion injury, which mimics the storage conditions of organs (e.g., kidneys) that have been removed from a donor in preparation for transplantation, is disclosed herein (see Example 3). Studies using this model revealed that inhibiting CCRl function can significantly inhibit ischemia reperfusion injury. Specifically, renal ischemia/reperfusion had no measurable effect on the renal function of CCRl KO mice with 100% of the mice surviving for the 72 hour follow-up period. In contrast, CCRl +/+ control mice experienced considerable impairment of renal function and 80% mortality at 72 hours (Table 3). Histological examination of kidneys removed for the CCRl KO and CCRl +/+ control mice 48 hours after ischemia/reperfusion revealed that inhibiting CCRl function can inhibit tubular necrosis and the infiltration of neutrophils into the kidney. Thus, by inhibiting CCRl function ischemia/reperfusion injury can be inhibited (reduced or prevented).

Accordingly, disruption of CCRl function can have significant beneficial actions in preventing injury to transplanted grafts (e.g., kidneys). For example, inhibiting CCRl function can inhibit (reduce or prevent) initial post-transplant graft injury, and thereby, lead to reduced acute and chronic allograft rejection. Furthermore, inhibiting CCRl function can enlarge the organ donor pool since many organs (e.g., kidneys) are not transplanted because they are thought to be at high risk of ischemia/reperfusion injury as a result of prolonged storage. Accordingly, another embodiment of the invention is a method for inhibiting ischemia/reperfusion injury comprising administering to a subject (e.g., a human) in need thereof an effective amount of an antagonist of CCRl function. In certain embodiments, the ischemia/reperfusion injury can be a consequence of a medical procedure, trauma or a result of a pathological condition. In a particular embodiment, the invention provides a method for inhibiting ischemia/reperfusion injury which can occur during graft transplantation. In a preferred embodiment, the invention provides a method for inhibiting ischemia/reperfusion injury which can occur during transplantation of a kidney. In another embodiment, the method of inhibiting ischemia/reperfusion injury comprises the administration of an antagonist of CCRl function, which is selected from the group consisting of small organic molecules, natural products, peptides, peptidomimetics and proteins, wherein said proteins are not chemokines or mutants or analogues thereof. In another embodiment, the method of inhibiting ischemia/reperfusion injury comprises administering to a subject in need thereof an effective amount of an antagonist of CCRl function and an effective amount of one or more additional therapeutic agents which can promote blood flow and/or inhibit leukocyte infiltration. For example, the additional agent can be selected from the group consisting of, a fibrinolytic agent (e.g., Retavase), a thrombolytic agent, such as a plasminogen activator (e.g., tissue plasminogen activator, urokinase, streptokinase, recombinant tissue plasminogen activator), an anticoagulant (e.g., a coumarin anticoagulant (e.g., warfarin, ethyledine dicoumarol), heparin, hirulog, hirudin, aspirin), a vasodilator (e.g., nitroglycerin, amotriphene, erythritol, prenylamine), an agent which stimulates or inhibits the production of nitric oxide (e.g., a stimulator or inhibitor of nitric oxide synthase, for example, the compounds disclosed in U.S. Patent Nos.: 5,811,437 issued to Singh et al, 5,854,234 issued to Hansen et al. and 5,854,251 issued to Hallinan et al), an immunosuppressive agent and a cell adhesion inhibitor. Cell adhesion inhibitors suitable for co-administration with an antagonists of CCRl function include, for example, proteins, such as, cytokines, antibodies which bind to cell adhesion molecules (e.g., integrins. selectins) and antigen-binding fragments thereof, and soluble adhesion molecules (e.g., chimeric adhesion molecules), small organic molecules, peptides and peptidomimetics, for example, the compounds disclosed in United States Patent Nos: 5,843, 441 issued to Gundel et al, 5,695,760 issued to Faanes et al, 5,843,425 issued to Tedder et al, 5,753,617 and 5,710,123, issued to Heavner et al, 5,837,689 issued to Anderson et al, 5,725,802 issued to Barrett et al, 5,510,332 issued to Kogan et al, 5,707,985 and 5,260,277 issued to McKenzie et al, International Patent Applications WO 97/03094, WO 98/04247, WO 96/22966 by Biogen Inc., WO 97/10839,

WO 96/00581 by Texas Biotechnology Corporation, WO 94/15958, WO 93/08823, WO 92/08464 by Tanabe Seiyaku Co. Ltd. The entire teaching of each of the above cited references in incorporated herein by reference.

In a preferred embodiment, the additional agent that is co-administered with the antagonist of CCRl function can be selected from the group consisting of an immunosuppressive agent and a cell adhesion inhibitor. In another preferred embodiment, the additional agent can be selected from the group consisting of a fibrinolytic agent, a thrombolytic agent, an anti-coagulant, a vasodilator and an agent which stimulates or inhibits the production of nitric oxide.

The invention further relates to an antagonist of CCRl function for use in therapy (including prophylaxis), for example, as described herein, and to the use of such an antagonist for the manufacture of a medicament for inhibiting graft rejection (e.g., acute rejection, chronic rejection) and/or ischemia/reperfusion injury as described herein. The invention also relates to a medicament for inhibiting graft rejection (e.g., acute rejection, chronic rejection) and/or ischemia/reperfusion injury wherein said medicament comprises an antagonist of CCRl function.

A "subject" is preferably a human, but can also be a mammal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, fowl, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

An effective amount of the antagonist of CCRl function can be administered to a subject to inhibit (reduce or prevent) graft rejection and/or ischemia/reperfusion injury. For example, an effective amount of the antagonist of CCRl function can be administered before, during and/or after transplant surgery or other medical procedure which can result in ischemia/reperfusion injury. When co-administration of an antagonist of CCRl function and an additional therapeutic agent is indicated or desired for inhibiting graft rejection and/or ischemia reperfusion injury, the antagonist of CCRl function can be administered before, concurrently with or after administration of the additional therapeutic agent. When the antagonist of CCRl function and additional therapeutic agent are administered at different times, they are preferably administered within a suitable time period to provide substantial overlap of the pharmacological activity (e.g., inhibition of CCRl function, immunosuppression) of the agents. The skilled artisan will be able to determine the appropriate timing for co-administration of an antagonist of CCRl function and an additional therapeutic agent depending on the particular agents selected and other factors.

An "effective amount" of a CCRl antagonist is an amount sufficient to achieve a desired therapeutic and/or prophylactic effect, such as an amount sufficient to inhibit inhibit graft rejectiona and/or ischemia/reperfusion injury. For Example, an effective amount is an amount sufficient to inhibit a (i.e., one or more) function of CCRl (e.g., CCRl ligand-induced leukocyte migration, CCRl ligand-induced integrin activation, CCRl ligand-induced transient increase in the concentration of intracellular free calcium [Ca^2*]_; and/or CCRl ligand-induced secretion (e.g. degranulation) of pro inflammatory mediators), and thereby, inhibit graft rejectiona and/or ischemia/reperfusion injury. An "effective amount" of an additional therapeutic agent (e.g., immunosuppressive agent) is an amount sufficient to achieve a desired therapeutic and/or prophylactic effect (e.g., immunosuppression).

The amount of agent (e.g., CCRl antagonist, additional therapeutic agent) administered to the individual will depend on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs as well as the degree, severity and type of rejection and/or ischemia/reperfusion injury. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Typically, an effective amount can range from about 0.1 mg per day to about 100 mg per day for an adult. Preferably, the dosage ranges from about 1 mg per day to about 100 mg per day.

The agent (e.g., CCRl antagonist, additional therapeutic agent) can be administered by any suitable route, including, for example, orally in capsules, suspensions or tablets or by parenteral administration. Parenteral administration can include, for example, intramuscular, intravenous, subcutaneous, or intraperitoneal administration. The agent (e.g., CCRl antagonist, additional therapeutic agent) can also be administered orally (e.g., dietary), transdermally, topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) or rectally. Administration can be local or systemic as indicated. The preferred mode of administration can vary depending upon the particular agent (e.g., CCRl antagonist, additional therapeutic agent) chosen, however, oral or parenteral administration is generally preferred. The agent (e.g., CCRl antagonist, additional therapeutic agent) can be administered as a neutral compound or as a salt. Salts of compounds containing an amine or other basic group can be obtained, for example, by reacting with a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like. Compounds with a quaternary ammonium group also contain a counteranion such as chloride, bromide, iodide, acetate, perchlorate and the like. Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base, for example, a hydroxide base. Salts of acidic functional groups contain a countercation such as sodium, potassium and the like. The antagonist of CCRl function can be administered to the individual as part of a pharmaceutical composition for inhibition of graft rejection and/or ischemia/reperfusion injury comprising a CCRl antagonist and a pharmaceutically acceptable carrier. Pharmaceutical compositions for co-therapy can comprise an antagonist of CCRl function and one or more additional therapeutic agents. An antagonist of CCRl function and an additional therapeutic agent can be components of separate pharmaceutical compositions which can be mixed together prior to administration or administered separately. Formulation will vary according to the route of administration selected (e.g., solution, emulsion, capsule). Suitable pharmaceutical carriers can contain inert ingredients which do not interact with the antagonist of CCRl function and/or additional therapeutic agent. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Methods for encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al, "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986).

The present invention will now be illustrated by the following Examples, which are not intended to be limiting in any way.

EXAMPLES Example 1 CCRl Targeting and Cardiac Transplantation

Experimental design

3 sets of data were generated:

(I) The tempo of acute rejection was determined in CCRl knockout (KO) vs. control mice. (ii) The effect of immunosuppression with cyclosporin A (CsA) on CCRl KO vs. control mice was determined.

(iii) The effects of CCRl deletion on development of chronic rejection was determined.

Methods Mice. CCRl KO mice (also refered to as CCRl -/-)(B6/129 strain, H-2^b), which are homozygous for a target gene disruption of CCRl, were produced by

Craig Gerard (Children's Hospital, Boston, MA; Gerard, C. et al, J. Clin. Invest.

100:2022-2021 (1997)) and were bred at LeukoSite (Cambridge, Massachusetts).

All other mice were obtained from Jackson Laboratory (Bar Harbor, ME). These included donor strains (BALB/c, H-2^d; B6.C-H2(bml2)/KhEg(bml2), H-2^b) and control recipients (B6/129). BALB/c and B6/129 differ at both class I and class II major histocompatibility complex (MHC) loci, whereas Bml2 and B6/129 differ at

MHC class II alone. Mouse cardiac allografting (Mottram, P.L. et al, Transplantation 59:559-565 (1995); Hancock, W.W., et al, Proc. Natl Acad. Sci (USA), 93: 13961- 3912 (1996)) was performed with the aid of an operating microscope (Nikon, 4x to 38x magnification) under clean conditions (rigorously sterile conditions were not required for studies involving CCRl KO or control mice, whereas they are required for use of other immunodeficient mice):

Preparation of the donor heart. Donor mice were anesthetized with Nembutal (50 mg/10 g body weight) and Atropine sulfate (0.17 mg/100 g body weight) i.p.; additional anaesthesia with Methoxyflurane supplementation was administered via a face mask as required during the procedure. Mice were shaved and cleansed with 70% alcohol. A midline abdominal incision was made in the donor animal and 1 ml of a 10% solution of heparin in saline was injected into the inferior vena cava. The incision was then extended cephalic to open the chest through a median sternotomy. The thorax was opened. The inferior vena cava was ligated with 6-0 silk and divided inferior to the tie. The superior vena cava was then similarly ligated and divided superior to the tie. The aorta and pulmonary artery were separated and divided as far distally as possible. At this point, blood was evacuated from the heart by applying pressure with applicator sticks. The aorta was transected just proximal to the brachiocephalic artery and the main pulmonary artery transected just proximal to its bifurcation. The pulmonary veins were then ligated and divided en mass and the heart placed in iced saline.

Preparation of the recipient. After being anesthetized in the same way as the donor, the recipient was brought under the microscope, a midline abdominal incision was made, and segments of the aorta and vena cava below the renal vessels were dissected free, but not separated from each other, over a length of about 2 mm. A clamp was placed on the proximal aorta and vena cava, and a distal tie of 6-0 silk was placed around both the aorta and vena cava in preparation for later occlusion of the vessels.

Transplantation of the heart. The tie that had been placed around the distal aorta and vena cava was secured by means of a single knot. An aortotomy and a venotomy in the vena cava were made adjacent to one another. The donor heart was then removed from the chilled saline, and the donor aorta and pulmonary artery were joined end-to-side to the recipient aorta and vena cava, respectively, with running suture, using 10-0 tipped with a BV-3 needle. Since the anastomoses were done adjacent to one another, the side of the pulmonary artery-cava suture line next to the aortic anastomosis was sutured from the inside with an everting running suture. During this period, chilled saline was dripped on the ischemic heart at frequent intervals. After the completion of the anastomoses, the inferior vascular occluding tie was released first, thus filling the inferior vena cava and donor pulmonary artery with recipient venous blood. Upon release of the proximal occluding tie, the aorta and coronary arteries of the transplant were perfused with oxygenated recipient blood. Blood loss was minimized by gradual release of the proximal tie. Warm saline was used externally to warm the heart immediately after establishing coronary perfusion. With warming and coronary perfusion, the heart began to fibrillate and usually within a few minutes it reverted spontaneously to a sinus rhythm. Occasionally, cardiac massage was required to re-establish a normal beat. The intestines were placed carefully back into the abdominal cavity around the auxiliary heart, and the abdomen was closed with a single running suture to all layers (saline with antibiotic was used to wash the peritoneal cavity as needed). The mouse was then placed in a constant temperature at 35°C for recovery from anesthesia.

Immunosuppression. The effect of cyclosporin A (CsA) (Sigma, St. Louis, MO) therapy (10 mg/kg/day, injected subcutaneously) was tested by using our standard cardiac allograft model of BALB/c - B6/129 or B6/129-CCR1 KO and injecting CsA daily until rejection or for a maximum of 21 days, beginning on the day of transplantation.

Monitoring of allograft survival. Cardiac allograft survival was monitored twice daily by palpation of ventricular contractions through the abdominal wall

(Mottram, P.L. et al, Transplantation, 59:559-565 (1995)), rejection was defined as the day of cessation of palpable heartbeat, and was verified by autopsy (Gerard, C, et al, J. Clin Invest., 100:2022-2021 (1997); Mottram, P.L.,et al, Transplantation, 59:559-565 (1995)). Once cardiac graft function ceased, mice were anesthetized as above, and grafts were surgically excised, subdivided into portions for (a) formalin fixation, paraffin embedding and subsequent light microscopy examination, or (b) snap-frozen in liquid nitrogen and stored at -70°C until processed for immunohistology or RNAse protection assays.

Immunopathology. For histology, paraffin sections were stained with hematoxylin and eosin (H&E) to evaluate graft morphology, and with Weigert's elastin stain so as to examine the extent of intimal proliferation in penetrating branches of myocardial arteries (a key feature of transplant arterioscelerosis). (Gerard, C, et al, J. Clin Invest. 100:2022-2021 (1997); Mottram, P.L., et al, Transplantation 59/559-565 (1995)). Chemokine and chemokine receptor mRNA expression was determined using RNAse protection assay kits (Pharmingen, San Diego, CA).

Results

Allograft survival data (mean + SD) are summarized in Table 1 (using 6-10 animals/group)

p values were determined by the Mann- Whitney U test.

The key points arising from Table 1 are:

CCR1⁺ cells contribute to the pathogenesis of allograft rejection. Disruption of CCRl function in a complete MHC mismatch significantly prolongs allograft rejection (group 1 vs. 2). As anticipated, rejection in control mice was associated with graft infiltration by CCRl^* mononuclear cells (mainly macrophages), and mRNA studies showed that rejection in control mice was associated with intragraft induction of expression of the mRNAs for the CCRl ligands, MIP-1 and RANTES. Though rejection in CCRl KO mice was accompanied by dense mononuclear cell infiltration and upregulation of CCRl ligand mRNA, allografts lacked CCRl mRNA or protein expression.

Addition of CsA (10 mg/kg/d) caused only a minor prolongation of allograft survival in control mice (2-3 days) as compared with untreated recipients (group 1 vs. 3). However, the same dose of CsA in CCRl KO mice (for a maximum of 21 days) led to permanent engraftment in all recipients (group 4, further evaluation revealed that mice of group 4 survived for >200 days following cardiac transplant). The beneficial actions of some experimental agents can be undermined by concomitant immunosuppression. However, CsA and inhibition of CCRl function are synergistic in efficacy. In addition, grafts harvested at day 100 from CCRl KO recipients showed only a minor mononuclear cell infiltrate and no evidence of transplant arteriosclerosis. These findings are in contrast to the severe arteriosclerosis observed in control allograft recipients which were treated with high dose CsA (30 mg/kg/d) or CD4 mAb therapy (Mottram, P.L., Han, W. R., et al, "Increased expression of IL-4 and IL-10 and decreased expression of IL-2 and IFN-γ in long-surviving mouse heart allografts after brief CD4-monoclonal antibody therapy," Transplantation 59:559-565 (1995); Hancock, W.W., Buelow, R., et al, "Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes," Nature Medicine 4: 1392-1396 (1998)). Disruption of CCRl function in class II-mismatch combinations is highly efficacious. Whereas untreated recipients rejected their allografts at around day 35 (group 5), disruption of CCRl function in this combination resulted in permanent engraftment (group 6). Moreover, as with group 4 (low dose CsA), allografts harvested at day 100 showed no evidence of transplant arteriosclerosis or other features of chronic rejection.

Example 2 CCRl and Chronic Rejection in Cardiac Allograft Recipients Administration of CD4 monoclonal antibody (mAb) can prolong the survival of cardiac allografts in the described murine model (Mottram et al, Transplantation 59:559-565 (1995)). However, the extended survival of grafts in anti-CD4 treated animals is complicated by the development of chronic rejection with florid transplant arteriosclerosis (Hancock et al, Nature Medicine 4: 1392-1396 (1998)).

Methods Cardiac allografts derived from Balb/c donors were transplanted into CCRl

KO or CCRl +/+ control mice as described in Example 1.

Immunosuppression. CD4 mAb (GK1.5, American Type Culture Collection, Manassas, VA; Accession No. TIB-207) was administered four times to CCRl -/- or CCRl +/+ allograft recipients (6/group); 250 μg by intraperitoneal injection on day 0 (time of transplantation) and on subsequent days 1, 2 and 3.

Monitoring of chronic rejection. Cardiac allograft survival was monitored twice daily by palpation of ventricular contractions through the abdominal wall. All grafts survived to day 60, and the readout was morphologic examination, particularly, the extent of development of transplant arteriosclerosis. Accordingly, grafts were fixed in formalin, embedded in paraffin and sections counterstained with Weigert's elastin stain. All intramyocardial arteries were scored for the extent of intimal proliferation as <5% occlusion (0), >5-20% (1), >20-40% (2), >40-60% (3), >60-80% (4) or >80-100% (5) (Murphy et al, Transplantation 64: 4-19 (1997)).

Results Results of scoring of vessels within cardiac allografts (6 grafts/group) and statistical evaluation (Mann- Whitney U test) are presented in Table 2.

Table 2 - Effect of CCRl KO on development of transplant arteriosclerosis

The key points arising from Table 2 are:

CCRl^" cells contribute to the pathogenesis of chronic allograft rejection.

Disruption of CCRl function blocks the development of transplant atherosclerosis. Disruption of CCRl function blocks the development of other features of chronic rejection.

Example 3 CCRl and Renal Ischemia/Reperfusion Injury

Prolonged interruption of blood supply to an organ will lead to its complete necrosis even if the circulation is eventually restored. Interruption for shorter periods can lead to death of specific cell types which are highly susceptible to hypoxia but the overall organ can recover to a partial or even complete extent depending upon the period of lack of blood flow (ischemia) and other factors. The damage, known as ischemia/reperfusion injury, which is apparent following restoration of blood flow to a transiently ischemic kidney or other commonly transplanted organs is primarily mediated by neutrophils, which are recruited to the organ upon revascularization. Though multiple pathways are implicated in such neutrophil recruitment, we hypothesized that the chemokine receptor, CCRl, might play a key role such that blockade of this pathway would ameliorate injury following ischemia/reperfusion. We established a new mouse model of cold renal ischemia/reperfusion injury in which maintaining the organ at 4°C during the period of ischemia mimics the storage conditions of a kidney which has been harvested in preparation for transplantation. Hence, these observations relate directly to ways in which clinical ischemia/reperfusion injury in the context or renal allografting might be decreased, or even eliminated, by blockade of the CCRl pathway.

Experimental design

Kidneys of inbred CCRl KO mice or control B6/129 were perfused in situ until pale, using cold saline, and packed in ice for varying periods prior to revascularization. After preliminary studies, we undertook 60 minutes of cold ischemia and followed-up for 48-72 hours, with analysis of renal function, histology, immunopathology (with quantitative image analysis) and mRNA analysis. Methods

Mice. CCRl KO mice (B6/129 strain, H-2^b) were produced by Craig Gerard (Children's Hospital, Boston, MA; Gerard, C. et al, J. Clin. Invest. 100:2022-2021 (1997)) and were bred at LeukoSite (Cambridge, Massachusetts). Control B6/129 mice were obtained from Jackson Laboratory (Bar Harbor, ME).

Ischemia/reperfusion model The ischemia/reperfusion procedure was performed under sterile conditions with the aid of an operating microscope (Zeiss, 2- 50x magnifications). Mice (CCRl KO or B6/129) were anesthetized with Nembutal (50 mg/100 g body weight); Methoxyflurane supplementation, administered via a facemask, was used to provide additional anesthesia, as required. After shaving and disinfecting the abdomen with 70% alcohol, the laparotomy was performed via a midline incision. After access to the right kidney's renal artery and vein, the suprarenal and infrarenal vena cava and aorta were separated, the peri-renal tissue was cut and the kidney was mobilized. To stop arterial bloodflow to the kidney infra- and suprarenal aorta were closed by application of microclips. An aortomy was then performed and the kidney was flushed with cold saline, until pale. To enable the backflow of the flushing solution in the abdomen, a small incision was performed at the renal vein, between clip and kidney after clamping the vein with a microclip. All incisions were sutured with 10-0 prolene sutures. After this procedure the kidneys were exposed for varying ischemic times of up to 75 minutes, throughout which times the kidneys were kept cold by packing them in ice. After removing the clips and reperfusion of the organ, a contralateral nephrectomy was performed. The abdomen was closed with 6-0 silk. The animals were kept post-operatively in a heated cage, under observation, until they were seen to have recovered from the procedure.

Monitoring of the effects of ischemia/reperfusion injury. Serum creatinine levels were measured daily. Since creatinine levels peaked at 48 hours post- ischemia/reperfusion, mice (8/group) were either sacrificed at 48 hours for histologic and mRNA studies of the kidneys, or were followed for 72 hours to determine effects on animal survival. For histology, kidneys were fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) and periodic acid/Schiff (PAS) stain. Chemokine and chemokine receptor mRNA expression were determined using RNAse protection assay kits (Pharmingen, San Diego, CA).

Results

The contrasting findings in control B6/129 vs. CCRl mice are shown in Table 3.

Table 3 - Effect of CCRl KO on mouse renal ischemia/reperfusion injury

p values were determined by the Mann- Whitney U test.

The key points arising from Table 3 are:

Disruption of CCRl function blocked neutrophil recruitment and completely prevented the impairment of renal function following ischemia/reperfusion injury. CCRl blockade also almost completely suppressed tubular cell necrosis.

Not shown in Table 3 are the results of mRNA analysis. Kidneys from both CCRl KO and B6/129 mice showed induction of the expression of mRNA for the CCRl -associated chemokines, MIP-1 α and RANTES. Thus, the effects of CCRl deletion left pro-inflammatory cell responses intact, but the absence of CCRl on host cells inhibited leukocyte recruitment and subsequent renal injury.

The study was expanded by increasing the number of animals in each group to 15, and extending the analysis period to 100 days. The results reported in Table 3 were included in the analysis of the expanded study. The expanded study demonstrated that CCRl-/- mice survived significantly longer after ischemia/reperfusion than B6/129 mice (92 ± 6.1 vs. 45 ± 12.4 days, mean survival time ± standard error of the mean, limited to 100 days, pO.OOl). 86% of CCR1-/- mice were alive 100 days following the ischemia/reperfusion procedure, while only 26%o of B6/129 mice survived for 100 days following the procedure (Figure 1). Measurements of serum creatinine were made 2, 7 and 100 days after the ischemia/reperfusion procedure to assess renal function. CCRl-/- mice were protected from renal injury and showed serum creatinine levels which were similar to those detected in sham animals (unilateral nephrectomy no ischemia/reperfusion). However, serum creatinine levels were significantly increased, by about 5-fold, in wild type B6/129 animals two days after ischemia reperfusion (p<0.005). Significantly elevated (4-fold) serum creatinine levels were also detected in wild type B6/129 animals seven days after ischemia/reperfusion (p<0.005). Serum creatinine was elevated in all groups, including sham animals, 100 days after the procedure (p<0.01)(Figure 2).

Example 4 CCRl and Hepatic Ischemia/Reperfusion Injury Methods

Male C57B6/129 (B6/129) mice or male CCRl-/- mice weighing 22-28 g were used. Partial hepatic ischemia was induced in mice anesthetized with sodium pentobarbital (60 mg/kg i.p.). A midline laparotomy was performed and an atraumatic clip was used to interrupt the blood supply to the middle and left lobes of the liver, thereby producing partial hepatic ischemia. After 90 minutes the clip was removed, initiating hepatic reperfusion. Sham control mice underwent the same surgical procedure without interruption of the blood supply to the middle and left lobes of the liver. Mice were sacrificed at serial intervals and hepatic tissue and blood samples were taken for analysis. Liver function was assessed by measuring serum alanine aminotransferase (SGPT) and/or serum aspartate aminotransferase (SGOT). Results

CCRl-/- mice were protected against liver dysfunction over 0.5-7 days following hepatic ischemia/reperfusion, as assessed by serum alanine aminotransferase (SGPT) (Figure 3) and serum aspartate aminotransferase (SGOT) assays. In addition, CCRl-/- mice survived significantly longer than B6/129 wild type mice following hepatic ischemia/reperfusion (13.4 ± 3.6 days compared to 8.7 ± 5.1 days, p<0.01). Immunohistological analysis of hepatic tissue demonstrated ischemia/reperfusion-induced expression of CCRl ligands in CCRl-/- and B6/129 mice. Widespread hepatic necrosis and neutrophil infiltration was observed in liver tissue removed from B6/129 mice following hepatic ischemia/reperfusion. However, only minor and focal hepatocyte injury and no neutrophil infiltration was seen in hepatic tissue of CCRl-/- mice after hepatic ischemia/reperfusion. Thus, the absence of CCRl in mice inhibited hepatic ischemia/reperfusion injury and subsequent liver dysfunction.

Example 5 Immunocompetence of CCRl-/- mice In Vitro T cell Proliferative Responses

Mixed lymphocyte responses (MLR) were assessed by culturing responder splenocytes (isolated from CCRl-/- or wild type B6/129 mice) with mitomycin-C inactivated allogeneic stimulator splenocytes (isolated from Balb/c mice) in RPMI- 1640 medium containing 5% FBS, 1% penicillin/streptomycin and 5 x 10^"5 M 2- mercaptoethanol, in 96 well flat-bottom plates. Cultures were incubated at 37°C in 5% CO₂ for 3 to 5 days and were pulsed with [³H]thymidine for 6 hours before harvesting. The amount of [³H]thymidine incorporated by the cultured cells was measured by scintillation counting. The mean amount of radioactivity incorporated (counts per minute) and standard deviations were calculated using 12 wells per group.

Mitogen-induced proliferation of T cells was measured using Concanavilin-A (Con-A, a T cell mitogen). Splenocytes isolated from CCRl-/- or wild type B6/129 mice were cultured in 96 well flat-bottom plates in RPMI-1640 medium containing 5% FBS, 1% penicillin/streptomycin, 5 x 10° M 2-mercaptoethanol and 1.25-10 μg/mL Con-A (Sigma Chemical Co., St. Louis, MO). The cultures were incubated at 37°C in 5% CO- for 72 hours and were pulsed with [³H]thymidine for 6 hours before harvesting. The amount of [³H] thymidine incorporated by the cultured cells was measured by scintillation counting. The mean amount of radioactivity incorporated (counts per minute) and standard deviation were calculated using 12 wells per group.

Results

The results of the in vitro T cell proliferation studies are presented in Figures 4 A and 4B. The mitogen-induced T cell proliferation detected in cultures of splenocytes from CCRl-/- mice was identical to that detected in cultures of splenocytes from wild type B6/129 mice (Figure 4A). CCRl-/- mice developed a vigorous response to allogeneic stimulator cells in MLR assays (p<0.001) (Figure 4B). However, the overall magnitude of the response in repeated 5-day MLR assays was consistently 20-25% lower in cultures containing responder splenocytes isolated from CCRl -/- mice in comparison to cultures containing B6/129 responder splenocytes (p<0.01). These studies of in vitro T cell responses demonstrate that CCRl-/- mice are immunocompetent.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of inhibiting graft rejection comprising administering to a subject in need thereof an effective amount of an antagonist of CCRl function.

2. The method of Claim 1 wherein said graft is an allograft.

3. The method of Claim 2 wherein said allograft is selected from the group consisting of kidney, liver, lung, heart- lung, pancreas, bowel and heart.

4. The method of Claim 3 wherein said allograft is a heart.

5. The method of Claim 1 wherein said antagonist of CCRl function is selected from the group consisting of small organic molecules, natural products, peptides, proteins and peptidomimetics.

6. The method of Claim 5 wherein said antagonist of CCRl function is a small organic molecule.

7. The method of Claim 5 wherein said antagonist of CCRl function is a natural product.

8. The method of Claim 5 wherein said antagonist of CCRl function is a peptide.

9. The method of Claim 5 wherein said antagonist of CCRl function is a peptidomimetic.

10. The method of Claim 5 wherein said antagonist of CCRl function is a protein.

11. The method of Claim 10 wherein said protein is an anti-CCRl antibody or antigen-binding fragment thereof.

12. A method of inhibiting graft rejection comprising administering to a subject in need thereof an effective amount of an antagonist of CCRl function and an immunosuppressive agent.

13. The method of Claim 12 wherein said immunosuppressive agent is one or more agents selected from the group consisting of calcineurin inhibitors, glucocorticoids, nucleic acid synthesis inhibitors, and antibodies which bind to lymphocytes or antigen-binding fragments thereof.

14. The method of Claim 13 wherein said immunosuppressive agent is a calcineurin inhibitor.

15. The method of Claim 14 wherein said calcineurin inhibitor is cyclosporin A.

16. The method of Claim 14 wherein said calcineurin inhibitor is FK-506.

17. The method of Claim 13 wherein said immunosuppressive agent is a glucocorticoid.

18. The method of Claim 17 wherein said glucocorticoid is prednisone or methy lpredniso lone .

19. A method of inhibiting graft versus host disease comprising administering an effective amount of an antagonist of CCRl function to a recipient of a transplanted graft.

20. The method of Claim 19 wherein said graft is bone marrow.

21. The method of Claim 20 further comprising administering an immunosuppressive agent.

22. The method of Claim 21 wherein said immunosuppressive agent is a calcineurin inhibitor.

23. The method of Claim 22 wherein said calcineurin inhibitor is cyclosporin A or FK-506.

24. A method of inhibiting ischemia/reperfusion injury comprising administering to a subject in need thereof an effective amount of an antagonist of CCRl function.

25. The method of Claim 24 wherein said antagonist of CCRl function is selected from the group consisting of small organic molecules, natural products, peptides, proteins and peptidomimetics.

26. The method of Claim 25 wherein said antagonist of CCRl function is a small organic molecule.

27. The method of Claim 25 wherein said antagonist of CCRl function is a natural product.

28. The method of Claim 25 wherein said antagonist of CCRl function is a peptide.

29. The method of Claim 25 wherein said antagonist of CCRl function is a peptidomimetic.

30. The method of Claim 25 wherein said antagonist of CCRl function is a protein.

31. The method of Claim 30 wherein said protein is an anti-CCRl antibody or antigen-binding fragment thereof.

32. The method of Claim 25 wherein said ischemia/reperfusion injury is a consequence of a medical procedure which stops, restricts or redirects the flow ofblood.

33. The method of Claim 32 wherein said medical procedure is angioplasty or surgery.

34. The method of Claim 32 wherein said medical procedure is surgery.

35. The method of Claim 34 wherein said surgery is graft transplantation.

36. The method of Claim 35 wherein said graft is selected from the group consisting of kidney, lung, liver, heart-lung, pancreas, bowel and heart.

37. The method of Claim 36 wherein said graft is a kidney.

38. The method of Claim 24 wherein said ischemia/reperfusion injury is the result of a pathological condition selected from the group consisting of arteriosclerosis, myocardial infarction, stroke and transient ischemic attack.

39. The method of Claim 38 wherein said pathological condition is myocardial infarction.

40. The method of Claim 38 wherein said pathological condition is stroke.

41. The method of Claim 38 wherein said pathological condition is arteriosclerosis.

42. The method of Claim 24 further comprising administering to said subject an effective amount of one or more additional therapeutic agents selected from the group consisting of fibrinolytic agents, thrombolytic agents, anticoagulants, cell adhesion inhibitors, anti-thrombotic agents, stimulators of nitric oxide synthase and inhibitors of nitric oxide synthase.

43. The method of Claim 42 wherein said additional therapeutic agent is a fibrinolytic agent.

44. The method of Claim 42 wherein said additional therapeutic agent is a thrombolytic agent.

45. The method of Claim 42 wherein said additional therapeutic agent is an anticoagulant.

46. The method of Claim 42 wherein said additional therapeutic agent is a cell adhesion inhibitor.

47. The method of Claim 42 wherein said additional therapeutic agent is an anti- thrombotic agent.

48. The method of Claim 42 wherein said additional therapeutic agent is an activator or inhibitor of nitric oxide synthase.