Antibodies that bind CCX-CKR2 are described.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of identifying an agent that binds to CCX-CKR2 on a cell. In some embodiments, the method comprises contacting a plurality of agents to a CCX-CKR2 polypeptide comprising an extracellular domain at least 95% identical to an extracellular domain of SEQ ID NO:2, or a SDF1 or I-TAC-binding fragment thereof; and selecting an agent that competes with I-TAC or SDF1 for binding to the CCX-CKR2 polypeptide or fragment thereof, thereby identifying an agent that binds to CCX-CKR2 on a cell.

In some embodiments, the cell is a cancer cell. In some embodiments, the method further comprises testing the selected agent for the ability to bind to, or inhibit growth of, a cell. In some embodiments, the cell is a cancer cell.

In some embodiments, the method further comprises testing the selected agent for the ability to alter kidney function. In some embodiments, the method further comprises testing the selected agent for the ability to alter brain or neuronal function. In some embodiments, the method further comprises testing the selected agent for the ability to change cell adhesion to endothelial cells.

In some embodiments, the agent is less than 1,500 daltons. In some embodiments, the agent is an antibody. In some embodiments, the CCX-CKR2 polypeptide comprises the sequence displayed in SEQ ID NO:2.

The present invention also provides methods for determining the presence or absence of a cancer cell. In some embodiments, the methods comprise contacting a sample comprising a cell with an agent that specifically binds with SEQ ID NO:2; and detecting binding of the agent to a polypeptide in the sample, wherein binding of the agent to the sample indicates the presence of a cancer cell.

In some embodiments, the agent is an antibody. In some embodiments, the agent is less than 1500 daltons. In some embodiments, the polypeptide detected is SEQ ID NO:2. In some embodiments, the sample is from a human. In some embodiments, the method is used to diagnose cancer in a human. In some embodiments, the method is used to provide a prognosis of cancer in a human. In some embodiments, the cancer is selected from the group consisting of cervical cancer, breast cancer, lymphoma, glioblastomas, prostate cancer, and leukemia. In some embodiments, the cancer is not Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associated primary effusion lymphoma. In some embodiments, the antibody competes with SDF1 and I-TAC for binding to SEQ ID NO:2.

The present invention also provides methods of providing a diagnosis or prognosis of an individual having cancer. In some embodiments, the methods comprise detecting the presence or absence of expression of a polynucleotide encoding a CCX-CKR2 polypeptide in a cell of an individual, wherein the CCX-CKR2 polypeptide binds I-TAC and/or SDF1 and the CCX-CKR2 polypeptide is at least 95% identical to SEQ ID NO:2, thereby diagnosing a cancer in the individual.

In some embodiments, the CCX-CKR2 polypeptide is displayed in SEQ ID NO:2. In some embodiments, the cancer is selected from the group consisting of cervical cancer, breast cancer, lymphoma, glioblastomas, prostate cancer, and leukemia. In some embodiments, the cancer is not Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associated primary effusion lymphoma.

The present invention also provides antibodies that specifically competes with SDF-1 and I-TAC for binding to SEQ ID NO:2. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody.

The present invention also provides methods comprising contacting a cell with an agent that specifically binds to SEQ ID NO:2, wherein the agent competes with SDF-1 and I-TAC for binding to a CCX-CKR2 polypeptide, and wherein the cell expresses a CCX-CKR2 polypeptide comprising an extracellular domain at least 95% identical to an extracellular domain of SEQ ID NO:2, thereby binding the agent to the CCX-CKR2 polypeptide on the cell.

In some embodiments, the agent is less than 1,500 daltons. In some embodiments, the agent is an antibody. In some embodiments, the CCX-CKR2 polypeptide is as displayed in SEQ ID NO:2. In some embodiments, the agent is identified by a method comprising contacting a plurality of agents to a CCX-CKR2 polypeptide comprising an extracellular domain at least 95% identical to an extracellular domain of SEQ ID NO:2, or a SDF1 or I-TAC-binding fragment thereof; and selecting an agent that competes with I-TAC or SDF-1 for binding to the CCX-CKR2 polypeptide or fragment thereof, thereby identifying an agent that binds to a cancer cell.

The present invention also provides methods of treating cancer in an individual. In some embodiments, the methods comprise administering to the individual a therapeutically effective amount of an agent that competes with SDF1 and I-TAC for binding to SEQ ID NO:2. In some embodiments, the agent is less than 1,500 daltons. In some embodiments, the agent is an antibody. In some embodiments, the agent is identified by a method comprising contacting a plurality of agents to a CCX-CKR2 polypeptide comprising an extracellular domain at least 95% identical to an extracellular domain of SEQ ID NO:2, or a SDF1 or I-TAC-binding fragment thereof; and selecting an agent that competes with I-TAC or SDF-1 for binding to the CCX-CKR2 polypeptide or fragment thereof, thereby identifying an agent that binds to a cancer cell. In some embodiments, the cancer is selected from the group consisting of cervical cancer, breast cancer, lymphoma, glioblastomas, prostate cancer, and leukemia. In some embodiments, the cancer is not Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associated primary effusion lymphoma.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides the discovery that the orphan receptor RDC1, referred to herein as CCX-CKR2, binds the chemokine ligands SDF1 and I-TAC. Moreover, the present invention provides the surprising discovery of CCX-CKR2's involvement in cancer. Thus, the invention provides methods of diagnosing cancer by detecting CCX-CKR2. The invention also provides methods of inhibiting cancer by administering a modulator of CCX-CKR2 to an individual with cancer.

II. CCX-CKR2 Polypeptides and Polynucleotides

In numerous embodiments of the present invention, nucleic acids encoding CCX-CKR2 polypeptides of interest will be isolated and cloned using recombinant methods. Such embodiments are used, e.g., to isolate CCX-CKR2 polynucleotides (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9)) for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from a CCX-CKR2 polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10)), to monitor CCX-CKR2 gene expression, for the isolation or detection of CCX-CKR2 sequences in different species, for diagnostic purposes in a patient, e.g., to detect mutations in CCX-CKR2 or to detect expression of CCX-CKR2 nucleic acids or CCX-CKR2 polypeptides. In some embodiments, the sequences encoding CCX-CKR2 are operably linked to a heterologous promoter. In some embodiments, the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a mouse, a rat, a dog, etc.

In some cases, the CCX-CKR2 polypeptides of the invention comprise the extracellular amino acids of the human CCX-CKR2 sequence (e.g., of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10)) while other residues are either altered or absent. In other embodiments, the CCX-CKR2 polypeptides comprise ligand-binding fragments of CCX-CKR2. For example, in some cases, the fragments bind I-TAC and/or SDF1. The structure of seven trans-membrane receptors (of which CCX-CKR2 is one) are well known to those skilled in the art and therefore trans-membrane domains can be readily determined. For example, readily available hydrophobicity algorithms can be found on the internet at the G Protein-Coupled Receptor Data Base (GPCRDB), e.g., http://www.gpcr.org/7tm/seq/DR/RDC1_HUMAN.TABDR.html or http://www.gpcr.org/7tm/seq/vis/swac/P25106.html.

This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

Appropriate primers and probes for identifying the genes encoding CCX-CKR2 from mammalian tissues can be derived from the sequences provided herein (e.g., SEQ ID NO: 1). For a general overview of PCR, see, Innis et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990).

III. Development of Specific Therapeutics

Molecules that bind to CCX-CKR2, including modulators of CCX-CKR2 function, i.e. agonists or antagonists or agents of CCX-CKR2 activity, are useful for treating a number of mammalian diseases, including cancer.

Diseases or conditions of humans or other species which can be treated with antagonists of a chemokine receptor or other inhibitors of chemokine receptor function, include, but are not limited to, e.g., carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer, cancer of the gallbladder, cancer of the small intestine, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma (see, CANCER: PRINCIPLES AND PRACTICE (DeVita, V. T. et al. eds 1997) for additional cancers); as well as brain and neuronal dysfunction, such as Alzheimer's disease and multiple sclerosis; kidney dysfunction; rheumatoid arthritis; cardiac allograft rejection; atherosclerosis; asthma; glomerulonephritis; contact dermatitis; inflammatory bowel disease; colitis; psoriasis; reperfusion injury; as well as other disorders and diseases described herein.

Alternatively, an agonist of CCX-CKR2 can be used to treat disease, e.g., in renal, brain or neuronal dysfunction as well as in cases where stem cell mobilization is therapeutic.

A. Methods of Identifying Modulators of Chemokine Receptors

A number of different screening protocols can be utilized to identify agents that modulate the level of activity or function of CCX-CKR2 in cells, particularly in mammalian cells, and especially in human cells. In general terms, the screening methods involve screening a plurality of agents to identify an agent that interacts with CCX-CKR2 (or an extracellular domain thereof), for example, by binding to CCX-CKR2, preventing a ligand (e.g., I-TAC and/or SDF1) from binding to CCX-CKR2 or activating CCX-CKR2. In some embodiments, an agent binds CCX-CKR2 with at least about 1.5, 2, 3, 4, 5, 10, 20, 50, 100, 300, 500, or 1000 times the affinity of the agent for another protein.

1. Chemokine Receptor Binding Assays

In some embodiments, CCX-CKR2 modulators are identified by screening for molecules that compete with a ligand of CCX-CKR2 such as SDF1 or I-TAC. Those of skill in the art will recognize that there are a number of ways to perform competition analyses. In some embodiments, samples with CCX-CKR2 are pre-incubated with a labeled CCX-CKR2 ligand and then contacted with a potential competitor molecule. Alteration (e.g., a decrease) of the quantity of ligand bound to CCX-CKR2 indicates that the molecule is a potential CCX-CKR2 modulator.

Preliminary screens can be conducted by screening for agents capable of binding to a CCX-CKR2, as at least some of the agents so identified are likely chemokine receptor modulators. The binding assays usually involve contacting CCX-CKR2 with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, immunohistochemical binding assays, flow cytometry, radioligand binding, europium labeled ligand binding, biotin labeled ligand binding or other assays which maintain the conformation of CCX-CKR2. The chemokine receptor utilized in such assays can be naturally expressed, cloned or synthesized.

2. Cells and Reagents

The screening methods of the invention can be performed as in vitro or cell-based assays. In vitro assays are performed for example, using membrane fractions or whole cells comprising CCX-CKR2. Cell based assays can be performed in any cells in which CCX-CKR2 is expressed.

Cell-based assays involve whole cells or cell fractions containing CCX-CKR2 to screen for agent binding or modulation of activity of CCX-CKR2 by the agent. Exemplary cell types that can be used according to the methods of the invention include, e.g., any mammalian cells including leukocytes such as neutrophils, monocytes, macrophages, eosinophils, basophils, mast cells, and lymphocytes, such as T cells and B cells, leukemias, Burkitt's lymphomas, tumor cells, endothelial cells, fibroblasts, cardiac cells, muscle cells, breast tumor cells, ovarian cancer carcinomas, cervical carcinomas, glioblastomas, liver cells, kidney cells, and neuronal cells, as well as fungal cells, including yeast. Cells can be primary cells or tumor cells or other types of immortal cell lines. Of course, CCX-CKR2 can be expressed in cells that do not express an endogenous version of CCX-CKR2.

In some cases, fragments of CCX-CKR2, as well as protein fusions, can be used for screening. When molecules that compete for binding with CCX-CKR2 ligands are desired, the CCX-CKR2 fragments used are fragments capable of binding the ligands (e.g., capable of binding I-TAC or SDF1). Alternatively, any fragment of CCX-CKR2 can be used as a target to identify molecules that bind CCX-CKR2. CCX-CKR2 fragments can include any fragment of, e.g., at least 20, 30, 40, 50 amino acids up to a protein containing all but one amino acid of CCX-CKR2. Typically, ligand-binding fragments will comprise transmembrane regions and/or most or all of the extracellular domains of CCX-CKR2.

3. Signaling Activity

In some embodiments, signaling triggered by CCX-CKR2 activation is used to identify CCX-CKR2 modulators. Signaling activity of chemokine receptors can be determined in many ways. For example, signaling can be determined by detecting chemokine receptor-mediated cell adhesion. Interactions between chemokines and chemokine receptors can lead to rapid adhesion through the modification of integrin affinity and avidity. See, e.g., Laudanna, Immunological Reviews 186:37-46 (2002).

Signaling can also be measured by determining, qualitatively and quantitatively, whether a modulator can induce calcium mobilization in a cell. Calcium mobilization assays are described in, e.g., Dairaghi et al., J. Biol. Chem. 272 (45): 28206-9 (1997). Other secondary messengers, such as cyclic AMP or inositol phosphates, as well as phosphorylation or dephosphorylation events can also be monitored. See, e.g., Premack, et al. Nature Medicine 2: 1174-1178 (1996) and Bokoch, Blood 86:1649-1660 (1995).

In addition, downstream molecular events can also be monitored to determine signaling activity. Downstream events include those activities or manifestations that occur as a result of stimulation of a chemokine receptor. Exemplary downstream events include, e.g., changed state of a cell (e.g., from normal to cancer cell or from cancer cell to non-cancerous cell). Cell responses include adhesion of cells (e.g., to endothelial cells).

4. Validation

Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Preferably such studies are conducted with suitable animal models. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a disease model for humans and then determining if the disease (e.g., cancer) is in fact modulated and/or the disease or condition is ameliorated. The animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice, rats and zebrafish.

B. Agents that Interact with CCX-CKR2

The agents tested as modulators of CCX-CKR2 can be any small chemical compound, or a biological entity, such as a polypeptide, sugar, nucleic acid or lipid. Alternatively, modulators can be genetically altered versions, or peptidomimetic versions, of a chemokine or other ligand. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.

In some embodiments, the agents have a molecular weight of less than 1,500 daltons, and in some cases less than 1,000, 800, 600, 500, or 400 daltons. The relatively small size of the agents can be desirable because smaller molecules have a higher likelihood of having physiochemical properties compatible with good pharmacokinetic characteristics, including oral absorption than agents with higher molecular weight. For example, agents less likely to be successful as drugs based on permeability and solubility were described by Lipinski et al. as follows: having more than 5H-bond donors (expressed as the sum of OHs and NHs); having a molecular weight over 500; having a LogP over 5 (or MLogP over 4.15); and/or having more than 10H-bond acceptors (expressed as the sum of Ns and Os). See, e.g., Lipinski et al. Adv Drug Delivery Res 23:3-25 (1997). Compound classes that are substrates for biological transporters are typically exceptions to the rule.

In one embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks." For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

CCX7923 (see, FIG. 4 (see Original Patent)) is commercially available and can be made by the condensation of N-[3-(dimethylamino)propyl]-N,N-dimethyl-1,3-propanediamine with bromomethyl-bicyclo(2,2,1)hept-2-ene by methods known in the art. CCX0803 (see, FIG. 4) is commercially available and can be made by condensation of 3-(2-bromoethyl)-5-phenylmethoxy-indole and 2,4,6-triphenylpyridine by methods well known in the art. See, e.g., Organic Function Group Preparations, 2nd Ed. Vol. 1, (S. R. Sandler & W. Karo 1983); Handbook of Heterocyclic Chemistry (A. R. Katritzky, 1985); Encyclopedia of Chemical Technology, 4th Ed. (J. I. Kroschwitz, 1996).

In one embodiment, the active compounds (i.e., CCX-CKR2 modulators) of the present invention have the general structure (I) -- see Original Patent.

The wavy bond connecting the olefin to the substituted phenyl ring signifies that the ring may be either cis or trans to R.sup.6. In a preferred embodiment, n is 1, 2, or 3. In another preferred embodiment, n is 2 or 3. In a further preferred embodiment, n is 3.

In another embodiment, preferred compounds have the general structure (I), where R.sup.6 is hydrogen. In a further embodiment, preferred compounds have the general structure (I), where R.sup.6 is methyl.

In another embodiment, preferred compounds have the general structure (I), where R.sup.3, R.sup.4, and R.sup.5 are independently hydrogen, hydroxy, alkyl, alkoxy, aryloxy, and halo substituted alkyl. More preferably, R.sup.3, R.sup.4, and R.sup.5 are independently alkoxy or hydrogen. In another embodiment, preferred compounds have the general structure (I), where R.sup.4 is hydrogen and R.sup.3 and R.sup.5 are alkoxy (--OR), including trifluoroalkoxy groups such as trifluoromethoxy and (--OCH.sub.2CF.sub.3). In a further embodiment, R.sup.3 is hydrogen and R.sup.4 and R.sup.5 are alkoxy. In either of these embodiments, the alkoxy group may be methoxy (--OCH.sub.3) or ethoxy (--OCH.sub.2CH.sub.3).

In another embodiment, preferred compounds have the general structure (I), where R.sup.4 and R.sup.5 together form a heterocyclic, aryl, or heteroaryl ring. In another preferred embodiment, R.sup.3 is hydrogen and R.sup.4 and R.sup.5 together are --O(CH.sub.2).sub.3O--, --(CH).sub.4--, or --N(CH).sub.2N--.

In another embodiment, preferred compounds have the general structure (I), where Z is nitrogen and Z in combination with R.sup.1 and R.sup.2 form a heteroaryl or heterocyclic group. In a preferred embodiment, compounds have the general structure (I), where Z is CH and Z in combination with R.sup.1 and R.sup.2 form a heteroaryl or heterocyclic group. More preferable compounds have the general structure (I), where Z is CH and Z in combination with R.sup.1 and R.sup.2 form a heterocyclic group containing nitrogen. In a further embodiment, Z in combination with R.sup.1 and R.sup.2 form a substituted or unsubstituted morpholinyl, pyrrolidinyl, piperidinyl, or piperazinyl group.

Preferred substituents for the heteroaryl or heterocyclic group include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, alkoxy, hydroxy, heteroatoms, and halides. In an especially preferred embodiment, the heteroaryl or heterocyclic group is substituted with benzyl, phenyl, methyl, ethyl, cyclohexyl, methoxy-methyl (--CH.sub.2OCH.sub.3), or cyclohexyl-methyl (--CH.sub.2(C.sub.6H.sub.11)) groups.

In one embodiment, a preferred compound has the general structure (I), where Z in combination with R.sup.1 and R.sup.2 is an alkyl- or methoxy-methyl-substituted pyrrolidinyl group; a benzyl-, phenyl-, methyl-, ethyl-, or substituted heteroatom substituted piperidinyl group; or a benzyl-, phenyl-, or sulfonyl-substituted piperazinyl group. Especially preferred substituted heteroatom groups include alkoxy, aminyl, cycloalkyl aminyl, alkyl aminyl, cyclopropyl aminyl, isopropyl aminyl, benzyl aminyl, and phenoxy. Preferably, the substituted heteroatom is at the 3 position of the piperidinyl ring.

In another aspect, preferred compounds have the general structure (I), where Z in combination with R.sup.1 and R.sup.2 is -- see Original Patent.

Preferred compounds having the general structure (I) can also have Z as a nitrogen atom, have R.sup.1 and R.sup.2 each as alkyl or methyl groups, or have R.sup.1 and R.sup.2 together forming --C(C(O)N(CH.sub.3).sub.2)(CH.sub.2).sub.3--.

In another embodiment, Z in combination with R.sup.1 and R.sup.2 form a 5-membered ring including nitrogen and optionally including one or more additional heteroatoms. In this embodiment, n is preferably I and Z is preferably --CH--. In an especially preferred embodiment of this type, Z in combination with R.sup.1 and R.sup.2 is -- see Original Patent.

In another preferred embodiment R.sup.7 can be a halogenated benzyl or phenyl group. In a further embodiment, R.sup.7 is preferably hydrogen, methyl, ethyl, benzyl, or para-fluoro-phenyl.

In another embodiment, the active compounds of the present invention have the general structure (II) -- see Original Patent.

As in structure (I) above, the wavy bond connecting the olefin to the substituted phenyl ring signifies that the ring may be either cis or trans.

In another embodiment, preferred compounds may have the general structure (II), where n is 3. In another embodiment, preferred compounds may have the general structure (II), where R.sup.3, R.sup.4, and R.sup.5 are substituted as described for structure (I) above. At present, especially preferred compounds have the general structure (II), where R.sup.3, R.sup.4, and R.sup.5 are alkoxy or methoxy.

While many synthetic routes known to those of ordinary skill in the art may be used to synthesize the active compounds of the present invention, a general synthesis method is given below in Scheme I -- see Original Patent.

In Scheme I, aldehyde (2) undergoes a condensation reaction with primary amine (3) via reductive amination. Suitable primary amines are commercially available from Aldrich, Milwaukee, Wis., for example, or may be synthesized by chemical routes known to those of ordinary skill in the art.

The amination reaction may be carried out with a reducing agent in any suitable solvent, including, but not limited to tetrahydrofuran (THF), dichloromethane, or methanol to form the intermediate (4). Suitable reducing agents for the condensation reaction include, but are not limited to, sodium cyanoborohydride (as described in Mattson, et al., J. Org. Chem. 1990, 55, 2552 and Barney, et al., Tetrahedron Lett. 1990, 31, 5547); sodium triacethoxyborohydride (as described in Abdel-Magid, et al., Tetrahedron Lett. 31:5595 (1990)); sodium borohydride (as described in Gribble; Nutaitis Synthesis. 709 (1987)); iron pentacarbonyl and alcoholic KOH (as described in Watabane, et al., Tetrahedron Lett. 1879 (1974)); and BH.sub.3-pyridine (as described in Pelter, et al., J. Chem. Soc., Perkin Trans. 1:717 (1984)).

The transformation of intermediate (4) to compound (5) may be carried out in any suitable solvent, such as tetrahydrofuran or dichloromethane, with a suitably substituted acyl chloride in presence of a base. Tertiary amine bases are preferred. Especially preferred bases include triethylamine and Hunnings base.

Alternatively, the transformation of intermediate (4) to compound (5) can also be obtained with a suitable coupling reagent, such as 1-ethyl-3-(3-dimethylbutylpropyl) carbodiimide or Dicyclohexyl-carbodiimide (as described in B. Neises and W. Steglich, Angew. Chem., Int. Ed. Engl. 17:522 (1978)), in the presence of a catalyst, such as 4-N,N-dimethylamino-pyridine, or in the presence of hydroxybenzotriazole (as described in K. Horiki, Synth. Commun. 7:251).

To demonstrate that the compounds described above are useful antagonists for SDF-1 and I-TAC chemokines, the compounds were screened in vitro to determine their ability to displace SDF-1 and I-TAC from the CCX-CKR2 receptor at multiple concentrations. The compounds were combined with mammary gland cells expressing CCX-CKR2 receptor sites in the presence of the .sup.125I-labeled SDF-1 and/or .sup.125I I-TAC chemokine. The ability of the compounds to displace the labeled SDF-1 or I-TAC from the CCX-CKR2 receptor cites at multiple concentrations was then determined with the screening process.

Compounds that were deemed effective SDF-1 and I-TAC antagonists were able to displace at least 50% of the SDF-1 and/or I-TAC chemokine from the CCX-CKR2 receptor at concentrations at or below 1.1 micromolar (.mu.M) and more preferably at concentrations at or below 300 nanomolar (nM). In some cases, it is desirable that compounds can displace at least 50% of the SDF-1 and/or I-TAC from the CCX-CKR2 receptor at concentrations at or below 200 nM. Exemplary compounds that met these criteria are reproduced in Table I below -- see Original Patent.

C. Solid Phase and Soluble High Throughput Assays

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.

The invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the function or activity of CCX-CKR2. Control reactions that measure CCX-CKR2 activity of the cell in a reaction that does not include a potential modulator are optional, as the assays are highly uniform. Such optional control reactions, however, increase the reliability of the assay.

In some assays it will be desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. First, a known activator or ligand of CCX-CKR2 can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased activity of CCX-CKR2 (e.g., as determined according to the methods herein). Second, an inhibitor or antagonist of CCX-CKR2 can be added, and the resulting decrease in signal for the activity of the chemokine receptor can be similarly detected. It will be appreciated that modulators can also be combined with activators or inhibitors to find modulators which inhibit the increase or decrease that is otherwise caused by the presence of the known modulator of CCX-CKR2.

IV. Diagnosis and Prognosis

The present invention provides methods of detecting a cancer cell, including methods of providing a prognosis or diagnosis of cancer. As demonstrated herein, CCX-CKR2 is expressed in nearly every cancer cell tested to date, whereas normal (non-cancer) expression of CCX-CKR2 appears to be limited to the kidney and some brain cells as well as in certain developmental stages of fetal liver. Therefore, expression of CCX-CKR2 in a cell, and in particular, in a non-fetal cell and/or a cell other than a kidney or brain cell, indicates the likely presence of a cancer cell. In some cases, samples containing CCX-CKR2-expressing cells are confirmed for the presence of cancer cells using other methods known in the art.

According to yet another aspect of the invention, methods for selecting a course of treatment of a subject having or suspected of having cancer are provided. The methods include obtaining from the subject a biological sample, contacting the sample with antibodies or antigen-binding fragments thereof that bind specifically to CCX-CKR2, detecting the presence or absence of antibody binding, and selecting a course of treatment appropriate to the cancer of the subject. In some embodiments, the treatment is administering CCX-CKR2 antagonists to the subject.

Detection methods using agents that bind a protein are well known and include, e.g., various immunoassays, flow cytometry, etc. Using flow cytometry, cells expressing a specific antigen of interest within a mixed population of cells can be identified. Briefly, cells are permitted to react with an antibody specific for the protein of interest (e.g., CCX-CKR2). The antibody can either be fluorescently labeled (direct method of staining), or if it is not labeled, a second antibody that reacts with the first can be fluorescently tagged (indirect method of staining). Cells are then passed through an instrument that can detect the fluorescent signal. Cells are aspirated and made into a single cell suspension. This cell suspension is passed by a laser that excites the fluorochrome labeled antibody now binding to the cells and acquires this data. Cells that are found to be bright (i.e. react with the fluorescently labeled antibody) express the protein of interest; cells that are dull (i.e. do not react with the fluorescently labeled antibody) do not express the protein of interest.

The present invention provides for methods of diagnosing human diseases including, but not limited to cancer, e.g., carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer, cancer of the gallbladder, cancer of the small intestine, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma (see, CANCER: PRINCIPLES AND PRACTICE (DeVita, V. T. et al. eds 1997) for additional cancers); as well as brain and neuronal dysfunction, such as Alzheimer's disease and multiple sclerosis; kidney dysfunction; rheumatoid arthritis; cardiac allograft rejection; atherosclerosis; asthma; glomerulonephritis; contact dermatitis; inflammatory bowel disease; colitis; psoriasis; reperfusion injury; as well as other disorders and diseases described herein. In some embodiments, the subject does not have Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associated primary effusion lymphoma. As provided herein, including in the examples, normal and diseased cells and tissues can be distinguished based on reactivity to an anti-CCX-CKR2 monoclonal antibody or SDF-1 and I-TAC. For example, cancer cells are detected by detecting on a cell a chemokine receptor for which SDF-1.alpha. and I-TAC compete for binding.

In addition, differences in ligand binding between chemokine receptors can be detected and such differences can be used to detect cells expressing CCX-CKR2. For example, no other chemokine receptor has both SDF1 and I-TAC as ligands. Chemokine binding can be determined using tissue samples (e.g., biopsies) or can be monitored directly in a tissue in situ (e.g., using radiolabelled chemokine imaging).

Immunoassays can also be used to qualitatively or quantitatively analyze CCX-CKR2. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988). Alternatively, non-antibody molecules with affinity for CCX-CKR2 can also be used to detect the receptor.

Methods for producing polyclonal and monoclonal antibodies that react specifically with a protein of interest are known to those of skill in the art (see, e.g. Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler and Milstein Nature, 256:495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors. For example, in order to produce antisera for use in an immunoassay, the protein of interest or an antigenic fragment thereof, is isolated as described herein. For example, a recombinant protein is produced in a transformed cell line. An inbred strain of mice, rats, guinea pigs or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used as an immunogen. A further option is to use a cell expressing the protein or a membrane fraction or liposome comprising CCX-CKR2 or a fragment thereof as an antigen. Antibodies raised against the cell, membrane fraction or liposome can then be selected for their ability to bind to the protein.

Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 10.sup.4 or greater are selected and tested for their crossreactivity against a different, and sometimes, homologous proteins, using a competitive binding immunoassay. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K.sub.D of at least about 0.1 mM, more usually at least about 1 .mu.M, preferably at least about 0.1 .mu.M or better, and most preferably, 0.01 .mu.M or better to CCX-CKR2.

For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO 92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are well known in the art. Such antibodies are useful for both detection and therapeutic applications. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

V. Methods of Treatment, Administration and Pharmaceutical Compositions

Modulators of CCX-CKR2 (e.g., antagonists or agonists) can be administered directly to the mammalian subject for modulation of chemokine receptor signaling in vivo. In some embodiments, the modulators compete with SDF1 and/or I-TAC for binding to CCX-CKR2. Modulation of CCX-CKR2 can include, e.g., antibodies (including monoclonal, humanized or other types of binding proteins that are known in the art), small organic molecules, siRNAs, etc.

In some embodiments, the CCX-CKR2 modulators are administered to a subject having cancer. In some cases, CCX-CKR2 modulators are administered to treat cancer, e.g., carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer, cancer of the gallbladder, cancer of the small intestine, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma (see, CANCER: PRINCIPLES AND PRACTICE (DeVita, V. T. et al. eds 1997) for additional cancers); as well as brain and neuronal dysfunction, such as Alzheimer's disease and multiple sclerosis; kidney dysfunction; rheumatoid arthritis; cardiac allograft rejection; atherosclerosis; asthma; glomerulonephritis; contact dermatitis; inflammatory bowel disease; colitis; psoriasis; reperfusion injury; as well as other disorders and diseases described herein. In some embodiments, the subject does not have Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associated primary effusion lymphoma. Since CCX-CKR2 if often expressed in cancer cells but not non-cancer cells, it is typically desirable to administer antagonists of CCX-CKR2 to treat subjects having cancer. In some cases, the modulators have a molecular weight of less than 1,500 daltons, and in some cases less than 1,000, 800, 600, 500, or 400 daltons.

Administration of the modulators can be by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17.sup.th ed. 1985)).

The modulators (e.g., agonists or antagonists) of the expression or activity of CCX-CKR2, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The modulators can also be administered as part of a prepared food or drug.

In some embodiments, CCX-CKR2 modulators of the present invention can be administered in combination with other appropriate therapeutic agents, including, e.g., chemotherapeutic agents, radiation, etc. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders such as, e.g., cancer, kidney dysfunction, brain dysfunction or neuronal dysfunction. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial response in the subject over time (e.g., to reduce tumor size or tumor load). The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of a particular disease. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administered a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies. In general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.

For administration, chemokine receptor modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side-effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.

VI. Compositions, Kits, Integrated Systems and Proteomic Applications

The invention provides compositions, kits and integrated systems for practicing the assays described herein using anti-CCX-CKR2 antibodies or other agents that specifically detect CCX-CKR2.

The invention provides assay compositions for use in solid phase assays; such compositions can include, for example, a CCX-CKR2 polypeptide (including, e.g., as part of a cell, membrane fractions or liposomes (see, e.g., Babcok et al., J. Biol. Chem. 276(42):38433-40 (2001); Mirzabekov et al., Nat. Biotechnol. 18(6):649-54 (2000))) immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. For example, the solid support can be, e.g., a petri plate, multi-well plate or microarray. In addition, microarrays of peptide libraries can be used to identify peptide sequences that specifically bind CCX-CKR2.

Agents that specifically bind to CCX-CKR2 can also be included in the assay compositions. For example, an antibody that specifically binds to CCX-CKR2 can be immobilized on a solid support. In some of these embodiments, the agent is used to detect the presence or absence of CCX-CKR2 or cells expressing CCX-CKR2. For example, the solid support can be petri plate, multi-well plate or microarray.

The invention also provides kits for carrying out the assays of the invention. The kits typically include an agent (e.g., an antibody or other small molecule) that specifically binds to CCX-CKR2 and a label for detecting the presence of the agent. The kits may include one or more other chemokine receptor polypeptides. Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on activity or function of chemokine receptors, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the function or activity of chemokine receptors, a robotic armature for mixing kit components or the like.

In some embodiments, the kits comprise SDF1 and/or I-TAC. In some embodiments, the kits comprise a labeled or tagged SDF-1 and cold competitor I-TAC or alternatively, a labeled or tagged I-TAC and cold competitor SDF-1. The labeled or tagged chemokine can be labeled or tagged in any way known to those of skill in the art. In some embodiments, the labeled chemokine is radiolabeled or tagged with biotin or a fluorescent label. Alternatively, or in addition, the kit can contain an anti-1-TAC binding reagent (e.g., an antibody) for detection of I-TAC. The kits can also contain the appropriate salt buffers and other reagents to perform a competitive binding assay, e.g., on intact cells or cell membranes. Such reagents are described in, e.g., the examples below. In some aspects, the kits also comprise a solid support or receptacle for measuring ligand binding to CCX-CKR2 (e.g., in a plate format for reactions compatible with scintillation counters or automated plate readers). In some aspects, the kits comprise instructions for using the kits, e.g., in the methods of the invention.

The invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the activity or function of potential CCX-CKR2 modulators. The systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety.

Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image.
 

Claim 1 of 3 Claims

1. An isolated antibody that specifically competes with SDF-1 or I-TAC for binding to the polypeptide of SEQ ID NO:2.

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