KR20060136373A - Compositions and methods for detecting and treating diseases and conditions related to chemokine receptors - Google Patents

Abstract

The present invention relates to ligands of CCX-CKR2 and the biological role of CCX-CKR2 in cancer.

Description

COMPOSITIONS AND METHODS FOR DETECTING AND TREATING DISEASES AND CONDITIONS RELATED TO CHEMOKINE RECEPTORS}

Cross-References to Related Applications

This application is a partial continuing application of US Application No. 10 / 698,541 (October 30, 2003), which is a partial continuing application of US Application No. 10 / 452,015 (May 30, 2003). Partial application of 10 / 245,850 (September 16, 2002), which is US Patent Application Serial Number (USSN) 60 / 338,100 (November 30, 2001) and USSN 60 / 337,961 (November 30, 2001). Claims of priority are made, each of which is hereby expressly incorporated by reference in its entirety for all purposes. The application is also directed to U.S. Provisional U.S. Pat. Claim priority to 60 / 434,912 (20 December 2002), which is hereby expressly incorporated by reference in its entirety for all purposes.

Chemokines are produced during inflammation and form a small group of cytokines that regulate the recruitment, activation and proliferation of white blood cells (Baggiolini, M. et. al ., Adv . Immunol . 55: 97-179 (1994); Springer, TA, Annul Rev. Physiol . 57: 827-872 (1995); And Schall, TJ and KB Bacon, Curr . Opin . Immunol . 6: 865-873 (1994)). Chemokines can selectively induce chemotaxis of components including leukocytes formed (except red blood cells) such as neutrophils, monocytes, macrophages, eosinophils, basophils, mast cells and lymphocytes such as T cells and B cells. In addition to also stimulate chemotaxis, other changes e. G., Cell shape change of the by responsive cells in a chemokine associated with leukocyte activation, intracellular free calcium ions (Ca ^{2 +)} transient increase in the concentration, granules extracellular discharge, integrin Upregulation, formation of bioactive lipids (eg, leukotrienes) and respiratory release may also be induced selectively. Therefore, chemokines cause an early inflammatory response, causing inflammatory mediator release at the site of infection or inflammation, causing chemotaxis and blood loss.

The two subgroups of chemokines, CXC and CC chemokines, are distinguished by the first two arrangements of the four conserved cysteine residues, which are one amino acid [CXC chemokine SDF-1, IL-8, IP- 10, MIG, PF4, ENA-78, GCP-2, GROα, GROβ, GROγ, NAP-2, NAP-4, as in I-TAC] or residues adjacent to each other [CC chemokine MIP -1α, MIP-1β, RANTES, MCP-1, MCP-2, MCP-3, I-309. Most CXC chemokines attract neutrophil leukocytes. For example, the CXC chemokine interleukin 8 (IL-8), platelet factor 4 (PF4) and neutrophil-activated peptide 2 (NAP-2) are potent chemotactic and activators of neutrophils. MIG (monocaine induced by gamma interferon) and IP-10 (inducible interferon-γ, 10 kDa protein), members of the CXC chemokines, are particularly active in inducing chemotaxis of activated peripheral blood lymphocytes. CC chemokines are generally less selective and attract various types of white blood cells such as monocytes, eosinophils, basophils, T-lymphocytes and natural killer cells. CC chemokines such as human monocyte chemotactic proteins 1-3 (MCP-1, MCP-2 and MCP-3), Regulated on Activation, Normal T Expressed and Secreted (RANTES), and macrophage inflammatory proteins 1α and 1β (MIP- 1α and MIP-1β) are known to be chemotactic and activating factors of monocytes or lymphocytes, but not chemotactic factors for neutrophils.

CC and CXC chemokines act through receptors belonging to a higher class of seven dura G protein-coupled receptors [Murphy, PM, Pharmacol Rev. 52: 145-176 (2000). This group of G protein-coupled receptors comprises a large group of integrating membrane proteins comprising seven transmembrane regions. The receptor is coupled to G protein, a heterotrimeric regulatory protein capable of binding GTP and mediating signal transduction from coupled receptors, for example, by the generation of intracellular mediators.

In general, the interaction of chemokine and chemokine receptors is coincidental in that one chemokine can bind multiple chemokine receptors and conversely one chemokine receptor can interact with several chemokines. However, there are some exceptions to this rule; One is that SDF-1 interacts with CXCR4 [Bleul et. al ., J Exp Med , 184 (3): 1101-9 (1996); Oberlin et al ., Nature, 382 (6594): 833-5 (1996). Inherently pre-B-cell growth stimulating factor [Nagasawa et al ., Proc Natl Acad Sci USA , 91 (6): 2305-9 (1994)], SDF-1 is reported only as a human ligand for CXCR4. The SDF-1 gene encodes two proteins, SDF-1α and SDF-1β by alternative splicing. These two proteins are identical except for the four amino acid residues present at the carboxy terminus of SDF-1β but not at SDF-1α.

Many aspects of chemokine receptor signal transduction and selectivity to ligands are not previously understood. For example, the function of numerous orphan receptors is not yet known. For example, RDC1, previously considered as a receptor for vasoactive intestinal peptide (VIP), is not currently considered an orphan receptor because its endogenous ligand has not been identified. See, eg, Cook et al ., FEBS Letts . 300 (2): 149-152 (1992).

The present invention addresses these and other issues.

Brief Description of the Invention

The present invention relates to a method for selecting a substance that binds to CCX-CKR2 on a cell. In some embodiments, the method comprises contacting a plurality of agents with a CCX-CKR2 polypeptide comprising an extracellular region at least 95% identical to the extracellular region of SEQ ID NO: 2, or an SDF-1 or I-TAC binding fragment thereof; Selecting a substance that competes with I-TAC or SDF-1 to bind to the CCX-CKR2 polypeptide or fragment thereof, thereby selecting a substance that binds to CCX-CKR2 on the cell.

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

In some embodiments, the method further comprises testing the ability of the selected material to change the function of the kidney. In some embodiments, the method further comprises testing the ability of the selected material to change the function of the brain or neurons. In some embodiments, the method further comprises testing the ability of the selected material to change cell adhesion to endothelial cells.

In some embodiments, the material is less than 1,500 Daltons. In some embodiments, the substance is an antibody. In some embodiments, the substance is a polypeptide. In some embodiments, the CCX-CKR2 polypeptide comprises the sequence set forth in SEQ ID NO: 2.

The present invention also provides a method for measuring the presence or absence of cancer cells. In some embodiments, the method comprises contacting a sample comprising a cell with a substance that specifically binds SEQ ID NO: 2; Detecting the binding of the substance to the polypeptide in the sample, wherein the binding of the substance to the sample indicates the presence of cancer cells.

In some embodiments, the substance is an antibody. In some embodiments, the material is less than 1500 Daltons. In some embodiments, the substance is a polypeptide. In some embodiments, the polypeptide detected is SEQ ID NO: 2. In some embodiments, the sample is human. In some embodiments, the method is used to diagnose cancer of a human. In some embodiments, the method is used to determine the prognosis of human cancer. In some embodiments, the cancer is selected from the group consisting of cervical cancer, breast cancer, lymphoma, glioblastoma, prostate cancer, and leukemia. In some embodiments, the cancer is not Kaposi's sarcoma, multiple Castleman's disease or AIDS-related primary effusion lymphoma. In some embodiments, the antibody competes with SDF-1 and 1-TAC to bind SEQ ID NO: 2.

The present invention also provides a method for diagnosing or prognosticing a subject with cancer. In some embodiments, the method comprises detecting the presence or absence of expression of a polynucleotide encoding a CCX-CKR2 polypeptide in a cell of the subject, thereby diagnosing the cancer of the subject, wherein the CCX-CKR2 polypeptide is I Binds to -TAC and / or SDF-1 and the CCX-CKR2 polypeptide is at least 95% identical to SEQ ID NO: 2.

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

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

The invention also includes contacting a cell with a substance that specifically binds SEQ ID NO: 2, thereby binding the substance with a CCX-CKR2 polypeptide on the cell, wherein the substance binds to the CCX-CKR2 polypeptide. To compete with SDF-1 and I-TAC, wherein the cell expresses a CCX-CKR2 polypeptide comprising an extracellular region that is at least 95% identical to the extracellular region of SEQ ID NO: 2.

In some embodiments, the material is less than 1,500 Daltons. In some embodiments, the substance is an antibody. In some embodiments, the substance is a polypeptide. In some embodiments, the CCX-CKR2 polypeptide is shown in SEQ ID NO: 2. In some embodiments, the agent comprises contacting a plurality of agents with a CCX-CKR2 polypeptide comprising at least 95% identical to the extracellular region of SEQ ID NO: 2, or an SDF-1 or I-TAC binding fragment thereof ; It is identified by a method comprising selecting a substance that competes with I-TAC or SDF-1 to bind to the CCK-CKR2 polypeptide or fragment thereof, thereby selecting a substance that binds to cancer cells.

The present invention also provides a method of treating cancer in a subject. In some embodiments, the method comprises administering to the individual a therapeutically effective amount of a polynucleotide that inhibits expression of the CCX-CKR2 polynucleotide. In some embodiments, the CCX-CKR2 polynucleotide encodes SEQ ID NO: 2. In some embodiments, the CCX-CKR2 polynucleotide comprises SEQ ID NO: 1. In some embodiments, the administered polynucleotides inhibit expression via siRNA.

The present invention also provides a method of treating cancer in a subject. In some embodiments, the method comprises administering to the individual a therapeutically effective amount of a substance that competes with SDF-1 and I-TAC to bind SEQ ID NO: 2. In some embodiments, the material is less than 1,500 Daltons. In some embodiments, the substance is an antibody. In some embodiments, the substance is a polypeptide. In some embodiments, the agent comprises contacting a plurality of agents with a CCX-CKR2 polypeptide comprising at least 95% identical to the extracellular region of SEQ ID NO: 2, or an SDF-1 or I-TAC binding fragment thereof ; It is identified by a method comprising selecting a substance that competes with I-TAC or SDF-1 to bind to the CCK-CKR2 polypeptide or fragment thereof, thereby selecting a substance that binds to cancer cells. In some embodiments, the cancer is selected from the group consisting of cervical cancer, breast cancer, lymphoma, glioblastoma, prostate cancer, and leukemia. In some embodiments, the cancer is not Kaposi's sarcoma, multiple Castleman's disease or AIDS-related primary effusion lymphoma.

Brief description of the drawings

1A-1I show binding data showing individual SDF-1 translocation binding 'fingerprints' for different cell types. In binding potential experiments with a comprehensive array of chemokine strains as 90 or more individual viruses, human and murine chemokines and unlabeled competitors, the binding competition profiles were (a) CEM-NKr (FIGS. 1A-1C) and (b) MCF-7 In addition to using ¹²⁵ I SDF-1α as the radioligand probe for (FIGS. 1D-1F), (c) ¹²⁵ II-TAC is used as the radioligand probe for MCF-7 (FIGS. 1G-1I). % Inhibition of radioligand binding is shown in bar graphs, revealing that SDF-1α and I-TAC are cross-potentially on MCF-7 rather than CEM-NKr cells. White bars have potential high affinity (more than 80% inhibition), gray bars have potential moderate to low affinity (60-79% inhibition), black bars have little or no affinity (less than 60% inhibition) ). The result is the average of three measurements. Error bars are omitted for clarity.

2 compares ligand binding affinity and specificity for CEM-NKr and MCF-7. Potential high affinity ligands selected in FIG. 2 were selected for competition in dose response to CEN-NKr (white square) and MCF-7 (black square). In each competition ¹²⁵ I SDF-1α competes with an unlabeled competitor chemokine as indicated.

3 shows that ¹²⁵ II-TAC binding on MCF-7 cells is not due to typical CXCR3 binding interactions. ^{125 The} ability to compete with the indicated chemokine for 1-TAC was due to the presence of only buffer (black squares), excess MIG (to inhibit any CXCR3-mediated binding; white triangles) or excess SDF-1α (asterix) Under investigation.

4 shows that the binding phenotypes described herein can be repeated in cell lines that do not endogenously express the receptor. CCX-CKR2 transfected breast cancer cell line MDA MB 435s shows binding to ¹²⁵ I SDF-1α. Such binding may be comparable to unlabeled SDF-1α and I-TAC. In comparison it does not produce wild type cells (untransfected) ¹²⁵ I SDF binding signal.

5 shows competitive binding data. Two small molecules, CCX0803 (black circle) and CCX7923 (white circle), compete specifically with ¹²⁵ I SDF-1α in distinct cell types, and no cross competition is observed. SDF-1α (Asterix) was also included as an unlabeled competitor of ¹²⁵ I SDF-1α that binds to MCF-7 and CEM-NKr. The chemical structures of CCX0803 and CCX7923 are shown in the inset. Expected IC50 values of the SDF-1α and CXCR4 antagonist competition are provided in the attached table.

FIG. 6 shows the therapeutic effect of mammalian carcinoma cells expressing CCX-CKR2 when treated with small molecule CCX-CKR2 antagonist compared to cells not treated with antagonist.

FIG. 7 shows that CCX-CKR2 antagonist 3451 (see Compound 49 in Table 1) inhibits attachment of CCX-CKR2 expressing cells to vascular endothelial monolayer.

8 shows that antagonism of CCX-CKR2 on mammalian carcinoma cells reduces tumor volume.

Justice

"Chemokine" or "chemokine ligand" means a small protein consisting of approximately 50-110 amino acids and sharing sequence homology with other known chemokines [eg, Murphy, PM, Pharmacol Rev. 52: 145-176 (2000). Chemokines are classified according to the relative position of the first pair of cysteines (Cs) found in the primary amino acid sequence. In CXCL chemokines, the first pair of cysteines are separated by any single amino acid. CCL chemokines have adjacent cysteines. For CX3CL chemokines, the first cysteine pair is separated by three amino acids. CL chemokines contain only one cysteine in the homologous position. Chemokines can promote biological function by binding to and activating chemokine receptors.

"Chemokine receptor" means a polypeptide that specifically interacts with a chemokine molecule. Chemokine receptors are G protein coupled receptors with seven transmembrane domains. Chemokine receptors include strongly acidic N-terminal domains; Amino acid sequence DRYLAIVHA (or minimal variation in that sequence) found in the second extracellular loop of a plurality of chemokine receptors; A third short intercellular loop that is entirely base charged; It possesses some common structural features, including cysteine residues present in each of the four extracellular domains. Typically, the total size of the chemokine receptor is about 340-370 amino acid residues (eg, Murphy, PM Chemokine Receptors ; Overview , Academic Press 2000; And Loetscher P. and Clark-Lewis IJ Leukocyte Biol . 69: 881 (2001). Examples of representative chemokine receptors include CC-chemokine receptors (including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 and CCR10), CXC-chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR6) -CKR2 (including eg SEQ ID NO: 2), CX3CR1, CXR1, CCXCKR (OCR11), virus encoded chemokine receptor, US28, ECRF3, Kaposi's sarcoma-associated herpes virus GPCR (ORF 74), pox virus membrane binding G Protein coupled receptors; D6 and DARC.

Defined responses that can be stimulated by chemokine receptors include dural signaling, activation of the cytoplasmic signaling cascade, cytoskeletal rearrangement, adhesion, chemotaxis, invasion, metastasis, cytokine production, gene induction, gene expression, induction of protein expression, or Regulation of cell proliferation and differentiation, including the occurrence of cancer.

"RDC1", referred to herein as "CCX-CKR2", is the seven transmembrane domains that are thought of as G-protein coupled receptors (GPCRs). The CCX-CKR2 dog ortholog was first discovered in 1991 (see, eg, Libert et. al . Science 244: 569-572 (1989). Dog sequences are described in Libert et. al ., Nuc . Acids Res . 18 (7): 1917 (1990). Mouse sequences are described, eg, in Heesen et al ., Immunogenetics 47: 364-370 (1998). Human sequences are described, for example, in Sreedharan et. al ., Proc . Natl . Acad . Sci . USA 88: 4986 4990 (1991), wherein the protein is erroneously described as the vasoactive intestinal peptide. "CCX-CKR2" is substantially the same as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 Or a sequence that is a conventionally modified variant thereof.

"Peptidomimetic" and "mock" means a synthetic chemical compound having structural and functional properties that are substantially the same as the antagonist or agonist of the chemokine receptor. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. Non-peptide compounds of this type are referred to as "peptide mimetics" or "peptidomimetics" [Fauchere, J. Adv . Drug , Res . 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); And Evans et al . J. Med . Chem . 30: 1229 (1987), all of which are incorporated herein by reference. Peptide mimetics that are structurally similar to therapeutically effective peptides can be used to exhibit the same or more enhanced therapeutic or prophylactic effects. In general, peptidomimetics are structurally similar to paradigm polypeptides (ie, polypeptides having biological or pharmacological activity), for example, -CH ₂ NH-, -CH ₂ S-, -CH ₂ -CH _2- . At least one peptide bond optionally substituted by a bond selected from the group consisting of -CH = CH- (cis and trans), -COCH _2- , -CH (OH) CH ₂ -and -CH ₂ SO-. The mimetics are chimeric molecules in which all are synthetic, non-natural amino acid analogs, or some are natural peptide amino acids and some are non-natural amino acid analogs. The mimetic can also introduce any amount of conservative substitutions of natural amino acids, provided such substitutions do not substantially alter the structure and / or activity of the mimetic. For example, the mimic can mimic the binding of SDF-1 or I-TAC to CCX-CKR2. For example, mock compositions are included within the scope of the present invention as long as they can inhibit or increase the activity or function of CCX-CKR2.

"Antibody" means an immunoglobulin gene or a polypeptide substantially encoded by immunoglobulin genes, or a fragment thereof, which binds to and recognizes an analyte (antigen) specifically. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a number of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which define the immunoglobulin groups, ie IgG, IgM, IgA, IgD and IgE, respectively.

Representative immunoglobulin (antibodies) structural units include tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region consisting of about 100 to 110 or more amino acids primarily involved in antigen recognition. The variable part light chain (V _L ) and the variable part heavy chain (V _H ) mean such light and heavy chains, respectively.

Antibodies exist, for example, as intact immunoglobulins, or as many widely characterized fragments generated by digestion with various peptidase. Thus, for example, pepsin cleaves the disulfide bond below the antibody's hinge to remove the dimer of F (ab) ' ₂ , a Fab that is itself bound to V _H -C _H 1 by disulfide bonds. To produce. The F (ab) ' ₂ is reduced under mild conditions to degrade disulfide bonds in the hinge moiety, whereby the F (ab)' ₂ dimer is converted to Fab 'monomers. The Fab 'monomer is essentially a Fab that retains a portion of the hinge (see Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of degradation of native antibodies, those skilled in the art will appreciate that such fragments may be synthesized chemically fresh or synthesized using recombinant DNA methods. Thus, as used herein, the term antibody also encompasses antibody fragments prepared by modifying whole antibodies, or antibody fragments (eg, single chain Fv) newly synthesized using recombinant DNA methods.

By “humanized” antibody is meant a molecule having an antigen binding site substantially derived from immunoglobulins from non-human species and the remaining immunoglobulin constructs of the molecule based on the structure and / or sequence of the human immunoglobulin. The antigen binding site may comprise complementary determining sites (CDRs) grafted onto an appropriate framework region within the variable region or a complete variable region fused onto a constant region. The antigen binding site can be wild type or modified by one or more amino acid substitutions to be more similar, for example, to human immunoglobulins. All forms of humanized antibodies preserve all CDR sequences (eg, humanized mouse antibodies containing all six CDRs from mouse antibodies). Another form of humanized antibody has one or more CDRs (1, 2, 3, 4, 5, 6) modified relative to the original antibody.

When referring to a protein or polypeptide, the words "specifically (or selectively) bind to an antibody" or "specifically (or selectively) immunoreact with" means the presence of a heterogeneous population of proteins and other organisms. Refers to a binding reaction that confirms the presence of the protein. Thus, under the specified immunoassay conditions, the specified antibody binds to a particular protein and does not bind in significant amounts to other proteins present in the sample. Specific binding to an antibody under these conditions may require the antibody to be selected for specificity for a particular protein. For example, an antibody against a protein having an amino acid sequence encoded by any of the polynucleotides of the present invention may be selected to at least 80% of that protein other than a protein other than a polymorphic variant, for example SEQ ID NO: 2. Antibodies that specifically immunoreact with the same protein, 85%, 90%, 95% or 99% can be obtained. Various immunoassay formats can be used to select antibodies that specifically immune to specific proteins. In order to select monoclonal antibodies that specifically immunoreact with one protein, for example, ELISA immunoassays, Western blots, or immunohistochemistry are routinely used. For a description of immunoassay formats and conditions that can be used to measure specific immunoreactivity, see, eg, Harlow and Lane Antibodies , A Laboratory. Manual , Cold Spring Harbor Publications, NY (1988). In general, the specific or selective response will be at least two times the background signal or noise, more generally at least 10-100 times.

By "ligand" is meant a substance, such as a polypeptide or other molecule, capable of binding to a chemokine receptor.

As used herein, "substance that binds a chemokine receptor" refers to a substance that binds a chemokine receptor with high affinity. "High affinity" means a natural chemokine ligand to bind to the chemokine receptor at affinity sufficient to elicit a pharmacologically appropriate response, e.g., at a pharmacologically appropriate concentration (e.g., less than about 10 ^-5 M). And ability to compete significantly with (see, eg, Example 1 and FIG. 5). And some representative substances having an affinity is to be combined with affinity chemokine receptor affinity of ^10-6 M of the excess, and sometimes 10 ^-7 M or 10 ^-8 M excess. A substance that fails to compete for binding to a natural receptor ligand when the concentration of the substance is less than 10-4M will be considered "unbound" for the purposes of the present invention.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in the form of single or double chains. Unless specifically limited, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless stated otherwise, it is evident that certain nucleic acid sequences also include conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences, as well as those shown explicitly. In particular, degenerate codon substitutions can be made by preparing a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue [Batzer e t al ., Nucleic Acid Res . 19: 5081 (1991); Ohtsuka et al ., J. Biol . Chem . 260: 2605-2608 (1985); And Cassol et al . (1992); Rossolini et al ., Mol . Cell . Probes 8: 91-98 (1994). The term nucleic acid is used interchangeably with genes, cDNAs encoded by genes and mRNA.

The terms "polypeptide", "peptide" and "protein" as used herein interchangeably refer to polymers of amino acid residues. The term applies to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. As used herein, the terms include amino acid chains of any length, for example full length proteins (ie, antigens), wherein the amino acid residues are joined by covalent peptide bonds.

The term "amino acid" means not only naturally occurring amino acids and synthetic amino acids, but also amino acid analogs and amino acid mimetics that function in a similar manner to these naturally occurring amino acids. Naturally occurring amino acids are not only encoded by the genetic code, but also later modified amino acids such as hydroxyproline, γ-carboxyglutamate and O-phosphoserine. An amino acid analog means a compound having the same basic chemical structure as a naturally occurring amino acid, i.e., a compound having an α carbon bonded to a hydrogen, carboxyl group, amino group and R group, and examples thereof include homoserine, norleucine, methionine sulfoxide and methionine. Methyl sulfonium. These analogs have a modified R group (eg, norleucine) or a modified peptide backbone, but have the same basic chemical structure as naturally occurring amino acids. By “amino acid mimic” is meant a chemical compound which has a structure different from the general chemical structure of an amino acid, but which acts in a manner similar to the naturally occurring amino phase.

The amino acids herein may be referred to by the generally known three letter representation, or as one letter representation, recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their generally accepted one letter codes.

"Conservatively modified variant" is a term that applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, "conservatively modified variant" means a nucleic acid that encodes an identical or essentially identical amino acid sequence, and if the nucleic acid does not encode an amino acid sequence, it means an essentially identical sequence. . Due to the degeneracy of the genetic code, many nucleic acid sequences will encode any given protein. For example, codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be modified with any of the codons described above without modifying the encoded polypeptide. Such nucleic acid variations are referred to as "silent variations", which are a kind of conservatively modified variations. All of the nucleic acid sequences herein that encode a polypeptide also exhibit all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to form functionally identical molecules. Thus, each silent variation of a nucleic acid encoding a polypeptide encompasses each sequence shown.

When an amino acid is replaced by its chemically similar amino acid through modification, those skilled in the art, with respect to the amino acid sequence, each skilled in the art will recognize that each of the nucleic acid, peptide, polypeptide or protein sequences that alters, adds or deletes a single amino acid or a small amount of amino acids in the encoded sequence. It will be appreciated that substitutions, deletions or additions are “conservatively modified variants”. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are not excluded in addition to the polymorphic variants, interspecies homologs and alleles of the present invention.

The eight groups containing amino acids that are conservatively substituted with each other are as follows:

1) Alanine (A), Glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); And

8) Cysteine (C), Methionine (M)

(See, eg, Creighton, Proteins (1984)).

"Sequence identity ratio" is determined by comparing two optimally aligned sequences against a comparison window, where a portion of the polynucleotide sequence in the comparison window is the reference sequence for the optimal alignment of the two sequences (additional Or addition or deletion (ie, 겝) relative to the sequence in which no deletion occurred. The ratio is obtained by measuring the number of positions with identical nucleic acid bases or amino acid residues in both sequences, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and then dividing the result. It can be calculated by multiplying by 100 to find the proportion of sequence identity.

The term "identical" or "identity" ratios for two or more nucleic acid or polypeptide sequences herein refers to a designated region determined by manual alignment and visual observation, or using one of the following sequence comparison algorithms, Or 60% identity, optionally with respect to the same (ie, the entire polypeptide sequence of the invention or a particular region of the extracellular domain of the polypeptide of the invention, when compared and aligned with respect to the maximum correspondence to the comparison window). By two or more sequences or internal sequences having a certain proportion of amino acid residues or nucleotides, which represent 65%, 70%, 75%, 80%, 85%, 90% or 95% identity, or are identical. These sequences are then referred to as "substantially identical." This definition also means the complementarity of the test sequence. Optionally, the identity is present over a region of about 50 or more nucleotides in length, but more preferably over a region of 100 to 500 or 1000 or more nucleotides in length.

The term "similarity" or "similarity ratio" in the context of two or more polypeptide sequences refers to a designated region, or comparison, as measured by manual alignment and visual observation, or using one of the following sequence comparison algorithms: When compared and aligned with respect to maximal correspondence to a window, it possesses a certain proportion of amino acid residues equal to or similar to the eight conservative amino acid substitutions as defined above (ie, for example, the entire polypeptide sequence of the invention For example 60%, optionally 65%, 70%, 75%, 80%, 85% relative to the specific region or the entire sequence of a polynucleotide of CCX-CKR2 (SEQ ID NO: 2) or the extracellular domain of a polypeptide of the invention , 90% or 95% similar to two or more sequences or internal sequences, which are hereinafter referred to as "substantially similar." In general, this equality is present with respect to about 50 or more amino acids in area, or more preferably a length of about 100-500 amino acids, or more than 1000 amino acid long region.

In sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are entered into the computer, internal sequence equivalents are designated if necessary, and parameters of the sequence algorithm program are specified. Default program parameters can be used, or alternative parameters can be specified. A sequence comparison algorithm is then used to calculate the sequence identity ratio of the test sequence to the reference sequence based on this program parameter.

As used herein, “comparative window” means a reference segment for a full length sequence or a segment consisting of any of a plurality of contiguous positions selected from the group consisting of 20 to 600, about 50 to about 200, or about 100 to about 150 Wherein the sequence can be compared with a reference sequence having the same number as adjacent positions after two sequences are optionally aligned. Methods for aligning comparative sequences are well known in the art. Optimal alignment of comparative sequences is described, for example, in Smith and Waterman (1970) Adv . Appl . Math . 2: Local homology algorithm of 482c, Needleman and Wunsch (1970) J Mol . Biol . 48: 443 homology alignment algorithm, Pearson and Lipman (1988) Proc . Natl . Acad . Sci . USA 85: 2444 search for similarity, computerized instruments of these algorithms [GAP, BESTFIT, FASTA and TFAST, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI], or manual sorting and visual observation It can be carried out by (see, for example, Ausubel et al ., Current Protocols in Molecular Biology (1995 supplement)].

Examples of suitable algorithms for measuring sequence identity ratio and sequence similarity ratio include the BLAST and BLAST 2.0 algorithms, as described in Altschul et al., (1977) Nuc . Acids . Res . 25: 3389-3402 and Altschul et al . (1990) J. Mol . Biol. 215: 403-410, respectively. Software for performing BLAST assays is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The algorithm uses a high scoring sequence by identifying short words of length W in a query sequence that initially meet or meet a threshold score T, which is some positive value when aligned with words of equal length in a database sequence. Identifying a pair (HSP). T means neighboring word score limit [Altschul et al. , Homology]. This initial neighboring word's hit acts as a seed to initiate the search process to find the longer HSP containing this hit. These word hits extend in both directions along each sequence to the extent that the cumulative alignment score can increase. Cumulative scores include parameter M [reward score for matching residue pairs; Always> 0] and N [penalty score for mismatched residues; Always <0] for the nucleotide sequence. In the amino acid sequence, a cumulative score is calculated using a scoring matrix. The extension of the word hit in each direction is when the cumulative alignment score falls by an amount X and its maximum value that can be obtained; The cumulative score is less than or equal to zero due to the accumulation of one or more negative scoring residue alignments; Or stop when reaching either end of the sequence. The BLAST algorithm parameters W, T, and X determine the accuracy and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the word length (W) of 11, expected value (E) of 10, M = 5, N = -4 as defaults, and is used for comparison of both chains. For amino acid sequences, the BLASTP program uses the word length 3, expected value (E) 10 and the BLOSUM62 scoring matrix as default values (Henikoff and Henikoff (1989) Proc . Natl . Acad . Sci . USA 89: 10915, alignment (B) = 50, expected (E) = 10, M = 5, N = -4, and both sequences comparison].

The BLAST algorithm also performs statistical analysis of the similarity between the two sequences (see, eg, Karlin and Altschul (1993) Proc . Natl . Acad . Sci . USA 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability method (P (N)), which indicates the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence when the minimum sum probability is less than about 0.2, more preferably less than about 0.01, most preferably less than about 0.001 in the comparison of the test nucleic acid to the reference nucleic acid.

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid sequence is immunologically cross reacted with the antibody to the polypeptide encoded by the second nucleic acid sequence as described below. Thus, for example, if two peptides differ only in conservative substitutions, the polypeptide is generally substantially identical to the second polypeptide. Another indication that the two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize under stringent conditions, as described below. Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

A “modulator” of CCX-CKR2 activity refers to a molecule that directly or indirectly increases or decreases the activity of CCX-CKR2, which is selected using in vitro and in vivo assays for CCX-CKR2 binding or signaling. It includes molecules. CCX-CKR2 activity can be achieved, for example, by contacting the CCX-CKR2 polypeptide with an agonist, and / or. In some cases it may be increased by expressing CCX-CKR2 in cells. An agonist refers to a molecule that increases the activity of CCX-CKR2. An agonist is a substance that binds to CCX-CKR2 and stimulates, increases, opens, activates, facilitates, increases activity, sensitizes or upregulates its activity. Modulators can compete with known CCX-CKR2 ligands such as SDF-1 and I-TAC and the small molecules described herein for binding to CCX-CKR2.

Antagonists refer to molecules that inhibit CCX-CKR2 activity, for example by blocking binding of agonists such as I-TAC or SDF-2. An agonist is, for example, a substance that binds to CCX-CKR2 and partially or globally blocks, reduces, prevents, delays activation, inactivates, desensitizes or downregulates stimulation for its activity.

Modulators include substances that alter, for example, the interaction of CCX-CKR2 with proteins that bind to receptors, activators or inhibitors, including G-protein coupled receptors (GPCRs), kinases and the like. Modulators include naturally occurring ligands and synthetic ligands, antagonists, agonists, chemical small molecules, siRNA and the like, as well as genetically modified forms of, for example, naturally occurring chemokine receptor ligands with altered activity. Assays for such inhibitors and activators include, for example, the addition of putative modulator compounds to cells expressing CCX-CKR2, followed by CCX-CKR2 signaling, eg, ERK1 and / or ERK2 phosphorylation or ERK1 or ERK2 signaling pathways. Measuring its functional effects on the activation of the component and / or other effects described herein. To investigate the extent of inhibition, a sample or assay comprising CCX-CKR2 treated with a potential activator, inhibitor or modulator is compared to a control sample without activator, inhibitor or modulator. Control samples (untreated with inhibitor) are set at 100% relative chemokine receptor activity. Inhibition of CCX-CKR2 is achieved when the CCX-CKR2 activity or expression for the control is about 85%, optionally about 90%, optionally about 80%, optionally about 50% or about 25-0%. CCX-CKR2 activity or expression for the control is at least about 105%, about 110%, optionally at least about 105%, about 150%, optionally at least about 105%, about 200-500%, or at least about 105%, about 1000- When 3000% or more, the activation of CCX-CKR2 is achieved.

"siRNA" may interfere with gene expression in cells such as mammalian cells (including human cells) and the body, such as the mammalian body (including the human body), resulting in inhibition of post-transcriptional expression of certain genes. Refers to small interfering RNA. RNA interference phenomena are described in Bass, Nature 411: 428-29 (2001); Elbahir et al., Nature 411: 494 98 (2001), and Fire et al., Nature 391: 806-11 (1998). And described in WO 01/75164, and methods of interference of RNA are also described Nucleic acid and sequence based siRNA encoding the gene products disclosed herein typically have less than 100 base pairs, for example For example, may be less than 30 base pairs, and may be prepared by approaches or synthetic methods known in the art, including the use of complementary DNA strands, said siRNA may be a cell, eg, a mammalian cell (including human cells). And interference with gene expression in the body, eg, in the mammalian body (including the human body), resulting in inhibition of post-transcriptional expression of a particular gene .. Typical siRNAs according to the present invention are 29 base pairs or less, 25 base pairs, 22 base pairs, 21 base pairs, 20 base pairs, 15 base pairs, 10 base pairs, 5 Base pairs, or any integer base pairs in or near the base pairs, The tools for preparing the best inhibitory siRNAs include those available from DNAengine Inc. (Seattle, WA) and Ambion, Inc. (Austin, TX). .

One RNAi technique utilizes a gene construct in which the sense and antisense sequences are located in regions adjacent to the intron sequence in an appropriate splicing orientation with donor and acceptor splicing sites. Alternatively, spacer sequences of various lengths can be used to separate self-complementary regions of the construct. In the course of the transcription of the genetic construct, the intron sequence is removed by splicing, so that the sense and antisense sequences, as well as the splice seam sequence, are bound to the double stranded RNA. The ribonuclease is then selected to bind and cleave two stranded RNAs to initiate inactivation of specific mRNA gene sequences and suppression of expression of specific genes.

"Compound" refers to a particular molecule and its enantiomers, diastereomers, polymorphs and salts.

"Heteroatom" refers to a nitrogen, oxygen, or sulfur atom bonded.

"Substituted" refers to a group attached to a parent molecule or group. Thus, the benzene ring with methyl substituent is methyl substituted benzene. Similarly, the benzene ring with five hydrogen substituents will be an unsubstituted phenyl group when bound to the parent molecule.

"Substituted heteroatom" refers to a group in which a heteroatom is substituted. The heteroatoms are halogen, alkyl, alkylene, alkenyl, alkynyl, aryl, arylene, cycloalkyl, cycloalkylene, heteroaryl, heteroarylene, heterocyclyl, carbocyclic, hydroxy, alkoxy, aryloxy And may be substituted with atoms or groups including, but not limited to, sulfonyl. Examples of representative substituted heteroatoms include cyclopropyl aminyl, isopropyl aminyl, benzyl aminyl, and phenoxy.

"Alkyl" refers to a monovalent saturated hydrocarbon group which may be linear or branched. Unless defined otherwise, the alkyl groups typically contain 1-10 carbon atoms. Examples of typical alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n- Nonyl, n-decyl, etc. are mentioned.

"Alkylene" refers to a divalent saturated hydrocarbon group that may be linear or branched. Unless defined otherwise, the alkylene groups typically contain 1 to 10 carbon atoms. Examples of representative alkylene groups include methylene, ethane-1,2-diyl ("ethylene"), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, pentane-1, 5-diyl etc. are mentioned.

"Alkenyl" may be linear or branched and refers to a monovalent unsaturated hydrocarbon group having one or more, and typically having 1, 2 or 3 carbon-carbon double bonds. Unless defined otherwise, the alkenyl groups typically contain 2 to 10 carbon atoms. Examples of representative alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and the like.

"Alkynyl" may be linear or branched and refers to a monovalent unsaturated hydrocarbon group having one or more, and typically having 1, 2 or 3 carbon-carbon triple bonds. Unless defined otherwise, the alkynyl groups typically contain 2 to 10 carbon atoms. Examples of representative alkynyl groups include ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like.

"Aryl" refers to a monovalent aromatic hydrocarbon having a single ring (ie phenyl) or fused ring (ie naphthalene). Unless defined otherwise, the aryl groups typically have 6 to 10 carbon ring atoms. Examples of representative aryl groups include phenyl and naphthalen-1-yl, naphthalen-2-yl and the like.

"Arylene" refers to a divalent aromatic hydrocarbon having a single ring (ie phenylene) or fused ring (ie naphthalenediyl). Unless defined otherwise, the arylene group contains 6 to 10 carbon ring atoms. Examples of the representative arylene groups include 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, naphthalene-1,5-yl, naphthalene-2,7-diyl and the like.

"Aralkyl" refers to an aryl substituted alkyl group. Benzyl is mentioned as an exemplary alkyl group.

"Cycloalkyl" refers to a monovalent saturated carbocyclic hydrocarbon group having a single ring or fused ring. Unless defined otherwise, the cycloalkyl groups typically contain 3 to 10 carbon atoms. Examples of representative cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

"Cycloalkylene" refers to a divalent saturated carbocyclic hydrocarbon group having a single ring or fused ring. Unless defined otherwise, the cycloalkylene groups typically contain 3 to 10 carbon atoms. Examples of representative cycloalkylene groups are cyclopropane-1,2-diyl, cyclobutyl-1,2-diyl, cyclobutyl-1,3-diyl, cyclopentyl-1,2-diyl, cyclopentyl-1,3 -Diyl, cyclohexyl-1,2-diyl, cyclohexyl-1,3-diyl, cyclohexyl-1,4-diyl, etc. are mentioned.

"Heteroaryl" refers to a substituted or unsubstituted monovalent aromatic group having a single ring or fused ring and containing one or more heteroatoms (typically one to three heteroatoms) selected from nitrogen, oxygen, or sulfur in the ring. . Unless defined otherwise, the heteroaryl groups typically contain 5 to 10 total ring atoms. Examples of representative heteroaryl groups include pyrrole, imidazole, thiazole, oxazole, furan, thiophene, thiazole, pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, indole , Benzofuran, benzothiophene, benzimidazole, benzthiazole, quinoline, isoquinoline, quinazoline, quinooxaline and the like, and a monovalent kind, and the bonding position is any available carbon or nitrogen ring atom. .

"Heteroarylene" refers to a divalent aromatic group having a single ring or fused ring and containing one or more heteroatoms (typically 1-3 heteroatoms) selected from nitrogen, oxygen, or sulfur in the ring. Unless defined otherwise, the heteroarylene group typically contains 5 to 10 total ring atoms. Examples of typical heteroarylene groups include pyrrole, imidazole, thiazole, oxazole, furan, thiophene, thiazole, pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, Monovalent types such as indole, benzofuran, benzothiophene, benzimidazole, benzthiazole, quinoline, isoquinoline, quinazoline, quinooxaline and the like, and the bonding position is any available carbon or nitrogen ring atom. to be.

A "heterocyclyl" or "heterocyclic group" has a single ring or a plurality of fused rings and contains one or more heteroatoms (typically 1-3 heteroatoms) selected from nitrogen, oxygen, or sulfur in the ring. Refers to a substituted or unsubstituted monovalent substituted or unsubstituted (non-aromatic) group. Unless defined otherwise, the heterocyclic groups typically contain 2 to 9 total ring atoms. Examples of representative heterocyclic groups include pyrrolidine, morpholine, imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, thiomorpholine, piperazine, 3-pyrroline and the like. And the bonding position is any available carbon or nitrogen ring atom.

"Carbocycle" refers to an aromatic or non-aromatic ring where each atom in the ring is carbon. Representative carbocyclic rings include cyclohexane, cyclohexene, and benzene.

"Halo" or "halogen" refers to fluoro-(-F), chloro-(-Cl), bromo-(-Br), and iodo-(-I).

"Hydroxy" or "hydroxyl" refers to an -OH group.

"Alkoxy" refers to an -OR group, where R can be substituted or unsubstituted alkyl, alkylene, cycloalkyl, or cycloalkylene. Suitable substituents include halo, cyano, alkyl, amino, hydroxy, alkoxy, and amido. Examples of representative alkoxy groups include methoxy, ethoxy, isopropoxy, and trifluoromethoxy.

"Aryloxy" refers to an -OR group, where R is a substituted or unsubstituted aryl or heteroaryl group. Phenoxy is mentioned as a representative aryloxy group.

"Sulfonyl" refers to the group -S (O) ₂ -or -S (O) ₂ R, wherein R is alkyl, alkylene, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylene, heteroaryl, hetero Arylene, heterocyclic, or halogen. Examples of representative sulfonyl groups include sulfonates, sulfonamides, sulfonyl halides, and dipropylamine sulfonates.

"Condensation" refers to a reaction in which two or more molecules are covalently bonded. Likewise, condensation products are products formed by condensation reactions.

Detailed description of the invention

I. Introduction

The present invention provides the discovery that orphan receptor RDC1, referred to herein as CCX-CKR2, binds to chemokine ligands SDF-1 and I-TAC. The present invention also provides surprising findings that CCX-CKR2 is associated with cancer. Accordingly, the present invention provides a method of diagnosing cancer by detecting CCX-CKR2. The present invention also provides a method of inhibiting cancer by administering a modulator of CCX-CKR2 to a patient with cancer.

II . CCX - CKR2  Polypeptides and Polynucleotides

In many embodiments of the invention, nucleic acids encoding CCX-CKR2 polypeptides of interest will be isolated and replicated using recombinant methods. The above embodiments provide for CCX-CKR2 polynucleotides (eg, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9) for the expression of proteins or during the generation of variants, derivatives, expression cassettes. Or isolating or detecting CCX-CKR2 in different species to isolate other sequences derived from CCX-CKR2 polypeptide (eg, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10). In order to monitor CCX-CKR2 gene expression in order to be used for diagnostic purposes of the patient, for example to detect mutations in CCX-CKR2 or to detect expression of CCX-CKR2 nucleic acid or CCX-CKR2 polypeptide. In some embodiments, the sequence encoding CCX-CKR2 is effectively bound to a heterologous promoter. In some embodiments, the nucleic acid of the present invention is derived from any mammal, particularly humans, mice, mice, dogs, and the like.

In some cases, CCX-CKR2 polypeptides of the invention comprise extracellular amino acids of a human CCX-CKR2 sequence (eg, 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 modified or absent. In other embodiments, the CCX-CKR2 polypeptide comprises a ligand binding fragment of CCX-CKR2. For example, in some cases, the fragment binds to I-TAC and / or SDF-1. The structure of the seven transmembrane receptors (eg, CCX-CKR2) is well known to those skilled in the art and thus can easily determine the transmembrane domain. For example, readily available hydrophobic algorithms are available on the Internet in the G-Protein Linked Receptor Database (GPCRDB), for example http: // www. gpcr. org / 7tm / seq / DR / RDCl_HUMAN.TABDR.html or http: //www.gpcr. org / 7tm / seq / vis / swac / P25 1 06.html.

The present invention relies on general techniques in the field of recombinant genetics. As a basic document describing the general method used in the present invention, 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)).

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

III. Development of Specific Therapies

Molecules that bind CCX-CKR2, including CCX-CKR2 modulators, ie agonists, antagonists or substances of CCX-CKR2 activity, are useful for the treatment of diseases in many mammals, including cancer.

Diseases or symptoms of humans or other species that may be treated by antagonists of chemokine receptors or other inhibitors of chemokine receptor function include carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, Glioblastoma, leukemia, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell cancer, small cell cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, 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, retinoblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma ( for other cancers literature [cANCER: PRINCTPEES aND PRACTICE (. DeVita, VT et al eds 1997] Note); as Amira brain and nerve cell function disorders, such as Alzheimer's disease and multiple sclerosis; new 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, including but not limited to It doesn't happen.

Alternatively, agonists of CCX-CKR2 can be used to treat diseases such as kidney, brain or neuronal dysfunction, as well as when treated with stem cell recruitment.

A. Methods for Selecting Modulators of Chemokine Receptors

Numerous different selection protocols can be used to select substances that modulate the level of activity or action of CCX-CKR2 in cells, particularly mammalian cells, and especially human cells. In general, the screening method selects a plurality of substances to bind CCX-CKR2 and to allow ligands (eg, I-TAC and / or SDF-1) to bind to or activate CCX-CKR2. Blocking the selection of substances that interact with CCX-CKR2 (or its extracellular domain). In some embodiments, the substance binds to CCX-CKR2 with an affinity of about 1.5, 2, 3, 4, 5, 10, 20, 50, 100, 300, 500, or 1000 times greater than the affinity of the substance for another protein. do.

1. Analysis of Chemokine Receptor Binding

In some embodiments, the CCX-CKR2 modulator is selected by selecting a molecule that competes with a ligand of CCX-CKR2, such as SDF-1 or I-TAC. Those skilled in the art will appreciate that there are a number of ways to perform competitive assays. In some embodiments, a sample with CCX-CKR2 is preincubated with a labeled CCX-CKR2 ligand and then contacted with a potential competitor molecule. Changes in the amount of ligand bound to CCX-CKR2 (eg, disease) indicate that the molecule is a potential CCX-CKR2 modulator.

Preliminary screening can be performed by selecting a material that can bind to CCX-CKR2, since at least some of the selected material may be chemokine receptor modulators. Binding assays usually involve contacting CCX-CKR2 with one or more test substances and allowing the protein and test substance to form a binding complex for a sufficient time. Any binding complex 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 assays, europium labeled ligand binding assays, biotin labeled ligand binding assays, or other assays that maintain the conformation of CCX-CKR2. It is not. The chemokine receptors used in the assay can be naturally expressed, or can be cloned or synthesized. For example, it is possible to select those molecules that stimulate CCX-CKR2 activity by contacting CCX-CKR2 with a potential agonist and measuring CCX-CKR2 activity.

2. Cells and Reagents

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

Cell-based assays include whole cells or cell parts containing CCX-CKR2 to select substances that bind to or modulate the activity of CCX-CKR2. Representative cell types that can be used according to the methods of the invention include, for example, any mammalian cells such as neutrophils, monocytes, macrophages, eosinophils, basophils, mast cells, and lymphocytes such as T cells and B cells, leukemia Fungi, including Burkitt's lymphoma, tumor cells, endothelial cells, fibroblasts, heart cells, muscle cells, breast tumor cells, ovarian cancer carcinoma, cervical carcinoma, glioblastoma, liver cells, kidney cells, and nerve cells, as well as yeast Cell. The cells may be primary cells, tumor cells or other types of immortal cell lines. Of course, CCX-CKR2 can be expressed in cells that do not express the endogenous form of CCX-CKR2.

In some cases, protein fusion as well as fragments of CCX-CKR2 can be used for selection. If a molecule that competes to bind a CCX-CKR2 ligand is desired, the CCX-CKR2 fragment used is a fragment capable of binding a ligand (eg, binding to I-TAC or SDF-1). Alternatively, fragments of CCX-CKR2 can be used as targets for selecting molecules that bind to CCX-CKR2. CCX-CKR2 fragments may include any fragment of a protein containing all amino acids except for example 20, 30, 40, 50 amino acids to 1 amino acid of CCX-CKR2. Typically, the ligand binding fragment will comprise most or all of the transmembrane region of CCX-CKR2 and / or the extracellular domain of CCX-CKR2.

3. Signaling active

In some embodiments, the signaling caused by CCX-CKR2 activity is used to select CCX-CKR2 modulators. The signaling activity of chemokine receptors can measure a number of methods. For example, signaling can be measured by detecting chemokine receptor mediated cell adhesion. Interactions between chemokines and chemokine receptors can lead to rapid attachment through regulation of integrin affinity and binding capacity (see, eg, Laudanna, Immunological Reviews 186: 37-46 (2002)).

Signaling can also be measured by quantitatively and qualitatively measuring messengers such as cyclic AMP or inositol phosphate, as well as monitoring phosphorylation or dephosphorylation [eg, Premack, et. al . Nature Medicine 2 : 1174-1178 (1996) and Bokoch, Blood 86: 1649-1660 (1995).

The examples provide results demonstrating that CCX-CKR2 activity is mediated by the MAPK pathway, specifically that CCX-CKR2 promotes phosphorylation of MARK proteins ERK1 and ERK2. A representative unique sequence ERK1 is provided in GenBank Accession No, p27361; Representative native sequence ERK2 is provided in GenBank Accession No, p28482. Thus, some assays are designed to detect signals in the MAPK pathway. The term "signal" as used for the MAPK pathway refers to components that become part of the pathway and / or some activity or expression associated with the pathway. The component can be any molecule included in (eg, produced, used or modified) a MAPK signal transduction pathway. In some cases the signal is to detect phosphorylation of MAPK proteins such as EPK1 or EPK2. Other signals that can be detected include components formed and / or used downstream of phosphorylation of ERK1 or ERK2, as well as activities associated with the formation and use of these components. Some assays may be performed to detect components formed and / or used upstream of phosphorylation of ERK1 or ERK2, as well as activities associated with the production or use of such upstream components. As indicated above, for ERK1 or ERK2, the upstream component may be, for example, Ras, MKKK (eg c-Raf1, B-Raf, and A-Raf) and MKK (eg, MKK1 and MKK2). Downstream components include, for example, proteins p90RSK, c-JUN, c-FOS CREB and STAT. Thus, each of these components can be detected in certain assays and / or modifications (eg, phosphorylation) of these components. Gene expression profiling and protein secretion can also be detected.

Some screening assays provided include detecting phosphorylation of ERK1 and / or ERK2. Phosphorylation of these proteins depends on ligands (eg SDF-1, ITAC). Thus, analysis in certain screening methods is performed in the presence of ITAC or SDF-1 to promote phosphorylation of ERK1 and ERK2. When assays are performed with ITAC and SDF-1, this assay can be simplified using cells that do not express CXCR2 or CXFR4 for the reasons described above.

Various methods can be used to determine whether ERK1 or ERK2 is phosphorylated. One method is to lyse the cells after incubating the cells with the CCX-CKR2 ligand and the test substance. The resulting lysate is then analyzed by Western blot by electrophoretic separation of the lysate into a separate protein on the gel and then probing the gel with an antibody that specifically binds to the phosphorylated form of ERK1 or ERK2. can do. The antibody is available from Cell Signaling Technologies, Beverly, Massachusetts. The total amount of ERK1 or ERK2 present can be optionally determined using antibodies that specifically bind to both phosphorylated and non-phosphorylated forms of ERK1 and ERK2. Further details about the method are described in the Examples. Another approach suitable for high efficiency screening is to use an ELISA method using an antibody that specifically binds to phosphorylated forms of ERK1 and ERK2. Kits for performing the assays in a high efficiency format may also be purchased from Cell Signaling Technologies, Beverly, Mass. (See, eg, PathScan Phospho- p44 / 42 MAPK (T202 / Y204) Sandwich ELISA Kit).

Similar western blotting and ELISA techniques can be used to detect the presence or absence of proteins that are components of the MAPK pathway downstream of the phosphorylation of ERK1 and ERK2, wherein the antibodies specifically capable of recognizing components contained in this pathway Use For example, phosphorylation of p90RSK can be assessed using commercially available phosphorus-specific antibodies, similar to Western detection of phosphorylated ERK1 or ERK2.

In addition, other events occurring downstream of CCX-CKR2 activation may be monitored to measure signaling activity. Downstream events include those activities or expressions that occur as a result of stimulation of chemokine receptors. Representative downstream events include, for example, the state of charge of a cell (eg, from normal cells to cancer cells or from cancer cells to non-cancer cells). An established signaling cascade (eg, VEGF-mediated signaling) involved in angiogenesis can also be monitored for consequences caused by CCX-CKR2 modulators. For example, chorionic uremic analysis (CAM) can be used to analyze the results at vasculature, or, for example, the Miles assay can be used to study the effect on vasculature permeability.

In another embodiment, cell survival can be measured instead of CCX-CKR2 activity. As described in more detail in the Examples, the expression of CCX-CKR2 is a cell expressing CCX-CKR2 that has been propagated in a low serum environment as compared to cells that do not express CCX-CKR2, which have been expanded under the same environment Prolong your survival. Thus, antagonism of CCX-CKR2 is expected to reduce cell survival, while activity (eg, via agonists) is said to increase cell survival. As a result, cell survival and apoptosis can act as readout information for CCX-CKR2 activity.

A wide variety of cell death assays and apoptosis assays can be introduced as screening methods for selecting CCX-CKR2 modulators. In general, this type of assay usually involves exposing the cell population to conditions that induce cell death or apoptosis with or without test compounds that are potential regulators of cell death or apoptosis. An assay is then performed with the cells, or extracts thereof, to analyze how the test substance affects cell death or apoptosis by comparing the range of cell death or apoptosis with or without the test substance. Instead of assaying for cell death or apoptosis, the opposite type of assay, ie, for cell survival, can be performed as well as for related activities such as cell growth and cell proliferation. Regardless of the particular type of assay, some assays are performed in the presence of a ligand that activates CCX-CKR2, such as I-TAC or SDF-1.

Various different parameters that characterize cell death or apoptosis can be analyzed for the selection methods of the invention. Non-limiting examples of such parameters include monitoring the activity of cell pathways for toxic responses by gene or protein expression analysis, DNA fragmentation, changes in cell membrane composition, membrane permeability, death receptors or components of downstream signaling pathways (eg For example, activation of caspase), general stress response, activation of NF-kappa B and response to mitogen.

Another method that takes into account the role that CCX-CKR2 plays in reducing apoptosis is to perform an assay for the reverse of apoptosis and cell death, i.e., a screening method that detects cell survival or cell proliferation. Cell survival can be measured, for example, by monitoring the length of time a cell is alive, the length of time a certain percentage of the original cell survives, or an increase in the number of cells. These parameters can be monitored visually using established techniques.

Another assay to assess apoptosis involves labeling cells with Annexin V (conjugated to Alexa Fluor (r) 488 pigment) and propidium iodide (PI) (Eugene Oregon, molecular probe). PI, a nucleic acid binding pigment of red fluorescence, is impermeable to both living and apoptotic cells. PI labels only necrotic cells by tightly binding to nucleic acid in the cell. Annexin V utilizes apoptosis cells to transfer phosphatylserine (PS) to the outer surface of the cell. Annexin V is a human anticoagulant with high affinity for (PS). Apoptotic cells that are not living cells express PS on their outer surface. Annexin V (labeled with Alexa Fluor (r) 488 pigment) labels these cells with green fluorescence. The cells were then analyzed on fluorescence activated cell separator (FACS) to analyze fluorescence in the red and green channels; Apoptotic cells (positive for Annexin, negative for PI) only fluoresce in the green channel; Living cells (negative to Annexin, positive to PI) showed low fluorescence in both red and green channels; Necrosis or dead cells (positive for Annexin, negative for PI) showed strong fluorescence in both red and green channels.

Other selection methods are based on the observation that expression of certain regulatory proteins is induced by the presence or activation of CCX-CKR2. Thus, the detection of these proteins can be used to indirectly measure the activity of CCX-CKR2. As described in more detail in the Examples below, a series of ELISA assays are performed to compare the relative concentrations of various secreted proteins in cells transfected with CCX-CKR2 and non-transfected cells in cell media. These studies have shown that CCX-CKR2 induces the production of numerous different regulatory proteins including growth factors, chemokines, metalloproteinases and inhibitors of metalloproteinases. Thus, some selection methods provided include revealing whether the test substance modulates the production of specific growth factors, chemokines, metalloproteinases and inhibitors of metalloproteinases by CCX-CKR2. In some cases, the assay is performed using cells (or extracts thereof) grown under restrictive serum conditions found to increase production of CCX-CKR2 derived proteins (see Examples).

The following proteins are examples of the various types of proteins detected, as well as specific proteins belonging to each class: (1) growth factors (eg, GM-CSF); (2) chemokines (eg, RANTES, MCP-1); (3) metalloproteinases (eg, MMP3); And (4) inhibitors of metalloproteinases (eg TIMP-1). It is predicted that other proteins belonging to these various kinds can also be detected.

These specific proteins can be detected using standard immune detection methods known to those skilled in the art. One method suitable for use in high efficiency formats is, for example, ELISA performed in multiple well plates. An ELISA kit for detecting TIMP-1 can be purchased from Dakositometry (product number EL513). Further examples of suppliers of antibodies that specifically bind to the proteins set forth above are provided below. Proteins such as metalloproteinases, which are enzymes, can also be detected by known enzyme assays.

In other embodiments, the ability to modulate cell adhesion is tested for potential modulators of CCX-CKR2. Adherence of tumor cells to endothelial cell monolayers has been studied as a model of metastasis invasion (see, eg, Blood and Zetter, Biovhem. Biophys. Acta, 1032, 89-119 (1990)). Monolayers mimic the lymphatic vascular system and can be stimulated by various cytokines and growth factors (ie, TNFalpha and IL-beta) The ability of cells expressing CCX-CKR2 to adhere to these monolayers is not only a static attachment assay but also a cell Can be evaluated both in assays under flow conditions that mimic the force of the vascular system in vivo, and tests to assess adhesion can also be performed in vivo (see, eg, von Andrian, UH Microcirculatiorm . 3 ( 3): 287-300 (1996)).

4. Validation

Further testing may be performed to ascertain whether the substance originally identified by any of the aforementioned screening methods has apparent activity. Preferably such studies are carried out using suitable animal models. The basic format of this method is the step of administering the lead compound identified during the initial screening to the animal serving as a model for humans and then whether the disease (eg cancer) is actually controlled and / or the disease or symptom is improved. Determining the step. Animal models used in validation studies are generally any kind of mammal. Specific examples of suitable animals include, but are not limited to, primates, mice, rats and zebrafish.

B. Substances that interact with CCX-CKR2

Modulators (eg, antagonists or agonists) of CCX-CKR2 include, for example, antibodies (including monoclonal binding proteins, humanized binding proteins or other types of binding proteins known in the prior art), small organic molecules. , siRNA, CCX-CKR2 polypeptide or variants thereof, chemokines (including but not limited to SDF-1 and / or I-TAC), chemokine mimetics, chemokine polypeptides, and the like.

Test substances as modulators of CCX-CKR2 can be any small compound, or biological moiety such as a polypeptide, sugar, nucleic acid or lipid. Alternatively, the modulator may be in the form of a genetically modified, peptidomimetic form of chemokine or other ligand. Typically, the test compound will be small chemical molecules and peptides. While essentially any compound may be used as a potential modulator or ligand in the assays of the present invention, compounds are used that are mostly soluble in aqueous or organic (especially DMSO based) solutions. Assays automate the assay steps and allow screening of large chemical libraries by providing compounds from any convenient source, typically in concurrently run assays (eg, microliter formats on microliter plates in assays using robots). Devise There are a number of suppliers of compounds including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), and others. Will understand.

In some embodiments, the material has a molecular weight of less than 1,500 Daltons, in some cases less than 1,000, 800, 600, 500, or 400 Daltons. Relatively small sized materials may be desirable for small molecules, as they are more likely to have good pharmacokinetic and affinity physicochemical properties, including oral absorption, than large molecular weight materials. For example, substances less likely to be successful as drugs on the basis of permeability and solubility have more than 5 H-binding donors (expressed as the sum of OH and NH) by Lipinski et al .; Has a molecular weight greater than 500; Have more than 5 LogPs (or more than 4.15 MLogPs); It is described as having more than 10 H-binding receptors (expressed as the sum of N and S). The class of compounds that are substrates for biological carriers is usually an exception to this rule.

In one embodiment, high efficiency screening methods include providing a combinatorial chemistry or peptide library containing a number of potential therapeutic compounds (potential modulators or ligand compounds). The "combination chemistry library" or "ligand library" is then screened in one or more assays as described above to select library members (especially chemical species or subtypes) that exhibit certain characteristic activities. Accordingly, the selected compounds can act as conventional "lead compounds" or can themselves be used as potential therapeutic or actual therapeutic agents.

A combinatorial chemical library is a collection of various compounds produced by chemical or biological synthesis, by combining multiple chemical “building blocks”. For example, linear combinatorial chemical libraries, such as polypeptide libraries, are formed by binding a set of chemical building blocks (amino acids) in all possible ways for a given compound length (ie, the number of amino acids in a polypeptide compound). Combinational mixing of the chemical building blocks enables the synthesis of millions of compounds.

Methods of making and screening combinatorial chemical libraries are well known to those skilled in the art. Such combinatorial chemistry libraries include peptide libraries (see, eg, US Pat . No. 5,010,175, Furka, Inert . J. Pept . Prot . Res . 37: 487-493 (1991)) and Houghton et al., Nature 354: 84-88 (1991)]. Other chemical structures can also be used that produce chemically diverse libraries. Such chemical structures may include peptoids (eg, PCT Publication WO 91/19735), encoded peptides (eg, PCT Publication WO 93/20242), random bio-oligomers (eg, PCT Publication WO 92/00091), benzodiazepines (eg, US Pat. No. 5,288,514), diversomers such as hydantoin, 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)), non-peptide peptidomimetics with glucose scaffolding (Hirschmann et al ., J. Amer . Chem . Soc . 114: 9217-9218 (1992), analogous organic compounds of small compound libraries (Chen et al ., J. Amer. Chem . Soc . 116: 2661 (1994)), oligocarmates (Cho et al ., Science 261: 1303 (1993)), and / or peptidyl phosphonate (Campbell et al ., J. Org . Chem . 59: 658 (1994)), nucleic acid libraries (see Berger and Sambrook, all homologs), peptide nucleic acid libraries (see, eg, US Pat. No. 5,593,083), antibody libraries (eg, Vaughn et al. , Nature I Biotechnology , 14 (3): 309-314 (1996) and PCT / US96 / 10287), carbohydrate libraries (eg, Liang et. al ., Science , 274: 1520-1522 (1996) and US Pat. No. 5,593,853), small organic molecular libraries (eg, benzodiazepines [Baum C & EN, Jan 18, page 33 (1993)]; isoprenoids (US Patent 5,569,588); thiazolidinone and metathiazanone (US Pat. No. 5,549,974); pyrrolidine (US Pat. Nos. 5,525,735 and 5,519,134); morpholino compounds (US Pat. No. 5,506,337); benzodiazepines (US Pat. No. 5,288,514, etc.) It is not limited.

Devices for the production of combinatorial libraries are commercially available (e.g., 357 MPS and 390 MPS from Advanced Chemical Technologies, Kentucky; Symphony, Wynn, Mass .; Symphony, Reynin, CA; 433A Applied Bio, Foster City, CA). System; see 9050 Plus in Massachusetts, Bedford, Millipore). Many combinatorial libraries themselves are also commercially available (e.g., New Jersey, Princeton's Comgenex; Missouri, St. Louis' Triforce Inc .; Pennsylvania, Exton's 3D Pharmaceuticals; Maryland, Columbia) See Martech Bioscience, etc.).

CCX7923 (eg, FIG. 4) is commercially available, which is N- [3- (dimethylamino) propyl] -N, N-dimethyl-1,3-propanediamine and bromo by methods known in the art. It can be prepared by condensation of methyl-bicyclo (2,2,1) -hept-2-ene. CCX0803 (eg, FIG. 4) is commercially available, which is 3- (2-bromoethyl) -5-phenylmethoxy-indole and 2,4,6-triphenylpyridine by methods known in the art. Can be prepared by condensation. See, eg, Organic Function Group Preparations , 2nd Ed. Vol. 1, (SR Sandler & W. Karo 1983); [ Handbook of Heterocyclic Chemistry (AR Katritzky, 1985); [ Encyclopedia of Chemical Technology , 4th Ed. (JI Kroschwitz, 1996).

In one embodiment, the active compound of the present invention (ie, CCX-CKR2 modulator) has formula (I):

Where

m is an integer from 1 to 5, and each Y substituting the benzyl ring is hydrogen, alkyl, halo substituted alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkylene, halogen, heterocyclic, aryl , Arylene, heteroaryl, heteroarylene, hydroxy, alkoxy, and aryloxy, independently selected from the group consisting of

n is 0, 1, 2 or 3;

Z is -CH- or -N-;

R ¹ and R ² are each independently alkyl or hydrogen,

Or Z together with R ¹ and R ² form a 5 or 6 membered ring comprising at least one nitrogen and at least one additional heteroatom, wherein said 5-6 membered ring is alkyl, alkenyl, phenyl, benzyl Optionally and independently substituted with one or more moieties selected from the group consisting of sulfonyl and substituted heteroatoms,

R ³ , R ⁴ , and R ⁵ are each hydrogen, alkyl, halo substituted alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkylene, heterocyclic, aryl, arylene, heteroaryl, heteroaryl Independently selected from the group consisting of ene, hydroxy, alkoxy, and aryloxy;

R ⁶ is alkyl or hydrogen;

Provided that when Z is nitrogen and R ¹ and R ² together with Z form a morpholinyl group, n is 3 and at least one of R ³ , R ⁴ , and R ⁵ is hydroxy, alkoxy, or aryloxy ego; or

Provided that when n is 1, Z is carbon and R ¹ and R ² are not —CH _{2 C} H ₂ NCH ₂ CH ₂ —; or

Provided that when R ¹ and R ² together are —CH (CH ₃ ) (CH ₂ ) ₄ —, then Z is —CH—; or

Provided that when R ⁵ is t-butyl, R ³ is hydrogen; or

When R ⁴ and R ⁵ form a five membered ring, at least one atom bonded to the phenyl ring is carbon. See US Provisional Application No. 60 / 434,912 (December 20, 2002) and US Provisional Application No. 60 / 516,151 (December 20, 2003).

Wavy bonds linking an olefin to a substituted phenyl ring mean that the ring can be cis or trans for R 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 Formula I, wherein R ⁶ is hydrogen. In further embodiments, preferred compounds have Formula I, wherein R ⁶ is methyl.

In another embodiment, preferred compounds have Formula I, wherein R ³ , R ⁴ , and R ⁵ are independently hydrogen, hydroxy, alkyl, alkoxy, aryloxy, and alkyl substituted with halo. More preferably, R ³ , R ⁴ , and R ⁵ are independently alkoxy or hydrogen. In another embodiment, preferred compounds have Formula I, wherein R ⁴ is hydrogen and R ³ and R ⁵ are alkoxy including trifluoroalkoxy groups such as trifluoromethoxy and (—CH ₂ CF ₃ ) . In further embodiments, R ³ is hydrogen and R ⁴ and R ⁵ are alkoxy. In these two embodiments, the alkoxy group can be methoxy (-OCH ₃ ) or ethoxy (-OCH ₂ CH ₃ ).

In another embodiment, preferred compounds have Formula I, wherein R ⁴ and R ⁵ together form a heterocyclic ring, an aryl ring or a heteroaryl ring. In another preferred embodiment, R ³ is hydrogen and R ⁴ and R ⁵ together are —O (CH ₂ ) ₃ O—, — (CH ₂ ) ₄ —, or —N (CH) ₂ N—.

In another embodiment, preferred compounds have Formula I, wherein Z is nitrogen and Z together with R ¹ and R ² form a heteroaryl or heterocyclic group. In a preferred embodiment, the compound has formula I, wherein Z is CH and Z and R ¹ and R ² form a heteroaryl or heterocyclic group. More preferred compounds have formula I, wherein Z is CH and Z together with R ¹ and R ² form a heterocyclic group containing nitrogen. In a further embodiment, Z forms together with R ¹ and R ² a substituted or unsubstituted morpholinyl, pyrrolidinyl, piperidinyl, or piperazinyl group.

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

In one embodiment, preferred compounds have Formula I, wherein Z is an alkyl- or methoxy-methyl-substituted pyrrolidinyl group with R ¹ and R ² ; Substituted heteroatoms substituted with benzyl-, phenyl-, methyl-, ethyl-, or piperidinyl groups; Or benzyl-, phenyl-, or sulfonyl-substituted piperazinyl groups. Particularly preferred substituted heteroatom groups include alkyl, aminyl, cycloalkyl aminyl, alkyl aminyl, cyclopropyl aminyl, isopropyl aminyl, benzyl aminyl, and phenoxy. Preferably, the substituted heteroatom is at position 3 of the piperidinyl ring.

In another embodiment, preferred compounds have Formula I, wherein Z is combined with R ¹ and R ²

to be.

Preferred compounds having the formula (I) can also have Z as a nitrogen atom and can have R ¹ and R ² as alkyl groups or methyl groups, respectively, or R ¹ and R ² together can be -C (C (O) N ( CH ₃ ) ₂ ) (CH ₂ ) ₃ −.

In another embodiment, Z together with R ¹ and R ² form a May ring comprising nitrogen and optionally one or more additional heteroatoms. In the above embodiment, n is preferably 1 and Z is preferably -CH-. In a particularly preferred embodiment of this type, Z is combined with R ¹ and R ²

Wherein R ⁷ is preferably hydrogen, alkyl, aryl or aralkyl.

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

In another embodiment, the active compounds of the invention have formula II:

In the above formula,

m is an integer of 1-5,

Each Y substituting benzyl ring is hydrogen, alkyl, halo substituted alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkylene, halogen, heterocyclic, aryl, arylene, heteroaryl, heteroaryl Independently selected from the group consisting of ene, hydroxy and alkoxy,

n is 0, 1, 2 or 3;

R ³ , R ⁴ , and R ⁵ are each halogen, alkyl, halo substituted alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkylene, heterocyclic, aryl, arylene, heteroaryl, heteroaryl Independently from lene, hydroxy, alkoxy, and aryloxy.

As in Formula I above, wavy bonds linking olefins to substituted phenyl rings means that the rings may be cis or trans.

In another embodiment, preferred compounds can have Formula II, wherein n is 3. In another embodiment, preferred compounds may have Formula II, wherein R ³ , R ⁴ , and R ⁵ are substituted as described in Formula I above. Particularly preferred compounds may have formula II, wherein R ³ , R ⁴ , and R ⁵ are alkoxy or methoxy.

A number of synthetic routes known to those skilled in the art can be used to synthesize the active compounds of the present invention, and general synthetic methods are shown in Scheme I below:

In Scheme I, aldehyde (2) is condensed with primary amine (3) via reduction amidation. Suitable primary amines are commercially available, for example, from Miluki Aldrich, Wisconsin, and can be synthesized by synthetic routes known to those skilled in the art.

The amidation reaction can be performed with a reducing agent in any suitable solvent, including but not limited to tetrahydrofuran (THF), dichloromethane, or methanol to form intermediates. As a non-limiting example of a suitable reducing agent for the condensation reaction, sodium cyanoborohydride (Mattson, et. al ., J. Org . Chem . 1990, 55 , 2552 and Barney, et. al ., Tetrahedron Lett. 1990, 31 , 5547); Sodium triacetoxyborohydride (Abdel-Magid, et al ., Tetrahedron Lett . 31: 5595 (1990)); Sodium borohydride (as described by Gribble; Nutaitis Synthesis . 709 (1987)); Pentacarbonyl iron (I) and alcoholic KOH (Watabane, et. al ., Tetrahedron Lett . 1879; (1974)); And BH ₃ -pyridine (Pelter, et. al ., J. Chem . Soc , Perkin Trans . 1: 717 (1984)).

The reaction for converting intermediate (4) to compound (5) can be carried out in a suitable solvent such as tetrahydrofuran or dichloromethane with a suitable substituted acyl chloride in the presence of a base. Tertiary amine bases are preferred. Particularly preferred bases include triethylamine and Hunnings base.

Alternatively, the reaction of converting intermediate (4) to compound (5) may also be carried out in the presence of a catalyst such as 4,4-dimethylamino-pyridine or hydroxybenzotriazole (K. Horiki, Synth. Commun . 7 : 251), in the presence of a suitable coupling reagent such as 1-ethyl-3- (3-dimethylbutylpropyl) carbodiimide or dicyclohexyl-carbodiimide (B. Neises and W Steglich, Angew. Chem ., Int . Ed. Ethel. 17: 522 (1978)).

To demonstrate that the compounds described above are useful antagonists for SDF-1 and I-TAC chemokines, the compounds were selected in vitro to determine their ability to substitute SDF-1 and I-TAC from the CCX-CKR2 receptor at various concentrations. do. The compound is combined with mammalian glandular cells expressing the CCX-CKR2 receptor in the presence of ¹²⁵ I-labeled SDF-1 and / or ¹²⁵ II-TAC chemokines. Thereafter, the ability of the compound to substitute labeled SDF-1 or I-TAC from the CCX-CKR2 receptor site at various concentrations is measured by a selection process.

Compounds considered to be effective SDF-1 and I-TAC antagonists include at least 50% SDF-1 from the CCX-CKR2 receptor at concentrations below 1.1 micromolar (μM) and more preferably at concentrations below 300 nanomolar (nM) and And / or I-TAC chemokine. In some cases, it is desirable for the compound to be capable of replacing at least 50% of SDF-1 and / or I-TAC from the CCX-CKR2 receptor at concentrations of 200 nM or less. Representative compounds that meet these criteria are shown in Table 1 below.

TABLE 1

C. Solid Phase and Soluble High Efficiency Analysis

The high efficiency assays of the present invention can screen up to thousands of different regulators or ligands per day. Specifically, each well of the microtiter plate can be used to perform an independent assay for selected potential regulators, or if a single regulator is to be tested every 5-10 wells, if one intends to observe the effect of concentration or incubation time Can be. Thus, a single standard microtiter plate can analyze about 100 (eg 96) modulators. If a 1536 well plate is used, a single plate can analyze about 100 to about 1,500 different compounds. It is possible to analyze several different plates per day, and with the integrated system of the present invention it is possible to carry out assay screening for up to about 6,000 to 20,000 different compounds. More recently, microfluidics have been developed for reagent manipulation.

The present invention provides in vitro assays for identifying compounds capable of modulating the function or activity of CCX-CKR2 in a high throughput format. Since the assay is very constant, a control response that measures the CCX-CKR2 activity of the cells in a response that does not include a potential modulator is optional. However, the selective control response increases the reliability of the assay.

In some assays, it will be desirable to have a positive control to confirm that the analytical component is working properly. At least two types of positive controls are appropriate. First, a known active agent or ligand of CCX-CKR2 can be incubated with one assay sample, resulting in amplification of the signal from the increased activity of CCX-CKR2 (eg, as measured according to the methods herein). do. Secondly, inhibitors or antagonists of CCX-CKR2 can be added, and as a result, a decrease in the signal on the activity of the chemokine receptor can be observed similarly. Modulators may also be combined with activators or inhibitors to find modulators that inhibit the increase or decrease caused by the presence of known modulators of chemokine receptors.

IV . In the object CCX - CKR2 Expression of

In some embodiments, CCX-CKR2 is expressed in a subject thus increasing expression of CCX-CKR2. Alternatively, inhibitory polynucleotides, including for example siRNA or antisense sequences, can be expressed in vitro or in vivo to inhibit the expression of CCX-CKR2. In some cases, polynucleotides encoding CCX-CKR2 are introduced into cells in vitro, which cells are then introduced into the patient. In some of these cases, cells are first isolated from the patient, and then polynucleotides are introduced and then reintroduced to the patient. In another embodiment, the polynucleotide encoding CCX-CKR2 is introduced directly into a cell of a patient in vivo.

In some cases, the CCX-CKR2 coding polypeptide is introduced into a cell from (i) the tissue of interest, (ii) exogenous cells introduced into the tissue, or (ii) surrounding cells not present inside the tissue. In some embodiments, polynucleotides of the invention are introduced into endothelial cells. The tissue to which endothelial cells will bind is any tissue in which it is desirable to promote migration or expansion of the endothelial cells.

Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids encoding the processed polypeptides of the invention into mammalian cells or target cells. The method can be used to administer a nucleic acid encoding a polypeptide of the invention (eg, CCX-CKR2) to an in vitro cell. In some embodiments, the nucleic acid encoding a polypeptide of the invention is administered for in vivo or ex vivo gene therapy use. Non-viral vector delivery systems include nucleic acids complexed with delivery media such as DNA plasmids, naked nucleic acids, and liposomes. Viral vector delivery systems include DNA and RNA viruses with episomal or integrated genomes after being delivered to cells. For a review of gene therapy methods, see, eg, Science 256: 808-813 (1992); Nabel & Feigner, TIBTECH 11: 211 217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357: 455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); [Vigne, Restorative Neurology and Neuroscience 8: 35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); Hadada et al ., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); And Yu et al ., Gene Therapy 1: 13-26 (1994).

Non-viral delivery methods of nucleic acids encoding processed polypeptides of the invention include lipofection, microinjection, gene gun, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, Artificial virions, and substance-enhanced absorption of DNA. Lipofections are described, for example, in US 5,049,368; US 4,946,787; And US 4,897,355, and lipofection reagents are commercially available (eg, Transfectam® and Lipofectin®). Suitable cationic and neutral lipids for effective receptor-cognitive lipofection of polynucleotides include the lipids of Felgner of WO 91/17424, WO91 / 16024. Delivery to cells (ex vivo administration) or target tissue (in vivo administration).

Methods for the preparation of lipid: nucleic acid complexes, such as immunolipids, comprising target liposomes are known in the prior art (see, eg, Crystal, Science 270: 404-410 (1995); Blaese et. al ., Cancer Gene Ther . 2: 291-297 (1995); Behr et al ., Biocorjugate Chew . 5: 382- 389 (1994); Remy et al ., Bioconjugate Chem . 5: 647 654 (1994); Gao et al ., Gene Therapy 2: 710-722 (1995); Ahmad et al ., Cancer Res . 52: 4817 4820 (1992); US Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, and 4,946,787).

The use of RNA or DNA viral systems for the delivery of nucleic acids encoding processed polypeptides of the present invention uses highly developed methods of targeting viruses to specific cells in the body and transporting viral loads into the nucleus. The viral vector can be administered directly to the subject (in vivo) or can be used to process the cells in vitro and the modified cells can be administered to the patient (ex vivo). Typical viral system systems for delivering polypeptides of the invention include vectors of retroviruses, lentiviruses, adenoviruses, adenovirus dependent viruses and simple herpes viruses for gene delivery. Viral vectors are currently the most effective and versatile method of delivering genes in target cells and tissues. Integration with retrovirus, lentiviral, and adenovirus dependent viral gene delivery methods in the host genome is possible, often resulting in long term expression of the inserted transgene. In addition, high effects of transduction have been found in many different cell types and target tissues.

The affinity of retroviruses can be changed by introducing foreign envelope proteins and expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transforming or infecting non-dividing cells and typically produce high viral titers. Thus, the selection of retroviral gene delivery systems depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with packaging capacity for foreign sequences up to 6-10 kb. Minimal cis-acting LTR is sufficient for replication and packaging of the vector, which vector is then used to integrate the therapeutic gene into the target cell so that the transgene is permanently expressed. Widely used retroviral vectors include bovine leukemia virus (MuLV), gibbon leukemia virus (GaLV), monkey immunodeficiency pyrus (SIV), human immunodeficiency virus (HIV), and combinations thereof (e.g., For example, Buchscher et al ., J. Virol. 66: 2731-2739 (1992); Johann et al ., J. Virol . 66: 1635-1640 (1992); Sommerfelt et al ., Virol . 176: 58-59 (1990); Wilson e t al ., J. Virol . 63: 2374-2378 (1989); Miller et al ., J. Virol. 65.2220-2224 (1991); See PCT / US94 / 05700)

Where transient expression of the polypeptides of the invention is desired, it is common to use adenovirus-based systems. Adenovirus-based vectors have very high transformation effects in many cell types and do not require cell division. With this vector, high expression titers and levels have been achieved. The vector can be generated in large quantities in a relatively simple system. Adenovirus dependent virus (“AAV”) vectors are also used, for example, for in vitro production of nucleic acids and peptides, and for transforming cells into target nucleic acids for in vivo and ex vivo gene therapy methods (eg For example, West et al ., Virology 160: 38-47 (1987); US Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5: 793-801 (1994); Muzyczka, J. Clin . Invest . 94.1351 (1994)]. Construction of recombinant AAV vectors has been described in a number of publications, such as US Pat. No. 5,173,414; Tratschin et al ., Mol . Cell . Biol . 5: 3251-3260 (1985); Tratschin, et al ., Mol . Cell . Biol . 4: 2072-2081 (1984); Hermonat & Muzyczka, PNAS 81: 6466-6470 (1984); And Samulski et al ., J. Virol . 63: 03822-3828 (1989).

pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et. al ., Blood 85: 3048-305 (1995); Kohn et al ., Nat . Med . 1: 1017-102 (1995); Malech et al ., PNAS 94:22 12133-12138 (1997). PA317 / pLASN was the first therapeutic vector used in gene therapy attempts (Blaese et. al ., Science 270: 475-480 (1995). Over 50% transformation efficiency was observed for MFG-S packaged vectors (Ellem et al. al ., Immunol Imrmunother . 44 (1): 10-20 (1997); Dranoff et al ., Hum . Gene Ther . 1: 111-2 (1997).

Recombinant adenovirus dependent virus vectors (rAAV) are promising alternative gene delivery systems based on defective and nonpathogenic parvovirus adeno dependent type 2 viruses. All vectors are derived from plasmids bearing only the AAV 145 base pair inverted terminal repeats adjacent to the transgene expression cassette. Effective gene delivery and stable transgene delivery through integration into the genome of transformed cells are key characteristics of the vector system (Wagner et. al ., Lances 351: 9117 1702 3 (1998); Kearns et al ., Gene Ther . 9: 748-55 (1996)).

Replication deficient recombinant adenovirus vectors (Ad) have a transgene replacing the Ad E1a, E1b, and E2 genes; Replication deficient vectors can then be engineered to proliferate in human 293 cells supplying the missing gene function in trans. Ad vectors can transform many types of in vivo tissues, including non-dividing differentiated cells, such as those found in liver, kidney and muscular tissues. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in clinical trials is the treatment of polynucleotides by intramuscular injection for anti-tumor immunization (Sterman et al. al ., Hum . Gene Iher . 7: 1083-9 (1998). Further examples of the use of adenovirus vectors for gene delivery in clinical trials are described in Rosenecker et. al ., Infection 24: 1 5-10 (1996); Sterman et al ., Hum . Gene Ther . 9: 7 1083 1089 (1998); Welsh et al ., Hum . Gene Iher . 2: 205-18 (1995); Alvarez et al ., Hum . Gene Ther . 5: 597-613 (1997); Topf et al ., Gene Iher . 5: 507-513 (1998); Sterman et al ., Hum . Gene Her . 7: 1083-1089 (1998).

Packaging cells are used to form viral particles that can infect host cells. Examples of the cells include 293 cells packaging adenoviruses, and ψ2 cells or PA317 cells packaging retroviruses. Viral vectors used in gene therapy are usually produced by producer cell lines that package nucleic acid vectors into viral particles. Such vectors typically contain the minimum viral sequence necessary for packaging and subsequent integration into the host and other viral sequences to be replaced by the expression cassette for the protein to be expressed. The lack of virus function is supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically have only ITR sequences in the AAV genome required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, where the cell line contains another AAP gene, rep And a helper plasmid encoding cap but without the ITR sequence. The cell line is also infected by the helper adenovirus. The helper virus promotes replication of the AAV vector and expression of the AAV gene from the helper plasmid. The helper plasmid is not packaged in significant amounts due to the absence of ITR sequences. Contamination by adenoviruses can be reduced, for example, by heat treatment in which adenoviruses are less sensitive than AAV.

For many gene therapy applications, it is desirable for gene therapy vectors to be highly specific for specific tissue types. Viral vectors are modified to have specificity for a given cell type by expressing ligands as fusion proteins with viral envelope proteins on the outer surface of the virus. The ligand is chosen to have an affinity for a receptor known to be present on the cell type of interest. For example, Han et al . , PNAS 92: 9747-9751 (1995)], the moroni murine leukemia virus can be modified to express human hergululin fused to gp70, and the recombinant virus can express specific human breast cancer cells expressing human epidermal growth factor receptor. It is reported to infect. The principle can also be applied to other pairs of viruses expressing ligand fusion proteins and target cells expressing receptors. For example, filamentous phage can be engineered to show antibody fragments (eg, FAB or Fv) that have specific binding affinity for substantially all selected cell receptors. While the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. The vector can be engineered to contain a specific acquisition sequence that is thought to favor acquisition by a particular target cell.

Gene therapy vectors are typically delivered in vivo by administration to systemic administration (e.g., by intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or by topical administration as described above. Can be. Alternatively, the vector is delivered to ex vivo cells, such as explanted cells (eg, lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells from individual patients, usually after selection of the cells into which the vector has been introduced. The cells are then transplanted to the patient.

In vitro cell transfection (eg, via re-perfection of a transfected cell into a host organism) for diagnosis, investigation, or gene therapy is well known to those skilled in the art. In a preferred embodiment, the cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA) encoding a polypeptide of the invention and re-perfused back to the subject organism (eg, patient). Various cell types suitable for ex vivo transfection are well known to those skilled in the art (see, eg, Freshney et. al ., Culture of Animal Cells , A Manual of Basic Technique (3rd ed. 1994)), cited above describes a method for isolating and culturing cells from a patient.

In one embodiment, the stem cells are used in ex vivo methods for cell transfection and gene therapy. The advantage of using stem cells is that they can be differentiated into other in vitro cell types, or they can be introduced into mammals (such as donors of cells) and engraft in bone marrow. Methods of differentiating in vitro CD34 + cells into clinically important immune cell types using cytokines such as GM-CSF, IFN-γ and TNF-α are known (eg, Inaba et al. al ., J. Exp . Med . 176: 1693-1702 (1992).

Stem cells are isolated using known methods for transformation and differentiation. For example, stem cells are combined with antibodies that bind bone marrow cells to unwanted cells, such as CD4 + and CD8 + (T cells), CD45 + (panB cells), GR-1 (granulocytes) and Iad (differentiated antibody donor cells). Isolation from bone marrow cells by panning (Inaba et al. al ., J. Exp . Med . 176: 1693-1702 (1992).

Therapeutic nucleic acids containing vectors (eg retroviruses, adenoviruses, liposomes, etc.) are also administered directly to the organism for transformation of cells in vivo. Alternatively, naked DNA is administered. Administration is by any route conventionally used for the final contact of a molecule with blood or tissue cells. Methods suitable for the administration of such nucleic acids are available and well known to those skilled in the art, and although one or more routes may be used to administer a particular composition, certain routes are often faster and more effective than other routes.

Pharmaceutically acceptable carriers can be determined, in part, by the particular composition being administered, as well as by the particular method of administration of the composition. Thus, as described below, there are a wide variety of materials suitable for the pharmaceutical compositions of the present invention (eg, Remington's Pharmaceutical Sciences , 17th ed., 1989).

V. Diagnosis and Prognosis Judgment

The present invention provides a method for detecting cancer cells, including a method for diagnosis or prognosis of cancer. As described herein, CCX-CKR2 is expressed in almost all cancer cells tested to date, whereas normal (non-cancer) expression of CCX-CKR2 is limited to specific stages of development between the kidney and some brain cells and the fetus. Seems to be. Thus, expression of CCX-CKR2 in cells, particularly non-fetal cells and / or cells other than renal cells or brain cells, indicates the presence of cancer cells. In some cases, for samples containing CCX-CKR2 expressing cells, other methods known in the art are used to confirm the presence of cancer cells.

According to another embodiment of the present invention, a method for screening a treatment course of a patient with or suspected of having cancer is provided. The method obtains a biological sample from the patient, contacts the sample with an antibody or antigen-binding fragment thereof that specifically binds CCX-CKR2, detects the presence or absence of antibody binding, and selects a course of treatment appropriate for the cancer of the patient. It involves doing. In some embodiments, the treatment is administering a CCX-CKR2 antagonist to the patient.

Detection methods using substances that bind proteins are well known and include, for example, various immunoassays, flow cytometry, and the like. Flow cytometry can be used to identify cells that express a particular antigen of interest in a mixed cell population. Briefly, cells are allowed to react with antibodies specific for the protein of interest (eg, CCX-CKR2). The antibody is fluorescently labeled (direct staining) or, if not labeled, fluorescently labels a second antibody that reacts with the first antibody (indirect staining). Thereafter, the cells are passed through a tool capable of detecting the fluorescent signal. The cells are aspirated and made into a single cell suspension. The cell suspension is now passed through a laser to excite fluorochrome labeled antibodies that bind to the cells to obtain data. Brightly observed cells (ie, cells that react with fluorescently labeled antibodies) express the protein of interest; Darkly observed cells (ie, cells that do not react with fluorescently labeled antibodies) do not express the protein of interest.

The invention includes carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, prostate cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell cancer, Small cell cancer, esophageal cancer, stomach cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penis cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, Adrenal cancer, interest endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (see CANCER: PRINCTPEES AND PRACTICE (DeVita, VT et. al . eds 1997); As well as Amira 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 methods of diagnosing human diseases, including but not limited to the diseases described herein. In some embodiments, the patient does not have Kaposi's sarcoma, multiple Castleman's disease or AIDS-related primary exudative lymphoma. As described herein, including the examples, normal cells and tissues and disease cells and tissues can be distinguished based on responsiveness to anti-CCX-CKR2 monoclonal antibodies or SDF-1 and I-TAC. For example, cancer cells can be detected by detecting chemokine receptors on the cell where SDF-1α and I-TAC compete for binding.

In addition, ligand binding differences between chemokine receptors can be detected, and the differences can detect cells expressing CCX-CKR2. For example, no chemokine receptor has both SDF-1 and I-TAC as ligands. Chemokine binding can be detected using a tissue sample (eg, biopsy), or directly monitored in the tissue in situ (eg, using radiolabeled chemokine imaging).

In addition, immunoassays can be used to qualitatively or quantitatively analyze CCX-CKR2. A general overview of applicable techniques is described in Harlow & Lane, Antibodies: A Laboratory Manual (1988). Alternatively, non-antibody molecules with affinity for CCX-CKR2 can be used to detect the receptor.

Methods for preparing polyclonal antibodies and monoclonal antibodies that specifically react with a protein of interest are known to those of skill in the art (see, eg, 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 MilsteinNature , 256: 495-497 (1975). The technique involves preparing an antibody by selecting the antibody from a library of recombinant antibodies that are phage or similar vectors. For example, to prepare antisera for use in immunoassays, proteins of interest or antigen fragments thereof are isolated as described herein. For example, recombinant proteins are prepared in transformed cell lines. Crossbreeding strains of mice, mice, guinea pigs or rabbits are immunized with proteins using standard adjuvant such as Freund's adjuvant, and standard immunization protocol. Alternatively, synthetic peptides derived from the sequences disclosed herein and conjugated to a carrier protein can be used as immunogens. A further alternative is to use as a antigen a cell expressing a protein or membrane fragment or liposome comprising CCX-CKR2 or a fragment thereof. Thereafter, the antibodies generated against cells, membrane fragments or liposomes are selected according to their ability to bind proteins.

Polyclonal serum is collected and titrated against the immunogen in an immunoassay, eg, a solid phase immunoassay with an immunogen immobilized on a solid support. Polyclonal antisera having a titer of at least 10 ⁴ are selected and tested for their cross-reactivity to different, and sometimes similar proteins, using competitive binding immunoassays. Certain monoclonal and polyclonal antibodies and antisera are usually present in CCX-CKR2 with a K _{D of} at least about 0.1 mM, more typically at least about 1 μM, preferably at least about 0.1 μM, and most preferably at least 0.01 μM. Will combine.

For the preparation of antibodies, for example recombinant antibodies, monoclonal antibodies or polyclonal antibodies, a number of techniques known in the art can be used. See, eg, 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, Cuf rent Protocols in Immcuology (1991); Harlow & Lane, Antibodies , A; Laboratory Manual (1988); And Goding, Monoclonal Antibodies : Principles and Practice (2d ed. 1986)]. Genes encoding the heavy and light chains of the antibody of interest can be cloned from the cell, for example, a gene encoding a monoclonal antibody can be used to clone from hybridomas to generate recombinant monoclonal antibodies. have. In addition, gene libraries encoding the heavy and light chains of monoclonal antibodies can be prepared from hybridoma or plasma cells. Random combinations of light and heavy chain genes produce a large pool of antibodies with different antigen specificities (see, eg, Kuby, Immunology (3rd ed. 1997)). Techniques for preparing single chain antibodies or recombinant antibodies (US Pat. No. 4,946,778, US Pat. No. 4,816,567) can be modified to produce antibodies against the polypeptides of the invention. In addition, transgenic mice, or other organisms, such as other mammals, can be used to express humanized or human antibodies (eg, US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 No., 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 expression techniques are used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (eg, McCafferty et. al ., Nature 348: 552-554 (1990); Marks et al ., Biotechnology 10: 779-783 (1992). Antibodies can also be made bispecific, ie can recognize two different antigens (eg, WO 93/08829, Traunecker et. al ., EMBO J. 10: 3655-3659 (1991); And Suresh et al., Methods in Enzymology 121: 210 (1986)). The antibody may be a heteroconjugate, for example two covalently linked antibodies, or immunotoxins (see, eg, US 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 prior art. Such antibodies are useful for both detection and therapeutic use. In general, humanized antibodies have one or more amino acid residues introduced thereto from a non-human source. These non-human amino residues are often referred to as import residues that are commonly taken from the influx variable domain. Humanization is essentially Winter's and colleague's method (eg, Jones et al. 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 replacing a corresponding sequence of a human antibody with a murine CDR or CDR sequence. Thus, the humanized antibody is a chimeric antibody (US Pat. No. 4,816,567), wherein substantially fewer sites than complete human variable regions are replaced by corresponding sequences of the non-human kind. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted with residues from analogous sites of murine antibodies.

IV . Treatment, Methods of Administration and Pharmaceutical Compositions

CCX-CKR2 modulators (eg, antagonists or agonists) can be administered directly to a mammalian subject for the regulation of chemokine receptor signaling in vivo. In some embodiments, the modulator competes with SDF-1 and / or I-TAC, to bind CCX-CKR2. Modulation of CCX-CKR2 may include, for example, regulation of antibodies (including monoclonal antibodies, humanized antibodies or other types of binding proteins known in the art), small organic molecules, siRNAs and the like.

In some embodiments, the CCX-CKR2 modulator is administered to a patient with cancer. In some cases, the CCX-CKR2 modulator is a cancer, for example, carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, prostate cancer, and Burkitt's lymphoma, Head and neck cancer, colon cancer, colorectal cancer, non-small cell cancer, small cell cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, 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, interest endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (CANCER: PRINCTPEES AND PRACTICE DeVita, VT et al . eds 1997); As well as Amira 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 the diseases described herein. In some cases, the patient does not have Kaposi's sarcoma, multiple Castleman's disease or AIDS-related primary exudative lymphoma. Since CCX-CKR2 is often expressed in cancer cells rather than non-cancer cells, it is desirable to administer the antagonist of CCX-CKR2 to patients with cancer. In some cases, modulators have a molecular weight of less than 1,500 Daltons, and in some cases have a molecular weight of less than 1,000, 800, 600, 500, or 400 Daltons.

Administration of the modulator can be carried out by any route conventionally used to finally introduce the modulator compound into the tissue to be administered, which is well known to those skilled in the art. Although one or more routes can be used to administer a particular composition, certain routes can often provide a faster and more effective response than another route.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be determined, in part, by the particular composition being administered, as well as by the particular method of administration of the composition. Thus, there are a wide variety of materials suitable for the pharmaceutical compositions of the present invention (eg, Remington's Pharmaceutical Sciences , 17th ed., 1989).

Modulators (eg, agonists or antagonists) of expression or activity of CCX-CKR2 may be prepared by aerosol substances (ie, they are “sprayable”) alone or in combination with other suitable ingredients and administered via inhalation. Aerosol materials can be encased in acceptable pressure propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Aqueous or non-aqueous solutions as materials suitable for administration; Isotonic sterile solutions that may contain antioxidants, buffers, bacteriostatics, and solutes that impart isotonicity to the material; And sterile aqueous and non-aqueous sterile suspensions which may include suspending agents, stabilizers, thickening agents, stabilizers, and preservatives. In the practice of the present invention, the composition may be administered orally, nasal, topical, intravenous, intraperitoneal, or intradural, for example. The compound material may be provided in sealed containers for unit or multiple administration, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Modulators can be administered as part of the prepared food or drug.

In some embodiments, the CCX-CKR2 modulators of the invention can be administered in conjunction with other suitable therapeutic agents such as chemotherapeutic agents, radiation, and the like. The selection of materials suitable for use in combination therapy can be selected by those skilled in the art according to conventional pharmaceutical principles. The combination of therapeutic agents will have a synergistic effect in the treatment or prevention of various disorders such as cancer, kidney dysfunction, brain dysfunction or neuronal dysfunction. Using this method, the possibility of side effects can be reduced by achieving a therapeutic effect with a small dosage of each substance.

In the context of the present invention, the dosage administered to a patient should be an amount sufficient to produce a beneficial response to the patient over a period of time (eg, to reduce tumor size or tumor load). The optimal level of administration for any patient will depend on various factors such as the efficacy of the particular modulator used, age, weight, physical activity, and the patient's diet, possible combinations with other drugs, and the severity of the particular disease. In addition, the level of administration will be determined by the presence, nature, and extent of side effects associated with the administration of a particular compound or vector in a particular patient.

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

For administration, the 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 in use, relative to the patient's mass and overall health. Administration can be via single or divided doses.

VII . Composition, Kit , Integrated systems and Proteomics  Applications

The present invention provides compositions, kits, and integrated systems for carrying out the assays disclosed herein using other materials that specifically detect CCX-CKR2 or CCX-CKR2.

The present invention provides an analytical composition for use in solid phase analysis; Such compositions include, for example, CCX-CKR2 polypeptides as part of cells, membrane fractions or liposomes immobilized on a solid support [eg, Babcok et. al ., J. Biol . Chem . 276 (42): 38433-40 (2001); Mirzabekov et al ., Nat . Biotechnol . 18 (6): 649-54 (2000)], and labeling reagents. In each case, the assay composition may also include additional substances desirable for hybridization. For example, the solid support can be a Petri plate, a multi well plate or a microarray. In addition, microarrays of peptide libraries can be used to identify peptide sequences that specifically bind to CCX-CKR2.

In addition, substances that specifically bind to CCX-CKR2 may be included in the assay composition. For example, antibodies that specifically bind to CCX-CKR2 can be immobilized on a solid support. In some of these embodiments, the substance is used to detect the presence or absence of cells expressing CCX-CKR2 or CCX-CKR2. For example, the solid support can be a Petri plate, a multi well plate or a microarray.

The present invention also provides a kit for carrying out the assay of the present invention. Kits typically include a substance (eg, an antibody or other small molecule) that specifically binds to CCX-CKR2 and a label for detecting the presence of the substance. The kit may comprise one or more chemokine receptor polypeptides. The kit may comprise any of the above-mentioned compositions, and in some cases, instructions for conducting a high efficiency assay for the effect on the activity or function of the chemokine receptor, one or more containers or compartments (eg, probes, labels, etc.) To control the function or activity of the chemokine receptor, a robot armature for mixing the kit components, and the like.

In some embodiments, the kit comprises SDF-1 and / or I-TAC. In some embodiments, the kit comprises a labeled or tagged SDF-1 and an unlabeled competitor I-TAC, or alternatively a labeled or tagged I-TAC and an unlabeled competitor SDF-1. do. Labeled or tagged chemokines can be labeled or tagged by any method known in the art. In some embodiments, labeled chemokines are tagged or radiolabeled with biotin or fluorescent labels. Alternatively or in parallel, the kit may comprise an anti-I-TAC binding agent (eg, an antibody) for detecting I-TAC. The kit may also include suitable salt buffers and other reagents for conducting competitive binding assays on intact cells or cell membranes. Such reagents are disclosed, for example, in the Examples below. In some aspects, the kit also includes a receptacle or solid support (eg, plate form for reactions suitable for scintillation counters or automated plate readers) that measure ligand binding to CCX-CKR2. In some aspects, the kit includes instructions for using the kit, such as in the method of the present invention.

The present invention also provides an integrated system for screening potential modulators at high throughput for effects on the activity or function of potential CCX-CKR2 modulators. The system typically includes a substrate or reaction comprising a robotic electron that transfers fluid from a source to a destination, a controller that controls the robotic electron, a label detector, a data storage unit that records the label detection, and an immobilized nucleic acid or immobilized portion. Analytical components such as microtiter dishes containing wells with mixtures.

An optical image viewed by a camera or other recording device (e.g., photodiode and data storage device) (e.g., recorded in some cases) is disclosed herein, for example by digitizing the image and storing and analyzing the image on a computer. In any embodiment optionally further processing. Various commercially available peripherals and software are available for digitizing, storing and analyzing digitalized video or digitalized optical images.

Example  One

This example shows that SDF-1 and I-TAC compete to bind new chemokine receptors.

Materials and methods

Reagents and Cells

Human, viral and murine recombinant chemokines were obtained from designated R & D Systems (Minneapolis, Minnesota) and Peprotech (Rocky Hill, NJ). ¹²⁵ I-labeled SDF-1a was purchased from Perkin Elmer Life Sciences Inc. (Boston, Massachusetts), and ¹²⁵ I-labeled I-TAC was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). It was. Monoclonal antibodies used for flow cytometry and ligand binding competition were obtained from R & D Systems, Minneapolis, Minnesota: anti-CXCR4 clones 12G5, 44708.111 (171), 44716.111 (172), 44717.111 (173), nmIgG2a, and nmIgG2b. Antibody binding was detected by flow cytometry using a secondary antibody goat anti-mouse IgG PE conjugate (Culter Immunotech, Miami, FL). The following cells were obtained from the American P. aureus culture collection (Manassas, VA): MCF-7 (adenocarcinoma; mammary gland), MDA MB-231 (adenocarcinoma; mammary gland), MDA MB-435s (mammary cancer; mammary gland), DU 4475 (Wired), ZR 75-1 (mammary carcinoma; mammary gland) and HEK 293 (human belly kidney), HUV-EC0-C (human navel vein; vasculature endothelial; normal). CEM-NKr (acute lymphocytic leukemia: peripheral blood; T lymphocytic) cells were obtained from the NIH AIDS Research and Reference Reagent program. Cell lines were incubated in DMEM (Meditech, Herdon, Va.) Supplemented with 10% Fetal Bovine Serum (FBS) (Hyclones, Logan, Utah) at 37 ° C. in a humidified thermostat of 5% CO ₂ / air mixture. Human peripheral blood monocytes (PBMCs) were obtained by centrifugation with a Ficoll-Hypaque density gradient from the leukocyte soft layer of a healthy donor (Stanford Blood Center, Palo Alto, CA). The isolated PBMCs were 2.5 μg / ml Phytohemagglutinin (PHA) in RPMI-1640 (Miditech, Herndon, VA) supplemented with 10% FBS at 37 ° C. in a humidified thermostat of 5% CO ₂ / air mixture. Sigma Chemical Co., St. Louis) and lOng / ml recombinant human IL-2 (Al and D Systems, Minneapolis, Minn.) Were activated for 3 days. After activation, cells were washed, cultured in RPMI supplemented with 10% FBS and 10 ng / ml IL-2 and fed every 3-4 days until the day the cells were used.

Binding analysis

"DisplaceMax" was used to investigate the overall profile of the interaction of chemokine ligands with SDF-1 receptor receptor MCF-7 and CEM-NKr cells. This method utilizes extended ligand binding to maximize the efficacy using the filtration protocol as described above (Dairaghi, et. al . J Biol Chem 274: 21569-74 (1999); Gosling, J. et al . J Immunol 164: 2851-6 (2000)). In these assays, DisplaceMax ™ is MCF-7 with at least 110 distinct purified chemokines for the ability to replace ¹²⁵ I radiolabeled SDF-1α or I-TAC as presented using the protocol described above. Or simultaneous questioning of CEN-NKr cells was used (Dairaghi, et. al . J Biol Chem 274: 21569-74 (1999); Gosling, J. et al . J Immnunol 164: 2851-6 (2000)). In brief, the chemokine components were incubated with the cells and then 3 hours in binding medium (25 mM HEPES, 140 mM NaCl, 1 mM CaCl ₂ , 5 mM MgCl ₂ and 0.2% bovine serum albumin, adjusted to pH 7.1). Radiolabeled chemokines ( ¹²⁵ I SDF-1a or ¹²⁵ I h I-TAC) were added at 4 ° C. during. Small molecules were included in some assays when indicated. In these assays compounds were added to the plates at the indicated concentrations followed by radiolabeled chemokines. All analytes were then incubated at 4 ° C. for 3 hours with gentle stirring. After incubation in all binding assays, the reaction was aspirated with PEI-treated GF / B glass filters (Packard) using a cell harvester (Packard) and washed twice (25 mM HEPES, 500 mM NaCl, 1). mM CaCl ₂ , 5 mM MgCl ₂ , adjusted to pH 7.1). Scintillant (MicroScint 10, Packard) was added to the wells and the filters were counted on a Packard Top Count Scintillation Counter. Data was analyzed and plotted using a prism (GraphPad Prism version 3.Oa for Macintosh, GraphPad software).

¹²⁵ I SDF -1α receptor binding measurement

Using the filter system assay described above, cells were preincubated with 1) buffer alone, 2) excess SDF-1β (final 90 nM) or 3) MIG (final 175 nM) as directed for 30 minutes at 4 ° C. It was. After incubation, the designated chemokine competitor and ¹²⁵ I h I-TAC, not at the stated concentrations, were added to the binding reaction. All analytes were incubated, harvested and analyzed as described above.

RT PCR

MRNA was isolated from the cells using standard methods. Complementary DNA was analyzed for expression of CXCR3 and CXCR4 by PCR. Specific primers were obtained from Integrated DNA Technologies, Coralville, Iowa. Specific PCR products were measured with Hybaid Omn-E (E & K Scientific Products, Inc., Saratoga, Calif.) For 35 cycles. GAPDH was measured as a control.

Adhesion analysis

HUVEC cells were grown overnight on tissue culture treated slides in the presence of TNFα (25 ng / ml) and IFNγ (50 ng / ml). The following day, wild type controls as well as NSO transfected CCX-CKR2 cells were labeled with calcein-AM. Calcein labeled cells were then cultured on endothelial monolayers with or without CCX-CKR2 antagonist (CCX3451). The slides were incubated at 37 ° C. for 40 minutes and then washed with PBS to remove unattached cells. Attached NSO cells were visualized by fluorescence microscopy. Cells treated with the compound or vehicle were visually counted at three viewing angles (fov) and indicated in the coordinates.

result

Recent reports have confirmed CXCR4 expression in some tumor cell types (Sehgal, et. al ., J Surg Oncol 69: 99-104 (1998); Sehgal, A., et al . J Surg Oncol 69: 239-48 (1998); Burger, et al . Blood 94: 3658-67 (1999); Rempel, et al. Clin Cancer Res 6: 102-11 (2000); Koshiba, T. et al . Clin Cancer Res 6: 3530-5 (2000); Muller, A. et al . Nature 410: 50-6 (2001); Robledo, et al . J Biol Chem 276: 45098-45105 (2001)), in some instances it has been found that this expression is associated with metastasis of breast tumor cells (Muller, A. et. al . Nature 410: 50-6 (2001). To further investigate the role of chemokine receptors on tumor cells, expression of CXCR4 was evaluated in several human breast tumor cell lines. Initially, CXCR4 expression patterns were assessed by flow cytometry. Primary IL-2 cultured T lymphocytes and two T cell lines (ie CEM-NKr and Jurkat) were examined to determine anti-CXCR4 staining of the T cell phenotype. Three breast tumor cell lines, MCF-7, MDA MB-231 and MDA MB-435s, were also tested (FIG. 1A). All four anti-CXCR4 clones tested stained T cells. Surprisingly, breast tumor cells have been reported to express CXCR4, but no widely used clone 12G5 detected any CXCR4 on breast tumor cells. Weak and variable reactivity was detected by three different clones tested on breast tumor cell lines. The assay also tested breast tumor cell lines DU 4475 and ZR 75-1 (data not shown) and was found to have an antibody staining profile similar to the other breast tumor cells tested. Thus, the staining pattern of the mAb panel for CXCR4 is characterized by two distinct types of reactivity: the “leukocyte” CXCR4 phenotype (exemplified by CEM-NKr, Jurkat and IL-2 lymphocyte staining) and the breast tumor cell phenotype (MCF). -7 and MDA MB-231 breast tumor cell lines).

Consistent lack of reactivity with the most widely used anti-CXCR4 mAb, clone 12G5, on breast tumor cells allows CPCCR4 expression to be investigated in these cells by RT PCR. MRNA was isolated from IL-2 cultured lymphocytes and T cell lines as positive controls for CXCR4 expression, namely CEM-NKr and Jurkat as well as three breast tumor cell lines tested in flow cytometry. The breast tumor cell lines MCF-7 and MDA MB-231 did not express the CXCR4 message, although there was no reactivity with 12G5 and variable reactivity with other anti-CXCR4 clones tested. However, MDA MB-435s was found to be negative for CXCR4 expression. In all cases, GAPDH was measured as a control. To investigate whether the difference in mAb reactivity can result in epitope changes in CXCR4 on various cell lines due to sequence differences, PCR products generated from MCF-7 as representative CXCR4 + breast tumor cells and CEM-NKr as representative T cells were sequenced. Decided. The sequences obtained from these two cell lines were identical to the published CXCR4 sequences, suggesting that despite the different CXCR4 antibody profiles, the gene structure and thus polypeptide structure of CXCR4 in both cell types is identical.

We have reported a set of techniques that allow simultaneous analysis of receptor binding to a comprehensive array of chemokine ligands (Dairaghi, et al. J Biol Chem 274: 21569-74 (1999); Gosling, J. et al. J lmmunol 164: 2851-6 (2000)). In this way, CXCR4 binding profiles on CEM-NKr were identified as compared to MCF-7 cells. More than 90 chemokine components were tested for the ability to replace the signal chemokine ¹²⁵ I SDF-1α for binding to CEM-NKr (FIG. 1) or MCF-7 cells (FIG. 1). As expected, potential high affinity competitors of ¹²⁵ I SDF-1α on CEM-NKr include hSDF-1β and mSDF-1, while hSDF-1α and HHV8 vMIP-II exhibit a potential moderate affinity competition. . This is consistent with the results of SDF-1 already reported as the only nonviral ligand for CXCR4. However, the overall pattern of competition on MCF-7 cells was significantly different. In this cell type hI-TAC and mI-TAC showed high affinity competition for the same signal ligand SDF-1. To further investigate this particular result, ¹²⁵ I I-TAC was tested as signal ligand on MCF-7 cells (FIG. 2). The high affinity substitution profile with ¹²⁵ I I-TAC on MCF-7 was identical to the profile obtained with ¹²⁵ I SDF-1α. Thus, I-TAC and SDF-1 on MCF-7 cells act indistinguishable in binding and competition for the same receptor site.

To further characterize the binding of I-TAC and SDF-1, dose response curves were obtained in competitive binding experiments with potential high affinity ligands selected on CEM-NKr and MCF-7. As suggested by DisplaceMax ™ data, I-TAC competes with ¹²⁵ I SDF-1α for binding to MCF-7, but not for CEM-NKr (FIG. 2). Homologous competition between ¹²⁵ I SDF-α and SDF-1 isoforms, SDF-1α or SDF-1β, results in complete competition in CEM-NKr and MCF-7 (FIG. 2). In particular, the affinity of SDF-1 for receptors expressed on MCF-7 is higher than that for receptors expressed on CEM-NKr. Thus, the sequence of CXCR4 is identical in both cell types, but the ligand binding specificity and affinity is different on T cells versus breast tumor cells.

Since CXCR3 has long been established as a major receptor for I-TAC, we investigated whether I-TAC binding detected on MCF-7 cells is CXCR3-mediated (Cole, KE et al. al . J Exp Med 187: 2009-21. (1998). To this end, it inhibits 'classic' CXCR3-mediated binding (ie, the reported CXCR3 ligands MIG, I-TAC and IP-10 to CXCR3), thereby inhibiting 'classic' CXCR4-mediated binding (ie, the reported CXCR4 ligand SDF- ¹²⁵ I I-TAC binding was tested under conditions and conversion conditions that allow for binding to CXCR4 of 1. MCF-7 cells were preincubated with medium alone, medium containing excess MIG (˜175 nM; inhibiting CXCR3 mediated binding) or medium containing excess SDF-1β (˜90 nM; inhibiting CXCR4 mediated binding) It was. I-TAC competed with ¹²⁵ I I-TAC for 1 nM IC50 for binding to MCF-7 cells (FIG. 3), confirming that I-TAC is a high affinity ligand for this receptor in these cells. Let it be. Similarly, cells pre-incubated with excess MIG first gave the same cognate I-TAC / ¹²⁵ I I-TAC binding curves with an IC50 of 1 nM (FIG. 3). However, when cells were first pretreated with excess SDF-1β, all ¹²⁵ I I-TAC binding was inhibited (FIG. 3), indicating that ¹²⁵ I I-TAC binding observed in breast tumor cells was expressed on these cells. Suggest that it is mediated by the SDF-1 receptor. ¹²⁵ I I-TAC binding to MCF-7 cells was not inhibited when IP-10 was tested as an unlabeled chemokine competitor. In addition, preincubation with excess MIG did not show this binding profile. However, preincubation of cells with SDF-1β completely inhibited ¹²⁵ I I-TAC binding. When the CXCR4 ligand MIG was tested as an unlabeled competitor, ¹²⁵ I I-TAC binding to cells was not inhibited (FIG. 3). As expected from the DisplaceMax ™ data presented in FIG. 1, SDF-1β competed with ¹²⁵ I I-TAC for binding to these cells with high affinity (IC 50 1 nM). Pretreatment of cells with excess MIG did not result in SDF-1β / ¹²⁵ I I-TAC competition, suggesting that the detected binding is not mediated by CXCR3.

This hypothesis was further investigated by PCR. Previously used isolated mRNA (see above) was used to explore evidence of CXCR3 transcripts. IL-2 cultured lymphocytes express CXCR3, while other cells tested did not express CXCR3. The absence of detection of CXCR3 expression by RT PCR supports the data in FIG. 3 and suggests that I-TAC binding is not CXCR3 mediated on MCF-7 cells.

Changes in anti-CXCR4 antibody reactivity, as well as changes in ligand binding specificity and affinity, mean that the receptor is not classical CXCR4. While some 'orphan' chemokine receptors have been identified, no chemokine ligands have been found. We considered several orphan receptors, one of which is called RDC1 (hereafter referred to as CCX-CKR2). When the protein sequence for RDC1 is transfected with MDA MB 435s (cell line that does not endogenously express CXCR4, CXCR3 or CCX-CKR2), the characteristics of the ligand binding phenotype are reproduced (FIG. 4). CCX-CKR2 expressed in MDA MB 435s binds to radiolabeled SDF-1. Unlabeled competitors SDF-1 and I-TAC compete for this binding.

To target the receptor as a low molecular weight organic compound (SMC) therapeutic, one is to assess the CXCR4-mediated leukocyte SDF-1 binding phenotype, and the other is to investigate the CCX-CKR2-mediated breast cancer SDF-1 binding phenotype. Small molecules (nearly 135,000) were screened using two high efficiency screenings to do. The results of these screenings show that a clear pharmacological distinction of the two binding phenotypes is possible (FIG. 5). For example, a small molecule designated CCX0803 competes with ¹²⁵ I SDF-1α for binding to MCF-7 with an IC50 value of 46 nM, while these small molecules have no binding of ¹²⁵ I SDF-1α to CEM-NKr. Do not suppress In contrast, CCX7923, a different small molecule antagonist, inhibits the binding of ¹²⁵ I SDF-1α to CEM-NKr with an IC50 value of 106 nM, whereas it does not inhibit the binding of ¹²⁵ I SDF-1α to MCF-7 cells (FIG. 5). ). These two compounds show a distinct and clear pattern of ligands inhibiting non-mutually binding to both receptors (breast cancer versus leukocytes).

Further determination was made after determining that breast cancer cells exhibit binding affinity for SDF-1 that is distinct from that shown for other non-tumor or non-cancerous tissues. These phenotypic investigations (using methods of antibody reactivity, ligand binding profiles, and pharmacological differences, see the methods described herein) provide for binding affinity (e.g., in which many cancer (or tumor) cell types are initially correlated with breast cancer cells) , Antibody reactivity, ligand binding and pharmacological differences), and thus clearly express CCX-CKR2. The following tumor cells were tested and showed binding affinity associated with cancer: human ovarian carcinoma, human cervical adenocarcinoma, human Burkitt's lymphoma, human breast carcinoma, human ductal carcinoma, human glioblastoma, and mouse carcinoma.

Tumors and other cancers are difficult to process in part because of their rapid growth rate. In this respect, tumors are known to share some growth characteristics with rapidly dividing early embryonic tissues. Some schools suggest that adult tumors represent 'returning mutants' for the embryonic growth phenotype. SDF-1 and CXCR4 gene knockout mice died in embryonic state, suggesting that ligand receptor pairs are important components for growth and development. About 50% of homozygous mutant SDF-1 folds died at 18.5 days of perinatal phase; The remaining homozygous litters died within one hour of birth (Kishimoto, et. al . Nature 382: 635-638 (1996)). Similarly, ˜1 / 3 of homozygous CXCR4 knockout mice died of perinatal E18.5 days (Ma, et. al . Proc . Natl . Acad. Sci . USA 95: 9448-9453). Defects in lymphocyte formation and hematopoietic action were observed in receptor and ligand knockout. Fetal liver is a major site of hematopoietic activity in mice on day 11 and continues to hematopoietic until 1 week of birth. Therefore, the expression of CCX-CKR2 in this compartment was determined. We investigated the expression of CCX-CKR2 in wild-type mouse embryos at E17 (point of occurrence near the knockout animal death) and E13 (apart from the death of the nutout animal, but after the onset of hematopoiesis). .

In SDF-1 binding assays, radiolabeled human-SDF-1 binds to E13 fetal liver cells and SDF and I-TAC (mouse and human proteins) can compete with radiolabeled tracers for binding. Altered ligand specificity, as exemplified by the binding of I-TAC to the SDF-1 receptor, is a hallmark of the binding phenotype first associated with cancer cells and now demonstrated as CCX-CKR2. In addition, CCX-CKR2 antagonists may compete with the binding of SDF-1 to E13 fetuses, while CXCR4 antagonists do not.

Later in development, E17 fetal liver cells express CXCR4, but these cells migrate intracellular calcium to respond to SDF-1. CXCR4 antagonists inhibit this SDF-1 mediated calcium migration, but CCX-CKR2 antagonists do not exhibit such effects. Thus, these data suggest that wild type fetal liver cells express both CXCR4 at E13 and E17, while CCX-CKR2 is expressed early (E11) but not at later time points (E15).

Although binding studies in an embryonic mouse model correlate with human study data, preliminary trials using mice with targeted disruption of the CXCR4 gene showed that SDF-1 and I-TAC observed in fetal liver cells at embryonic day 13 (E13). Indicates that the binding profile did not change. This further demonstrates that the gene encoding the polypeptide having cancer related SDF-1 binding affinity is not CXCR4.

In addition, experiments have demonstrated that the CCX-CKR2 receptor can provide a stimulus signal to growing tumor cells. Tumor cells may upregulate certain genes involved in transcription or cell cycle in response to SDF-1 stimulation. More importantly, apoptosis (cell death) begins to progress when tumor cells lack serum in the medium overnight. Supplementing these cultures with the addition of SDF-1 allows cell recovery from asa as compared to untreated controls. Thus, SDF-1 acts as an antiapoptotic signal. Cancer cells are often characterized as cells that have lost their ability to progress apoptosis.

Example  2

This example demonstrates that the binding phenotype associated with cancer, described in Example 1, is mediated by CCX-CKR2 (formerly known as orphan receptor RDC1).

In general, CCX-CKR2 is preferentially expressed in transformed cells. As shown in Table 1 (left column), the various different cancer cells tested were positive for the expression of CCX-CKR2. In contrast, most normal (non-tumor) cells do not express CCX-CKR2 (see right column of Table 1).

TABLE 1

CCX-CKR2 positive CCX-CKR2 voice Human Breast Carcinoma (MCF-7, MDA MB 361) Human Glioblastoma (T98G) Human Prostate Carcinoma (LN Cap) Human B Cell Lymphoma (Raji, IM9) Human Ovarian Carcinoma (HeLa) Human Lung Carcinoma (A549) Mouse Carcinoma (4T1) ) Mouse pancreatic endothelial cells, SV 40 transformed (SVR) mouse B cell lymphoma (BCL1) mouse normal kidney ^* mouse normal brain ^* mouse fetal liver (E11-E13) activated endothelial cells Normal Human PBMC Human T Cell Leukemia (MOLT4, Jurkat, CEM-NKr) Unstimulated Endothelial Cells Mouse Thoracic Mouse Lung Mouse Spleen Mouse Heart Mouse PBL Mouse Liver Mouse Total Adult Bone Marrow Mouse Lineage Negative Adult Bone Mice Fetal Liver (E15-Birth) ^* Expression in these organs is weak when measured by radioligand defect signals

However, CCX-CKR2 also appears to play a role in some normal cells. CCX-CKR2 receptor is expressed during fetal development. CCX-CKR2 is expressed in mouse fetal liver until day 11 of embryonic development (E11), but is no longer observed by E15 (as measured by radiolabeled SDF-1 binding and I-TAC substitution), as well as Northern analysis No CCX-CKR2 transcript was also observed when measured by. In adult mice, it is expressed in normal kidneys. Compared with renal expression, the expression level is low in normal brain. Since experiments were performed using whole brain homogenate, this low signal in radioligand binding assay means that the number of brain cells expressing CCX-CKR2 is small.

To further demonstrate the role of CCX-CKR2 in cancer, it was demonstrated that cancer cell proliferation can be inhibited by antagonizing CCX-CKR2 in cancer cells. Antagonism of CCX-CKR2 expressed in breast carcinoma by CCX-CKR2 antagonist inhibited cell proliferation in vitro. In vitro treated cells showed decreased cell proliferation over time compared to untreated controls (see FIG. 6).

CCX-CKR2 is also concerned with attachment. Leukocyte migration involves several steps, including the step of cell attachment followed by migration to a given tissue. These results can be observed by in vitro static adhesion analysis. Vascular endothelial monolayers grow on the surface. Cells expressing CCX-CKR2 are then labeled with fluorescent dyes for visualization. When CCX-CKR2 cells are allowed to attach to the endothelial surface, more CCX-CKR2 expressing cells attach to the endothelial layer as compared to the CCX-CKR2 cell control. In addition, the addition of CCX-CKR2 antagonist inhibits adhesion as compared to the mediator treated control (see FIG. 7).

In vivo evidence further supports the role of CCX-CKR2 in tumor proliferation. Tumors are formed when human B cell lymphoma cells expressing CCX-CKR2 are injected into immunodeficient mice. Treatment of these mice with CCX-CKR2 antagonists inhibits angiogenic tumor formation. In this trial, one of 17 mice treated with CCX-CKR2 antagonist developed encapsulated angiogenic tumors, whereas 11 of 17 mice in the mediated control developed an angiogenic tumor. It became. These data suggest that CCX-CKR2 is involved in the ability of tumors to differentiate and form vascular layers and provide evidence that the antagonism of CCX-CKR2 is useful in treating cancer.

The effect of antagonism of CCX-CKR2 in the breast cancer model was also tested. In a breast cancer growth model, immunodeficient mice were injected with human breast carcinoma. Tumors were measured three times a week, and the volumes were plotted. Mice treated with CCX-CKR2 antagonists had a reduced tumor volume compared to mediated macrophages, demonstrating that CCX-CKR2 plays a role in tumor growth (see FIG. 8).

Example  3

This example demonstrates that CCX-CKR2 promotes cell survival by reducing apoptosis.

Interactions between chemokines and chemokine receptors are typically assessed by measuring intracellular calcium migration and chemotaxis. However, CCX-CKR2 does not cause transient calcium migration or allow cells to migrate in response to their ligands CXCL12 or CXCL11. However, cells expressing CCX-CKR2 have increased adhesion to activated endothelial cell monolayers. Moreover, under low serum supplementation environment of culture medium (1% instead of 10%), recovery of adherent cells that survived after 3 days was CCX-CKR2 MDA MB transfectant compared to untransfected WT cells (WT 435s). (Indicated by CCX-CKR2 435s) was much larger. Consistent with this observation, the frequency of killed cells recovered from the supernatants collected from these media was much greater for WT compared to CCX-CKR2 transformants. These results can be visualized by fluorescence using the DNA insert pigment 7AAD (7 aminoactinomycin D). 435 s cells of CCX-CKR2-435s transformant or DITODGUDD were harvested after growing at different serum concentrations and incubated with 7AAD (1 μg / ml in DMSO) for 15-30 minutes at room temperature. FACS analysis demonstrated more dead cells / apoptotic cells (ie 7AAD-positive) in wild type 435s cells compared to CCX-CKR2-435s transformants.

In an extension of these results, we found that cultured CCX-CKR2 transfectants or non-transfected WT cells, only annexin for detecting apoptotic cells, and iodinated propri, for detecting dead cells but not apoptotic cells. Co staining with sodium (PI). By this method, the cell population is known to induce cell apoptosis, using substances which serve as good controls in these assays, for example campotesine (CMP), or TNFalpha plus cycloheximide (CHX). Easily identify the percentage of hepatic apoptosis cells.

By this assay, the incidence of apoptosis cells over time in either CCX-CKR2-435s transformants or wild type 435s cells grown in optimal serum (10%) or marginal serum (1%) was measured. All of these cell types grown in 10% serum showed excellent viability over 4 days of incubation time. In contrast, WT cells grown in 1% serum showed a rapid decrease in viable cells after 3 and 4 days of culture. Co-staining by annexin and PI shows that this reflects the development of both apoptotic and dead cells. Interestingly, CCX-CKR2-435s cells grown in 1% serum showed excellent viability over 4 days of incubation time, which suggests that introducing CCX-CKR2 into 435s is a key factor in the development of these cells under suboptimal serum supplementation. To prevent rapid cellular apoptosis.

The same results were obtained in the second experiment using the same CCX-CKR2-435s transformant, and in addition a separate non-replicating group of 435s cells transfected with CCX-CKR2. This experiment indicates that the decrease in apoptosis in early replicating transformants is by CCX-CKR2 expression rather than by specific mutations in the transform clone.

Example 4

This example demonstrates that CCX-CKR2 mediates the phosphorylation of p44 / 42 MARK (ERK1 and ERK2).

CCX-CKR2 is unique in that ligand binding (eg, by ITAC or SDF-1) does not cause the migration of calcium that is common to many chemokine receptors. This prompted the evaluation of other potential signaling pathways, including investigating whether CCX-CKR2 activity is mediated through ERK phosphorylation. Studies conducted with lysates from MCF7 cell lines expressing CCX-CKR2, stimulated by ITAC or SDF-1, showed that ligand-dependent phosphorylation of ERK1 and ERK2 (sometimes referred to in the literature as p44 / 42 MAPK) Proved to exist. Phosphorylated ERK1 and ERK2 are detected by Western blot analysis, where lysates from MCF7 cell lines are initially electrophoresed to separate proteins in lysate. Phosphorylated ERK1 and ERK2 on electrophoretic gels are detected by probing with antibodies specific for the phosphorylated form of these proteins. These antibodies are available from Cell Signaling Technology, Beverly, Massachusetts.

Similar experiments were performed using Hela cells expressing CCX-CKR2. Similar results were obtained when these cells were stimulated with ITA or SDF-1.

ITAC and SDF-1 bind to other chemokine receptors, for example, to CXCR3 for ITAC and to CXCR4 for SDF-1, so these receptors are ERK phosphorylated by these ligands in MCF7 cells. It is important to consider potential contributions to FACS analysis of MCF7 cells using antibodies specific for CXCR3 or CXCR4 showed that these receptors were completely absent in MCF7 cells. Positive controls using cell lines expressing CXCR3 or CXCR4 demonstrated that the antibodies used in these tests bind to these receptors. Thus, these data indicate that binding ligands for CCX-CKR2, such as ITAC or SDF-1, cause intracellular signaling through phosphorylation of ERK, which will result in activation of additional downstream components. The discovery that CCX-CKR2 mediates phosphorylation of ERK1 and ERK2 indicates that CCX-CKR2 is involved in various biological processes, since ERK phosphorylation has been demonstrated to mediate the regulation of cell growth and cell differentiation.

Example  5

This example demonstrates that cell expression of CCX-CKR2 results in the induction of a number of regulatory proteins.

As an alternative method for investigating CCX-CKR2 mediated signaling events, supernatants collected from CCX-CKR2 transfected MDA MB 435s cells were compared to supernatants collected from wild-type MDA MB 435s (435s) cells and analyzed for specific ELISA. Was evaluated for the presence of a large family of secreted proteins. CCX-CKR2 expressing 435s cells produced substantially higher amounts of GM-CSF, RANTES, MCP-1, TIMP-1, and MMP3 than wild type 435s cells, especially when grown under limiting serum environments. Interestingly, all these factors have been reported to be involved in growth, vascular remodeling and chemotaxis associated with tumorigenesis. In addition, they may be related to the apoptosis deficient phenotype of CCX-CKR2 described above.

Example  6

This example demonstrates that CCX-CKR2 is inhibited by siRNA.

SMARTpool ™ siRNA (Pharmacon) specific for CXCR4 or CCX-CKR2 was obtained. SMARTpool ™ siRNA is a pool of four different siRNA sequences, each targeting a different region of the specified mRAN. These siRNA pools were tested in HeLa cells. CXCR4 expression was assessed by 12G5 or 173 Mab staining and FACS, while CCX-CKR2 expression was measured by binding assay using ¹²⁵ I-SDF-1. CXCR4 was expressed on HeLa cells in a conformation that did not exhibit detectable ¹²⁵ I-SDF-1 binding, thus allowing detection of CCX-CKR2 expression. CCX-CKR2 SMARTpool® siRNA (25-100 nM) significantly inhibited ¹²⁵ I-SDF-1 binding (≧ 50%), whereas CXCR4 SMARTpool® siRNA did not. Similar results were obtained with 293 CCX-CKR2 transformants.

In addition, each of the following three siRNA sequences was found to reduce binding of SDF-1 when introduced into cells at low concentrations of 4 nM.

siRNA #l: GCCGTTCCCTTCTCCATTATT

siRNA # 2: GAGCTCACGTGCAAAGTCATT

siRNA # 3: GACATCAGCTGGCCATGCATT

Example  7

This example demonstrates the expression of CCX-CKR2 in the brain.

CCX-CKR2 is expressed in many tumor cell lines, while rarely expressed in normal tissues. One exception to this pattern has been demonstrated in binding studies that brain cells from normal adult mice express CCX-CKR2. In an extension of this observation, field hybridization studies were performed using CCX-CKR2 specific probes to limit the region of CCX-CKR2 expression to the brain. Brain samples were collected from normal adult mice and fixed overnight at 4 ° C. with 4% PFA in PBS and then overnight at 4 ° C. with 30% sucrose in PBS. The tissue was then inserted into the OCT, cut into 20 μm slices and placed on a superfrost plus slide. For in situ hybridization studies, antisense riboprobes and sense riboprobes were prepared by in vitro transcription, respectively, using T7 and SP6 RNA polymerase using the DIG cRNA label kit (Roche) after linearization. 20 μm frozen sections were fixed in fresh 4% PFA and then treated with protease K (2 μm / ml, for 20 minutes at 37 ° C.). The slides were prehybridized at 55 ° C. for 1 hour and then hybridized at 55 ° C. O / N in a sealed container. The slides were then washed at 65 ° C. with 50% formamide, 5 × SSC pH4.5 and 1% SDS, then masked with 5% sheep serum for 1 hour and 1% sheep serum at 4 ° C. O / N. Incubated with 1: 1000 anti-Dig antibody. Slides were washed with TBST and detected with NBT / BCIP.

The results clearly demonstrate strong expression of CCX-CKR2 by neurons in the cerebellum, hippocampus and cortex. There is little or no detectable expression in glial cells, such as the region containing the white matter tube and the corpus callosum in the cerebellum. The cardiac conducting muscle cells showed a uniformly strong positive signal and a subset of granule cells in the inner granular cell layer showed a positive signal. In addition, the cortex is generally quite positive and individual neurons within the cortex exhibited clear positive staining. The hippocampus exhibited strong CCX-CKR2 signals in the tooth gyrus and neurodegeneration of CA1-3. In addition, the overlapping cortex was also strongly positive.

These data provide insight into the potential relevance of CCX-CKR2 to brain tumors. CCX-CKR2 is not expressed in ventricular cells or glial cells that are thought to develop astrocytoma. In contrast, stromal blastoma is partially expressed in a subset of cerebellar granulocytes, the cell type resulting therefrom. The expression profile of CCX-CKR2 is very different from that seen in other SDF-1 receptor CXCR4.

Example  8

This example demonstrates the effect of the CCX-CKR2 ligand competitor in a mouse xenograft model of lung carcinoma.

Lung carcinoma is the leading cause of death in the United States. CCX-CKR2 is expressed in activated endothelial as well as lung carcinoma. The effect of the administration of 700 series of compounds of the CCX-CKR2 ligand competitor in a xenograft model of lung carcinoma was evaluated.

In lung carcinoma xenograft studies, A549 tumor fragments (30-40 mg) were implanted into the subcutaneous space of nude mice. Tumors were allowed to grow until they were approximately 150 mg (100-200 mg) in size. Mice received CCX-CKR2 ligand competitor (25 mpk; sc administration, Q1D) or mediator controls. Melphalan (9 mpk / administration, ip administration, Q4Dx3) was included as a positive control. Were measured twice a week for tumor in a two-dimensional caliper, the formula for the pyeonjang ellipsoid (axb ^2/3, a further and longer length, b is assuming a shorter length of a unit density ^{(1 mm 3 = 1 ㎎)} ) Was converted to tumor mass. In addition, body weights were measured twice per week to assess any adverse effects upon compound administration. Anticancer activity was assessed by the delay of tumor proliferation in the treated group compared to the mediator treated control.

Mice treated with competitors had reduced tumor loads as compared to the vehicle treated group. This difference in tumor volume is statistically significant between these groups, with a 32% decrease in mean tumor volume. Melphalan also reduced tumor volume with an average tumor volume reduction of 60%. In addition, in this study, daily dosing of competitors was well tolerated as measured by weight gain in animals treated with the compound, which was equivalent to weight gain in the vehicle treated group.

Tumor weight was assessed on the last day of compound treatment (day 49). Mice treated with the CCX-CKR2 competitor showed statistically smaller tumors than tumors of the vehicle control. Similarly, mice receiving melphalan also had significantly smaller tumors compared to the mediator treated group.

All publications and patent applications cited herein are incorporated by reference to the extent that each individual publication or patent application is specifically and individually designated to be incorporated by reference.

Although the foregoing invention has been described in detail by way of illustration and example for purposes of understanding, it will be apparent to those skilled in the art from the teachings of the invention that some changes and modifications may be made therein without departing from the spirit and scope of the appended claims. Will be self explanatory.

                               SEQUENCE LISTING <110> Burns, Jennifer M.       Summers, Bretton       Howard, Maureen C.       Schall, Thomas J.       ChemoCentryx, Inc. <120> Compositions and Methods for Detecting and Treating       Diseases and Conditions Related to Chemokine Receptors <130> 019934-003360PC <140> WO PCT / US04 / 34807 <141> 2004-10-19 <150> US 10 / 698,541 <151> 2003-10-30 <150> US 10 / 912,638 <151> 2004-08-04 <160> 14 <170> Patent In Ver. 2.1 <210> 1 <211> 1089 <212> DNA <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2 (RDC1)       coding sequence <400> 1 atggatctgc atctcttcga ctactcagag ccagggaact tctcggacat cagctggcca 60 tgcaacagca gcgactgcat cgtggtggac acggtgatgt gtcccaacat gcccaacaaa 120 agcgtcctgc tctacacgct ctccttcatt tacattttca tcttcgtcat cggcatgatt 180 gccaactccg tggtggtctg ggtgaatatc caggccaaga ccacaggcta tgacacgcac 240 tgctacatct tgaacctggc cattgccgac ctgtgggttg tcctcaccat cccagtctgg 300 gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg agctcacgtg caaagtcaca 360 cacctcatct tctccatcaa cctcttcggc agcattttct tcctcacgtg catgagcgtg 420 gaccgctacc tctccatcac ctacttcacc aacaccccca gcagcaggaa gaagatggta 480 cgccgtgtcg tctgcatcct ggtgtggctg ctggccttct gcgtgtctct gcctgacacc 540 tactacctga agaccgtcac gtctgcgtcc aacaatgaga cctactgccg gtccttctac 600 cccgagcaca gcatcaagga gtggctgatc ggcatggagc tggtctccgt tgtcttgggc 660 tttgccgttc ccttctccat tatcgctgtc ttctacttcc tgctggccag agccatctcg 720 gcgtccagtg accaggagaa gcacagcagc cggaagatca tcttctccta cgtggtggtc 780 ttccttgtct gctggctgcc ctaccacgtg gcggtgctgc tggacatctt ctccatcctg 840 cactacatcc ctttcacctg ccggctggag cacgccctct tcacggccct gcatgtcaca 900 cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc tctacagctt catcaatcgc 960 aactacaggt acgagctgat gaaggccttc atcttcaagt actcggccaa aacagggctc 1020 accaagctca tcgatgcctc cagagtctca gagacggagt actctgcctt ggagcagagc 1080 accaaatga 1089 <210> 2 <211> 362 <212> PRT <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2 (RDC1) <400> 2 Met Asp Leu His Leu Phe Asp Tyr Ser Glu Pro Gly Asn Phe Ser Asp   1 5 10 15 Ile Ser Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr Val              20 25 30 Met Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr Leu Ser          35 40 45 Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala Asn Ser Val      50 55 60 Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly Tyr Asp Thr His  65 70 75 80 Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp Leu Trp Val Val Leu Thr                  85 90 95 Ile Pro Val Trp Val Val Ser Leu Val Gln His Asn Gln Trp Pro Met             100 105 110 Gly Glu Leu Thr Cys Lys Val Thr His Leu Ile Phe Ser Ile Asn Leu         115 120 125 Phe Gly Ser Ile Phe Phe Leu Thr Cys Met Ser Val Asp Arg Tyr Leu     130 135 140 Ser Ile Thr Tyr Phe Thr Asn Thr Pro Ser Ser Arg Lys Lys Met Val 145 150 155 160 Arg Arg Val Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser                 165 170 175 Leu Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser Asn Asn             180 185 190 Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser Ile Lys Glu Trp         195 200 205 Leu Ile Gly Met Glu Leu Val Ser Val Val Leu Gly Phe Ala Val Pro     210 215 220 Phe Ser Ile Ile Ala Val Phe Tyr Phe Leu Leu Ala Arg Ala Ile Ser 225 230 235 240 Ala Ser Ser Asp Gln Glu Lys His Ser Ser Arg Lys Ile Ile Phe Ser                 245 250 255 Tyr Val Val Val Phe Leu Val Cys Trp Leu Pro Tyr His Val Ala Val             260 265 270 Leu Leu Asp Ile Phe Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg         275 280 285 Leu Glu His Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser     290 295 300 Leu Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile Asn Arg 305 310 315 320 Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile Phe Lys Tyr Ser Ala                 325 330 335 Lys Thr Gly Leu Thr Lys Leu Ile Asp Ala Ser Arg Val Ser Glu Thr             340 345 350 Glu Tyr Ser Ala Leu Glu Gln Ser Thr Lys         355 360 <210> 3 <211> 1089 <212> DNA <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2.2       coding sequence <400> 3 atggatctgc acctcttcga ctacgccgag ccaggcaact tctcggacat cagctggcca 60 tgcaacagca gcgactgcat cgtggtggac acggtgatgt gtcccaacat gcccaacaaa 120 agcgtcctgc tctacacgct ctccttcatt tacattttca tcttcgtcat cggcatgatt 180 gccaactccg tggtggtctg ggtgaatatc caggccaaga ccacaggcta tgacacgcac 240 tgctacatct tgaacctggc cattgccgac ctgtgggttg tcctcaccat cccagtctgg 300 gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg agctcacgtg caaagtcaca 360 cacctcatct tctccatcaa cctcttcagc ggcattttct tcctcacgtg catgagcgtg 420 gaccgctacc tctccatcac ctacttcacc aacaccccca gcagcaggaa gaagatggta 480 cgccgtgtcg tctgcatcct ggtgtggctg ctggccttct gcgtgtctct gcctgacacc 540 tactacctga agaccgtcac gtctgcgtcc aacaatgaga cctactgccg gtccttctac 600 cccgagcaca gcatcaagga gtggctgatc ggcatggagc tggtctccgt tgtcttgggc 660 tttgccgttc ccttctccat tatcgctgtc ttctacttcc tgctggccag agccatctcg 720 gcgtccagtg accaggagaa gcacagcagc cggaagatca tcttctccta cgtggtggtc 780 ttccttgtct gctggctgcc ctaccacgtg gcggtgctgc tggacatctt ctccatcctg 840 cactacatcc ctttcacctg ccggctggag cacgccctct tcacggccct gcatgtcaca 900 cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc tctacagctt catcaatcgc 960 aactacaggt acgagctgat gaaggccttc atcttcaagt actcggccaa aacagggctc 1020 accaagctca tcgatgcctc cagagtgtcg gagacggagt actccgcctt ggagcaaaac 1080 gccaagtga 1089 <210> 4 <211> 362 <212> PRT <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2.2 <400> 4 Met Asp Leu His Leu Phe Asp Tyr Ala Glu Pro Gly Asn Phe Ser Asp   1 5 10 15 Ile Ser Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr Val              20 25 30 Met Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr Leu Ser          35 40 45 Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala Asn Ser Val      50 55 60 Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly Tyr Asp Thr His  65 70 75 80 Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp Leu Trp Val Val Leu Thr                  85 90 95 Ile Pro Val Trp Val Val Ser Leu Val Gln His Asn Gln Trp Pro Met             100 105 110 Gly Glu Leu Thr Cys Lys Val Thr His Leu Ile Phe Ser Ile Asn Leu         115 120 125 Phe Ser Gly Ile Phe Phe Leu Thr Cys Met Ser Val Asp Arg Tyr Leu     130 135 140 Ser Ile Thr Tyr Phe Thr Asn Thr Pro Ser Ser Arg Lys Lys Met Val 145 150 155 160 Arg Arg Val Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser                 165 170 175 Leu Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser Asn Asn             180 185 190 Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser Ile Lys Glu Trp         195 200 205 Leu Ile Gly Met Glu Leu Val Ser Val Val Leu Gly Phe Ala Val Pro     210 215 220 Phe Ser Ile Ile Ala Val Phe Tyr Phe Leu Leu Ala Arg Ala Ile Ser 225 230 235 240 Ala Ser Ser Asp Gln Glu Lys His Ser Ser Arg Lys Ile Ile Phe Ser                 245 250 255 Tyr Val Val Val Phe Leu Val Cys Trp Leu Pro Tyr His Val Ala Val             260 265 270 Leu Leu Asp Ile Phe Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg         275 280 285 Leu Glu His Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser     290 295 300 Leu Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile Asn Arg 305 310 315 320 Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile Phe Lys Tyr Ser Ala                 325 330 335 Lys Thr Gly Leu Thr Lys Leu Ile Asp Ala Ser Arg Val Ser Glu Thr             340 345 350 Glu Tyr Ser Ala Leu Glu Gln Asn Ala Lys         355 360 <210> 5 <211> 1089 <212> DNA <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2.3       coding sequence <400> 5 atggatctgc atctcttcga ctactcagag ccagggaact tctcggacat cagctggcca 60 tgcaacagca gcgactgcat cgtggtggac acggtgatgt gtcccaacat gcccaacaaa 120 agcgtcctgc tctacacgct ctccttcatt tacattttca tcttcgtcat cggcatgatt 180 gccaactccg tggtggtctg ggtgaatatc caggccaaga ccacaggcta tgacacgcac 240 tgctacatct tgaacctggc cattgccgac ctgtgggttg tcctcaccat cccagtctgg 300 gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg agctcacgtg caaagtcaca 360 cacctcatct tctccatcaa cctcttcggc agcattttct tcctcacgtg catgagcgtg 420 gaccgctacc tctccatcac ctacttcacc aacaccccca gcagcaggaa gaagatggta 480 cgccgtgtcg tctgcatcct ggtgtggctg ctggccttct gcgtgtctct gcctgacacc 540 tactacctga agaccgtcac gtctgcgtcc aacaatgaga cctactgccg gtccttctac 600 cccgagcaca gcatcaagga gtggctgatc ggcatggagc tggtctccgt tgtcttgggc 660 tttgccgttc ccttctccat tgtcgctgtc ttctacttcc tgctggccag agccatctcg 720 gcgtccagtg accaggagaa gcacagcagc cggaagatca tcttctccta cgtggtggtc 780 ttccttgtct gctggttgcc ctaccacgtg gcggtgctgc tggacatctt ctccatcctg 840 cactacatcc ctttcacctg ccggctggag cacgccctct tcacggccct gcatgtcaca 900 cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc tctacagctt catcaatcgc 960 aactacaggt acgagctgat gaaggccttc atcttcaagt actcggccaa aacagggctc 1020 accaagctca tcgatgcctc cagagtctca gagacggagt actctgcctt ggagcagagc 1080 accaaatga 1089 <210> 6 <211> 362 <212> PRT <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2.3 <400> 6 Met Asp Leu His Leu Phe Asp Tyr Ser Glu Pro Gly Asn Phe Ser Asp   1 5 10 15 Ile Ser Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr Val              20 25 30 Met Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr Leu Ser          35 40 45 Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala Asn Ser Val      50 55 60 Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly Tyr Asp Thr His  65 70 75 80 Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp Leu Trp Val Val Leu Thr                  85 90 95 Ile Pro Val Trp Val Val Ser Leu Val Gln His Asn Gln Trp Pro Met             100 105 110 Gly Glu Leu Thr Cys Lys Val Thr His Leu Ile Phe Ser Ile Asn Leu         115 120 125 Phe Gly Ser Ile Phe Phe Leu Thr Cys Met Ser Val Asp Arg Tyr Leu     130 135 140 Ser Ile Thr Tyr Phe Thr Asn Thr Pro Ser Ser Arg Lys Lys Met Val 145 150 155 160 Arg Arg Val Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser                 165 170 175 Leu Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser Asn Asn             180 185 190 Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser Ile Lys Glu Trp         195 200 205 Leu Ile Gly Met Glu Leu Val Ser Val Val Leu Gly Phe Ala Val Pro     210 215 220 Phe Ser Ile Val Ala Val Phe Tyr Phe Leu Leu Ala Arg Ala Ile Ser 225 230 235 240 Ala Ser Ser Asp Gln Glu Lys His Ser Ser Arg Lys Ile Ile Phe Ser                 245 250 255 Tyr Val Val Val Phe Leu Val Cys Trp Leu Pro Tyr His Val Ala Val             260 265 270 Leu Leu Asp Ile Phe Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg         275 280 285 Leu Glu His Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser     290 295 300 Leu Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile Asn Arg 305 310 315 320 Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile Phe Lys Tyr Ser Ala                 325 330 335 Lys Thr Gly Leu Thr Lys Leu Ile Asp Ala Ser Arg Val Ser Glu Thr             340 345 350 Glu Tyr Ser Ala Leu Glu Gln Ser Thr Lys         355 360 <210> 7 <211> 1089 <212> DNA <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2.4       coding sequence <400> 7 atggatctgc atctcttcga ctactcagag ccagggaact tctcggacat cagctggcca 60 tgcaacagca gcgactgcat cgtggtggac acggtgatgt gtcccaacat gcccaacaaa 120 agcgtcctgc tctacacgct ctccttcatt tacattttca tcttcgtcat cggcatgatt 180 gccaactccg tggtggtctg ggtgaatatc caggccaaga ccacaggcta tgacacgcac 240 tgctacatct tgaacctggc cattgccgac ctgtgggttg tcctcaccat cccagtctgg 300 gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg agctcacgtg caaagtcaca 360 cacctcatct tctccatcaa cctcttcggc agcattttct tcctcacgtg catgagcgtg 420 gaccgctacc tctccatcac ctacttcacc aacaccccca gcagcaggaa gaagatggta 480 cgccgtgtcg tctgcatcct ggtgtggctg ctggccttct gcgtgtctct gcctgacacc 540 tactacctga agaccgtcac gtctgcgtcc aacaatgaga cctactgccg gtccttctac 600 cccgagcaca gcatcaagga gtggctgatc ggcatggagc tggtctccgt tgtcttgggc 660 tttgccgttc ccttctccat tatcgctgtc ttctacttcc tgctggccag agccatctcg 720 gcgtccagtg accaggagaa gcacagcagc cggaagatca tcttctccta cgtggtggtc 780 ttccttgtct gctggctgcc ctaccacgtg gcggtgctgc tggacatctt ctccatcctg 840 cactacatcc ctttcacctg ccggctggag cacgccctct tcacggccct gcatgtcaca 900 cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc tctacagctt catcaatcgc 960 aactacaggt acgagctgat gaaggccttc atcttcaagt actcggccaa aacagggctc 1020 accaagctca tcgatgcctc cagagtctca gagacggagt actctgcctt ggagcagagc 1080 accaaatga 1089 <210> 8 <211> 362 <212> PRT <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2.4 <400> 8 Met Asp Leu His Leu Phe Asp Tyr Ser Glu Pro Gly Asn Phe Ser Asp   1 5 10 15 Ile Ser Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr Val              20 25 30 Met Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr Leu Ser          35 40 45 Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala Asn Ser Val      50 55 60 Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly Tyr Asp Thr His  65 70 75 80 Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp Leu Trp Val Val Leu Thr                  85 90 95 Ile Pro Val Trp Val Val Ser Leu Val Gln His Asn Gln Trp Pro Met             100 105 110 Gly Glu Leu Thr Cys Lys Val Thr His Leu Ile Phe Ser Ile Asn Leu         115 120 125 Phe Gly Ser Ile Phe Phe Leu Thr Cys Met Ser Val Asp Arg Tyr Leu     130 135 140 Ser Ile Thr Tyr Phe Thr Asn Thr Pro Ser Ser Arg Lys Lys Met Val 145 150 155 160 Arg Arg Val Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser                 165 170 175 Leu Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser Asn Asn             180 185 190 Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser Ile Lys Glu Trp         195 200 205 Leu Ile Gly Met Glu Leu Val Ser Val Val Leu Gly Phe Ala Val Pro     210 215 220 Phe Ser Ile Ile Ala Val Phe Tyr Phe Leu Leu Ala Arg Ala Ile Ser 225 230 235 240 Ala Ser Ser Asp Gln Glu Lys His Ser Ser Arg Lys Ile Ile Phe Ser                 245 250 255 Tyr Val Val Val Phe Leu Val Cys Trp Leu Pro Tyr His Val Ala Val             260 265 270 Leu Leu Asp Ile Phe Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg         275 280 285 Leu Glu His Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser     290 295 300 Leu Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile Asn Arg 305 310 315 320 Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile Phe Lys Tyr Ser Ala                 325 330 335 Lys Thr Gly Leu Thr Lys Leu Ile Asp Ala Ser Arg Val Ser Glu Thr             340 345 350 Glu Tyr Ser Ala Leu Glu Gln Ser Thr Lys         355 360 <210> 9 <211> 1089 <212> DNA <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2.5       coding sequence <400> 9 atggatctgc atctcttcga ctactcagag ccagggaact tctcggacat cagctggccg 60 tgcaacagca gcgactgcat cgtggtggac acggtgatgt gtcccaacat gcccaacaaa 120 agcgtcctgc tctacacgct ctccttcatt tacattttca tcttcgtcat cggcatgatt 180 gccaactccg tggtggtctg ggtgaatatc caggccaaga ccacaggcta tgacacgcac 240 tgctacatct tgaacctggc cattgccgac ctgtgggttg tcctcaccat cccagtctgg 300 gtggtcagtc tcgtgcagca caaccagtgg cccatgggcg agctcacgtg caaagtcaca 360 cacctcatct tctccatcaa cctcttcagc agcattttct tcctcacgtg catgagcgtg 420 gaccgctacc tctccatcac ctacttcacc aacaccccca gcagcaggaa gaagatggta 480 cgccgtgtcg tctgcatcct ggtgtggctg ctggccttct gcgtgtctct gcctgacacc 540 tactacctga agaccgtcac gtctgcgtcc aacaatgaga cctactgccg gtccttctac 600 cccgagcaca gcatcaagga gtggctgatc ggcatggagc tggtctccgt tgtcttgggc 660 tttgccgttc ccttctccat tatcgctgtc ttctacttcc tgctggccag agccatctcg 720 gcgtccagtg accaggagaa gcacagcagc cggaagatca tcttctccta cgtggtggtc 780 ttccttgtct gctggttgcc ctaccacgtg gcggtgctgc tggacatctt ctccatcctg 840 cactacatcc ctttcacctg ccggctggag cacgccctct tcacggccct gcatgtcaca 900 cagtgcctgt cgctggtgca ctgctgcgtc aaccctgtcc tctacagctt catcaatcgc 960 aactacaggt acgagctgat gaaggccttc atcttcaagt actcggccaa aacagggctc 1020 accaagctca tcgatgcctc cagagtctca gagacggagt actccgcctt ggagcagagc 1080 accaaatga 1089 <210> 10 <211> 362 <212> PRT <213> Homo sapiens <220> G-protein coupled receptor (GPCR) CCX-CKR2.5 <400> 10 Met Asp Leu His Leu Phe Asp Tyr Ser Glu Pro Gly Asn Phe Ser Asp   1 5 10 15 Ile Ser Trp Pro Cys Asn Ser Ser Asp Cys Ile Val Val Asp Thr Val              20 25 30 Met Cys Pro Asn Met Pro Asn Lys Ser Val Leu Leu Tyr Thr Leu Ser          35 40 45 Phe Ile Tyr Ile Phe Ile Phe Val Ile Gly Met Ile Ala Asn Ser Val      50 55 60 Val Val Trp Val Asn Ile Gln Ala Lys Thr Thr Gly Tyr Asp Thr His  65 70 75 80 Cys Tyr Ile Leu Asn Leu Ala Ile Ala Asp Leu Trp Val Val Leu Thr                  85 90 95 Ile Pro Val Trp Val Val Ser Leu Val Gln His Asn Gln Trp Pro Met             100 105 110 Gly Glu Leu Thr Cys Lys Val Thr His Leu Ile Phe Ser Ile Asn Leu         115 120 125 Phe Ser Ser Ile Phe Phe Leu Thr Cys Met Ser Val Asp Arg Tyr Leu     130 135 140 Ser Ile Thr Tyr Phe Thr Asn Thr Pro Ser Ser Arg Lys Lys Met Val 145 150 155 160 Arg Arg Val Val Cys Ile Leu Val Trp Leu Leu Ala Phe Cys Val Ser                 165 170 175 Leu Pro Asp Thr Tyr Tyr Leu Lys Thr Val Thr Ser Ala Ser Asn Asn             180 185 190 Glu Thr Tyr Cys Arg Ser Phe Tyr Pro Glu His Ser Ile Lys Glu Trp         195 200 205 Leu Ile Gly Met Glu Leu Val Ser Val Val Leu Gly Phe Ala Val Pro     210 215 220 Phe Ser Ile Ile Ala Val Phe Tyr Phe Leu Leu Ala Arg Ala Ile Ser 225 230 235 240 Ala Ser Ser Asp Gln Glu Lys His Ser Ser Arg Lys Ile Ile Phe Ser                 245 250 255 Tyr Val Val Val Phe Leu Val Cys Trp Leu Pro Tyr His Val Ala Val             260 265 270 Leu Leu Asp Ile Phe Ser Ile Leu His Tyr Ile Pro Phe Thr Cys Arg         275 280 285 Leu Glu His Ala Leu Phe Thr Ala Leu His Val Thr Gln Cys Leu Ser     290 295 300 Leu Val His Cys Cys Val Asn Pro Val Leu Tyr Ser Phe Ile Asn Arg 305 310 315 320 Asn Tyr Arg Tyr Glu Leu Met Lys Ala Phe Ile Phe Lys Tyr Ser Ala                 325 330 335 Lys Thr Gly Leu Thr Lys Leu Ile Asp Ala Ser Arg Val Ser Glu Thr             340 345 350 Glu Tyr Ser Ala Leu Glu Gln Ser Thr Lys         355 360 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: chemokine       receptor second extracellular loop common       structural feature <400> 11 Asp Arg Tyr Leu Ala Ile Val His Ala   1 5 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: small       interfering RNA siRNA # 1 <400> 12 gccgttccct tctccattat t 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: small       interfering RNA siRNA # 2 <400> 13 gagctcacgt gcaaagtcat t 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: small       interfering RNA siRNA # 3 <400> 14 gacatcagct ggccatgcat t 21

Claims (45)

As a method of identifying a substance that binds to CCX-CKR2 on a cell, Contacting the plurality of materials with a CCX-CKR2 polypeptide comprising at least 95% identical extracellular domain to the extracellular domain of SEQ ID NO: 2, or an SDF-1 or I-TAC binding fragment thereof; And Identifying a substance that binds to CCX-CKR2 on the cell by selecting a substance that competes with I-TAC or SDF-1 to bind to the CCX-CKR2 polypeptide or fragment thereof How to include. The method of claim 1, wherein said cell is a cancer cell. The method of claim 1, further comprising testing the ability of the selected material to bind to the cell or inhibit the growth of the cell. The method of claim 3, wherein the cells are cancer cells. The method of claim 1, further comprising testing the ability of the selected material to alter kidney function. The method of claim 1, further comprising testing the ability of the selected material to alter brain function or neuronal function. The method of claim 1, further comprising testing the ability of the selected material to alter cell adhesion to endothelial cells. The method of claim 1 wherein the material is less than 1,500 Daltons. The method of claim 1, wherein the substance is an antibody. The method of claim 1, wherein the substance is a polypeptide. The method of claim 1, wherein the CCX-CKR2 polypeptide comprises the sequence set forth in SEQ ID NO: 2. Contacting a sample comprising cells with a substance that specifically binds SEQ ID NO: 2; And Detecting binding of the substance to the polypeptide in the sample, wherein binding of the substance to the sample indicates the presence of cancer cells Including, the method of determining the presence or absence of cancer cells. The method of claim 12, wherein the substance is an antibody. The method of claim 12, wherein the material is less than 1500 Daltons. The method of claim 12, wherein the substance is a polypeptide. The method of claim 12, wherein the detected polypeptide is SEQ ID NO: 2. The method of claim 12, wherein the sample is of human origin. The method of claim 12, wherein the method is used to diagnose cancer in humans. The method of claim 12, wherein the method is used to determine the prognosis of human cancer. The method of claim 12, wherein the cancer is selected from the group consisting of cervical cancer, breast cancer, lymphoma, glioblastoma, prostate cancer, and leukemia. The method of claim 12, wherein the cancer is not Kaposi's sarcoma, multiple Castleman's disease or AIDS-related primary effusion lymphoma. The method of claim 12, wherein the antibody competes with SDF-1 and I-TAC for binding to SEQ ID NO: 2. A method of diagnosing or prognosticing a subject with cancer, comprising: diagnosing the cancer of the subject by detecting the presence or absence of expression of a polynucleotide encoding a CCX-CKR2 polypeptide in a subject's cells, wherein the CCX-CKR2 polypeptide is I -TAC and / or SDF-1 and the CCX-CKR2 polypeptide is at least 95% identical to SEQ ID NO: 2. The method of claim 23, wherein the CCX-CKR2 peptide is set forth in SEQ ID NO: 2. The method of claim 23, wherein the cancer is selected from the group consisting of cervical cancer, breast cancer, lymphoma, glioblastoma, prostate cancer, and leukemia. The method of claim 23, wherein the cancer is not Kaposi's sarcoma, multiple Castleman's disease or AIDS-related primary effusion lymphoma. An antibody that specifically competes with SDF-1 and I-TAC to bind SEQ ID NO: 2. The antibody of claim 27, wherein said antibody is a monoclonal antibody. The antibody of claim 27, wherein said antibody is a humanized antibody. Contacting a cell that specifically binds SEQ ID NO: 2 with a cell to bind the material with a CCX-CKR2 polypeptide on the cell, wherein the material is bound to SDF-1 and to bind the CCX-CKR2 polypeptide. Wherein the cell expresses a CCX-CKR2 polypeptide comprising an extracellular domain that is at least 95% identical to the extracellular domain of SEQ ID NO: 2. 31. The method of claim 30, wherein the material is less than 1,500 Daltons. The method of claim 30, wherein said substance is an antibody. 31. The method of claim 30, wherein said substance is a polypeptide. The method of claim 30, wherein said CCX-CKR2 polypeptide is as set forth in SEQ ID NO: 2. The method of claim 30, wherein the material Contacting the plurality of materials with a CCX-CKR2 polypeptide comprising at least 95% identical extracellular domain to the extracellular domain of SEQ ID NO: 2, or an SDF-1 or I-TAC binding fragment thereof; And Identifying a substance that binds to cancer cells by selecting a substance that competes with I-TAC or SDF-1 to bind to the CCX-CKR2 polypeptide or fragment thereof How to identify by the method comprising a. A method of treating cancer in an individual comprising administering to the individual a therapeutically effective amount of a polynucleotide that inhibits expression of the CCX-CKR2 polynucleotide. The method of claim 36, wherein said CCX-CKR2 polynucleotide encodes SEQ ID NO: 2. The method of claim 36, wherein said CCX-CKR2 polynucleotide comprises SEQ ID NO: 1. A method of treating cancer in an individual comprising administering to the individual a therapeutically effective amount of a substance competing with SDF-1 and I-TAC to bind SEQ ID NO: 2. 40. The method of claim 39, wherein the material is less than 1,500 Daltons. The method of claim 39, wherein the substance is an antibody. The method of claim 39, wherein the substance is a polypeptide. The method of claim 39, wherein the substance is Contacting the plurality of materials with a CCX-CKR2 polypeptide comprising at least 95% identical extracellular domain to the extracellular domain of SEQ ID NO: 2, or an SDF-1 or I-TAC binding fragment thereof; And Identifying a substance that binds to cancer cells by selecting a substance that competes with I-TAC or SDF-1 to bind to the CCX-CKR2 polypeptide or fragment thereof How to identify by the method comprising a. The method of claim 39, wherein the cancer is selected from the group consisting of cervical cancer, breast cancer, lymphoma, glioblastoma, prostate cancer, and leukemia. The method of claim 39, wherein the cancer is not Kaposi's sarcoma, multiple Castleman's disease, or AIDS-related primary effusion lymphoma.

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