USOO7964194B2

(12) United States Patent (10) Patent No.: US 7.964,194 B2 Lillard, Jr. et al. (45) Date of Patent: Jun. 21, 2011

(54) ANTI- AND ASSOCIATED (52) U.S. Cl...... 424/145.1; 424/141.1 RECEPTOR ANTIBODIES AND USES FOR (58) Field of Classification Search ...... None INHIBITION OF INFLAMMATION See application file for complete search history. (75) Inventors: James W. Lillard, Jr., Smyrna, GA (56) References Cited (US); Udai P. Singh, Atlanta, GA (US); Shailesh Singh, Atlanta, GA (US); U.S. PATENT DOCUMENTS Jonathan K. Stiles, Powder Springs, GA 6,329,510 B1 12/2001 Qin et al. (US) 2003/0166589 A1* 9, 2003 Karin ...... 514,44 (73) Assignee: Morehouse School of Medicine, FOREIGN PATENT DOCUMENTS Atlanta, GA (US) WO WO O2/O15932 A1 * 2, 2002 (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 Angostini et al., (Am J Path. May 2001:158(5):1703-1711).* U.S.C. 154(b) by 0 days. International Search Report, Form PCT/ISA/210 of International Application No. PCT/US03/36556. (21) Appl. No.: 10/712,393 * cited by examiner (22) Filed: Nov. 14, 2003 Primary Examiner — Cherie M Woodward (65) Prior Publication Data (74) Attorney, Agent, or Firm — Ping Wang: Andrews Kurth, LLP US 2004/O19 1255A1 Sep. 30, 2004 (57) ABSTRACT Related U.S. Application Data It is possible to inhibit inflammatory processes by adminis (60) Provisional application No. 60/426,350, filed on Nov. tration of antibodies to . Identification of 15, 2002. chemokines which are over-produced makes it possible to block specific chemokine activity using antibodies to the (51) Int. Cl. over-expressed chemokines. A6 IK39/00 (2006.01) A 6LX39/395 (2006.01) 14 Claims, No Drawings US 7,964,194 B2 1. 2 ANT-CHEMOKNE AND ASSOCATED Exemplified are anti -CXCL9, -CXCL10, -CXCL11, RECEPTOR ANTIBODIES AND USES FOR -CCRL1, -CCRL2, -CCR5, -CCL1, -CCL2, -CCL3, -CCL4, INHIBITION OF INFLAMMATION -CCL4L1, -CCL5, -CCL7, -CCL8, -CCL14-1, -CCL14-2, -CCL14-3, -CCL15-1, -CCL15-2, -CCL16, -CCL19, This application takes priority from Provisional Patent 5 -CCL23-1, -CCL23-2, -CCL24, -CCL26, -CCR6, -CCL20, Application 60/426,350 filed Nov. 15, 2002. and -CCL25, -CCL25-1, -CCL25-2 antibodies. These This work was partially supported by the U.S. Govern chemokines are known. The Genbank assession numbers are ment. Hence, certain rights belong to the government. provided herein. The invention utilizes antibodies or func tional fragments thereof that bind to the target chemokines. FIELD OF THE INVENTION 10 The antibodies or antigen-binding fragment(s) bind to epitope(s) or peptide(s) that consists of 10 to 15 amino acids This invention relates to antibodies or the use of antibodies from sequences 1 through 30 (accession numbers in NIH directed against certain chemokines. The antibodies block NCBIGenebank given below) of the target chemokines. Anti high-affinity interactions leading to inflammation. bodies which act as modulators can administered mucosally 15 or systemically. BACKGROUND OF THE INVENTION The antibody, antibodies, or antigen-binding fragment or Despite recent advances in Studies related to the inflamma fragments can be isolated from the serum of immunized tion process, therapies for treatment of chronic inflammatory mammalian hosts, from cultures of immortalized cell lines diseases have remained elusive. This is perhaps a result of the and tissues such as hybridomas, lymphoblastoid or cells gen many and complex factors in the host that initiate and main erated by methods of recombinant molecule biology. For tain inflammatory conditions. Current therapies have disad increased effectiveness, fragments may be conjugated or Vantages associated with them, including the Suppression of linked to other peptides, proteins, nucleic acid sequences, the immune system that can render the host more Susceptible Vitamins, complex or simple carbohydrates, or other Suitable to bacterial, viral and parasitic infections. For example, use of 25 carrier molecules. steroids is a traditional approach to chronic inflammation Antibodies or antigen-binding fragments with specificity treatment. Such treatment can lead to changes in weight and for functional mutant or variant mammalian chemokines are Suppression of protective immunity. Advances in biotechnol appropriate. These mutations or polymorphisms occur in ogy have promoted the development of targeted biologicals nature or can be induced by recombinant molecular biologi with fewer side effects. To improve inflammatory disease 30 cal methods to generate single, multiple, or continuous amino treatment, technologies that alter and control the factors gen erated by cells of both innate and adaptive immunity systems acid residues, described in sequences 1 through 30, that are need to be developed. deleted, added, and/or substituted for other or no amino acids. Host cells have surface receptors that associate with The mucosal means of application include oral, intranasal, ligands to signal and regulate host cell activities. Administra 35 ocular, intravaginal, rectal, and/or intraurethral administra tion of anti-TNF-C. antibody or soluble TNF-C. receptor has tion in liquid or particulate form or on Solid Supports. Sys been shown to inhibit inflammatory diseases. Unfortunately, temic means of application include parenteral, intravenous, the side effects associated with this treatment can result in an or intramuscular administration in liquid or particulate form. increased risk of infections (e.g., tuberculosis) and other adverse reactions by mechanisms not fully understood. Simi 40 DETAILED DESCRIPTION OF THE INVENTION larly, antibody therapies focused on membrane bound mol ecules like CD40 have a propensity for inhibiting inflamma The invention provides methods of identifying, evaluating tion and graft-host diseases. While other targeted host cell and treating a subject Suffering from conditions arising from therapies to prevent inflammatory diseases are being devel inflammatory processes, including anaphylaxis and septic oped, there is no known single surface or secreted factor that 45 shock, arthritis (e.g., osteoarthritis, rheumatoid, psoriatic), will stop all inflammatory diseases. Consequently, the devel asthma, allergies (e.g., drug, insect, plant, food), atheroscle opment of therapies to exploit newly identified specific host rosis, delayed type hypersensitivity, dermatitis, diabetes (e.g., cell targets is required. mellitus, juvenile onset), graft rejection, inflammatory bowel A variety of pathogens or toxins activate macrophages, diseases (e.g., Crohn's disease, ulcerative colitis, enteritis), , T cells, B cells, , NK cells, Paneth and 50 interstitial cystitis, multiple Sclerosis, myasthemia gravis, crypt cells, as well as epithelial cells shortly after entry into Grave's disease, Hashimoto's thyroiditis, pneumonitis, pros the mucosa. Chemokines are a Superfamily of Small, cytok tatitis, psoriasis, nephritis, pneumonitis, chronic obstructive ine-like proteins that are resistant to hydrolysis, promote pulmonary disease, chronic bronchitis rhinitis, spondyloar neovascularization or endothelial cell growth inhibition, thropathies, scheroderma, systemic lupus erythematosus, or induce cytoskeletal rearrangement, activate or inactivate lym 55 throiditis using the said embodiments. As a result one or more phocytes, and mediate through interactions with of inflammatory processes including host cell migration or G-protein-coupled receptors. Chemokines can mediate the growth and migration of host cells that express their recep excretion. tors. The cellular mechanisms responsible for the function of It has now been demonstrated, using as examples anti chemokines are often, but not entirely, Ca" flux dependent 60 -CXCL9, -CXCL10, -CXCL11, -CCRL1, -CCRL2, -CCR5, and pertussis toxin-sensitive. However, the precise mecha -CCL1, -CCL2, -CCL3, -CCL4, -CCL4L1, -CCL5, -CCL7, nisms for chemokine-mediated events are not known. -CCL8, -CCL14-1, -CCL14-2, -CCL14-3, -CCL15-1, -CCL15-2, -CCL16, -CCL19, -CCL23-1, -CCL23-2, SUMMARY OF THE INVENTION -CCL24, -CCL26, -CCR6, -CCL20, and -CCL25, -CCL25-1, 65 -CCL25-2 antibodies, that it is possible to inhibit the inflam The present invention provides a means of inhibiting matory cell activation, migration or chemotaxis of the by inflammation by administering anti-chemokine antibodies. inflammatory processes. US 7,964,194 B2 4 Materials and Methods were collected when antibody titers reached 1:1,000,000. Primer Design Subsequently, normal or anti-sera were heat-inactivated and Messenger RNA sequences for CXCL9, CXCL10, diluted 1:50 in PBS. CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL3, Monoclonal Antibody Preparation CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL14-2, The 15 amino acid peptides from chemokines CXCL9, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, CCL23-2, CCL24, CCL26, CCR6, CCL20, and CCL25, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, CCL25-1, CCL25-2 were obtained from the NIH-NCBI gene CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, bank database (Sequences 31 through 60, accession numbers CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and 10 CCL25, CCL25-1, CCL25-2 (Sequences 1 through 30) were given later). Primers were designed using the Beacon.J 2.0 synthesized (Sigma Genosys) and conjugated to hen egg computer program. Thermodynamic analysis of the primers lysozyme (Pierce) to generate the Antigen?a) for Subsequent was conducted using computer programs: Primer Premier immunizations for anti-sera preparation or monoclonal anti and MIT Primer 3. The resulting primer sets were compared body generation. The endotoxin levels of chemokine peptide against the entire human genome to confirm specificity. 15 conjugates were quantified by the chromogenic Limulus RealTime PCR Analysis amebocyte lysate assay (Cape Cod, Inc., Falmouth, Miss.) Lymphocytes or inflamed tissues were cultured in RMPI and shown to be <5 EU/mg. 100 ug of the antigen was used as 1640 containing 10% fetal calf serum, 2% human serum, the immunogen together with complete Freund's adjuvant Supplemented with non-essential amino acids, L-glutamate, Ribi Adjuvant system (RAS) for the first immunization in a and Sodium pyruvate (complete media). Additionally, pri final volume of 200 ul. This mixture was subcutaneously mary inflammatory and normal-paired matched tissues were administered in 100 ulaliquots at two sites of the back of a rat, obtained from clinical isolates (Clinomics Biosciences, Fre mouse, or immunoglobulin-humanized mouse. Two weeks derick, Md. and UAB Tissue Procurement, Birmingham, later, animals received 100 ug of the antigen in addition to Ala.). Messenger RNA (mRNA) was isolated from 10° cells incomplete Freund's adjuvant for 3 Subsequent immuniza using TriReagent (Molecular Research Center, Cincinnati, 25 tions. Serum were collected and when anti -CXCL9, Ohio) according to manufacturers protocols. Potential -CXCL10, -CXCL11, -CCRL1, -CCRL2, -CCR5, -CCL1, genomic DNA contamination was removed from these -CCL2, -CCL3, -CCL4, -CCL4L1, -CCL5, -CCL7, -CCL8, samples by treatment with 10 U/ul of RNase free DNase -CCL14-1, -CCL14-2, -CCL14-3, -CCL15-1, -CCL15-2, (Invitrogen, San Diego, Calif.) for 15 minutes at 37°C. RNA -CCL16, -CCL19, -CCL23-1, -CCL23-2, -CCL24, -CCL26, was then precipitated and resuspended in RNA Secure (Am 30 -CCR6, -CCL20, and -CCL25, -CCL25-1, -CCL25-2 anti body titers reached 1:2,000,000, hosts were sacrificed and bion, Austin, Tex.). cDNA was generated by reverse tran splenocytes were isolated for hybridoma generation. scribing approximately 2 ug of total RNA using Taqman 7 B cells from the or lymph nodes of immunized hosts reverse transcription reagents (AppliedBiosystems, Foster were fused with immortal myeloma cell lines (e.g., YB2/0). City, Calif.) according to manufacturers protocols. Subse 35 Hybridomas were next isolated after selective culturing con quently, cDNAs were amplified with specific human cINA ditions (i.e., HAT-Supplemented media) and limiting dilution primers, to CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, methods of hybridoma cloning. Cells that produce antibodies CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, with the desired specificity were selected using ELISA. CCL8, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, Hybridomas from normal rats or mice were humanized with CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, 40 molecular biological techniques in common use. After clon CCR6, CCL20, and CCL25, CCL25-1, CCL25-2, using ing a high affinity and prolific hybridoma, antibodies were SYBR7 Green PCR master mix reagents (Applied Biosys isolated from ascites or culture Supernatants and adjusted to a tems) according to manufacturers protocol. The level of cop titer of 1:2,000,000 and diluted 1:50 in PBS. ies of mRNA of these targets were evaluated by real-time Anti-Sera or Monoclonal Antibody Treatment PCR analysis using the BioRad Icycler and software (Her 45 Knockout or transgenic mice (8 to 12 weeks old, Charles cules, Calif.). Anti-sera preparation River Laboratory, Wilmington, Mass.) that spontaneous—or The 15 amino acid peptides from chemokines CXCL9, when treated—develop inflammatory diseases were treated CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, with 200 ul intraperitoneal injections of either anti-sera or CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, monoclonal antibodies specific for each of the chemokines CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, 50 every three days. The inflammatory disease state of the host CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, and was next monitored for progression or regression of disease. CCL25, CCL25-1, CCL25-2 (Sequences 1 through 30) were Analysis by ELISA synthesized (Sigma Genosys, The Woodlands,Tex.) and con The serum level of IL-2, -IL-6, -TNF-C., and -IFN-y were jugated to hen egg lysozyme (Pierce, Rockford, Ill.) to gen determined by ELISA, following the manufacturers instruc erate the antigens for Subsequent immunizations for anti-sera 55 tions (E-Biosciences, San Diego, Calif.). Plates were coated preparation or monoclonal antibody generation. The endot with 100 ul of the respective capture antibody in 0.1 Mbicar oxin levels of chemokine peptide conjugates were quantified bonate buffer (pH 9.5) and incubated O/N at 4° C. After by the chromogenic Limulus amebocyte lysate assay (Cape aspiration and washing with wash buffer, the wells were Cod, Inc., Falmouth, Miss.) and shown to be <5 EU/mg. 100 blocked with assay diluent for 1 hour at RT. Samples and ug of the antigen was used as the immunogen together with 60 standards were added and incubated for 2 hours at RT. Next, complete Freund's adjuvant Ribi Adjuvant system (RAS) for 100 ul of detection antibody solutions were added and incu the first immunization in a final volume of 1.0 ml. This mix bated for 1 hour. 100 ul of avidin-HRP solution was added and ture was administered in 100 ml aliquots on two sites of the incubated for 30 minutes. Subsequently, 100 ul Tetramethyl back of the rabbit subcutaneously and 400 ml intramuscularly benzidine (TMB) substrate solution was added and allowed to in each hind leg muscle. Three to four weeks later, rabbits 65 react for 20 minutes. 50 ul of the stop solution was added and received 100 g of the antigen in addition to incomplete plates were read at 450 nm. The cytokine ELISA assays were Freund's adjuvant for 3 Subsequent immunizations. Anti-sera capable of detecting >15 pg/ml for each assay. US 7,964,194 B2 5 6 Cytokine Analysis by Multiplex Cytokine ELISA mouse. The disease score could range from 0 (no change in The T helper cell derived , IL-1C., IL-1B, IL-2, any segment) to a maximum of 12 with Grade 4 lesions of IL-12, IFN-Y, TNF-C. in serum were also determined by Segments. Beadlyte mouse multi-cytokine detection system kit provided Data Analysis by BioRad, following manufacturer instructions. Filter bot 5 SigmaStat 2000 (Chicago, Ill.) software was used to ana tom plates were rinsed with 100 ul of bio-plex assay buffer lyze and confirm the statistical significance of data. The data and removal using a Millipore Multiscreen Separation were Subsequently analyzed by the Student's t-test, using a Vacuum Manifold System (Bedford, Mass.), set at 5 in Hg. two-factor, unpaired test. In this analysis, treated Samples IL-1C., IL-1B, IL-2, IL-12, IFN-Y, TNF-C. beads in assay were compared to untreated controls. The significance level 10 was set at p-0.05. buffer were added into wells. Next, 50 ul of serum or standard Results solution were added and the plates were incubated for 30 Semiquantitative RT-PCRIdentification of Molecular Targets minutes at RT with continuous shaking (setting 3) using a RT-PCR products obtained using CXCL9-, CXCL10-, Lab-Line Instrument Titer Plate Shaker (Melrose, Ill.), after CXCL11-, CCRL1-, CCRL2-, CCR5-, CCL1-, CCL2-, sealing the plates. The filter bottom plates were washed 2 15 CCL3-, CCL4-, CCL4L1-, CCL5-, CCL7- CCL8-, CCL14 times, as before, and centrifuged at 300x g for 30 seconds. 1-, CCL14-2-, CCL14-3-, CCL15-1-, CCL15-2-, CCL16-, Subsequently, 50 ul of anti-mouse IL-1C., IL-1B, IL-2, IL-12, CCL19-, CCL23-1-, CCL23-2-, CCL24-, CCL26-, CCR6-, IFN-y, TNF-C. antibody-biotin reporter solution was added in CCL20-, and CCL25-. CCL25-1-. CCL25-2-specific primer each well followed by incubation with continuous shaking for sets did not cross react with other gene targets due to exclu 30 minutes followed by centrifugation at 300xg for 30 sec sion of primers that annealed to host sequences 31-60. The onds. The plates were washed 3 times with 100 ul of bio-plex primers used produced different size amplicon products rela assay bufferas before. Next, 50ll streptavidin-phycoerythrin tive the polymorphisms that resulted in CCL4 versus solution was added to each well and incubated with continu CCL4L1, CCL14-1, CCL14-2, versus CCL14-3, CCL15-1 ous shaking for 10 minute at RT. 125 ul of bio-plex assay versus CCL15-2, CCL23-1 versus CCL23-2, and CCL25, buffer was added and Beadlyte readings were measured using 25 CCL25-1, versus CCL25-2. To this end, RT-PCR analysis of a Luminexl instrument (Austin,Tex.). The resulting data was tissue from Subjects exhibiting anaphylaxis, arthritis (e.g., collected and calculated using Bio-plexl software (Bio-Rad). rheumatoid, psoriatic), asthma, allergies (e.g., drug, insect, The cytokine Beadlyte assays were capable of detecting >5 plant, food), atherosclerosis, delayed type hypersensitivity, pg/ml for each analyte. dermatitis, diabetes (e.g., mellitus, juvenile onset), graft Serum Amyloid Protein A (BAA) ELISA 30 rejection, inflammatory bowel diseases (e.g., Crohn's dis The SAA levels were determined by ELISA using a kit ease, ulcerative colitis, enteritis), multiple Sclerosis, myas supplied by Biosource International, (Camarillo, Calif.). themia gravis, pneumonitis, psoriasis, nephritis, rhinitis, Briefly, 50 ul of SAA-specific monoclonal antibody solution spondyloarthropathies, Scheroderma, systemic lupus, or was used to coat micro-titer strips to capture SAA. Serum throiditis revealed that CXCL9, CXCL10, CXCL11, CCRL1, samples and standards were added to wells and incubated for 35 CCRL2, CCR5, CCL1, CCL2, CCL3, CCL4, CCL4L1, 2 hours at RT. After washing in the assay buffer, the HRP CCL5, CCL7, CCL8, CCL14-1, CCL14-2, CCL14-3, conjugated anti-SAA monoclonal antibody solution was CCL15-1, CCL15-2, CCL16, CCL19, CCL23-1, CCL23-2, added and incubated for 1 hour at 37°C. After washing, 100 CCL24, CCL26, CCR6, CCL20, and CCL25, CCL25-1, ul Tetramethylbenzidine (TMB) substrate solution was added CCL25-2 were differentially expressed by inflammatory host and the reaction was stopped after incubation for 15 minutes 40 cells. at RT. After the stop solution was added, the plates were read In Vivo Inflammatory Disease Inhibition at 450 nm. Mammals that develop anaphylaxis, septic shock, arthritis Histology and Pathology Scoring (e.g., rheumatoid, psoriatic), asthma, allergies (e.g., drug, Fixed tissues were sectioned at 6 um, and stained with insect, plant, food), atherosclerosis, bronchitis, chronic pull hematoxylin and eosin for light microscopic examination. 45 monary obstructive disease, delayed type hypersensitivity, The intestinal lesions were multi-focal and of variable sever dermatitis, diabetes (e.g., mellitus, juvenile onset), graft ity, the grades given to any section of intestine took into rejection, Grave's disease, Hashimoto's thyroiditis, inflam account the number of lesions as well as their severity. A score matory bowel diseases (e.g., Crohn's disease, ulcerative coli (0 to 4) was given, based on the following criteria: (Grade 0) tis, enteritis), interstitial cystitis, multiple Sclerosis, myas no change from normal tissue. (Grade 1) 1 or a few multi 50 themia gravis, pneumonitis, psoriasis, nephritis, rhinitis, focal mononuclear cell infiltrates, minimal hyperplasia and spondyloarthropathies, scheroderma, systemic lupus erythe no depletion of mucus. (Grade 2) lesions tended to involve matosus, or throiditis were allowed to develop the inflamma more of the mucosa and lesions had several multi-focal, yet tory disease of interest. Antibodies directed against CXCL9, mild, inflammatory cell infiltrates in the lamina propria com CXCL10, CXCL11, CCRL1, CCRL2, CCR5, CCL1, CCL2, posed of mononuclear cells, mild hyperplasia, epithelial ero 55 CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL14-1, sions were occasionally present, and no inflammation was CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL19, noticed in the Sub-mucosa. (Grade 3) lesions involved a large CCL23-1, CCL23-2, CCL24, CCL26, CCR6, CCL20, or area of mucosa or were more frequent than Grade 2, where CCL25, CCL25-1, CCL25-2 differentially affected the pro inflammation was moderate and often involved in the sub gression and regression of inflammatory disease as deter mucosa as well as moderate epithelial hyperplasia, with a 60 mined by histological scoring and comparing pre- and post mixture of mononuclear cells and neutrophils. (Grade 4) treatment serum levels of IFN-Y, IL-1C., IL-1B, IL-6, IL-12, lesions usually involved most of the section and were more TNF-C. amyloid protein A. Antibodies directed towards severe than Grade 3 lesions. Additionally, Grade 4 inflamma CXCL9, CXCL10, CXCL11, CCRL1, CCRL2, CCR5, tions were more severe and included mononuclear cell and CCL1, CCL2, CCL3, CCL4, CCL4L1, CCL5, CCL7, CCL8, neutrophils; epithelial hyperplasia was marked with crowd 65 CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, ing of epithelial cells in elongated glands. The Summation of CCL16, CCL19, CCL23-1, CCL23-2, CCL24, CCL26, these score provide a total inflammatory disease score per CCR6, CCL20, or CCL25, CCL25-1, CCL25-2 effectively US 7,964,194 B2 7 8 lead to the both regression and impeding progression of CXCL11 (ACCESSION# NM 005409), CCRL1 (ACCES inflammatory disease as determined by histological scoring SION# NM 016557), and comparing pre- and post-treatment serum levels of IFN-y, CCRL2 (ACCESSION# NM 003965), CCR5 (ACCES IL-1C., IL-1B, IL-6, IL-12, TNF-C. amyloid protein A. SION# NM 000579), As indicated previously, the chemokines used in the meth CCL1 (ACCESSION# NM 002981), CCL2 (ACCES ods of the invention are known. The assession numbers for the SION# NM 002982), protein sequences are as follows: CCL3 (ACCESSION# XM 008450, NM 002983), CXCL9, (ACCESSION# NM 002416), CXCL10(ACCES CCL4 (ACCESSION# NM 002984), CCL4L1 (ACCES SION# NM 001565), SION# AY079147), CXCL11 (ACCESSION# NM 005409), CCRL1 (ACCES 10 SION# NM 016557), CCL5 (ACCESSION# NM 002985), CCL7 (ACCES CCRL2 (ACCESSION# NM 003965), CCR5 (ACCES SION# NM 006273), SION# NM 060579), CCL8 (ACCESSION# NM 005623), CCL14-1 (ACCES CCL1 (ACCESSION# NM 002981), CCL2 (ACCES SION# NM 004166), SION# NM 002982), 15 CCL14-2 (ACCESSION# NM 032962), CCL3 (ACCESSION# XM 008450, NM 002983), CCL14-3 (ACCESSION# NM 032963), CCL4 (ACCESSION# NM 002984), CCL4L1 (ACCES CCL15-1 (ACCESSION# NM 032964), SIONHAY079147), CCL15-2 (ACCESSION# NM 004167), CCL5 (ACCESSION# NM 002985), CCL7 (ACCES CCL16 (ACCESSION# NM 004590), CCL19 (ACCES SION# NM 006273), SION# NM 006274), CCL8 (ACCESSION# NM 005623), CCL14-1 (ACCES CCL23-1 (ACCESSION# NM 005064), SION# NM 004166), CCL23-2 (ACCESSION# NM 145898), CCL14-2 (ACCESSION# NM 032962), CCL24 (ACCESSION# NM 002991), CCL26 (ACCES CCL14-3 (ACCESSION# NM 032963), SION# NM 006072), CCL15-1 (ACCESSION# NM 032964), 25 CCR6 (ACCESSION# U45984), CCL20 (ACCESSION: CCL15-2 (ACCESSION# NM 004167), NM 004591), CCL16 (ACCESSION# NM 004590), CCL19 (ACCES CCL25 (ACCESSION# 015444), CCL25-1 (ACCESSION: SION# NM 006274), NM 005624), CCL23-1 (ACCESSION# NM 005064), As shown in the table, the particular chemokines which CCL23-2 (ACCESSION# NM 145898), 30 give rise to inflammatory diseases differ with the disease. CCL24 (ACCESSION# NM 002991), CCL26 (ACCES They also differ among individuals. Hence, it is wise, when SION# NM 006072), treating an individual, to identify the particular chemokines CCR6 (ACCESSION# U45984), CCL20 (ACCESSION: which are increased in the tissues of the patient. Using the NM 004591) antibodies produced against each of the chemokines and CCL25 (ACCESSION# 015444), CCL25-1 (ACCESSION: 35 exposing the tissue samples from the patient to the particular NM 005624), antibodies, then evaluating the amount of antibody/chemok and CCL25-2 (ACCESSION# NM 148888). ine binding, it is possible to evaluate the level of expression For the cDNA sequences, the following GenBank acces for each chemokine and to administer to the patient the par sion numbers apply: ticular antibodies that will bind the excessive chemokine. CXCL9 (ACCESSION# NM 002416), CXCL10(ACCES 40 This tailored approach to treatment of inflammatory disease SION# NM 001565), is novel, and a particularly valuable aspect of the invention. TABLE 1 Chemokine, and Inflammatory Disease Association (dependent of stage of disease Disease Chemokine Chemokine Receptor Allergies CCL1, CCL2, CCL5, CCL7, CCL8, CCR3, CCR4, CCR8, CCR9 (Skin, Food & Respiratory) CCL11, CCL13, CCL17, CCL22, CCL24, CCL25, CCL26 Asthma CCL3, CCLA, CCL5, CCL7, CCL8, CCR3, CCR4, CCR5, CCL11, CCL15, CCL17, CCL22, CCL24, CCL26, Septic Shock, Anaphylaxis CXCL1, CXCL2, CXCL3, CXCL5, CXCR1, CXCR2, CXCR3 CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CCL5 Arthritis CXCL9, CXCL10, CXCL11, CXCR3, CXCR4, CXCR5 Septic,p rheumatoid, ppsoriatic CXCL12, CXCL13 CCL20 CCR6 XCL XCR1 CX3CL1 CX3CR1 Osteoarthritis CXCL1, CXCL2, CXCL3, CXCL5, CXCR1, CXCR2, CXCL6, CXCL7, CXCL8, CXCL12, CCR2, CCR5 CXCL13, CCL2, CCL3, CCL4, CCL7, CCL8, CCL13, CCL5, CCL18 Atherosclerosis CXCL1, CXCL2, CXCL3, CXCL4, CXCR1, CXCR2 US 7,9 64,194 B2 10 TABLE 1-continued Chemokine, Chemokine Receptor and Inflammatory Disease Association (dependent of stage of disease Disease Chemokine Chemokine Receptor CCL2, CCL3, CCL4, CCL8, CCR2, CCR8 CCL12, CCL13, CCL17, CCL22 CX3CL1 CX3CR1 Dermatitis & Delayed-Typed CXCL9, CXCL10, CXCL11, CXCR3 Hypersensitivity CCL2, CCL3, CCL4, CCL5, CCR4, CCR5, CCR6, CCR10 CCL17, CCL2O, CCL22, CCL27 Diabetes CXCL9, CXCL10, CXCL11, CXCR3 CCL2, CCL9 CCR2, CCR4 CX3CL1 CX3CR1 Graft rejection CXCL9, CXCL10, CXCL11, CXCR3 CCL3, CCL4, CCL5 CCR5 XCL XCR Inflammatory Bowel DiseaseS CXCL9, CXCL10, CXCL11, CXCR3 CCL3, CCL4, CCL5 CCR5 Interstitial Cystitis CXCL9, CXCL10, CXCL11, CXCR3 CCL3, CCL4, CCL5 CCR5 Multiple Sclerosis CXCL9, CXCL10, CXCL11, CXCR3 CCL3, CCL4, CCL5, CCL7, CCR1, CCR5 CCL14, CCL15, CCL23 Myasthemia gravis, Grave's CXCL9, CXCL10, CXCL11, CXCR3 disease, & Hashimoto thyroiditis CCL3, CCL4, CCL5 CCR5 XCL XCR Nephritis & Systemic Lupus CXCL9, CXCL10, CXCL11, CXCR3, CXCR5 Erthematosus CXCL13 CCL2, CCL3, CCL4, CCL5, CCL8, CCL12, CCL13, CX3CL1 Pneumonitis, Chronic Obstructive CXCL1, CXCL2, CXCL3, CXCL5, CXCR2, CXCR3 Pulmonary Disease, & Chronic CXCL7, CXCL8 Bronchitis CCL3, CCL5, CCL7, CCL8, CCR3 CCL11, CCL13, CCL24, CCL26

The method consists of 1) exposing tissues from a mammal 35 b) determining a level of expression of CXCL10 based on suffering from and inflammatory condition to several differ binding of said antibody to said tissue sample; and ing antibodies which bind to specific chemokines, 2) identi c) if over-expression of CXCL10 is detected in said tissue fying level of expression of each chemokine based on binding sample of said mammal, administering to said mammal of the each of the differing antibodies, and 3) administering to an effective amount of an antibody which binds specifi said mammal those specific antibodies which bind to the 40 cally to CXCL10 for the treatment of said inflammatory over-expressed chemokines. Additionally, the level of expres disease. sion of any particular chemokine may be determined using 2. The method of claim 1, wherein said mammal is a PCR, as disclosed herein. human. Antibodies of the invention may be administered directly 3. The method of claim 1, wherein in step c, said antibody to target tissue. For example, compositions containing the 45 is a human, humanized, or chimeric antibody. compositions containing antibodies as prepared under the 4. The method of claim 1, wherein in step c, said antibody heading “anti-sera preparation' can be administered intrave is administered directly into an inflamed tissue. nously, rectally vaginally, intrathecally, by inhalation, trans 5. The method of claim 1, wherein in step c, said antibody vaginally, transurethrally or directly to tissue during Surgery. is administered parenterally. The anti-sera preparations may also be placed on a solid 50 6. The method of claim 1, wherein in step c, said antibody Support such as a sponge or gauze for administration to the is administered in a liquid carrier. mucosa. The antibodies may also be administered using 7. The method of claim 1, wherein in step c, said antibody microspheres or liposomes. The compositions for adminis is administered on a solid Support. tration are prepared in the usual pharmaceutically acceptable 8. The method of claim 1, wherein said inflammatory dis carriers such as saline, water, buffered saline, glucose in 55 ease is selected from the group consisting of anaphylaxis, saline, etc. septic shock, arthritis, asthma, allergies, atherosclerosis, Liquid compositions will most often be prepared contain bronchitis, chronic pulmonary obstructive disease, delayed ing 0.1 to 1000 g of antibody in each ml of solution. All type hypersensitivity, dermatitis, diabetes, graft rejection, compositions will be administered in an inflammatory inhib Grave's disease, Hashimoto's thyroiditis, inflammatory iting effective amount with the amount of dosage given 60 bowel diseases, interstitial cystitis, multiple Sclerosis, myas depending on the size and condition of the individual in need themia gravis, pneumonitis, psoriasis, nephritis, rhinitis, of anti-inflammatory treatment. spondyloarthropathies, scheroderma, systemic lupus erythe What is claimed is: matosus, and throiditis. 1. A method of treating a mammal with inflammatory 9. A method of treating a mammal with inflammatory disease, comprising: 65 disease, comprising: a) exposing a tissue sample from said mammal to an anti a) determining the level of CXCL10 in a tissue sample in body which binds specifically to CXCL10; said mammal using PCR; and US 7,964,194 B2 11 12 if over-expression of CXCL10 is detected in said tissue 14. The method of claim 9, wherein said inflammatory sample of said mammal, administering to said mammal disease is selected from the group consisting of anaphylaxis, an effective amount of an antibody which binds specifi- septic shock, arthritis, asthma, allergies, atherosclerosis, cally to CXCL10 for the treatment of said inflammatory bronchitis, chronic pulmonary obstructive disease, delayed disease. 5 type hypersensitivity, dermatitis, diabetes, graft rejection, 10. The method of claim.9, wherein said antibody is admin- Grave's disease, Hashimoto's thyroiditis, inflammatory istered directly into an inflamed tissue. bowel diseases, interstitial cystitis, multiple Sclerosis, myas 11. The method of claim 9, wherein said antibody is admin- themia gravis, pneumonitis, psoriasis, nephritis, rhinitis, istered systemically. spondyloarthropathies, scheroderma, systemic lupus erythe 12. The method of claim 9, wherein said antibody is admin- matosus, and throiditis. istered on a solid Support. 13. The method of claim 9, wherein said antibody is a human, humanized, or chimeric antibody. k . . . .