Pan-ELR+ CXC chemokine antibodies

Abstract

Antibodies are provided that specifically bind seven human ELR.sup.+ CXC chemokines. The antibodies of the invention are useful for treating various inflammatory/autoimmune diseases, such as inflammatory bowel disease (IBD), plaque psoriasis, and palmoplantar pustulosis; and cancer, such as renal cancer or ovarian cancer.

Claims

1. A method of treating ulcerative colitis in a patient, comprising administering to the patient a therapeutically effective amount of an antibody that binds human growth-regulated oncogene (“Gro”)-alpha, human Gro-beta, human Gro-gamma, human epithelial neutrophil activating peptide-78, human granulocyte chemotactic protein-2, human neutrophil activating protein-2, and human interleukin-8, the antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region (LCVR) and the heavy chain comprises a heavy chain variable region (HCVR), wherein the LCVR comprises light chain complementarity determining regions (“LCDR”) LCDR1, LCDR2, LCDR3 and the HCVR comprises heavy chain complementarity determining regions (“HCDR”) HCDR1, HCDR2, HCDR3, wherein LCDR1 is RASQSISNNLH (SEQ ID NO: 7), LCDR2 is YTSRSVS (SEQ ID NO: 8), LCDR3 is GQNNEWPEV (SEQ ID NO: 9), HCDR1 is GYEFTSYWIH (SEQ ID NO: 10), HCDR2 is NISPNSGSANYNEKFKS (SEQ ID NO: 11), and HCDR3 is EGPYSYYPSRXaaYYGSDL (SEQ ID NO: 20) wherein Xaa is E or Q.

2. The method of claim 1, wherein the amino acid sequence of the HCVR is SEQ ID NO: 2 or SEQ ID NO: 14 and the amino acid sequence of the LCVR is SEQ ID NO: 4 or 16.

3. The method of claim 1, wherein the amino acid sequence of the HCVR is SEQ ID NO: 2 and the amino acid sequence of the LCVR is SEQ ID NO: 4.

4. The method of claim 1, wherein the amino acid sequence of the heavy chain is SEQ ID NO: 1 and the amino acid sequence of the light chain is SEQ ID NO: 3.

5. The method of claim 1, wherein the antibody comprises two heavy chains having the amino acid sequence of SEQ ID NO: 1, and two light chains having the amino acid sequence of SEQ ID NO: 3.

6. A method of treating ulcerative colitis in a patient, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition, said pharmaceutical composition comprising an antibody and one or more pharmaceutically acceptable carriers, diluents or excipients, the antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region (LCVR) and the heavy chain comprises a heavy chain variable region (HCVR), wherein the LCVR comprises light chain complementarity determining regions (“LCDR”) LCDR1, LCDR2, LCDR3 and the HCVR comprises heavy chain complementarity determining regions (“HCDR”) HCDR1, HCDR2, HCDR3, wherein LCDR1 is RASQSISNNLH (SEQ ID NO: 7), LCDR2 is YTSRSVS (SEQ ID NO: 8), LCDR3 is GQNNEWPEV (SEQ ID NO: 9), HCDR1 is GYEFTSYWIH (SEQ ID NO: 10), HCDR2 is NISPNSGSANYNEKFKS (SEQ ID NO: 11), and HCDR3 is EGPYSYYPSRXaaYYGSDL (SEQ ID NO: 20) wherein Xaa is E or Q.

7. The method of claim 6, wherein the amino acid sequence of the HCVR is SEQ ID NO: 2 or SEQ ID NO: 14 and the amino acid sequence of the LCVR is SEQ ID NO: 4 or 16.

8. The method of claim 6, wherein the amino acid sequence of the HCVR is SEQ ID NO: 2 and the amino acid sequence of the LCVR is SEQ ID NO: 4.

9. The method of claim 6, wherein the amino acid sequence of the heavy chain is SEQ ID NO: 1 and the amino acid sequence of the light chain having is SEQ ID NO: 3.

10. The method of claim 6, wherein the antibody comprises two heavy chains having the amino acid sequence of SEQ ID NO: 1, and two light chains having the amino acid sequence of SEQ ID NO: 3.

Description

EXAMPLES

(1) Expression and Production of Antibodies

(2) Antibodies of the present invention can be expressed and purified as follows. An expression vector containing the DNA sequence of SEQ ID NO: 5 (encoding a heavy chain polypeptide of SEQ ID NO: 1) and SEQ ID NO: 6 (encoding a light chain polypeptide of SEQ ID NO: 3) is used to transfect NS0 cells. An antibody resulting from this expression vector is “Antibody 1.”

(3) Additionally, an expression vector containing the DNA sequence of SEQ ID NO: 17 (encoding a heavy chain polypeptide of SEQ ID NO: 13) and SEQ ID NO: 18 (encoding a light chain polypeptide of SEQ ID NO: 15) is used to transfect NS0 cells. An antibody resulting from this expression vector is “Antibody 2.”

(4) For either Antibody 1 or Antibody 2, transfected pools are plated at low density to allow for close-to-clonal outgrowth of stable expressing cells. The masterwells are screened for antibody expression and then scaled up in serum-free, suspension cultures to be used for production. Clarified media, into which the antibody has been secreted, is applied to a Protein A or G column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed to remove nonspecific binding components. The bound antibody is eluted by pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium citrate buffer pH 2.5). Antibody fractions are detected by SDS-PAGE and then are pooled. Further purification is optional, depending on the intended use. The antibody may be concentrated and/or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps may be greater than 99%. The product may be immediately frozen at −70° C. or may be lyophilized.

(5) Binding Affinity to Human ELR.sup.+ CXC Chemokines

(6) Biacore 2000 instrument and Biacore 2000 Evaluation Software Version 4.1 are used for surface plasmon resonance analysis. A CM5 chip is prepared using manufacturer's EDC/NHS amine coupling method. Briefly, the surfaces of all four flow cells are activated by injecting a 1:1 mixture of EDC/NHS for 7 minutes at 10 μL/min. Protein A is diluted to 100 μg/mL in 10 mM acetate, pH 4.5 buffer, and immobilized to achieve approximately 10,000 RU onto all 4 flow cells by 7-minute injection at a flow rate of 10 μL/minute. Unreacted sites are blocked with a 7-minute injection of ethanolamine at 10 μL/minute. Two injections of glycine (pH 1.5) for 30 seconds at 10 μL/minute are used to remove any non-covalently associated protein. Running buffer is HBS-EP+.

(7) Antibody 1 or Antibody 2 is diluted to 2 μg/mL in running buffer, and approximately 400-600 RU is captured in flow cell (Fe). Ligands are diluted from 100 μg/mL to 50 nM in running buffer and then two-fold serially diluted in running buffer to 3.125 nM. Duplicate injections of each ligand concentration are injected at 100 μL/min for 150 seconds followed by a dissociation phase. The dissociation phase is 1800 seconds for all ligands. Regeneration is performed by injecting 10 mM glycine (pH 1.5) for 60 seconds at 50 μL/min over all flow cells. Reference-subtracted data is collected as Fc2−Fc1, Fc3−Fc1, and Fc4−Fc1. The measurements are obtained at 25° C. The on-rate (k.sub.on) and off-rate (k.sub.off) for each ligand are evaluated using a “1:1 (mass transfer) Binding” binding model. The affinity (K.sub.D) is calculated from the binding kinetics according to the relationship: K.sub.D=k.sub.off/k.sub.on.

(8) The human ELR.sup.+ CXC chemokines produce a concentration-dependent binding response with Antibody 1 and Antibody 2. Tables 1 and 2 summarize the k.sub.on, k.sub.off, and K.sub.D values for Antibody 1 and Antibody 2. These results demonstrate that Antibody 1 and Antibody 2 bind all seven human ELR.sup.+ CXC chemokines with high affinity.

(9) TABLE-US-00001 TABLE 1 In Vitro Binding Affinities to Human ELR.sup.+ CXC Chemokines for Antibody 1 Chemokine k.sub.on (1/Ms) × 10.sup.5 k.sub.off (1/s) × 10.sup.−5 K.sub.D (pM) human CXCL1 (Gro alpha) 9.30 ± 0.45 10.50 ± 0.14 113 ± 4  human CXCL2 (Gro Beta) 7.73 ± 0.86 13.15 ± 0.64 171 ± 11 human CXCL3 (Gro gamma) 7.16 ± 0.08 12.35 ± 1.06 172 ± 13 human CXCL5 (ENA-78) 5.43 ± 0.61 12.20 ± 0.00 226 ± 25 human CXCL6 (GCP-2) 7.07 ± 0.11 57.80 ± 3.68 818 ± 40 human CXCL7 (NAP-2) 9.00 ± 0.75 16.15 ± 1.77 181 ± 35 human CXCL8 (IL-8) 3.39 ± 0.03 13.00 ± 0.57 384 ± 13

(10) TABLE-US-00002 TABLE 2 In Vitro Binding Affinities to Human ELR.sup.+ CXC Chemokines for Antibody 2 Chemokine k.sub.on (1/Ms) × 10.sup.5 k.sub.off (1/s) × 10.sup.−5 K.sub.D (pM) human CXCL1 (Gro alpha) 6.28 1.53 243 human CXCL2 (Gro beta) 1.14 2.05 180 human CXCL3 (Gro gamma) 4.65 1.76 379 human CXCL5 (ENA-78) 4.27 1.51 354 human CXCL6 (GCP-2) 7.19 6.11 849 human CXCL7 (NAP-2) 5.21 2.18 418 human CXCL8 (IL-8) 2.61 1.24 473
Physical Stability Evaluation

(11) Protein aggregation and self-association is an undesirable property in antibodies as it could potentially exacerbate unwanted effects, such as triggering an immune response. Thus, maintaining the antibody in a monomeric state is highly desirable. Percent high molecular weight (% HMW) aggregate is an indicator of protein aggregation and self-association. A higher % HMW aggregate indicates increased protein aggregation/self-association and increased physical instability. Physical stability of Antibody 1 and Antibody 2 are determined as follows.

(12) Antibody is dialyzed overnight at 4° C. into 10 mM Citrate, pH 6+/−150 mM NaCl. The next morning, the samples are concentrated to 50 mg/mL, filtered through 0.2 micron filters, and then Tween-80 is added to a final concentration of 0.02%. Each sample is incubated at 25 C. for the specified times. Soluble aggregate formation is followed by analytical SEC using a TSK3000SWXL, 5 micron column with dimensions 30 cm×0.78 cm. The mobile phase is 50 mM Sodium Phosphate, pH 7, 175 mM NaCl, at a flow rate of 0.5 mL/min. Samples are applied as 1 μL injections and monitored at 280 nm to determine the increase % HMW aggregate (Table 3).

(13) The 50 mg/mL formulations are incubated for 1 and 4 weeks at 25° C. to assess longer-term stability under stress conditions. The delta % HMW aggregate is determined by subtracting the % HMW aggregate at time zero (designated as ‘Initial’ in Table 3) from the % HMW at the 1 week or 4 week time point. After 1 and 4 weeks, both Antibody 1 and Antibody 2 demonstrated delta % HMW below 1%, thus demonstrating good physical stability.

(14) TABLE-US-00003 TABLE 3 % High Molecular Weight Aggregate 50 mg/mL 50 mg/mL Initial % HMW % HMW (4° C.) (t = 1 week) (t = 4 weeks) Antibody 1 10 mM Citrate pH7, 150 mM NaCl 0.78% 25° C. 1.24% 1.41% Δ % HMW 0.46% 0.63% 10 mM Citrate pH7, 150 mM NaCl, 1.05 0.02% Tween-80 25° C. 1.46% 1.75% Δ % HMW 0.41%  0.7% Antibody 2 10 mM Citrate pH7, 150 mM NaCl 0.37% 25° C. 0.48% 0.59% Δ % HMW 0.11% 0.22% 10 mM Citrate pH7, 150 mM NaCl, 0.42% 0.02% Tween-80 25° C. 0.55% 0.74% Δ % HMW 0.13% 0.32%
Antibody Epitope Mapping for Antibody 1

(15) Multiple approaches are undertaken to characterize the epitope for Antibody 1, including Western blot analysis, co-crystallization of the antibody with several ELR.sup.+ CXC chemokines, and mutational analysis for binding and neutralization.

(16) Western Blot Analysis

(17) To determine if Antibody 1 is able to bind a linear or conformational epitope, Western blot analysis is performed using reducing and non-reducing conditions. Electrophoresis of the proteins is performed using pre-cast NuPAGE® 4-12% Bis-Tris gels. NuPAGE® MES SDS running buffer is added to both the inner (200 mL) and outer (at least 600 mL) chambers of the mini-cells. Serial dilutions of human CXCL8 (400 ng, 100 ng, or 25 ng per lane) are made in NuPAGE® LDS 4× Sample Buffer with or without NuPAGE® 10× Sample Reducing Agent. Samples are heated at 95° C. for 2 minutes. Load volumes are 10 μL per lane for the samples, and 5 μL per lane for the SeeBlue Plus2 Prestained Standard marker. The gels are run at 200V for 35 minutes at room temperature.

(18) Proteins are transferred to PVDF using the iBlot® Dry Blotting System with iBlot® Transfer Stack, Nitrocellulose, Mini. The membrane is blocked in blocking solution (3% nonfat milk in phosphate buffered saline) for 1 hour at room temperature. Antibody 1 is added to the blocking solution to a final concentration of 1 μg/mL and then is incubated for 2 hours at room temperature. Following primary incubation, the antibody/block solution is removed, and the membrane is washed 3 times for 15 minutes in wash buffer (phosphate buffered saline+0.05% Tween 20). The membrane is then incubated with HRP conjugated donkey anti-human Fc specific IgG secondary antibody (0.1 μg/mL in blocking solution) for 1 hour at room temperature. After removal of the secondary antibody, the membrane is washed 4 times for 10 minutes with wash buffer. The membrane is then incubated with working solution of the Stable Peroxide Solution and the Luminol/Enhancer Solution (Super Signal West Pico Chemiluminescent Substrate) for 5 minutes. The membrane is placed in a plastic membrane protector in an X-ray film cassette with CL-X Posure™ Film for 30 seconds. Film is developed using a Konica SRX-101 system.

(19) Under non-reducing conditions, at a CXCL8 concentration of 400 ng, two bands appeared at about 17 kDa and about 10 kDa. At a CXCL8 concentration of 100 ng, one band appeared at about 10 kDa. At a CXCL8 concentration of 25 ng, no bands appeared. Under reducing conditions, no bands appeared at any of the tested concentrations.

(20) The results demonstrated that Antibody 1 is able to bind non-reduced, denatured human CXCL8, but is unable to bind reduced, denatured human CXCL8. Therefore, the two disulfide bonds in CXCL8 are needed to maintain the antibody epitope. These results demonstrate a conformational epitope for Antibody 1.

(21) Crystal Structure Analysis

(22) Crystal structures of the Fab/antigen complexes for human CXCL8, and cynomolgus monkey CXCL2, CXCL3, and CXCL7 are obtained to determine the complete binding surface of Antibody 1. Wild type truncated human CXCL8 1-66, human CXCL8 point mutants, and cynomolgus monkey CXCL7 are all expressed in E. coli with N-terminal His-SUMO tags. These proteins are refolded; the tags are cleaved; and the proteins are purified by standard purification techniques. Cynomolgus monkey CXCL2 and CXCL3 are expressed in HEK293 EBNA cells and are purified by standard purification techniques. Formation of disulfide bonds is confirmed by tryptic digest and mass fingerprint analysis, and activity is confirmed by neutrophil chemotaxis assay. A Fab of Antibody 1 is expressed in HEK293 EBNA cells and is purified by standard purification techniques. Fab/antigen complexes are formed by adding slight molar excess of antigen to the Fab, followed by size exclusion chromatography purification to remove excess free antigen. Complexes are crystallized, and the crystal structures are solved by molecular replacement using Buster 2.9.5 (Global Phasing Ltd.).

(23) These crystal structures confirmed that the epitope for Antibody 1 included the N-terminus of the ELR.sup.+ CXC chemokines, but they also showed contacts between the Fab and the β1-β2 loop and the β2 and β3 strands of the ELR.sup.+ CXC chemokines. The crystal structures also demonstrate that the antibody specifically recognizes the fold of the ELR.sup.+ CXC chemokines, since the crystal structures were superimposable. Numerous hydrogen bonding and Van der Waals interactions were also observed. Most notably, the conserved R6 side chain of the ELR motif sat in a deep binding pocket formed by the Fab heavy chain at residues W33, Y102, and Y110, and the Fab light chain at residue W94. The wild type truncated human CXCL8 chemokine also hydrogen bonds with both the Fab heavy chain at residue E99 and the Fab light chain at the N91 backbone carbonyl. Other hydrogen bonds observed were between the L5 backbone carbonyl of the wild type truncated human CXCL8 chemokine and the Fab light chain W94 backbone amide; the I10 backbone carbonyl of the wild type truncated human CXCL8 chemokine and the Fab heavy chain S52 side chain; the K11 side chain of the wild type truncated human CXCL8 chemokine and the Fab heavy chain T30 and S31 side chains; the H33 side chain of the wild type truncated human CXCL8 chemokine and the Fab light chain W94 backbone carbonyl; the A35 backbone amide and the Fab heavy chain N59 side chain; and the C50 backbone amide of the wild type truncated human CXCL8 chemokine and the Fab heavy chain Y104 backbone carbonyl. Further, the N-terminal residues 5 through 13 of the wild type truncated human CXCL8 chemokine made numerous contacts with the Fab as the N-terminus sat in a groove between the Fab heavy chain CDR2 and CDR3. Additionally, the Fab heavy chain CDR3 loop extended away from this groove and interacted with the non-N-terminal residue I40 on the β2 strand, and residues Glu48, Leu49, and Cys50 on the β3 strand of the wild type truncated human CXCL8 chemokine. Finally, residues 33-36 of the β1-β2 loop in the wild type truncated human CXCL8 chemokine packs against the Fab heavy chain CDR2.

(24) Mutational Analysis

(25) Several key contacts between the Antibody 1 Fab and wild type human CXCL8 are observed in the crystal structure and are further tested through binding kinetic studies and U937-huCXCR2 fluorescent imaging plate reader (FLIPR) neutralization of human CXCL8 point mutants. To study these key contacts, several point mutants (R6A, I10A, A35P, I40A, and L49A) are made based on the human CXCL8 sequence (SEQ ID NO: 27). Wild type, R6A, I10A, A35P, I40A, and L49A human CXCL8 point mutants are expressed, refolded, and purified according to standard techniques. Formation of disulfide bonds is confirmed by tryptic digest and mass fingerprint analysis, and activity is confirmed by neutrophil chemotaxis assay.

(26) Binding kinetics are tested on a Biacore 2000 instrument with Biacore 2000 Evaluation Software Version 4.1 as described above.

(27) Wild type and mutant human CXCL8 are tested for biologic activity and for neutralization by Antibody 1 using the U937-huCXCR2 assay. U937-huCXCR2 is a monocytic cell line transduced with retrovirus for expression of human CXCR2. A description of the 11937-huCXCR2 assay is found below under Neutralizing Assays.

(28) CXCL8 mutants are serially diluted in Assay Buffer containing 0.2% BSA in wells of v-bottom 96-well polypropylene plates. Ligand concentrations are 3 times the final assay concentration (final assay concentrations range from 300 to 0.0051 nM). A cell plate and a ligand plate are loaded into a Fluorescent Imaging Plate Reader (FLIPR-3, Molecular Devices) programmed to transfer 50 μL of ligand to wells of the cell plate. Fluorescence is recorded at 1 second intervals for 90 seconds. The change in fluorescence [delta relative fluorescence units (DRFU). Max RFU-Min RFU] is calculated from images 10 to 90. DRFU versus log (ligand concentration) is plotted and EC.sub.50 values are determined by nonlinear regression using Graph Pad Prism. Assays are performed in triplicate over three assay plates.

(29) Antibody 1 is serially diluted in Assay Buffer containing 0.2% BSA. Antibody concentrations are 3 times the final assay concentration (final concentrations range from 10 to 0.0195 μg/ml). Stock solutions of wild type human CXCL8 and CXCL8 mutants are prepared in Assay Buffer+0.2% BSA at 240 nM (30× the final assay concentration of 8 nM). 20 μL of ligand is mixed with 180 μL of Antibody 1 in wells of v-bottom 96-well polypropylene plates. Ligand and antibody are incubated at room temperature for 30 minutes. A cell plate and a ligand-antibody plate are loaded into a Fluorescent Imaging Plate Reader (FLIPR-3, Molecular Devices) programmed to transfer 50 μL, of ligand-antibody to wells of the cell plate. Fluorescence is recorded at 1 second intervals for 90 seconds. The change in fluorescence (DRFU) is calculated from images 10 to 90. DRFU versus log (antibody concentration) is plotted and IC.sub.50 values are determined by nonlinear regression using Graph Pad Prism. Assays are performed in triplicate over three assay plates.

(30) The binding kinetics, biologic activity, and neutralization results are summarized in Table 4. The affinity measurements are obtained at 25° C. The on-rate (k.sub.on) and off-rate (k.sub.off) for each ligand are evaluated using a “1:1 (mass transfer) Binding” binding model. The affinity (K.sub.D) is calculated from the binding kinetics according to the relationship: K.sub.D=k.sub.offk.sub.on.

(31) Several of the mutations knocked out activity to the receptor (EC.sub.50) and, therefore, could not be tested for neutralization. It is noted that the A35P mutation completely abolished neutralizing activity despite having full activity to the receptor. These results highlight key contacts (R6, I10, A35, I40, and L49) within the binding interface of the CXCL8 antigen that are important for antibody binding.

(32) TABLE-US-00004 TABLE 4 Binding kinetics, U937 FLIPR activity (EC.sub.50), and U937 FLIPR neutralization (IC.sub.50) of human CXCR8 wild type and mutants. Variant k.sub.on (M.sup.−1 s.sup.−1) k.sub.off (s.sup.−1) K.sub.D (pM) EC.sub.50 (μg/mL) IC.sub.50 (μg/mL) WT 3.03 ± 3.78 × 10.sup.6 4.41 ± 4.55 × 10.sup.−4 236 ± 144 1.3 ± 0.8 (n = 4) 0.9 R6A No Binding Not Active I10A 2.77 ± 0.39 × 10.sup.5 3.44 ± 0.01 × 10.sup.−4 1260 ± 184  Not Active A35P 1.06 ± 0.07 × 10.sup.6 4.97 ± 1.72 × 10.sup.−3 4740 ± 1920 1.9 ± 0.3 (n = 2) No neutralization I40A 1.61 ± 0.21 × 10.sup.4 4.24 ± 1.16 × 10.sup.−4 27100 ± 10700 Not Active L49A 1.43 ± 0.24 × 10.sup.5 5.44 ± 0.42 × 10.sup.−4 3830 ± 354  5.6 ± 1.3 (n = 2) 1.5

(33) Overall, the epitope mapping analysis for Antibody 1 demonstrated that the binding interface of the antigen includes the N-terminus of the ELR.sup.+ CXC chemokines (amino acids 5-13), the β1-β2 loop (amino acids 33-36), and the β2 and β3 strands (amino acids 40, 48-50). Key contacts within this interface that are important for antibody binding include the amino acids R6, I10, A35, I40, and L49 in CXCL8 (SEQ ID NO: 27).

(34) Neutralization Assays

(35) In Vitro Neutralization of Human ELR.sup.+ CXC Chemokines Using Human CXCR2-Transfected HMEC Cells

(36) Since all of the ELR.sup.+ CXC chemokines can bind CXCR2 receptor, cells expressing CXCR2 were selected for in vitro studies. HMEC-huCXCR2 is an immortalized human endothelial cell line transduced with retrovirus for expression of human CXCR2 receptor. HMEC cells expressing human CXCR2 are able to induce intracellular Ca.sup.2+ influx in response to human, cynomolgus monkey, rat, and mouse ELR.sup.+ CXC chemokines. Intracellular Ca.sup.2+ influx can be detected by a Fluorescent Imagine Plate Reader (FLIPR). Chemokine neutralization should therefore also neutralize intracellular Ca.sup.2+ influx induced by these chemokines.

(37) HMEC-huCXCR2 is maintained in MCDB 31 medium supplemented with 10% fetal bovine serum, 2× GlutaMax, 1× non-essential amino acids, 1 μg/mL hydrocortisone, 10 ng/mL human Epidermal Growth Factor, and 0.4 μg/mL puromycin at 37° C. in 5% CO.sub.2. Cultures are maintained at sub-confluent densities (50-80% confluent). Cells are harvested with TrypLE Express, cell density is adjusted to 3×10.sup.5 cells/mL in complete culture medium, and 100 μL of the cell suspension are seeded into wells of black clear-bottom assay plates. Cell plates are incubated at room temperature for 30 minutes to allow cells to settle to the bottom of the wells before plates are incubated overnight at 37° C. in 5% CO.sub.2. For each assay plate, the contents of one vial of Fluo-4NW reagent is suspended in 10 mL Assay Buffer and 100 μL probenecid to make 1× Fluo-4NW reagent. After incubation, culture medium is aspirated and 100 μL of the 1× Fluo-4NW solution is added to each well of the assay plate. Plates are incubated for 30 minutes at 37° C. followed by an additional 30 minutes at room temperature and protected from light. Antibody 1 is serially diluted in Assay Buffer containing 0.2% BSA. Antibody concentrations are 3× the final assay concentration (final concentrations range from 10-0.0195 μg/mL). Stock solutions of ligands are prepared in Assay Buffer+0.2% BSA at 300 nM (30× the final assay concentration of 10 nM). 20 μL of ligand is mixed with 180 μL of antibody in wells of v-bottom 96-well polypropylene plates. Ligand and antibody are incubated at room temperature for 30 minutes. A cell plate and a ligand-antibody plate are loaded into a Fluorescent Imagine Plate Reader (FLIPR-3, Molecular Devices) programmed to transfer 50 μL of ligand-antibody to wells of the cell plate. Fluorescence is recorded every second for 90 seconds. The change in fluorescence (DRFU) is calculated from images 10 to 90. DRFU versus log (antibody concentration) is plotted and IC.sub.50 values are determined by nonlinear regression using Graph Pad Prism. Assays are performed in triplicate over three assay plates. Data are expressed as the mean of replicates.

(38) The results are summarized in Table 5. These results demonstrate that Antibody 1 was able to neutralize all seven human ELR.sup.+ CXC chemokines. IC.sub.50 values are expressed as μg/mL of antibody (standard deviations are in parentheses). Both the 72 amino acid and 77 amino acid forms of CXCL8 were used. Data are average of 2-5 replicates.

(39) TABLE-US-00005 TABLE 5 In vitro FLIPR study using HMEC cells expressing human CXCR2 IC.sub.50 (μg/mL) CXCL1 0.867 (±0.153) CXCL2 1.281 (±0.449) CXCL3 0.731 (±0.187) CXCL5 0.681 (±0.347) CXCL6 1.122 (±0.523) CXCL7 1.068 (±0.324) CXCL8 (72) 0.951 (±0.416) CXCL8 (77) 0.358 (±0.078)

(40) In Vitro Neutralization of Human CXCL8 or CXCL1-induced Chemotaxis Using Primary Human Neutrophils

(41) A chemotaxis assay using human neutrophils was chosen to determine the neutralizing activity of Antibody 1 in cells naturally expressing both CXCR1 and CXCR2. Peripheral blood from healthy volunteers is drawn into two 10 mL sodium heparin tubes. To isolate neutrophils, 5 mL of blood is layered over 5 mL of Polymorphprep in four 15 mL tubes. The tubes are centrifuged for 30 minutes at 470×g, 18° C. The plasma and top cell band (mononuclear cells) are removed and discarded. The second band (neutrophils) is pooled from the 4 tubes and an equal volume of PBS is added. The tube is centrifuged for 10 minutes at 400×g, 18° C. The pellet is washed with 12 mL of PBS, centrifuged as before, and the pellet is re-suspended with 11 mL HBSS/BSA (7.5 mg/mL BSA, HBSS). 60×10.sup.6 cells are suspended in 12 mL HBSS/BSA and 5 μM CMFDA and incubated for 30 min at 37° C. Post-incubation, the tube is centrifuged to pellet the cells, washed one time with 12 mL HBSS/BSA, and then the cells are re-suspended in 12 mL HBSS/BSA (5×10.sup.6 cells/mL).

(42) Antibody 1 and isotype control (human IgG4 antibody) are diluted to 1495 nM using HBSS/BSA (Dilution 1) and then serially diluted 1:5 with HBSS/BSA. CXCL8 is diluted to 20 nM with HBSS/BSA. CXCL1 is diluted to 10.1 nM with HBSS/BSA.

(43) 70 μL of Antibody 1 or HBSS/BSA are mixed with 70 μL of either the CXCL8 or CXCL1 solution and incubated at room temperature for ˜30 minutes. 30 μL of the mixture are dispensed into the lower chamber wells of a ChemoTx plate in triplicate. Wells containing only HBSS/BSA (no chemokine or antibody) will show the background signal. The ChemoTx filter is placed over the lower chamber and 50 μL (250,000 cells) is dispensed above each well. The ChemoTx plate is incubated for 3 hours at 37° C. 5% CO.sub.2. After incubation, the cells are rinsed from the top surface with PBS and the ChemoTx filter is removed. Fluorescence is read (Wallac Victor.sup.3 1420 counter) 485/535 using only the bottom detector. The mean fluorescence of the background wells (HBSS/BSA only) is subtracted from the test well fluorescence, and the mean and standard deviations are calculated in Excel.

(44) At a CXCL8 concentration of 5 nM, the IC.sub.50 for Antibody 1 (MW 150,000 kDa) was 26.4 (±0.236) nM. At a CXCL8 concentration of 10 nM, the IC.sub.50 for Antibody 1 was 43.7 (±0.086) nM. At a CXCL1 concentration of 5 nM, the IC.sub.50 for Antibody 1 was 18.5 (±0.158) nM. At a CXCL1 concentration of 20 nM, the IC.sub.50 for Antibody 1 was 40.3 (±0.112) nM. At all tested concentrations of CXCL1 and CXCL8, the isotype control antibody did not affect chemotaxis. The data show that Antibody 1 can block the chemotactic activity of human CXCL8 or CXCL1 in a dose dependent manner while chemotactic activity was unaffected by the isotype control antibody.

(45) In Vivo Acute DSS Colitis Model in Mice

(46) Dextran sulfate sodium (DSS) is the most commonly used model of Ulcerative Colitis (UC). In this model, DSS is a chemical irritant that is added to drinking water to induce acute disease that resembles UC. The acute phase of DSS colitis is characterized by the recruitment of neutrophils to the mucosa and submucosa, and increased expression of ELR.sup.+ CXC chemokines. However, chronic exposure to DSS causes severe gut damage and significant weight loss, which is not suitable in a colitis model. To accommodate the acute nature of this model, Antibody 1 was used in a prophylactic manner to test its ability to inhibit the recruitment of neutrophils and the development of colitis. Of note in this model, mouse CXCL5 (LIX) protein is significantly increased in colon tissue (Kwon 2005); however, Antibody 1 does not neutralize this mouse chemokine.

(47) C57BL/6 mice, 8-10 wks old, weighing 18-22 g, are obtained. Blood is collected by cardiac puncture and analyzed to establish a baseline. To induce colitis, mice receive 2.5% DSS (MW=36.000-50,000) in the drinking water for 5 days (Days 1-5) followed by 6 days of no DSS water (reflecting acute inflammation). Control healthy mice receive water only (“no DSS” group). Mice receiving DSS are dosed by subcutaneous injection on Day 0, 2, 4, and 8 with human IgG4 control antibody (25 mg/kg) or Antibody 1 (25 mg/kg). Body weight is recorded daily. The number of mice used for each treatment is 9 (except 5 healthy mice are used in the healthy “no DSS” group.) The study is performed in quadruplicate.

(48) As shown in Table 6, DSS mice that received human IgG4 control antibody lost weight dramatically between Day 5 and Day 8. DSS mice that received Antibody 1 before induction of colitis and during the acute phase of the disease displayed less weight loss between Day 5 and Day 8 than those DSS mice treated with human IgG4 control antibody (94.0% initial body weight for Antibody 1 versus 85.3% initial body weight for IgG4 control on Day 8). These results demonstrate that systemic administration of Antibody I effectively mitigated weight loss in DSS-induced colitis, supporting the conclusion that Antibody 1 neutralizes the activity of certain mouse ELR.sup.+ CXC chemokines and decreases neutrophil recruitment to the colon.

(49) TABLE-US-00006 TABLE 6 % of Initial Body Weight Day No DSS IgG4 Antibody 1 1 104.4 99.9 100.0 2 103.9 99.8 102.9 3 103.0 100.3 101.4 4 101.0 99.5 100.9 5 101.2 97.5 96.9 8 102.2 85.3 94.0 9 99.8 89.6 93.5 10 100.7 93.7 93.6 11 101.3 98.2 97.0
In Vivo Neutralization in 786-O Clear Cell Renal Cell Xenograft Model

(50) 786-O renal cell carcinoma (RCC) cells are mixed 1:1 with matrigel and implanted subcutaneously in the right rear flank of athymic nude female mice at 3.0×10.sup.6 cells per injection. 786-O xenograft-bearing mice having tumor volumes that reached 100 mm.sup.3 were orally gavaged with 10 mg/kg of sunitinib two times a day under a continuous dosing regimen until the mice started progressing with tumor growth even under sunitinib treatment like control-treated mice (IgG4 and 10% vehicle). Mice that were progressing with tumor growth on sunitinib therapy were randomly divided into 2 groups. One group receives sunitinib at 10 mg/kg two times a day plus control IgG4 antibody at 20 mg/kg once a week. The other group receives suninitib at 10 mg/kg two times a day plus Antibody 1 at 20 mg/kg once a week. Average tumor volumes (standard error in parentheses) are shown in Table 7. The addition of Antibody 1 to sunitinib treatment reduced tumor growth significantly over time (p<0.0001), indicating that Antibody 1 resensitized clear cell RCC tumors to sunitinib treatment.

(51) TABLE-US-00007 TABLE 7 Average Tumor Volume (mm.sup.3) IgG4 and IgG4 and Antibody 1 and Day 10% vehicle Sunitinib Sunitinib 11  82.84 (±18.52) 73.21 (±8.91) 72.54 (±40.89) 17 112.12 (±25.06) 95.34 (±11.6) 95.99 (±54.11) 24 148.41 (±33.17) 124.86 (±15.19) 138.32 (±77.97)  27 180.24 (±40.29) 138.08 (±16.8)  152.87 (±86.18)  34 205.22 (±45.87)  183.2 (±22.29) 181.08 (±102.08) 38 221.66 (±49.55) 207.46 (±25.25) 211.1 (±119)   41 255.46 (±57.1)  210.04 (±25.56) 222.01 (±125.15) 46 267.75 (±59.85) 267.53 (±32.55) 248.74 (±140.22) 48 292.17 (±65.31) 268.24 (±32.64) 276.78 (±156.03) 52 325.13 (±72.68)  301.1 (±36.64) 286.57 (±161.55) 55 331.39 (±74.37) 328.55 (±39.98)  262.9 (±148.21) 59 373.91 (±84.32) 371.36 (±45.19) 304.06 (±171.41) 62 413.09 (±93.47) 387.79 (±47.19) 313.16 (±176.54) 66  479.4 (±108.92) 417.33 (±50.78) 285.85 (±161.14) 69  537.74 (±122.53) 494.68 (±60.19) 276.64 (±155.95) 73  520.11 (±118.92) 527.88 (±64.23) 244.07 (±137.59) 76  532.57 (±122.53) 595.93 (±72.52) 228.91 (±129.04) 81  597.7 (±137.49) 601.51 (±73.19) 196.06 (±110.52) 84  720.91 (±166.16) 652.01 (±79.34) 193.08 (±108.84) 87  713.64 (±164.79) 663.62 (±80.75) 181.85 (±102.51) 90  785.88 (±181.79) 775.05 (±94.31) 175.88 (±99.15)  94  891.11 (±206.57)  836.89 (±101.84) 192.57 (±108.56) 97 1073.86 (±249.3)  1010.26 (±122.93) 210.16 (±118.47)
In Vivo Neutralization in SKOV3-Luc Ovarian Cancer Xenograft Model

(52) SKOV3-Luc is a human ovary cancer cell line that expresses firefly luciferase gene. SKOV3-Luc cells are often used in vivo to establish human ovarian adenocarcinoma tumor growth.

(53) SKOV3-Luc ovarian cancer cells were mixed 1:1 with matrigel and implanted subcutaneously in the right rear flank of athymic nude female mice at 3.0×10.sup.6 cells per injection. Mice were randomized into 4 groups at baseline according to tumor volume after the xenografts were grown to an average tumor volume of 200 mm.sup.3. Mice received either control IgG4 antibody (2.5 mg/kg once a week), cisplatin (2.5 mg/kg once a week), Antibody 1 (20 mg/kg once a week), or a combination of cisplatin (2.5 mg/kg once a week) and Antibody 1 (20 mg/kg once a week) by intraperitoneal injection. Tumor growth is shown in Table 8. Cisplatin monotherapy did not show statistically significant tumor growth inhibition compared to isotype control. However, the combination of cisplatin and Antibody 1 showed statistically significant tumor growth inhibition (p≦0.001) compared to isotype control and cisplatin monotherapy, indicating that Antibody 1 synergistically augments chemotherapy in the SKOV3-Luc ovarian cancer xenograft model.

(54) TABLE-US-00008 TABLE 8 Average Tumor Volume (mm.sup.3) Antibody 1 and Day IgG4 Cisplatin Antibody 1 Cisplatin 14 128.45 (±7.25)  122.65 (±12.2)  109.35 (±12.7)  127.17 (±26.72) 19 182.92 (±10.33) 190.65 (±18.97) 173.95 (±20.2)  167.94 (±35.29) 22 269.83 (±15.23) 289.15 (±28.77) 234.58 (±27.25) 218.81 (±45.98) 26 507.66 (±28.66) 484.72 (±48.23) 364.42 (±42.33) 349.81 (±73.51) 32 904.66 (±51.07) 806.31 (±80.23) 739.26 (±85.86) 530.63 (±111.5) 35 1052.88 (±59.44)  923.02 (±91.85) 821.83 (±95.45) 579.7   (±121.81) 40 1143.39 (±64.55)  941.42 (±93.68) 1026.68 (±119.25) 585.37 (±123)   43 1382.03 (±78.03)  1047.34 1098.72 626.99 (±104.22) (±127.61) (±131.75)

(55) TABLE-US-00009 Sequences Antibody 1 Heavy chain amino acid sequence: SEQ ID NO: 1 QVQLVQSGAEVKKPGASVKVSCKASGYEFTSYWIHWVIZQAPGQGLEWMG NISPNSGSANYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARE GPYSYYPSREYYGSDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG Antibody 1 Heavy chain variable region: SEQ ID NO: 2 QVQLVQSGAEVKKPGASVKVSCKASGYEFTSYWIHWVRQAPGQGLEWMGN ISPNSGSANYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREG PYSYYPSREYYGSDLWGQGTLVTVSS Antibody 1 Light chain amino acid sequence: SEQ ID NO: 3 EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYY TSRSVSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGQNNEWPEVFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Antibody 1 Light chain variable region: SEQ ID NO: 4 EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYY TSRSVSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGQNNEWPEVFGG GTKVEIK Antibody 1 Heavy chain DNA sequence: SEQ ID NO: 5 CAGGTGCAGCTGGTGCAGTCTGGTGCTGAAGTGAAGAAGCCTGGGGCCTC AGTGAAGGTGTCCTGCAAGGCATCTGGCTACGAGTTCACCAGCTACTGGA TTCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAAT ATTTCTCCTAATAGTGGTAGTGCTAACTACAATGAGAAGTTCAAGAGCAG AGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGA GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGC CCTTACAGTTATTATCCGAGTAGGGAGTACTATGGCTCTGACCTCTGGGG GCAAGGGACCCTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGG TCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTG GAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC AGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAA CACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCAC CCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCC CCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCC TCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA GAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAA CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCG CCGTGGAGTGGGAAAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAAC CGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGA TGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCT CTGGT Antibody 1 Light chain DNA sequence: SEQ ID NO: 6 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA AAGAGCCACCCLCTCCMCAGGGCCAGTCAAAGTATCAGCAATAACCTACA CTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATTATA CTTCCCGGTCCGTCTCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCT GGGACAGATTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTGGACAGAATAACGAGTGGCCTGAGGTGTTCGGCGGAGG GACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCT TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGC CTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGA TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Antibody 1/Antibody 2 LCDR1: SEQ ID NO: 7 RASQSISNNLH Antibody 1/Antibody 2 LCDR2: SEQ ID NO: 8 YTSRSVS Antibody 1/Antibody 2 LCDR3: SEQ ID NO: 9 GQNNEWPEV Antibody 1/Antibody 2 HCDR1: SEQ ID NO: 10 GYEFTSYWIH Antibody 1/Antibody 2 HCDR2: SEQ ID NO: 11 NISPNSGSANYNEKFKS Antibody 1 HCDR3: SEQ ID NO: 12 EGPYSYYPSREYYGSDL Antibody 2 Heavy chain amino acid sequence: SEQ ID NO: 13 QVQLVQSGAEVKKPGASVKVSCKASGYEFTSYWIHWVRQAPGQGLEWMGN ISPNSGSANYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREG PYSYYPSRQYYGSDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG Antibody 2 Heavy chain variable region: SEQ ID NO: 14 QVQLVQSGAEVKKPGASVKVSCKASGYEFTSYWIHWVRQAPGQGLEWMGN ISPNSGSANYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREG PYSYYPSRQYYGSDLWGQGTLVTVSS Antibody 2 Light chain amino acid sequence: SEQ ID NO: 15 EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYY TSRSVSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGQNNEWPEVFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Antibody 2 Light chain variable region: SEQ ID NO: 16 EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYY TSRSVSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGQNNEWPEVFGG GTKVEIK Antibody 2 Heavy chain DNA sequence: SEQ ID NO: 17 CAGGTGCAGCTGGTGCAGTCTGGTGCTGAAGTGAAGAAGCCTGGGGCCTC AGTGAAGGTGTCCTGCAAGGCATCTGGCTACGAGTTCACCAGCTACTGGA TTCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAAT ATTTCTCCTAATAGTGGTAGTGCTAACTACAATGAGAAGTTCAAGAGCAG AGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGA GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGGC CCTTACAGTTATTATCCGAGTAGGCAGTACTATGGCTCTGACCTCTGGGG GCAAGGGACCCTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGG TCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTG GAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC AGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAA CACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCAC CCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCC CCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGT ACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCC TCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA GAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAA CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCG CCGTGGAGTGGGAAAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAAC CGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGA TGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCT CTGGGT Antibody 2 Light chain DNA sequence: SEQ ID NO: 18 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA AAGAGCCACCCTCTCCTGCAGGGCCAGTCAAAGTATCAGCAATAACCTAC ACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATTAT ACTTCCCGGTCCGTCTCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC TGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTG CAGTTTATTACTGTGGACAGAATAACGAGTGGCCTGAGGTGTTCGGCGGA GGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCAT CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTG GATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGA CAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Antibody 2 HCDR3: SEQ ID NO: 19 EGPYSYYPSRQYYGSDL HCDR3 Consensus Sequence: SEQ ID NO: 20 EGPYSYYPSRXaaYYGSDL wherein Xaa is E or Q Human Gro-alpha (CXCL1): SEQ ID NO: 21 ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKA CLNPASPIVKKIIEKMLNSDKSN Human Gro-beta (CXCL2): SEQ ID NO: 22 APLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKA CLNPASPMVKKIIEKMLKNGKSN Human Gro-gamma (CXCL3): SEQ ID NO: 23 ASVVTELRCQCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKA CLNPASPMVQKIIEKILNKGSTN Human ENA-78 (CXCL5): SEQ ID NO: 24 AAVLRELRCVCLQTTQGVHRKMISNLQVFAIGPQCSKVEVVASLKNGKEI CLDPEAPFLKKVIQKILDGGNKEN Human GCP-2 (CXCL6): SEQ ID NO: 25 VSAVLTELRCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQ VCLDPEAPFLKKVIQKILDSGNKKN Human NAP-2 (CXCL7): SEQ ID NO: 26 AELRCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDP DAPRIKKIVQKKLAGDESAD Human IL-8 (CXCL8): SEQ ID NO: 27 SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELC LDPKENWVQRVVEKFLKRAENS