Neutralizing Antibodies and Methods of Use Thereof

Abstract

This invention provides monoclonal antibodies that recognize the Toll-like Receptor 4/MD-2 receptor complex, and monoclonal antibodies that recognize the TLR4/MD2 complex as well as TLR4 when not complexed with MD-2. The invention further provides methods of using the humanized monoclonal antibodies as therapeutics. This invention also provides soluble chimeric proteins, methods of expressing and purifying soluble chimeric proteins, and methods of using soluble chimeric proteins as therapeutics, in screening assays and in the production of antibodies.

What is claimed is: 
     
         1 . An antibody that immunospecifically binds a Toll-like receptor 4 (TLR4)/MD-2 complex, wherein the antibody binds to an epitope comprising one or more amino acid residues on human TLR4 between residues 289 and 375 of SEQ ID NO: 54. 
     
     
         2 . The antibody of  claim 1 , wherein said antibody specifically binds to an epitope selected from the group consisting of:
 (a) an epitope that comprises at least residues 328 and 329 of SEQ ID NO: 54;   (b) an epitope that comprises at least residues 349 through 351 of SEQ ID NO: 54;   (c) an epitope that comprises at least residues 369 through 371 of SEQ ID NO: 54;   (d) an epitope that comprises at least residues 328, 329, 349 through 351 and 369 through 371 of SEQ ID NO: 54;   (e) an epitope that comprises at least residues 293 through 295 of SEQ ID NO: 54;   (f) an epitope that comprises at least residues 296 and 297 of SEQ ID NO: 54;   (g) an epitope that comprises at least residues 319 through 321 of SEQ ID NO: 54; and   (h) an epitope that comprises at least residues 293 through 295, 296, 297 and 319 through 321 of SEQ ID NO: 54.   
     
     
         3 . An antibody that immunospecifically binds a Toll-like receptor 4 (TLR4)/MD-2 complex, wherein the antibody binds to an epitope on human MD-2 between residues 19 and 57 of SEQ ID NO: 44. 
     
     
         4 . The antibody of  claim 3 , wherein said antibody specifically binds to an epitope that comprises at least residues 53 of SEQ ID NO: 44. 
     
     
         5 . A humanized antibody that immunospecifically binds to a Toll-like receptor 4 (TLR4)/MD-2 complex, wherein the antibody comprises a heavy chain with three complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of GGYSWH (SEQ ID NO: 23); YIHYSGYTDFNPSLKT (SEQ ID NO: 24); KDPSDGFPY (SEQ ID NO: 25); DSYIH (SEQ ID NO: 3); WTDPENVNSIYDPRFQG (SEQ ID NO: 4), GYNGVYYAMDY (SEQ ID NO: 5); TYNIGVG (SEQ ID NO: 33); HIWWNDNIYYNTVLKS (SEQ ID NO: 34); and MAEGRYDAMDY (SEQ ID NO: 35), and a light chain with three CDRs comprising an amino acid sequence selected from the group consisting of the amino acid sequence of RASQSISDHLH (SEQ ID NO: 28); YASHAIS (SEQ ID NO: 29); QNGHSFPLT (SEQ ID NO: 30); SASSSVIYMH (SEQ ID NO: 8); RTYNLAS (SEQ ID NO: 9); HQWSSFPYT (SEQ ID NO: 10); RASQDITNYLN (SEQ ID NO: 38); YTSKLHS (SEQ ID NO: 39); and QQGNTFPWT (SEQ ID NO: 40). 
     
     
         6 . The humanized antibody of  claim 5 , wherein the antibody comprises a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 46, 49, 51 and 52 and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 48, 50 and 53. 
     
     
         7 . A method of alleviating a symptom of a pathology associated with aberrant TLR4 signaling, the method comprising administering an antibody of  claim 1  to a subject in need thereof in an amount sufficient to alleviate the symptom of the pathology in the subject. 
     
     
         8 . The method of  claim 7 , wherein the subject is a human. 
     
     
         9 . The method of  claim 7 , wherein the amount of said antibody sufficient to alleviate the symptom of the pathology associated with aberrant TLR4 signaling is an amount sufficient to reduce LPS-induced pro-inflammatory cytokine production. 
     
     
         10 . The method of  claim 7 , wherein said pathology is selected from the group consisting of sepsis, ventilator-induced lung injury, acute inflammation, chronic inflammation, autoimmune diseases and disorders induced by endogenous soluble stress factors. 
     
     
         11 . The method of  claim 10 , wherein said chronic inflammation is associated with an allergic condition, or asthma. 
     
     
         12 . The method of  claim 10 , wherein said pathology is inflammatory bowel disorder or atherosclerosis. 
     
     
         13 . The method of  claim 10 , wherein said disorder induced by endogenous soluble stress factors is osteoarthritis or rheumatoid arthritis. 
     
     
         14 . The method of  claim 10 , wherein said endogenous soluble stress factor is Hsp60, fibronectin, heparan sulphate, hyaluronan, gp96, β-Defensin-2 or surfactant protein A. 
     
     
         15 . A pharmaceutical composition comprising an antibody of  claim 1  and a carrier.