Human antibodies against tissue factor and methods of use thereof

Abstract

Isolated human monoclonal antibodies which bind to human TF and related antibody-based compositions and molecules, are disclosed. Also disclosed are pharmaceutical compositions comprising the antibodies, and therapeutic and diagnostic methods for using the antibodies.

1 - 88 . (canceled) 
     
     
         89 . A human antibody which binds human Tissue Factor wherein said antibody:
 a) does not compete for Tissue Factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:65, and   b) competes for Tissue Factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.   
     
     
         90 . The antibody of  claim 89 , wherein the antibody binds to the extracellular domain of Tissue Factor with an apparent affinity (EC 50 ) of 0.50 nM or less, when determined as described in the assay in Example 13. 
     
     
         91 . The antibody of  claim 89 , wherein the antibody binds to mammalian cells expressing Tissue Factor, such as A431 cells transfected with a construct encoding Tissue Factor, with an apparent affinity (EC 50 ) of 8 nM or less, when determined as described in the assay in Example 14. 
     
     
         92 . The antibody of  claim 89 , wherein the antibody is capable of inducing antibody-dependent cellular cytotoxicity in A431 cells, preferably with an EC 50  value of 2 nM or less, when determined as described in the assay in Example 20. 
     
     
         93 . (canceled) 
     
     
         94 . The antibody of  claim 89 , wherein the antibody inhibits tissue factor induced blood coagulation, with a median inhibition concentration of less than 10 nM, when determined as described in the assay in Example 19. 
     
     
         95 . The antibody of  claim 89 , wherein the antibody inhibits FVIIa-induced IL-8 release by MDA-MB-231 cells, with a maximum inhibition value of inhibition of more than 40%, when determined as described in the assay in Example 17. 
     
     
         96 . The antibody of  claim 89 , wherein the antibody inhibits conversion of FX into FXa by the TF/FVIIa complex, in the range of 1-30%, when determined as described in the assay in Example 18. 
     
     
         97 . The antibody of  claim 89 , wherein the antibody is capable of inducing C3c and C4c deposition, and wherein the antibody is capable of inducing C3c and C4c deposition as determined in Example 21. 
     
     
         98 . The antibody of  claim 89 , wherein the antibody binds to human tissue factor and not murine tissue factor and shows reduced binding as compared to binding to human TF to the shuffle construct 85-122 mm, containing the human sequence for TF except for amino acid 85-122, which has been replaced with mouse sequence, as described in Example 27. 
     
     
         99 . (canceled) 
     
     
         100 . The antibody of  claim 89 , wherein the antibody comprises:
 a) a VH region comprising the CDR1, 2, and 3 sequences of SEQ ID NOs:30, 31, and 32, and a VL region comprising the CDR1, 2, and 3 sequences of SEQ ID NOs:86, 87, and 88,   b) a VH region comprising the CDR1, 2, and 3 sequences of SEQ ID NOs:34, 35, and 36, and a VL region comprising the CDR1, 2, and 3 sequences of SEQ ID NOs:90, 91, and 92,   c) a VH region comprising the CDR1, 2, and 3 sequences of SEQ ID NOs:38, 39, and 40, and a VL region comprising the CDR1, 2, and 3 sequences of SEQ ID NOs:94, 95, and 96,   d) a VH region comprising the CDR1, 2, and 3 sequences of SEQ ID NOs:54, 55, and 56, and a VL region comprising the CDR1, 2, and 3 sequences of SEQ ID NOs:110, 111, and 112, or   e) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2, or 3 amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino-acid substitutions in said sequences.   
     
     
         101 . (canceled) 
     
     
         102 . (canceled) 
     
     
         103 . The antibody of  claim 89  comprising:
 a) a VH region comprising the sequence of SEQ ID NO:29 and a VL region comprising the sequence of SEQ ID NO:85, 
 b) a VH region comprising the sequence of SEQ ID NO:33 and a VL region comprising the sequence of SEQ ID NO:89, 
 c) a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93, 
 d) a VH region comprising the sequence of SEQ ID NO:53 and a VL region comprising the sequence of SEQ ID NO:109, or 
 e) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2, or 3 amino-acid substitutions. 
 
     
     
         104 . The antibody of  claim 89 , wherein the antibody has an affinity to tissue factor which is less than 5 nM when determined by the method described in Example 22 herein. 
     
     
         105 . The antibody of  claim 89 , wherein the antibody is effective in inhibiting growth of established BX-PC3 tumors when determined by the method described in Example 26 herein. 
     
     
         106 . The antibody of  claim 89 , wherein the antibody further has one or more of the following properties: inhibition of proliferation, inhibition of tumor angiogenesis, induction of apoptosis of tumor cells, binding to alternatively spliced Tissue Factor. 
     
     
         107 . (canceled) 
     
     
         108 . The antibody of  claim 89 , wherein the antibody is a full-length antibody. 
     
     
         109 . The antibody of  claim 89 , wherein the antibody is conjugated to another moiety. 
     
     
         110 . The antibody of  claim 89 , wherein the antibody is an effector-function-deficient antibody. 
     
     
         111 . The antibody of  claim 110 , wherein the effector-function-deficient anti-Tissue-Factor antibody is a stabilized human IgG4 antibody. 
     
     
         112 . The antibody of  claim 89 , wherein the antibody is a monovalent antibody. 
     
     
         113 . The antibody of  claim 112 , wherein said monovalent antibody is constructed by a method comprising:
 i) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen specific antibody and a nucleotide sequence encoding the constant CL region of an Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen specific antibody and said nucleotide sequence encoding the CL region of an Ig are operably linked together, and wherein, in case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that the CL region does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human being;   ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of a selected antigen specific antibody and a nucleotide sequence encoding a constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to an animal human being, wherein said nucleotide sequence encoding the VH region of a selected antigen specific antibody and said nucleotide sequence encoding the CH region of said Ig are operably linked together;   iii) providing a cell expression system for producing said monovalent antibody; and   iv) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).   
     
     
         114 . The antibody of  claim 112 , wherein the monovalent antibody comprises:
 (i) a variable region of an antibody which binds human TF or an antigen binding part of the said region, and   (ii) a C H  region of an immunoglobulin or a fragment thereof comprising the C H 2 and C H 3 regions, wherein the C H  region or fragment thereof has been modified such that the region corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, other regions of the C H  region, do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical C H  region or other covalent or stable non-covalent inter-heavy chain bonds with an identical C H  region in the presence of polyclonal human IgG.   
     
     
         115 . The antibody of  claim 112 , wherein the heavy chain has been modified such that the entire hinge has been deleted. 
     
     
         116 . A bispecific molecule comprising an antibody of  claim 89  and a second binding specificity. 
     
     
         117 . (canceled) 
     
     
         118 . An expression vector comprising a nucleotide sequence encoding one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs:29-40 and SEQ ID NOs: 53-56. 
     
     
         119 . (canceled) 
     
     
         120 . A recombinant eukaryotic or prokaryotic host cell which produces the antibody of  claim 89 . 
     
     
         121 . A pharmaceutical composition comprising an antibody of  claim 89 , and a pharmaceutically acceptable carrier. 
     
     
         122 . A method of treating cancer comprising administering to a subject in need thereof an effective amount of the antibody of  claim 89 . 
     
     
         123 . The method of  claim 122 , wherein the cancer is selected from the group consisting of: tumors of the central nervous system, head and neck cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, liver and biliary cancer, pancreatic cancer, colorectal cancer, bladder cancer, kidney cancer, prostate cancer, endometrial cancer, ovarian cancer, malignant melanoma, sarcoma, tumors of unknown primary origin, bone marrow cancer, acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma, skin cancer, glioma, cancer of the brain, uterus, and rectum. 
     
     
         124 - 130 . (canceled) 
     
     
         131 . A method for inhibiting growth and/or proliferation of a tumor cell expressing Tissue Factor, comprising administration, to an individual in need thereof, of the antibody of  claim 89 . 
     
     
         132 . A method for producing the antibody of  claim 89 , said method comprising the steps of:
 a) culturing a host cell which produces the antibody, and   b) purifying the antibody from the culture media.   
     
     
         133 . A diagnostic composition comprising the antibody of  claim 89 . 
     
     
         134 . A method for detecting the presence of Tissue Factor in a sample, comprising:
 contacting the sample with the antibody of  claim 89  under conditions that allow for formation of a complex between the antibody or bispecific molecules and Tissue Factor; and   analyzing whether a complex has been formed.   
     
     
         135 . A kit for detecting the presence of Tissue Factor in a sample comprising:
 the antibody of  claim 89 ; and   instructions for use of the kit.