Endoglin Antibodies

Abstract

The present application relates to compositions of humanized and humanized/deimmunized anti-endoglin antibodies and antigen-binding fragments thereof. One aspect relates to antibodies having one or more modifications in at least one amino acid residue of at least one of the framework regions of the variable heavy chain, the variable light chain or both. Another aspect relates to antibodies which bind endoglin and inhibit angiogenesis. Another aspect relates to the deimmunization of humanized antibodies to reduce immunogenicity. Another aspect relates to the use of humanized and humanized/deimmunized antibodies which bind endoglin for the detection, diagnosis or treatment of a disease or condition associated with endoglin, angiogenesis or a combination thereof.

What is claimed is: 
     
         1 . An antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region having an amino acid sequence set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93. 
     
     
         2 . An antibody, or antigen-binding fragment thereof, that binds endoglin, comprising a heavy chain variable region having an amino acid sequence set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93, wherein:
 said heavy chain variable region further comprises one or more modifications selected from the group consisting of a substitution of glycine (G) by alanine (A) or serine (S) at position 49; a substitution of alanine (A) by isoleucine (I) at position 51; a substitution of lysine (K) by arginine (R) or glutamine (Q) at position 52b; a substitution of leucine (L) by valine (V) at position 78 utilizing the Kabat numbering system; and   the light chain variable region further comprises one or more modifications selected from the group consisting of a substitution of methionine (M) by leucine (L) at position 4; a substitution of alanine (A) by valine (V) at position 19; a substitution of threonine (T) by serine (S) at position 22; a substitution of alanine (A) by isoleucine (I) at position 48; and a substitution of threonine (T) by serine (S) at position 51 utilizing the Kabat numbering system.   
     
     
         3 . An antibody, or antigen-binding fragment thereof, of  claim 2  comprising a heavy chain variable region having an amino acid sequence set forth as SEQ ID NO: 88, 89, 90, 91 or 92; and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93, 94, 95, 96, 97, 100, 102, or 103. 
     
     
         4 . An antigen-binding fragment of  claim 1  wherein the antigen-binding fragment is a Fab fragment, a Fab′ fragment, a F(ab′) 2  fragment, an Fv fragment, an scFv fragment, a single chain binding polypeptide, a Fd fragment, a variable heavy chain, a variable light chain or a dAb fragment. 
     
     
         5 . A composition comprising an antibody or antigen-binding fragment of  claim 1 , and an acceptable carrier or excipient. 
     
     
         6 . A nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment of  claim 1 . 
     
     
         7 . An antibody or antigen-binding fragment thereof, of  claim 1 , further labeled with a therapeutic label, a diagnostic label, or both. 
     
     
         8 . A method of treating an angiogenesis-related disease in a subject comprising administering a composition of an antibody or antigen-binding fragment thereof, of  claim 1 . 
     
     
         9 . The method of  claim 8 , wherein said antibody or antigen-binding fragment thereof is labeled with a therapeutic label, a detectable label or both. 
     
     
         10 . The method of  claim 8 , wherein the angiogenesis-related disease is an ocular disease characterized by angiogenesis/neovascularization, diabetic nephropathy, IBD, rheumatoid arthritis, osteoarthritis, a cancer, or a metastasis. 
     
     
         11 . The method of  claim 10 , wherein the ocular disease is macular degeneration. 
     
     
         12 . The method of  claim 10 , wherein the ocular disease is diabetic retinopathy. 
     
     
         13 . The method of  claim 10 , wherein the cancer is a solid tumor. 
     
     
         14 . The method of  claim 10 , wherein the cancer is an epithelial based tumor 
     
     
         15 . The method of  claim 10 , wherein the cancer is selected from a lung cancer, a gynecologic malignancy, a melanoma, a breast cancer, a pancreatic cancer, an ovarian cancer, a uterine cancer, a colorectal cancer, a prostate cancer, a kidney cancer, a head cancer, a pancreatic cancer, a liver cancer (hepatocellular cancer), a uterine cancer, a neck cancer, a kidney cancer (renal cell cancer), a sarcoma, a myeloma, and a lymphoma. 
     
     
         16 . The method of  claim 8 , further comprising administering one or more angiogenesis inhibitors. 
     
     
         17 . The method of  claim 16 , wherein the angiogenesis inhibitor is chemotherapy, a VEGF receptor inhibitor, a VEGF inhibitor, or a combination thereof. 
     
     
         18 . The method of  claim 8  wherein the composition of an antibody or antigen-binding fragment thereof is administered in an amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, or about 200 mg/kg. 
     
     
         19 . A diagnostic method comprising:
 a) providing a sample of cancer cells of a solid tumor or plasma from a patient to be tested;   b) detecting in the sample the expression of at least one gene or gene product chosen from a panel of genes or gene products whose expression has been correlated with sensitivity or resistance to an angiogenesis inhibitor, wherein the at least one gene or gene product is chosen from one or more genes or gene products selected from the group consisting of VEGF, VEGF receptor, HIF-1α, placental growth factor receptor, and CD105; and   c) comparing the level of expression of at least one gene or gene product detected in the patient sample to a level of expression of at least one gene or gene product that has been correlated with sensitivity or resistance to the angiogenesis inhibitor.   
     
     
         20 . The method of  claim 19 , wherein the angiogenesis inhibitor is chosen from VEGF receptor inhibitors, VEGF inhibitors, and endoglin inhibitors. 
     
     
         21 . A kit comprising reagents for the detection of expression levels that have been correlated with sensitivity or resistance to an angiogenesis inhibitor of one or more genes selected from VEGF, VEGF receptor, HIF-1α, placental growth factor receptor, and endoglin in a sample of cancer cells or human plasma. 
     
     
         22 . The kit of  claim 21 , wherein said kit contains an antibody or antigen-binding fragment thereof. 
     
     
         23 . An antibody, or antigen-binding fragment thereof, that binds endoglin, that comprises a heavy chain variable region having an amino acid sequence set forth as SEQ ID NO: 88, 89, 90, 91 or 92 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93, 94, 95, or 102, wherein the antibody, or antigen-binding fragment thereof, has an affinity of from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM) or from about 100 nM to about 1 femtomolar (fM). 
     
     
         24 . The antibody, or antigen-binding fragment thereof, of  claim 23 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93, 94, 95, or 102. 
     
     
         25 . The antibody, or antigen-binding fragment thereof, of  claim 23 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93. 
     
     
         26 . The antibody, or antigen-binding fragment thereof, of  claim 23 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 94. 
     
     
         27 . The antibody, or antigen-binding fragment thereof, of  claim 23 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 95. 
     
     
         28 . The antibody, or antigen-binding fragment thereof, of  claim 23 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 102. 
     
     
         29 . A pharmaceutical composition that comprises the antibody, or antigen-binding fragment thereof, of  claim 23 , and one or more pharmaceutically acceptable carriers or excipients, wherein the one or more pharmaceutically acceptable carriers or excipients comprise a surfactant, a buffer, a polyol, an additive, an antibacterial agent, an antifungal agent, a preservative, a stabilizer, an antioxidant, or a combination thereof. 
     
     
         30 . A method of treating an angiogenesis-related disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of  claim 29 . 
     
     
         31 . The method of  claim 30 , wherein the angiogenesis-related disease comprises an ocular disease. 
     
     
         32 . The method of  claim 31 , wherein the ocular disease comprises age-related macular degeneration (AMD), choroidal neovascularization, or diabetic retinopathy. 
     
     
         33 . The method of  claim 32 , wherein the ocular disease comprises age-related macular degeneration (AMD) that is wet AMD or dry AMD. 
     
     
         34 . The method of  claim 30 , wherein the angiogenesis-related disease comprises diabetic nephropathy. 
     
     
         35 . The method of  claim 30 , wherein the angiogenesis-related disease comprises a cancer or a metastasis thereof. 
     
     
         36 . The method of  claim 35 , wherein the cancer or a metastasis thereof is a solid tumor. 
     
     
         37 . The method of  claim 35 , wherein the cancer or a metastasis thereof is an epithelial based tumor. 
     
     
         38 . The method of  claim 35 , wherein the cancer or the metastasis thereof is a lung cancer, a melanoma, a breast cancer, a pancreatic cancer, an ovarian cancer, a colorectal cancer, a prostate cancer, a kidney cancer, a liver cancer, a uterine cancer, a neck cancer, a sarcoma, a myeloma, a brain cancer, or a lymphoma. 
     
     
         39 . The method of  claim 38 , wherein the cancer or the metastasis thereof is a brain cancer that is a glioblastoma multiforme or a glioma. 
     
     
         40 . The method of  claim 35 , wherein treatment results in partial elimination of the cancer or the metastasis thereof, total elimination of the cancer or the metastasis thereof, prolongation of survival of the subject, or a combination thereof. 
     
     
         41 . The method of  claim 30 , wherein the pharmaceutical composition is administered to the subject one or more times. 
     
     
         42 . The method of  claim 30 , wherein the pharmaceutical composition is administered to the subject once per day, once per week, once per month, once bi-monthly, once every two months, once every three months, once every four months, once every 5 months, or once every 6 months. 
     
     
         43 . An antibody, or antigen-binding fragment thereof, that binds endoglin, that comprises a heavy chain variable region having an amino acid sequence set forth as SEQ ID NO: 88, 89, 90, 91 or 92 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93, 94, 95, or 102, wherein the antibody, or antigen-binding fragment thereof, has a K D  of from about 5×10 −10  or greater. 
     
     
         44 . The antibody, or antigen-binding fragment thereof, of  claim 43 , wherein the antibody, or antigen-binding fragment thereof, has a K D  of from about 5×10 −10  to about 9×10 −10 . 
     
     
         45 . The antibody, or antigen-binding fragment thereof, of  claim 43 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93, 94, 95, or 102. 
     
     
         46 . The antibody, or antigen-binding fragment thereof, of  claim 43 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 93. 
     
     
         47 . The antibody, or antigen-binding fragment thereof, of  claim 43 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 94. 
     
     
         48 . The antibody, or antigen-binding fragment thereof, of  claim 43 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 95. 
     
     
         49 . The antibody, or antigen-binding fragment thereof, of  claim 43 , wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region set forth as SEQ ID NO: 89 and a light chain variable region having an amino acid sequence set forth as SEQ ID NO: 102. 
     
     
         50 . A pharmaceutical composition that comprises the antibody, or antigen-binding fragment thereof, of  claim 43 , and one or more pharmaceutically acceptable carriers or excipients, wherein the one or more pharmaceutically acceptable carriers or excipients comprise a surfactant, a buffer, a polyol, an additive, an antibacterial agent, an antifungal agent, a preservative, a stabilizer, an antioxidant, or a combination thereof. 
     
     
         51 . A method of treating an angiogenesis-related disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of  claim 50 . 
     
     
         52 . The method of  claim 51 , wherein the angiogenesis-related disease comprises an ocular disease. 
     
     
         53 . The method of  claim 52 , wherein the ocular disease comprises age-related macular degeneration (AMD), choroidal neovascularization, or diabetic retinopathy. 
     
     
         54 . The method of  claim 53 , wherein the ocular disease comprises age-related macular degeneration (AMD) that is wet AMD or dry AMD. 
     
     
         55 . The method of  claim 51 , wherein the angiogenesis-related disease comprises diabetic nephropathy. 
     
     
         56 . The method of  claim 51 , wherein the angiogenesis-related disease comprises a cancer or a metastasis thereof. 
     
     
         57 . The method of  claim 56 , wherein the cancer or a metastasis thereof is a solid tumor. 
     
     
         58 . The method of  claim 56 , wherein the cancer or a metastasis thereof is an epithelial based tumor. 
     
     
         59 . The method of  claim 56 , wherein the cancer or the metastasis thereof is a lung cancer, a melanoma, a breast cancer, a pancreatic cancer, an ovarian cancer, a colorectal cancer, a prostate cancer, a kidney cancer, a liver cancer, a uterine cancer, a neck cancer, a sarcoma, a myeloma, a brain cancer, or a lymphoma. 
     
     
         60 . The method of  claim 59 , wherein the cancer or the metastasis thereof is a brain cancer that is a glioblastoma multiforme or a glioma. 
     
     
         61 . The method of  claim 56 , wherein treatment results in partial elimination of the cancer or the metastasis thereof, total elimination of the cancer or the metastasis thereof, prolongation of survival of the subject, or a combination thereof. 
     
     
         62 . The method of  claim 51 , wherein the pharmaceutical composition is administered to the subject one or more times. 
     
     
         63 . The method of  claim 51 , wherein the pharmaceutical composition is administered to the subject once per day, once per week, once per month, once bi-monthly, once every two months, once every three months, once every four months, once every 5 months, or once every 6 months.