Anti-VEGF antibodies
Anti-VEGF antibodies and variants thereof, including those having high affinity for binding to VEGF, are disclosed. Also provided are methods of using phage display technology with naïve libraries to generate and select the anti-VEGF antibodies with desired binding and other biological activities. Further contemplated are uses of the antibodies in research, diagnostic and therapeutic applications.
1. An isolated monoclonal antibody, or an Fab fragment thereof, capable of binding to mouse VEGF and human VEGF with Kd values within 10-fold of the other, wherein the antibody, or Fab fragment thereof, is capable of inhibiting the binding of VEGF to a VEGF receptor, and wherein the antibody binds to an epitope comprising residue F17, Y21, and Y25 of human VEGF.
2. An isolated monoclonal antibody, or an Fab fragment thereof, capable of binding to mouse VEGF and human VEGF with Kd values within 10-fold of the other, wherein the antibody, or Fab fragment thereof, is capable of inhibiting the binding of VEGF to a VEGF receptor, and wherein the antibody binds to an epitope comprising residues F17, I83, and Q89 of human VEGF.
3. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 , wherein said epitope further comprises residue M18 of human VEGF.
4. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 , wherein said epitope further comprises residues Q22, D63, and I83 of human VEGF.
5. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 , wherein said epitope further comprises residue Q89 of human VEGF.
6. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 3 , wherein said epitope further comprises residues D19, Q89, I91, K101, E103, and C104 of human VEGF.
7. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 2 , wherein said epitope further comprises residues Y21, Q22, Y25, and D63 of human VEGF.
8. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 , wherein said epitope further comprises any one or more additional residues selected from the group consisting of M18, Q22, K48, D63, L66, M81, I83, H86, Q89, I91, C104, and P106 of human VEGF.
9. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 8 , wherein the antibody, or Fab fragment thereof, binds to an epitope comprising the residues F17, M18, Y21, Q22, Y25, K48, D63, L66, M81, I83, H86, Q89, I91, C104, and P106 of human VEGF.
10. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 or 2 wherein the receptor is a VEGF receptor 2 (KDR).
11. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 or 2 , wherein the receptor is a VEGF receptor 1 (Flt-1).
12. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 or 2 , wherein the antibody, or Fab fragment thereof, is capable of inhibiting the binding of VEGF to VEGF receptor 1 and VEGF receptor 2.
13. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 or 2 , wherein the antibody, or Fab fragment thereof, binds to mouse and human VEGF with Kd values of no more than 10 nM.
14. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 or 2 , wherein the antibody, or Fab fragment thereof, binds to mouse and human VEGF with Kd values of no more than 2 nM.
15. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 or 2 , wherein the antibody, or Fab fragment thereof, binds to human VEGF with an on-rate (k on ) of at least 1.0×10 5 M −1 S −1 .
16. An isolated monoclonal antibody, or an Fab fragment thereof, capable of binding to mouse VEGF and human VEGF with Kd values within 10-fold of the other, wherein the antibody, or Fab fragment thereof, comprises a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a CDR H1, CDR H2, and CDR H3, wherein:
(a) CDR H1 comprises the contiguous amino acid sequence DYWIH (SEQ ID NO: 261);
(b) CDR H2 comprises the contiguous amino acid sequence GITPAGGYTYYADSVKG (SEQ ID NO: 474); and
(c) CDR H3 comprises the contiguous amino acid sequence FVFFLPYAMDY (SEQ ID NO: 475);
and wherein the light chain variable domain comprises a CDR L1, CDR L2, and CDRL3, wherein:
(d) CDR L1 comprises the contiguous amino acid sequence RASQDVSTAVA (SEQ ID NO: 48);
(e) CDR L2 comprises the contiguous amino acid sequence SASFLYS (SEQ ID NO: 49; and
(f) CDR L3 comprises the contiguous amino acid sequence QQSYTTPPT (SEQ ID NO: 363).
17. The isolated monoclonal antibody, or Fab fragment thereof, of claim 16 , wherein the antibody, or Fab fragment thereof, comprises the following amino acid sequence as its heavy chain variable domain (VH) and first heavy chain constant domain (CH1):
(SEQ ID NO. 29)
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAG
ITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFV
FFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTH
and wherein the antibody comprises the following amino acid sequences as its light chain variable domain (VL):
(SEQ ID NO. 28)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
18. An isolated monoclonal antibody, or an Fab fragment thereof, capable of binding to mouse VEGF and human VEGF with Kd values within 10-fold of the other, wherein the antibody, or Fab fragment thereof, comprises a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a CDR H1, CDR H2, and CDR H3, wherein:
(a) CDR H1 comprises the contiguous amino acid sequence DYWIH (SEQ ID NO: 261);
(b) CDR H2 comprises the contiguous amino acid sequence GITPAGGYTYYADSVKG (SEQ ID NO: 474); and
(c) CDR H3 comprises the contiguous amino acid sequence FVFFLPYAMDY (SEQ ID NO: 475);
and wherein the light chain variable domain comprises a CDR L1, CDR L2, and CDRL3, wherein:
(d) CDR L1 comprises the contiguous amino acid sequence RASQDVSTAVA (SEQ ID NO: 48);
(e) CDR L2 comprises the contiguous amino acid sequence SASFLYS (SEQ ID NO: 49); and
(f) CDR L3 comprises the contiguous amino acid sequence KQGYANPWT (SEQ ID NO: 143).
19. The isolated monoclonal antibody, or Fab fragment thereof, of claim 18 , wherein the antibody, or Fab fragment thereof, comprises the following amino acid sequence as its heavy chain variable domain (VH) and first heavy chain constant domain (CH1):
(SEQ ID NO. 29)
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAG
ITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFV
FFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTH
and wherein the antibody comprises the following amino acid sequences as its light chain variable domain (VL):
(SEQ ID NO. 30)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQGYANPWTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
20. An isolated monoclonal antibody, or an Fab fragment thereof, capable of binding to mouse VEGF and human VEGF with Kd values within 10-fold of the other, wherein the antibody, or Fab fragment thereof, comprises a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a CDR H1, CDR H2, and CDR H3, wherein:
(a) CDR H1 comprises the contiguous amino acid sequence DYWIH (SEQ ID NO: 261);
(b) CDR H2 comprises the contiguous amino acid sequence GITPAGGYTYYADSVKG (SEQ ID NO: 474); and
(c) CDR H3 comprises the contiguous amino acid sequence FVFFLPYAMDY (SEQ ID NO: 475);
and wherein the light chain variable domain comprises a CDR L1, CDR L2, and CDRL3, wherein:
(d) CDR L1 comprises the contiguous amino acid sequence RASQDVSTAVA (SEQ ID NO: 48);
(e) CDR L2 comprises the contiguous amino acid sequence SASFLYS (SEQ ID NO: 49); and
(f) CDR L3 comprises the contiguous amino acid sequence QQGYGNPFT (SEQ ID NO: 52).
21. The isolated monoclonal antibody, or Fab fragment thereof, of claim 20 , wherein the antibody, or Fab fragment thereof, comprises the following amino acid sequence as its heavy chain variable domain (VH) and first heavy chain constant domain (CH1):
(SEQ ID NO. 29)
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAG
ITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFV
FFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTH
and wherein the antibody comprises the following amino acid sequences as its light chain variable domain (VL):
(SEQ ID NO: 32)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYGNPFTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
22. An isolated monoclonal antibody, or an Fab fragment thereof, capable of binding to mouse VEGF and human VEGF with Kd values within 10-fold of the other, wherein the antibody, or Fab fragment thereof, comprises a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a CDR H1, CDR H2, and CDR H3, wherein:
(a) CDR H1 comprises the contiguous amino acid sequence DYWIH (SEQ ID NO: 261);
(b) CDR H2 comprises the contiguous amino acid sequence GITPAGGYTYYADSVKG (SEQ ID NO: 474); and
(c) CDR H3 comprises the contiguous amino acid sequence FVFFLPYAMDY (SEQ ID NO: 475);
and wherein the light chain variable domain comprises a CDR L1, CDR L2, and CDRL3, wherein:
(d) CDR L1 comprises the contiguous amino acid sequence RASQDVSTAVA (SEQ ID NO: 48);
(e) CDR L2 comprises the contiguous amino acid sequence SASFLYS (SEQ ID NO: 49); and
(f) CDR L3 comprises the contiguous amino acid sequence QQGAGSPLT (SEQ NO:
68).
23. The isolated monoclonal antibody, or Fab fragment thereof, of claim 22 , wherein the antibody, or Fab fragment thereof, comprises the following amino acid sequence as its heavy chain variable domain (VH) and first heavy chain constant domain (CH1):
(SEQ ID NO. 29)
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAG
ITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFV
FFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTH
and wherein the antibody comprises the following amino acid sequences as its light chain variable domain (VL):
(SEQ ID NO: 33)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGAGSPLTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
24. An isolated monoclonal antibody, or an Fab fragment thereof, capable of binding to mouse VEGF and human VEGF with Kd values within 10-fold of the other, wherein the antibody, or Fab fragment thereof, comprises a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a CDR H1, CDR H2, and CDR H3, wherein:
(a) CDR H1 comprises the contiguous amino acid sequence DYWIH (SEQ ID NO: 261);
(b) CDR H2 comprises the contiguous amino acid sequence GVTPAGGYTYYADSVK G (SEQ ID NO: 942); and
(c) CDR H3 comprises the contiguous amino acid sequence FVFFLPYAMDY (SEQ ID NO: 475);
and wherein the light chain variable domain comprises a CDR L1, CDR L2, and CDRL3, wherein:
(d) CDR L1 comprises the contiguous amino acid sequence RASQDVSTAVA (SEQ ID NO: 48);
(e) CDR L2 comprises the contiguous amino acid sequence SASFLYS (SEQ ID NO: 49); and
(f) CDR L3 comprises the contiguous amino acid sequence KQGFANPFT (SEQ ID NO: 943).
25. The isolated monoclonal antibody, or Fab fragment thereof, of claim 24 , wherein the antibody, or Fab fragment thereof, comprises the following amino acid sequence as its heavy chain variable domain (VH) and first heavy chain constant domain (CH1):
(SEQ ID NO: 35)
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAG
VTPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFV
FFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTH
and wherein the antibody comprises the following amino acid sequences as its light chain variable domain (VL):
(SEQ ID NO: 34)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQGFANPFTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
26. An isolated monoclonal antibody, or an Fab fragment thereof, capable of binding to mouse VEGF and human VEGF with Kd values within 10-fold of the other, wherein the antibody, or Fab fragment thereof, comprises a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a CDR H1, CDR H2, and CDR H3, wherein:
(a) CDR H1 comprises the contiguous amino acid sequence DYWIH (SEQ ID NO: 261);
(b) CDR H2 comprises the contiguous amino acid sequence GVTPAGGYTAYADSVKG (SEQ ID NO: 944); and
(c) CDR H3 comprises the contiguous amino acid sequence FVFFLPYAMDY (SEQ ID NO: 475);
and wherein the light chain variable domain comprises a CDR L1, CDR L2, and CDRL3, wherein:
(d) CDR L1 comprises the contiguous amino acid sequence RASQDVSTAVA (SEQ ID NO: 48);
(e) CDR L2 comprises the contiguous amino acid sequence SASFLYS (SEQ ID NO: 49); and
(f) CDR L3 comprises the contiguous amino acid sequence KQGFANPFT (SEQ ID NO: 943).
27. The isolated monoclonal antibody, or Fab fragment thereof, of claim 26 , wherein the antibody, or Fab fragment thereof, comprises the following amino acid sequence as its heavy chain variable domain (VII) and first heavy chain constant domain (CH1):
(SEQ ID NO: 36)
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAG
VTPAGGYTAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFV
FFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTH
and wherein the antibody comprises the following amino acid sequences as its light chain variable domain (VL):
(SEQ ID NO: 34)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQGFANPFTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
28. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 , 2 , 16 , 18 , 20 , or 22 , wherein the antibody is a bispecific antibody.
29. The isolated monoclonal antibody, or Fab fragment thereof, according to claim 1 , 2 , 16 , 18 , 20 , or 22 , wherein the antibody is a synthetic antibody.
30. The isolated monoclonal antibody, or Fab fragment thereof, of claim 1 , 2 , 16 , 18 , 20 , or 22 , wherein the antibody is capable of binding hVEGF with a Kd value of no more than about 2 nM.
31. The isolated monoclonal antibody, or Fab fragment thereof, of claim 30 , wherein the Kd value is no more than about 1 nM.
32. The isolated monoclonal antibody, or Fab fragment thereof, of claim 31 , wherein the Kd value is no more than about 500 pM.
33. A method of inhibiting angiogenesis in a human or a mouse, said method comprising the step of administering the isolated monoclonal antibody, or Fab fragment thereof, of claim 1 , 2 , 16 , 18 , 20 , or 22 , to the human or mouse.
34. The method of claim 33 , wherein the human or mouse is suffering from cancer or a disease caused by ocular neovascularization.
35. The method of claim 33 , wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and glioblastoma.
36. The method of claim 33 , wherein the disease caused by ocular neovascularization is diabetic blindness, retinopathy, primary diabetic retinopathy or age-induced macular degeneration.
37. A method of alleviating cancer in a human or mouse, said method comprising the step of administering the isolated monoclonal antibody, or Fab fragment thereof, of claim 1 , 2 , 16 , 18 , 20 , or 22 to the human or mouse.
38. The method of claim 37 , wherein said cancer is selected from the group consisting of breast cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma and glioblastoma.
39. A method of alleviating a disorder caused by ocular neovascularization, said method comprising the step of administering the isolated monoclonal antibody, or Fab fragment thereof, of claim 1 , 2 , 16 , 18 , 20 , or 22 to the human or mouse.
40. The method of claim 39 , wherein the disease caused by ocular neovascularization is diabetic blindness, retinopathy, primary diabetic retinopathy or age-induced macular degeneration.