Anti-vegf antibodies

Abstract

Humanized and variant anti-VEGF antibodies and various uses therefor are disclosed. The anti-VEGF antibodies have strong binding affinities for VEGF; inhibit VEGF-induced proliferation of endothelial cells in vitro; and inhibit tumor growth in vivo.

1 . A humanized anti-vascular endothelial growth factor (VEGF) antibody which binds human VEGF with a K d  value of no more than about 1×10 −8 M. 
     
     
         2 . A humanized anti-vascular endothelial growth factor (VEGF) antibody which binds human VEGF with a K d  value of no more than about 5×10 −9 M. 
     
     
         3 . A humanized anti-vascular endothelial growth factor (VEGF) antibody which has an ED50 value of no more than about 5 nM for inhibiting VEGF-induced proliferation of endothelial cells in vitro. 
     
     
         4 . A humanized anti-vascular endothelial growth factor (VEGF) antibody which inhibits VEGF-induced angiogenesis in vivo. 
     
     
         5 . The humanized anti-VEGF antibody of  claim 4  wherein 5 mg/kg of the antibody inhibits tumor growth by at least about 50% in tumor weight in vivo in nude mice transformed with human A673 rhabdomyosarcoma cells. 
     
     
         6 . The humanized anti-VEGF antibody of  claim 1  having a heavy chain variable domain comprising the following complementarity determining region (CDR) amino acid sequences: CDRH1 (GYX 1 FTX 2 YGMN, wherein X 1  is T or D and X 2  is N or H; SEQ ID NO:130), CDRH2 (WINTYTGEPTYAADFKR; SEQ ID NO:2) and CDRH3 (YPX 1 YYGX 2 SHWYFDV, wherein X 1  is Y or H and X 2  is S or T; SEQ ID NO:131). 
     
     
         7 . The humanized anti-VEGF antibody of  claim 6  comprising the amino acid sequence of SEQ ID NO:7. 
     
     
         8 . The humanized anti-VEGF antibody of  claim 6  having a heavy chain variable domain comprising the following hypervariable region amino acid sequences: CDRH1 (GYTFTNYGMN; SEQ ID NO:1), CDRH2 (WINTYTGEPTYAADFKR; SEQ ID NO:2) and CDRH3 (YPHYYGSSHWYFDV; SEQ ID NO:3). 
     
     
         9 . The humanized anti-VEGF antibody of  claim 1  having a light chain variable domain comprising the following hypervariable region amino acid sequences: CDRL1 (SASQDISNYLN; SEQ ID NO:4), CDRL2 (FTSSLHS; SEQ ID NO:5) and CDRL3 (QQYSTVPWT; SEQ ID NO:6). 
     
     
         10 . The humanized anti-VEGF antibody of  claim 9  comprising the amino acid sequence of SEQ ID NO:8. 
     
     
         11 . The humanized anti-VEGF antibody of  claim 1  having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:8. 
     
     
         12 . An anti-VEGF antibody light chain variable domain comprising the amino acid sequence: DIQX 1 TQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKR (SEQ ID NO:126), wherein X 1  is M or L. 
     
     
         13 . An anti-VEGF antibody heavy chain variable domain comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYX 1  FTX 2 YGMNWVRQAPGKGLEWVGWINTYTGEPTY AADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPX 3 YYGX 4 SHWYFDVWGQGTLVTV SS (SEQ ID NO:127), wherein X 1  is T or D; X 2  is N or H; X 3  is Y or H and X 4  is S or T. 
     
     
         14 . A variant of a parent anti-vascular endothelial growth factor (VEGF) antibody, wherein said variant binds human VEGF and comprises an amino acid substitution in a complementarity determining region (CDR) of a heavy chain variable domain of said parent antibody. 
     
     
         15 . The variant of  claim 14  wherein said parent antibody is a human or humanized antibody. 
     
     
         16 . The variant of  claim 14  which binds human VEGF with a K d  value of no more than about 1×10 −8  M. 
     
     
         17 . The variant of  claim 14  which binds human VEGF with a K d  value of no more than about 5×10 −9  M. 
     
     
         18 . The variant of  claim 14  wherein the substitution is in CDRH1 of the heavy chain variable domain. 
     
     
         19 . The variant of  claim 14  wherein the substitution is in CDRH3 of the heavy chain variable domain. 
     
     
         20 . The variant of  claim 14  which has amino acid substitutions in both CDRH1 and CDRH3. 
     
     
         21 . The variant of  claim 14  which binds human VEGF with a K d  value less than that of said parent antibody. 
     
     
         22 . The variant of  claim 14  which has an ED50 value for inhibiting VEGF-induced proliferation of endothelial cells in vitro which is at least about 10 fold lower than that of said parent antibody. 
     
     
         23 . The variant of  claim 18  wherein the CDRH1 comprises the amino acid sequence: 
       
         
           
                 
                 
                 
                 
               
                     
                   GYDFTHYGMN 
                   (SEQ ID NO:128) 
                     
                 
             
                
               
            
           
         
       
     
     
         24 . The variant of  claim 19  wherein the CDRH3 comprises the amino acid sequence: 
       
         
           
                 
                 
                 
                 
               
                     
                   YPYYYGTSHWYFDV. 
                   (SEQ ID NO:129) 
                     
                 
             
                
               
            
           
         
       
     
     
         25 . The variant of  claim 14  wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:118. 
     
     
         26 . The variant of  claim 25  further comprising the light chain variable domain amino acid sequence of SEQ ID NO:126. 
     
     
         27 . The variant of  claim 26  comprising the light chain variable domain amino acid sequence of SEQ ID NO:117. 
     
     
         28 . The humanized anti-VEGF antibody of  claim 1  which is a full length antibody. 
     
     
         29 . The humanized anti-VEGF antibody of  claim 28  which is a human IgG. 
     
     
         30 . The humanized anti-VEGF antibody of  claim 1  which is an antibody fragment. 
     
     
         31 . The antibody fragment of  claim 30  which is a Fab. 
     
     
         32 . A composition comprising the humanized anti-VEGF antibody of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         33 . A composition comprising the variant anti-VEGF antibody of  claim 14  and a pharmaceutically acceptable carrier.