Humanized anti-egfl7 antibodies and methods using same

Abstract

The present invention concerns antibodies to EGFL7 and the uses of same.

What is claimed is: 
     
         1 . A method of reducing or inhibiting perfusion and permeability of a tumor in a subject, the method comprising administering to an individual in need of such treatment an effective amount of an anti-EGFL7 antibody, the antibody comprising the following hypervariable region (HVR) sequences:
 HVR-L1 comprising KX 1 SX 2 SX 3 DYX 4 GDSYX 5 S, wherein X 1  is A or R; X 2  is H or Q; X 3  is G or V; X 4  is selected from the group consisting of D, L, R, S, and W; and X 5  is M or V (SEQ ID NO: 210);   (ii) HVR-L2 comprising GASX 1 X 2 EX 3 , wherein X 1  is N or Y; X 2  is selected from the group consisting of L, R and Y; and X 3  is Q or S (SEQ ID NO: 211);   (iii) HVR-L3 comprising QQNNEX 1 PX 2 T, wherein X 1  is D or E; and X 2  is F or Y (SEQ ID NO: 212);   (iv) HVR-H1 comprising GX 1 X 2 X 3 X 4 TYGX 5 S, wherein X 1  is H or V; X 2  is R or T; X 3  is selected from the group consisting of F, G, R, and S; X 4  is selected from the group consisting of D, G, R, and T; and X 5  is M or Y (SEQ ID NO: 213);   (v) HVR-H2 comprising GWINX 1 X 2 SGVPTX 3 AX 4 X 5 X 6 X 7 X 8 , wherein X 1  is selected from the group consisting of I, M, T, and W; X 2  is H or R; X 3  is selected from group consisting of I, M, T, and Y; X 4  is D or H; X 5  is selected from group consisting of D, M and T; X 6  is F or Y; X 7  is K or S; and X 8  is G or R (SEQ ID NO: 214, and   (vi) HVR-H3 comprising AX 1 LGSX 2 AVDX 3 , wherein X 1  is N or R; X 2  is selected from the group consisting of C, R, H, and Y; and X 3  is A or Y (SEQ ID NO: 215).   
     
     
         2 . A method of reducing or inhibiting perfusion and permeability of a tumor in a subject, the method comprising administering to an individual in need of such treatment an effective amount of an anti-EGFL7 antibody, the antibody comprising the following HVR sequences: HVR-L1 comprising an amino acid sequence selected from SEQ ID NOs: 37-43, HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-47, HVR-L3 comprising the amino acid sequence SEQ ID NO: 48, HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-57, HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58-73, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-77. 
     
     
         3 . The method of  claim 1 , wherein HVR-L1 comprises the amino acid sequence KASQSVDYSGDSYMS (SEQ ID NO: 234), HVR-L2 comprises the amino acid sequence GASYRES (SEQ ID NO: 235), HVR-L3 comprises the amino acid sequence QQNNEEPYT (SEQ ID NO: 236), HVR-Hl comprises the amino acid sequence GHTFTTYGMS (SEQ ID NO: 34), HVR-H2 comprises the amino acid sequence GWINTHSGVPTYADDFKG (SEQ ID NO: 35), and HVR-H3 comprises the amino acid sequence ARLGSYAVDY (SEQ ID NO: 237). 
     
     
         4 . The method of  claim 1 , wherein the heavy chain comprises the following framework sequences: FR-H1 comprises EX 1 QLVESGGGLVQPGGSLRLSCAAS, wherein X 1  is I or V (SEQ ID NO: 216); FR-H2 comprises WVRQAPGKGLEWX 1 , wherein X 1  is I, M, or V (SEQ ID NO: 217); FR-H3 comprises RFTX 1 SX 2 DX 3 SX 4 X 5 TX 6 YLQMNSLRAEDTAVYX 7 CAR, wherein X 1  is F or I; X 2  is L or R; X 3  is N or T, X 4  is selected from the group consisting of A, E, K and T; X 5  is N or S; X 6  is selected from the group consisting of A, L, M, T and V; and X 7  is F or Y (SEQ ID NO: 218); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO: 219). 
     
     
         5 . The method of  claim 4 , wherein the heavy chain comprises the following framework sequences: FR-H1 comprises EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 197); FR-H2 comprises WVRQAPGKGLEWV (SEQ ID NO: 198); FR-H3 comprises RFTISX 1 DNSKNTX 2 YLQMNSLRAEDTAVYYCAR, wherein X 1  L or R; X 2  is selected from the group consisting of A, L, M, T and V (SEQ ID NO: 220); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO: 200). 
     
     
         6 . The method of  claim 4 , wherein the heavy chain comprises the following framework sequences: FR-H1 comprises EIQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 238); FR-H2 comprises WVRQAPGKGLEWM (SEQ ID NO: 239); FR-H3 comprises RFTISX 1 DNSKSTX 2 YLQMNSLRAEDTAVYFCAR, wherein X 1  L or R; X 2  is selected from the group consisting of A, L, M, T and V (SEQ ID NO: 240); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO: 219). 
     
     
         7 . The method of  claim 1 , wherein the light chain comprises the following framework sequences: FR-L1 comprises DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 201), FR-L2 comprises WYQQKPGKAPKLLIY (SEQ ID NO: 202), FR-L3 comprises GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203), FR-L4 comprises FGQGTKVEIK (SEQ ID NO: 221) or FGQGTKVEIKR (SEQ ID NO: 204). 
     
     
         8 . The method of  claim 1 , wherein the light chain comprises the variable domain sequence of 4F11.v17 as shown in  FIG. 15  (SEQ ID NO: 82) or the variable domain sequence of 4F11.v22 as shown in  FIG. 15  (SEQ ID NO: 83). 
     
     
         9 . The method of  claim 1 , wherein the heavy chain comprises the variable domain sequence of 4F11.v17 as shown in  FIG. 16  (SEQ ID NO: 84) or the variable domain sequence of 4F11.v22 as shown in  FIG. 16  (SEQ ID NO: 85). 
     
     
         10 . The method of  claim 1 , wherein the light chain comprises the variable domain sequence of 4F11.v17 as shown in  FIG. 15  (SEQ ID NO: 82) and the heavy chain comprises the variable domain sequence of 4F11.v17 as shown in  FIG. 16  (SEQ ID NO: 84). 
     
     
         11 . The method of  claim 1 , wherein the light chain comprises the variable domain sequence of 4F11.v22 as shown in  FIG. 15  (SEQ ID NO: 83) and the heavy chain comprises the variable domain sequence of 4F11.v22 as shown in  FIG. 16  (SEQ ID NO: 85). 
     
     
         12 . The method of  claim 1 , wherein at least a portion of the framework sequence is a human consensus framework sequence. 
     
     
         13 . The method of  claim 12 , comprising human κ subgroup 1 consensus framework sequence or heavy chain human subgroup III consensus framework sequence. 
     
     
         14 . A method of reducing or inhibiting perfusion and permeability of a tumor in a subject, the method comprising administering to an individual in need of such treatment an effective amount of an anti-EGFL7 antibody, the antibody comprising a variable domain comprising the following HVR sequences:
 HVR-L1 comprising X 1 X 2 X 3 X 4 X 5 X 6 VX 7 X 8 X 9 X 10 ITYLX 11 , wherein X 1  is selected from the group consisting of L, Q, R, S, and T; X 2  is selected from the group consisting of P, T, and W; X 3  is H or S; X 4  is D or Q; X 5  is G or S; X 6  is L or V; X 7  is H or P; X 8  is selected from the group consisting of I, L, P, T, and Y; X 9  is selected from the group consisting of N, Q or S; X 10  is selected from the group consisting of A, G, and S; and X 11  is G or H (SEQ ID NO: 222);   (ii) HVR-L2 comprising RVSNX 1 X 2 S, wherein X 1  is D or R; and X 2  is selected from the group consisting of A, G, F, I, and T (SEQ ID NO: 223);   (iii) HVR-L3 comprising X 1 QSX 2 X 3 VPLT, wherein X 1  is selected from the group consisting of A, G, I, K, L, N, T, and V; X 2  is C or T; and X 3  is F or H (SEQ ID NO: 224);   (iv) HVR-H1 comprising GYX 1 X 2 X 3 DX 4 YX 5 N, wherein X 1  is N or T; X 2  is F or V; X 3  is selected from the group consisting of I, M, R, and S; X 4  is selected from the group consisting of Y, Q, and K; and X 5  is I or M (SEQ ID NO: 225);   (v) HVR-H2 comprising GDINX 1 X 2 X 3 X 4 X 5 X 6 HX 7 X 8 X 9 X 10 X 11 X 12 X 13 , wherein X 1  is selected from the group consisting of A, L, N, and P; X 2  is selected from the group consisting of D, L, and R; X 3  is selected from the group consisting of G, K, N, R, S, and Y; X 4  is G or S; X 5  is selected from the group consisting of G, I, K, R, S, T, and V; X 6  is selected from the group consisting of G, R, and T; X 7  is selected from the group consisting of I, V, L, and Y; X 8  is N or S; X 9  is selected from the group consisting of A, N, and Q; X 10  is K or V; X 11  is F or Q; X 12  is K or T; and X 13  is selected from the group consisting of G, H, R, and S (SEQ ID NO: 226); and   (vi) HVR-H3 comprising X 1 REGVYHX 2 YDDYAX 3 DY, wherein X 1  is selected from the group consisting of A, N, and T; X 2  is D or P; and X 3  is M or W (SEQ ID NO: 227).   
     
     
         15 . A method of reducing or inhibiting perfusion and permeability of a tumor in a subject, the method comprising administering to an individual in need of such treatment an effective amount of an anti-EGFL7 antibody, the antibody comprising the following HVR sequences: HVR-L1 comprising an amino acid sequence selected from SEQ ID NOs: 106-124 and 243-246, HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 125-129, HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130-145, HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 146-153, HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 154-187 and 247, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 188-192. 
     
     
         16 . The method of  claim 14 , wherein HVR-L1 comprises the amino acid sequence RTSQSLVHINAITYLH (SEQ ID NO: 241), HVR-L2 comprises the amino acid sequence RVSNRFS (SEQ ID NO: 101), HVR-L3 comprises the amino acid sequence GQSTHVPLT (SEQ ID NO: 131), HVR-H1 comprises the amino acid sequence GYTFIDYYMN (SEQ ID NO: 103), HVR-H2 comprises the amino acid sequence GDINLDNSGTHYNQKFKG (SEQ ID NO: 242), and HVR-H3 comprises the amino acid sequence AREGVYHDYDDYAMDY (SEQ ID NO: 105). 
     
     
         17 . The method of  claim 14 , wherein the heavy chain comprises the following framework sequences: FR-H1 comprises EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 197); FR-H2 comprises WVRQAPGKGLEWX 1 , wherein X 1  is I or V (SEQ ID NO: 228); FR-H3 comprises RX 1 TX 2 SX 3 DX 4 SX 5 X 6 TX 7 YX 8 QMNSLRAEDTAVYYC, wherein X 1  is F or V; X 2  is I or L; X 3  is selected from the group consisting of L, R, and V; X 4  is K or N; X 5  is selected from the group consisting of K, N, R, and S; X 6  is N or S; X 7  is selected from the group consisting of A, L, and V; and X 8  is L or M (SEQ ID NO: 229); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO: 200). 
     
     
         18 . The method of  claim 17 , wherein the heavy chain comprises the following framework sequences: FR-H1 comprises EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 197); FR-H2 comprises WVRQAPGKGLEWV (SEQ ID NO: 198); FR-H3 comprises RFTISRDX 1 SKNTX 2 YLQMNSLRAEDTAVYYCAR, wherein X 1  is N or K; and X 2  is selected from the group consisting of A, L, and V (SEQ ID NO: 230); and FR-H4 comprises WGQGTLVTVSS (SEQ ID NO: 200). 
     
     
         19 . The method of  claim 14 , wherein the light chain comprises the following framework sequences: FR-L1 comprises DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 201), FR-L2 comprises WYQQKPGKAPKLLIY (SEQ ID NO: 202), FR-L3 comprises GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203), FR-L4 comprises FGQGTKVEIK (SEQ ID NO: 221) or FGQGTKVEIKR (SEQ ID NO: 204). 
     
     
         20 . The method of  claim 14 , wherein the light chain comprises the variable domain sequence of 18F7.v6 as shown in  FIG. 27  (SEQ ID NO: 193) or the variable domain sequence of 18F7.v6k as shown in  FIG. 27  (SEQ ID NO: 194). 
     
     
         21 . The method of  claim 14 , wherein the heavy chain comprises the variable domain sequence of 18F7.v6 as shown in  FIG. 28  (SEQ ID NO: 195) or the variable domain sequence of 18F7.v6k as shown in  FIG. 28  (SEQ ID NO: 196). 
     
     
         22 . The method of  claim 14 , wherein the light chain comprises the variable domain sequence of 18F7.v6 as shown in  FIG. 27  (SEQ ID NO: 193) and the heavy chain comprises the variable domain sequence of 18F7.v6 as shown in  FIG. 28  (SEQ ID NO: 195). 
     
     
         23 . The method of  claim 14 , wherein the light chain comprises the variable domain sequence of 18F7.v6k as shown in  FIG. 27  (SEQ ID NO: 194) and the heavy chain comprises the variable domain sequence of 18F7.v6k as shown in  FIG. 28  (SEQ ID NO: 196). 
     
     
         24 . The method of  claim 14 , wherein at least a portion of the framework sequence is a human consensus framework sequence. 
     
     
         25 . The method of  claim 24 , comprising human κ subgroup 1 consensus framework sequence or heavy chain human subgroup III consensus framework sequence. 
     
     
         26 . The method of  claim 1  or  14 , wherein said antibody is a bispecific antibody. 
     
     
         27 . The method of  claim 26 , wherein said bispecific antibody binds to vascular endothelial growth factor (VEGF). 
     
     
         28 . The method of  claim 1  or  14 , wherein the tumor is selected from the group consisting of breast, colorectal, lung, esophageal, bladder, ovarian, pancreatic, and hepatocellular tumor. 
     
     
         29 . The method of  claim 28 , wherein the tumor is a colorectal or lung tumor. 
     
     
         30 . The method of  claim 1  or  14 , further comprising administering to the individual an effective amount of a second medicament, wherein the anti-EGFL7 antibody is a first medicament. 
     
     
         31 . The method of  claim 30 , wherein the second medicament is another antibody, a chemotherapeutic agent, a cytotoxic agent, an anti-angiogenic agent, an immunosuppressive agent, a prodrug, a cytokine, a cytokine antagonist, cytotoxic radiotherapy, a corticosteroid, an anti-emetic, a cancer vaccine, an analgesic, or a growth-inhibitory agent. 
     
     
         32 . The method of  claim 31 , wherein the second medicament is an anti-VEGF antibody. 
     
     
         33 . The method of  claim 32 , where the second medicament is bevacizumab or ranibizumab. 
     
     
         34 . The method of  claim 30 , further comprising administering to the individual an effective amount of a further medicament, wherein the anti-EGFL7 antibody is a first medicament and an anti-VEGF antibody is the second medicament. 
     
     
         35 . The method of  claim 34 , wherein the further medicament is a chemotherapeutic agent. 
     
     
         36 . The method of  claim 35 , wherein the chemotherapeutic agent is FOLFOX. 
     
     
         37 . The method of  claim 35 , wherein the chemotherapeutic agent is carboplatin and paclitaxel.