Humanized antibodies with anti-tumor activity
The present invention provides humanised antibodies and binding domains thereof with anti-tumor activity. In particular the humanised antibodies have specific binding to and direct killing of human colon tumor cells and display potent immune-mediated cytotoxic activity against human colon cancer cells in vitro using antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays and in vivo using mouse tumor models.
1. A humanized antibody, comprising a VH binding domain having an amino acid sequence selected from the group consisting of the amino acid sequence of residues 1 to 120 of SEQ ID NO: 50, the amino acid sequence of residues 1 to 120 of SEQ ID NO: 38, an amino acid sequence having at least 95% identity with the amino acid sequence of residues 1 to 120 of SEQ ID NO: 50 provided that the residue at position 72 is Arg or Lys and that the VH CDR1 has the amino acid sequence of SEQ ID NO: 96, the VH CDR2 has the amino acid sequence of SEQ ID NO: 98, and the VH CDR3 has the amino acid sequence of SEQ ID NO: 100, and an amino acid sequence having at least 95% identity with the amino acid sequence of residues 1 to 120 of SEQ ID NO: 38 provided that the residue at position 72 is Arg or Lys and that the VH CDR1 has the amino acid sequence of SEQ ID NO: 96, the VH CDR2 has the amino acid sequence of SEQ ID NO: 98, and the VH CDR3 has the amino acid sequence of SEQ ID NO: 100, and a VL binding domain having an amino acid sequence selected from the group consisting of the amino acid sequence of residues 1 to 107 of SEQ ID NO: 7, the amino acid sequence of residues 1 to 107 of SEQ ID NO: 8, an amino acid sequence having at least 95% identity with the amino acid sequence of residues 1 to 107 of SEQ ID NO: 7 provided that the VL CDR1 has the amino acid sequence of SEQ ID NO: 188, the VL CDR2 has the amino acid sequence of SEQ ID NO: 190, and the VL CDR3 has the amino acid sequence of SEQ ID NO: 192, and an amino acid sequence having at least 95% identity with the amino acid sequence of residues 1 to 107 of SEQ ID NO: 8 provided that the VL CDR1 has the amino acid sequence of SEQ ID NO: 188, the VL CDR2 has the amino acid sequence of SEQ ID NO: 190, and the VL CDR3 has the amino acid sequence of SEQ ID NO: 192.
2. The humanized antibody of claim 1 , in which the amino acid sequence of the VH binding domain comprises the amino acid sequence of residues 1 to 120 of SEQ ID NO: 50 or an amino acid sequence having at least 95% identity with the amino acid sequence of residues 1 to 120 of SEQ ID NO: 50 provided that the residue at position 72 is Arg or Lys and that the VH CDR1 has the amino acid sequence of SEQ ID NO: 96, the VH CDR2 has the amino acid sequence of SEQ ID NO: 98, and the VH CDR3 has the amino acid sequence of SEQ ID NO: 100.
3. The humanized antibody of claim 2 , in which the amino acid sequence of the VH binding domain CDR2 comprises HIHFSGRPTYNPSLKS (SEQ ID NO: 136).
4. The humanized antibody of claim 2 , in which the amino acid sequence of the VH binding domain comprises residues 1 to 120 of SEQ ID NO: 50.
5. The humanized antibody of claim 1 , in which the amino acid sequence of the VH binding domain comprises the amino acid sequence of residues 1 to 120 of SEQ ID NO: 38 or an amino acid sequence having at least 95% identity with the amino acid sequence of residues 1 to 120 of SEQ ID NO: 38 provided that the residue at position 72 is Arg or Lys and that the VH CDR1 has the amino acid sequence of SEQ ID NO: 96, the VH CDR2 has the amino acid sequence of SEQ ID NO: 98, and the VH CDR3 has the amino acid sequence of SEQ ID NO: 100.
6. The humanized antibody of claim 5 , in which the amino acid sequence of the binding domain VH CDR2 comprises HIHFSGRPTYNPSLKS (SEQ ID NO: 136).
7. The humanized antibody of claim 5 , in which the amino acid sequence of the VH binding domain comprises residues 1 to 120 of SEQ ID NO: 38.
8. The humanized antibody of claim 2 , in which the VH binding domain further comprises a constant domain.
9. The humanized antibody of claim 8 , in which the constant domain has the amino acid sequence:
(SEQ ID NO: 52)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
or
(SEQ ID NO: 92)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
or
(SEQ ID NO: 53)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
10. The humanized antibody of claim 5 , in which the VH binding domain further comprises a constant domain.
11. The humanized antibody of claim 10 , in which the constant domain has the amino acid sequence:
(SEQ ID NO: 52)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
or
(SEQ ID NO: 92)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
or
(SEQ ID NO: 53)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
12. The humanized antibody of claim 2 , in which the amino acid sequence of the VL binding domain comprises the amino acid sequence of residues 1 to 107 of SEQ ID NO: 7 or an amino acid sequence having at least 95% identity with the amino acid sequence of residues 1 to 107 of SEQ ID NO: 7 provided that the VL CDR1 has the amino acid sequence of SEQ ID NO: 188, the VL CDR2 has the amino acid sequence of SEQ ID NO: 190, and the VL CDR3 has the amino acid sequence of SEQ ID NO: 192.
13. The humanized antibody of claim 12 , in which the amino acid sequence of the VL binding domain comprises residues 1 to 107 of SEQ ID NO: 7.
14. The humanized antibody of claim 5 , in which the amino acid sequence of the VL binding domain comprises the amino acid sequence of residues 1 to 107 of SEQ ID NO: 8 or an amino acid sequence having at least 95% identity with the amino acid sequence of residues 1 to 107 of SEQ ID NO: 8 provided that the VL CDR1 has the amino acid sequence of SEQ ID NO: 188, the VL CDR2 has the amino acid sequence of SEQ ID NO: 190, and the VL CDR3 has the amino acid sequence of SEQ ID NO: 192.
15. The humanized antibody of claim 14 , in which the amino acid sequence of the VL binding domain comprises residues 1 to 107 of SEQ ID NO: 8.
16. The humanized antibody of claim 12 , in which the VL binding domain further comprises a constant domain.
17. The humanized antibody of claim 16 , in which the constant domain has the amino acid sequence:
(SEQ ID NO: 93)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
18. The humanized antibody of claim 14 , in which the VL binding domain further comprises a constant domain.
19. The humanized antibody of claim 18 , in which the constant domain has the amino acid sequence:
(SEQ ID NO: 93)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
20. A humanized antibody, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain comprising the amino acid sequence of SEQ ID NO:50.
21. A humanized antibody, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 8 and a heavy chain comprising the amino acid sequence of SEQ ID NO:38.
22. The humanized antibody of claim 1 , in which the antibody has a reduced level of fucose.
23. The humanized antibody of claim 20 , in which the antibody has a reduced level of fucose.
24. The humanized antibody of claim 21 , in which the antibody has a reduced level of fucose.
25. A pharmaceutical preparation, comprising the humanized antibody of claim 1 and a pharmaceutically acceptable carrier.
26. A pharmaceutical preparation, comprising the humanized antibody of claim 20 and a pharmaceutically acceptable carrier.
27. A pharmaceutical preparation, comprising the humanized antibody of claim 21 and a pharmaceutically acceptable carrier.