Humanised antibodies with anti-tumour activity
The present invention provides humanized antibodies and binding domains thereof with anti-tumor activity. In particular the humanized 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 V H binding domain and a V L binding domain wherein the V H binding domain comprises, in sequence, a first framework region (V H FR1), a first CDR (V H CDR1), a second framework region (V H FR2), a second CDR (V H CDR2), a third framework region (V H FR3), a third CDR (V H CDR3) and a fourth framework region (V H FR4), wherein: (i) the sequence of V H FR1 is X 1 VQLQESGPGLVKPSETLSLTCTVX 2 GYSX 3 X 4 (SEQ ID: 95) wherein; X i is Q or E X 2 is S or T X 3 is I, L or V X 4 is S or T; (ii) the sequence of V H CDR1 is SGYSWH (SEQ ID NO: 96); (iii) the sequence of V H FR2 is WIRQX 5 PGKGLX 6 WX 7 G (SEQ ID NO: 97) wherein X 5 is S or P X 6 is Q or E X 7 is M or I; (iv) the sequence of V H CDR2 is HIHX 8 SGRPTYX 9 PSLX 10 S (SEQ ID NO: 98) wherein X 8 is F, Y or W X 9 is N or D X 10 is K, L, H, F, R or S; (v) the sequence of V H FR3 is RX 11 X 12 ISX 13 X 14 TAKNQFSLKLTSMTAADTAVYYCAR (SEQ ID NO: 99) wherein X 11 is V or I X 12 is T or S X 13 is R or K X 14 is E or D; (vi) the sequence of V H CDR3 is KGKGSDDGLNY (SEQ ID NO: 100); (vii) the sequence of V H FR4 is WGQGTLVTVSS (SEQ ID NO: 101).
2. The humanized antibody as claimed in claim 1 , in which the sequence of the V H framework regions comprises the sequence (SEQ ID NO: 102) QVQLQESGPGLVKPSETLSLTCTVSGYSX 3 SWIRQPPGKGLQWIGRVTIS X 13 ETAKNQFSLKLTSMTAADTAVYYCARWGQGTLVTVSS; and wherein the humanized antibody binds to human colon cancer cell line COLO205.
3. The humanized antibody as claimed in claim 1 in which the sequence of V H FR1 is selected from the group consisting of
QVQLQESGPGLVKPSETLSLTCTVSGYSIS,
(SEQ ID NO: 103)
QVQLQESGPGLVKPSETLSLTCTVSGYSIT,
(SEQ ID NO: 104)
QVQLQESGPGLVKPSETLSLTCTVSGYSLS,
(SEQ ID NO: 105)
QVQLQESGPGLVKPSETLSLTCTVSGYSLT,
(SEQ ID NO: 106)
QVQLQESGPGLVKPSETLSLTCTVSGYSVS,
(SEQ ID NO: 107)
QVQLQESGPGLVKPSETLSLTCTVSGYSVT,
(SEQ ID NO: 108)
QVQLQESGPGLVKPSETLSLTCTVTGYSIS,
(SEQ ID NO: 109)
QVQLQESGPGLVKPSETLSLTCTVTGYSIT,
(SEQ ID NO: 110)
QVQLQESGPGLVKPSETLSLTCTVTGYSLS,
(SEQ ID NO: 111)
QVQLQESGPGLVKPSETLSLTCTVTGYSLT,
(SEQ ID NO: 112)
QVQLQESGPGLVKPSETLSLTCTVTGYSVS,
(SEQ ID NO: 113)
QVQLQESGPGLVKPSETLSLTCTVTGYSVT,
(SEQ ID NO: 114)
EVQLQESGPGLVKPSETLSLTCTVSGYSIS,
(SEQ ID NO: 115)
EVQLQESGPGLVKPSETLSLTCTVSGYSIT,
(SEQ ID NO: 116)
EVQLQESGPGLVKPSETLSLTCTVSGYSLS,
(SEQ ID NO: 117)
EVQLQESGPGLVKPSETLSLTCTVSGYSLT,
(SEQ ID NO: 118)
EVQLQESGPGLVKPSETLSLTCTVSGYSVS,
(SEQ ID NO: 119)
EVQLQESGPGLVKPSETLSLTCTVSGYSVT,
(SEQ ID NO: 120)
EVQLQESGPGLVKPSETLSLTCTVTGYSIS,
(SEQ ID NO: 121)
EVQLQESGPGLVKPSETLSLTCTVTGYSIT,
(SEQ ID NO: 122)
EVQLQESGPGLVKPSETLSLTCTVTGYSLS,
(SEQ ID NO: 123)
EVQLQESGPGLVKPSETLSLTCTVTGYSLT,
(SEQ ID NO: 124)
EVQLQESGPGLVKPSETLSLTCTVSTGYSVS,
(SEQ ID NO: 125)
and
EVQLQESGPGLVKPSETLSLTCTVTGYSVT,
(SEQ ID NO: 126).
4. The humanized antibody as claimed in claim 1 in which the sequence of V H CDR1 is SGYSWH (SEQ ID NO: 96).
5. The humanized antibody as claimed in claim 1 in which the sequence of V H FR2 is selected from the group consisting of WIRQSPGKGLQWIG (SEQ ID NO: 127), WIRQSPGKGLQWMG (SEQ ID NO: 128), WIRQSPGKGLEWIG (SEQ ID NO: 129), WIRQSPGKGLEWMG (SEQ ID NO: 130), WIRQPPGKGLQWIG (SEQ ID NO: 131), WIRQPPGKGLQWMG (SEQ ID NO: 132), WIRQPPGKGLEWIG (SEQ ID NO: 133), and WIRQPPGKGLEWMG (SEQ ID NO: 134).
6. The humanized antibody as claimed in claim 1 in which the sequence of V H CDR2 is selected from the group consisting of HIHFSGRPTYNPSLSS (SEQ ID NO: 135), HIHFSGRPTYNPSLKS (SEQ ID NO: 136), HIHFSGRPTYNPSLLS (SEQ ID NO: 137), HIHFSGRPTYNPSLHS (SEQ ID NO: 138), HIHFSGRPTYNPSLFS (SEQ ID NO: 139), HIHFSGRPTYNPSLRS (SEQ ID NO: 140), HIHWSGRPTYNPSLSS (SEQ ID NO: 141), HIHWSGRPTYNPSLKS (SEQ ID NO: 142), HIHWSGRPTYNPSLLS (SEQ ID NO: 143), HIHWSGRPTYNPSLHS (SEQ ID NO: 144), HIHWSGRPTYNPSLFS (SEQ ID NO: 145), HIHWSGRPTYNPSLRS (SEQ ID NO: 146), HIHYSGRPTYNPSLSS (SEQ ID NO: 147), HIHYSGRPTYNPSLKS (SEQ ID NO: 148), HIHYSGRPTYNPSLLS (SEQ ID NO: 149), HIHYSGRPTYNPSLHS (SEQ ID NO: 150), HIHYSGRPTYNPSLFS (SEQ ID NO: 151), HIHYSGRPTYNPSLRS (SEQ ID NO: 152), HIHFSGRPTYDPSLSS (SEQ ID NO: 153), HIHFSGRPTYDPSLKS (SEQ ID NO: 154), HIHFSGRPTYDPSLLS (SEQ ID NO: 155), HIHFSGRPTYDPSLHS (SEQ ID NO: 156), HIHFSGRPTYDPSLFS (SEQ ID NO: 157), HIHFSGRPTYDPSLRS (SEQ ID NO: 158), HIHWSGRPTYDPSLSS (SEQ ID NO: 159), HIHWSGRPTYDPSLKS (SEQ ID NO: 160), HIHWSGRPTYDPSLLS (SEQ ID NO: 161), HIHWSGRPTYDPSLHS (SEQ ID NO: 162), HIHWSGRPTYDPSLFS (SEQ ID NO: 163), HIHWSGRPTYDPSLRS (SEQ ID NO: 164), HIHYSGRPTYNPSLSS (SEQ ID NO: 165), HIHYSGRPTYDPSLKS (SEQ ID NO: 166), HIHYSGRPTYDPSLLS (SEQ ID NO: 167), HIHYSGRPTYDPSLHS (SEQ ID NO: 168), HIHYSGRPTYDPSLFS (SEQ ID NO: 169), and HIHYSGRPTYDPSLRS (SEQ ID NO: 170).
7. The humanized antibody as claimed in claim 1 in which the sequence of V H FR3 is selected from the group consisting of
RVTISRETAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 171)
RVTISRDTAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 172)
RVTISKETAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 173)
RVTISKDTAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 174)
RVSISRETAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 175)
RVSISRDTAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 176)
RVSISKETAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 177)
RVSISKDTAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 178)
RITISRETAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 179)
RITISRDTAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 180)
RITISKETAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 181)
RITISKDTAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 182)
RISISRETAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 183)
RISISRDTAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 184)
RISISKETAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 185)
and
RISISKDTAKNQFSLKLTSMTAADTAVYYCAR, (SEQ ID NO: 186).
8. The humanized antibody as claimed in claim 1 in which the sequence of V H CDR3 is KGKGSDDGLNY (SEQ ID NO: 100).
9. The humanized antibody as claimed in claim 1 in which the sequence of V H FR4 is WGQGTLVTVSS (SEQ ID NO: 101).
10. The humanized antibody as claimed in claim 1 further comprising a constant domain.
11. The humanized antibody as claimed in claim 10 in which the constant domain has the sequence:
(SEQ ID NO: 52)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
or
(SEQ ID NO: 92)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
or
(SEQ ID NO: 53)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
12. The humanized antibody as claimed in claim 1 wherein the V L binding domain comprises, in sequence, a first framework region (V L FR1), a first CDR (V L CDR1), a second framework region (V L FR2), a second CDR (V L CDR2), a third framework region (V L FR3), a third CDR (V L CDR3) and a fourth framework region (V L FR4), wherein: (i) the sequence of V L FR1 is EX 13 VLTQSPGTLSLSX 14 GERATLSC (SEQ ID NO: 187) wherein, X 13 is I or N, X 14 is A or P; (ii) the sequence of V L CDR1 is SASSSLSYIH (SEQ ID NO: 188); (iii) the sequence of V L FR2 is WYQQKPGQAPRLLIY (SEQ ID NO: 189); (iv) the sequence of V L CDR2 is DTSNLAS (SEQ ID NO: 190); (v) the sequence of V L FR3 is GIPDRFSGSGSGX 15 DFTLTISRVEPEDFAVYYC (SEQ ID NO: 191) wherein, X 15 is T or N; (vi) the sequence of V L CDR3 is FQGSEYPLT (SEQ ID NO: 192); (vii) the sequence of V L FR4 is FGQGTKLEIKR (SEQ ID NO: 193).
13. The humanized antibody as claimed in claim 1 , in which the sequence of the V L framework regions comprises the sequence (SEQ ID NO: 194) EIVLTQSPGTLSLSPGERATLSCWYQQKPGQAPRLLIYGIPDRFSGSGSG TDFTLTISRVEPEDFAVYYCFGQGTKLEIKR; and wherein the humanized antibody binds to human colon cancer cell line COLO205.
14. The humanized antibody as claimed in claim 1 in which the sequence of V L FR1 is selected from the group consisting of EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 195), EIVLTQSPGTLSLSAGERATLSC (SEQ ID NO: 196), ENVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 197), and ENVLTQSPGTLSLSAGERATLSC (SEQ ID NO: 198).
15. The humanized antibody as claimed in claim 1 in which the sequence of V L CDR1 is SASSSLSYIH (SEQ ID NO: 188).
16. The humanized antibody as claimed in claim 1 in which the sequence of V L FR2 is WYQQKPGQAPRLLIY (SEQ ID NO: 189).
17. The humanized antibody as claimed in claim 1 in which the sequence of V L CDR2 is DTSNLAS (SEQ ID NO: 190).
18. The humanized antibody as claimed in claim 1 in which the sequence of V L FR3 is GIPDRFSGSGSGTDFTLTISRVEPEDFAVYYC (SEQ ID NO: 199) or GIPDRFSGSGSGNDFTLTISRVEPEDFAVYYC (SEQ ID NO: 200).
19. The humanized antibody as claimed in claim 1 in which the sequence of V L CDR3 is FQGSEYPLT (SEQ ID NO: 192).
20. The humanized antibody as claimed in claim 1 in which the sequence of V L FR4 is FGQGTKLEIKR (SEQ ID NO: 193).
21. The humanized antibody as claimed in claim 1 further comprising a constant domain.
22. The humanized antibody as claimed in claim 21 in which the constant domain has the sequence
(SEQ ID NO: 93)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC.
23. The humanized antibody as claimed in claim 1 in which the antibody has a reduced level of fucose.
24. A method of treating cancer in a subject wherein the cancer is selected from the group consisting of colorectal, ovarian, pancreatic, prostatic and lung, the method comprising administering to the subject a therapeutically effective quantity of an antibody according to claim 1 .
25. A method of detecting the presence of cancer cells in a sample, the method comprising contacting the cell sample with an antibody according to claim 1 and detecting binding of the antibody to the cells.
26. A humanized antibody comprising a V H binding domain and a V L binding domain wherein the V H binding domain comprises, in sequence, a first framework region (V H FR1), a first CDR (V H CDR1), a second framework region (V H FR2), a second CDR (V H CDR2), a third framework region (V H FR3), a third CDR (V H CDR3) and a fourth framework region (V H FR4), wherein: (i) the sequence of V H FR1 is EVQLQQWGAGLLKPSETLSLTCAVYGYSX 18 X 19 (SEQ ID NO: 201) wherein; X 18 is I, L or V X 19 is S or T; (ii) the sequence of V H CDR1 is SGYSWH (SEQ ID NO: 96; (iii) the sequence of V H FR2 is WIRQPPGKGLEWX 20 G (SEQ ID NO: 202) wherein X 20 is M or I; (iv) the sequence of V H CDR2 is HIHX 21 SGRPTYX 22 PSLX 23 S (SEQ ID NO: 98) wherein X 21 is F, Y or W X 22 is N or D X 23 is K, L, H, F, R or S; (v) the sequence of V H FR3 is RX 24 X 26 ISX 26 DTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 203) wherein X 24 is V or I X 25 is S or T X 26 is R or K; (vi) the sequence of V H CDR3 is KGKGSDDGLNY (SEQ ID NO: 100); (vii) the sequence of V H FR4 is WGQGTLVTVSS (SEQ ID NO: 101).
27. The humanized antibody as claimed in claim 26 , in which the sequence of the framework regions comprises the sequence (SEQ ID NO: 204) EVQLQQWGAGLLKPSETLSLTCAVYGYSX 18 SWIRQPPGKGLEWIGRVTI SX 26 DTSKNQFSLKLSSVTAADTAVYYCARWGQGTLVTVSS; and wherein the humanized antibody binds to human colon cancer cell line COLO205.
28. The humanized antibody as claimed in claim 26 in which the sequence of V H FR1 is selected from the group consisting of
EVQLQQWGAGLLKPSETLSLTCAVYGYSIS,
(SEQ ID NO: 205)
EVQLQQWGAGLLKPSETLSLTCAVYGYSIT,
(SEQ ID NO: 206)
EVQLQQWGAGLLKPSETLSLTCAVYGYSLS,
(SEQ ID NO: 207)
EVQLQQWGAGLLKPSETLSLTCAVYGYSLT,
(SEQ ID NO: 208)
EVQLQQWGAGLLKPSETLSLTCAVYGYSVS,
(SEQ ID NO: 209)
and
EVQLQQWGAGLLKPSETLSLTCAVYGYSVT
(SEQ ID NO: 210).
29. The humanized antibody as claimed in claim 26 in which the sequence of V H CDR1 is SGYSWH (SEQ ID NO: 96).
30. The humanized antibody as claimed in claim 26 in which the sequence of V H FR2 is WIRQPPGKGLEWIG (SEQ ID NO: 133) or WIRQPPGKGLEWMG (SEQ ID NO: 134).
31. The humanized antibody as claimed in claim 26 in which the sequence of V H CDR2 is selected from the group consisting of HIHFSGRPTYNPSLSS (SEQ ID NO: 135), HIHFSGRPTYNPSLKS (SEQ ID NO: 136), HIHFSGRPTYNPSLLS (SEQ ID NO: 137), HIHFSGRPTYNPSLHS (SEQ ID NO: 138), HIHFSGRPTYNPSLFS (SEQ ID NO: 139), HIHFSGRPTYNPSLRS (SEQ ID NO: 140), HIHWSGRPTYNPSLSS (SEQ ID NO: 141), HIHWSGRPTYNPSLKS (SEQ ID NO: 142), HIHWSGRPTYNPSLLS (SEQ ID NO: 143), HIHWSGRPTYNPSLHS (SEQ ID NO: 144), HIHWSGRPTYNPSLFS (SEQ ID NO: 145), HIHWSGRPTYNPSLRS (SEQ ID NO: 146), HIHYSGRPTYNPSLSS (SEQ ID NO: 147), HIHYSGRPTYNPSLKS (SEQ ID NO: 148), HIHYSGRPTYNPSLLS (SEQ ID NO: 149), HIHYSGRPTYNPSLHS (SEQ ID NO: 150), HIHYSGRPTYNPSLFS (SEQ ID NO: 151), HIHYSGRPTYNPSLRS (SEQ ID NO: 152), HIHFSGRPTYDPSLSS (SEQ ID NO: 153), HIHFSGRPTYDPSLKS (SEQ ID NO: 154), HIHFSGRPTYDPSLLS (SEQ ID NO: 155), HIHFSGRPTYDPSLHS (SEQ ID NO: 156), HIHFSGRPTYDPSLFS (SEQ ID NO: 157), HIHFSGRPTYDPSLRS (SEQ ID NO: 158), HIHWSGRPTYDPSLSS (SEQ ID NO: 159), HIHWSGRPTYDPSLKS (SEQ ID NO: 160), HIHWSGRPTYDPSLLS (SEQ ID NO: 161), HIHWSGRPTYDPSLHS (SEQ ID NO: 162), HIHWSGRPTYDPSLFS (SEQ ID NO: 163), HIHWSGRPTYDPSLRS (SEQ ID NO: 164), HIHYSGRPTYDPSLSS (SEQ ID NO: 165), HIHYSGRPTYDPSLKS (SEQ ID NO: 166), HIHYSGRPTYDPSLLS (SEQ ID NO: 167), HIHYSGRPTYDPSLHS (SEQ ID NO: 168), HIHYSGRPTYDPSLFS (SEQ ID NO: 169), and HIHYSGRPTYDPSLRS (SEQ ID NO: 170).
32. The humanized antibody as claimed in claim 26 in which the sequence of V H FR3 is selected from the group consisting of
RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR, (SEQ ID NO: 218)
RVTISKDTSKNQFSLKLSSVTAADTAVYYCAR, (SEQ ID NO: 219)
RVSISRDTSKNQFSLKLSSVTAADTAVYYCAR, (SEQ ID NO: 220)
RVSISKDTSKNQFSLKLSSVTAADTAVYYCAR, (SEQ ID NO: 221)
RITISRDTSKNQFSLKLSSVTAADTAVYYCAR, (SEQ ID NO: 222)
RITISKDTSKNQFSLKLSSVTAADTAVYYCAR, (SEQ ID NO: 223)
RISISRDTSKNQFSLKLSSVTAADTAVYYCAR, (SEQ ID NO: 224)
RISISKDTSKNQFSLKLSSVTAADTAVYYCAR, (SEQ ID NO: 225).
33. The humanized antibody as claimed in claim 26 in which the sequence of V H CDR3 is KGKGSDDGLNY (SEQ ID NO: 100).
34. The humanized antibody as claimed in claim 26 in which the sequence of V H FR4 is WGQGTLVTVSS (SEQ ID NO: 101).
35. The humanized antibody as claimed in claim 26 further comprising a constant domain.
36. The humanized antibody as claimed in claim 35 in which the constant domain has the sequence:
(SEQ ID NO: 52)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
or
(SEQ ID NO: 92)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
or
(SEQ ID NO: 53)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
37. The humanized antibody as claimed in claim 26 , wherein the V L binding domain comprises, in sequence, a first framework region (V L FR1), a first CDR (V L CDR1), a second framework region (V L FR2), a second CDR (V L CDR2), a third framework region (V L FR3), a third CDR (V L CDR3) and a fourth framework region (V L FR4), wherein: (i) the sequence of V L FR1 is EX 23 VLTQSPATLSLSPGERATLSC (SEQ ID NO: 211) wherein, X 23 is I or N; (ii) the sequence of V L CDR1 is SASSSLSYIH (SEQ ID NO: 188); (iii) the sequence of V L FR2 is WYQQKPGQAPRLLIY (SEQ ID NO: 189); (iv) the sequence of V L CDR2 is DTSNLAS (SEQ ID NO: 190); (v) the sequence of V L FR3 is GIPDRFSGSGSGX 24 DFTLTISRLEPEDFAVYYC (SEQ ID NO: 212) wherein, X 24 is T or N; (vi) the sequence of V L CDR3 is FQGSEYPLT (SEQ ID NO: 192); (vii) the sequence of V L FR4 is FGGGTKVEIKR (SEQ ID NO: 193).
38. The humanized antibody as claimed in claim 26 , in which the sequence of the framework regions comprises the sequence (SEQ ID NO: 213) EIVLTQSPATLSLSPGERATLSCWYQQKPGQAPRLLIYGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCFGGGTKVEIKR; and wherein the humanized antibody binds to human colon cancer cell line COLO205.
39. The humanized antibody as claimed in claim 26 in which the sequence of V L FR1 is selected from the group EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 214) or ENVLTQSPATLSLSPGERATLSC (SEQ ID NO: 215).
40. The humanized antibody as claimed in claim 26 in which the sequence of V L CDR1 is SASSSLSYIH (SEQ ID NO: 188).
41. The humanized antibody as claimed in claim 26 in which the sequence of V L FR2 is WYQQKPGQAPRLLIY (SEQ ID NO: 189).
42. The humanized antibody as claimed in claim 26 in which the sequence of V L CDR2 is DTSNLAS (SEQ ID NO: 190).
43. The humanized antibody as claimed in claim 26 in which the sequence of V L FR3 is GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 216) or GIPDRFSGSGSGNDFTLTISRLEPEDFAVYYC (SEQ ID NO: 217).
44. The humanized antibody as claimed in claim 26 in which the sequence of V L CDR3 is FQGSEYPLT (SEQ ID NO: 192).
45. The humanized antibody as claimed in claim 26 in which the sequence of V L FR4 is FGGGTKVEIKR (SEQ ID NO: 193).
46. The humanized antibody as claimed in claim 26 further comprising a constant domain.
47. The humanized antibody as claimed in claim 46 in which the constant domain has the sequence
(SEQ ID NO: 93)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC.
48. The humanized antibody as claimed in claim 26 in which the antibody has a reduced level of fucose.
49. A method of treating cancer in a subject wherein the cancer is selected from the group consisting of colorectal, ovarian, pancreatic, prostatic and lung, the method comprising administering to the subject a therapeutically effective quantity of an antibody according to claim 26 .
50. A method of detecting the presence of cancer cells in a sample, the method comprising contacting the cell sample with an antibody according to claim 26 and detecting binding of the antibody to the cells.