Framework residue substituted humanized COL-1 antibodies and their use
The present disclosure provides humanized COL-1 monoclonal antibodies that retain CEA binding affinity, compared to a parent antibody. Also disclosed herein are humanized COL-1 monoclonal antibodies that have reduced immunogenicity, compared to a parent antibody. The disclosed humanized COL-1 antibodies include substitution of framework residues with residues from the corresponding positions of a homologous human sequence. In several embodiments, methods are disclosed for the use of a humanized COL-1 antibody in the detection or treatment of a CEA-expressing tumor or cell in a subject. Also disclosed is a kit including the humanized COL-1 antibodies described herein.
1. An isolated nucleic acid encoding a humanized COL-1 monoclonal antibody, wherein the humanized COL-1 monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 8 and a light chain comprising the amino acid sequence of SEQ ID NO: 4, but for:
(a) a valine substitution at position 20, an arginine substitution at position 38, a methionine substitution at position 48, an arginine substitution at position 67, a valine substitution at position 79 and a methionine substitution at position 81 of SEQ ID NO: 8;
(b) a valine substitution at position 20, an arginine substitution at position 38, a methionine substitution at position 48, an arginine substitution at position 67, a valine substitution at position 79, a methionine substitution at position 81 and a tyrosine substitution at position 27 of SEQ ID NO: 8;
(c) a valine substitution at position 20, an arginine substitution at position 38, a methionine substitution at position 48, an arginine substitution at position 67, a valine substitution at position 79, a methionine substitution at position 81 and a valine substitution at position 68 of SEQ ID NO: 8;
(d) a valine substitution at position 20, an arginine substitution at position 38, a methionine substitution at position 48, an arginine substitution at position 67, a valine substitution at position 79, a methionine substitution at position 81, at tyrosine substitution at position 27 and a valine substitution at position 68 of SEQ ID NO: 8;
(e) a valine substitution at position 20, an arginine substitution at position 38, a methionine substitution at position 48, an arginine substitution at position 67, a valine substitution at position 79, a methionine substitution at position 81 and a glutamine substitution at position 1 of SEQ ID NO: 8;
(f) a valine substitution at position 20, an arginine substitution at position 38, a methionine substitution at position 48, an arginine substitution at position 67, a valine substitution at position 79, a methionine substitution at position 81 and a lysine substitution at position 12 of SEQ ID NO: 8;
(g) a valine substitution at position 20, an arginine substitution at position 38, a methionine substitution at position 48, an arginine substitution at position 67, a valine substitution at position 79, a methionine substitution at position 81, a glutamine substitution at position 1 and a lysine substitution at position 12 of SEQ ID NO: 8; or
(h) a valine substitution at position 20, an arginine substitution at position 38, a methionine substitution at position 48, an arginine substitution at position 67, a valine substitution at position 79, a methionine substitution at position 81, a glutamine substitution at position 1, a lysine substitution at position 12, a tyrosine substitution at position 27 and a valine substitution at position 68 of SEQ ID NO: 8,
wherein the humanized COL-1 antibody retains binding affinity for carcinoembryonic antigen (CEA).
2. The isolated nucleic acid of claim 1 , wherein the humanized COL-1 monoclonal antibody encoded by the nucleic acid further comprises a detectable label conjugated to the humanized COL-1 monoclonal antibody.
3. The isolated nucleic acid of claim 2 , wherein the detectable label is a fluorescent or radioactive molecule.
4. A vector comprising a promoter operably linked to the nucleic acid of claim 1 .
5. An isolated host cell transformed with the vector of claim 4 .
6. The isolated host cell of claim 5 , wherein the cell is a eukaryotic cell.
7. An isolated nucleic acid encoding a humanized COL-1 monoclonal antibody, wherein the humanized COL-1 monoclonal antibody comprises a heavy and a light chain variable region, wherein
the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 9 and the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 12; and
wherein the humanized antibody retains binding affinity for CEA, as compared to HuCOL-1 AbrCDR , deposited as ATCC Accession No. PTA-4644.
8. The isolated nucleic acid of claim 7 , wherein the humanized COL-1 monoclonal antibody encoded by the nucleic acid further comprises a detectable label conjugated to the humanized COL-1 monoclonal antibody.
9. The isolated nucleic acid of claim 8 , wherein the detectable label is a fluorescent or radioactive molecule.
10. A vector comprising a promoter operably linked to the nucleic acid of claim 7 .
11. An isolated host cell transformed with the vector of claim 10 .
12. The isolated host cell of claim 11 , wherein the cell is a eukaryotic cell.
13. The isolated nucleic acid of claim 1 , wherein the humanized COL-1 monoclonal antibody encoded by the nucleic acid comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 4.
14. The isolated nucleic acid of claim 13 , wherein the humanized COL-1 monoclonal antibody encoded by the nucleic acid further comprises a detectable label conjugated to the humanized COL-1 monoclonal antibody.
15. The isolated nucleic acid of claim 14 , wherein the detectable label is a fluorescent or radioactive molecule.
16. A vector comprising a promoter operably linked to the nucleic acid of claim 13 .
17. An isolated host cell transformed with the vector of claim 16 .
18. The isolated host cell of claim 17 , wherein the cell is a eukaryotic cell.
19. The isolated nucleic acid of claim 1 , wherein the humanized COL-1 monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 4.
20. The isolated nucleic acid of claim 19 , wherein the humanized COL-1 monoclonal antibody encoded by the nucleic acid further comprises a detectable label conjugated to the humanized COL-1 monoclonal antibody.
21. The isolated nucleic acid of claim 20 , wherein the detectable label is a fluorescent or radioactive molecule.
22. A vector comprising a promoter operably linked to the nucleic acid of claim 19 .
23. An isolated host cell transformed with the vector of claim 22 .
24. The isolated host cell of claim 23 , wherein the cell is a eukaryotic cell.
25. The isolated nucleic acid of claim 1 , wherein the humanized COL-1 monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 4.
26. The isolated nucleic acid of claim 25 , wherein the humanized COL-1 monoclonal antibody encoded by the nucleic acid further comprises a detectable label conjugated to the humanized COL-1 monoclonal antibody.
27. The isolated nucleic acid of claim 26 , wherein the detectable label is a fluorescent or radioactive molecule.
28. A vector comprising a promoter operably linked to the nucleic acid of claim 25 .
29. An isolated host cell transformed with the vector of claim 28 .
30. The isolated host cell of claim 29 , wherein the cell is a eukaryotic cell.