Anti-GD2 antibodies
In this application are described chimeric, humanized, affinity matured, stability enhanced, and bispecific Anti-GD2 antibodies and fragments thereof. Also provided are methods of using individual antibodies or compositions thereof for the detection, prevention, and/or therapeutical treatment of GD2-related diseases, in particular, neuroblastoma.
1. An isolated nucleic acid molecule encoding a humanized or chimeric antibody or fragment thereof capable of binding GD2, wherein the humanized or chimeric antibody or fragment thereof comprises any of the following:
(i) a variable heavy chain domain of SEQ ID NO: 1 and a variable light chain domain of SEQ ID NO:2;
(ii) a variable heavy chain domain of SEQ ID NO:3 and a variable light chain domain of SEQ ID NO:2;
(iii) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO:5;
(iv) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO:7;
(v) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:5;
(vi) a variable heavy chain domain of SEQ ID NO:9 and a variable light chain domain of SEQ ID NO: 10;
(vii) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO: 5; and
(viii) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO: 7.
2. A recombinant vector comprising the nucleic acid molecule of claim 1 .
3. A host cell comprising the recombinant vector of claim 2 .
4. The host cell of claim 3 , wherein the host cell is a prokaryotic or eukaryotic cell.
5. The host cell of claim 3 , wherein the host cell is selected from the group consisting of COS-1, COS-7, HEK293, BHK21, CHO, CHO-S, DG44, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, and lymphoma cells.
6. The host cell of claim 5 , wherein the host cell is an engineered or variant host cell.
7. A method for the production of a humanized or chimeric antibody or fragment thereof capable of binding GD2, the method comprising
culturing a host cell comprising a recombinant vector according to claim 2 in a culture medium under conditions allowing the expression of the humanized or chimeric antibody or fragment thereof, and
recovering the humanized or chimeric antibody or fragment thereof from the culture medium.
8. The method of claim 7 , wherein the humanized or chimeric antibody comprises a variant Fc region.
9. The method of claim 8 , wherein the variant Fc region comprises a substitution of S239D, A330L and I332E.
10. The method of claim 8 , wherein the variant Fc region comprises a N297A substitution.
11. The method of claim 8 , wherein the host cell is an engineered or variant host cell.
12. The method of claim 11 , wherein the humanized or chimeric antibody is or comprises an engineered glycoform.
13. The method of claim 12 , wherein the humanized or chimeric antibody is glycosylated with terminal mannose, N-acetylglucose or glucose, but no fucose.
14. The method of claim 13 , wherein the humanized or chimeric antibody comprises a variant Fc region.
15. The method of claim 14 , wherein the variant Fc region comprises a substitution of S239D, A330L and I332E.
16. The method of claim 14 , wherein the variant Fc region comprises a N297A substitution.
17. The method of claim 13 , wherein the humanized or chimeric antibody has an altered affinity for an FcγR.
18. The method of claim 13 , wherein the humanized or chimeric antibody has increased affinity for an FcγR as compared to a reference humanized or chimeric antibody.
19. The method of claim 13 , wherein the humanized or chimeric antibody is characterized by about 10-fold higher antibody-dependent cellular cytotoxicity (ADCC) as compared to a reference humanized or chimeric antibody.
20. The method of claim 13 , wherein the humanized or chimeric antibody is characterized by about 100-fold higher antibody-dependent cellular cytotoxicity (ADCC) as compared to a reference humanized or chimeric antibody.
21. The method of claim 13 , wherein the humanized or chimeric antibody is characterized by an activating:inhibiting FcγR receptor ratio of about 100 or more as compared to a reference humanized or chimeric antibody.
22. An isolated nucleic acid molecule encoding a bispecific antibody having first and second antigen-binding sites, which first antigen-binding site comprises a light chain and a heavy chain of a humanized 3F8 antibody, wherein the humanized 3F8 antibody comprises any of the following:
(i) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO:5;
(ii) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO:7;
(iii) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:5;
(iv) a variable heavy chain domain of SEQ ID NO:9 and a variable light chain domain of SEQ ID NO: 10;
(v) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO: 5; and
(vi) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO: 7.
23. The isolated nucleic acid molecule of claim 22 , wherein the second antigen-binding site is specific for CD3 or for a DOTA (metal).
24. The isolated nucleic acid molecule of claim 23 , wherein the second antigen-binding site comprises a peptide of SEQ ID NO:23 that includes the second antigen-binding site.
25. The isolated nucleic acid molecule of claim 23 , wherein the second antigen-binding site comprises a peptide of SEQ ID NO:24 that includes the second antigen-binding site.
26. A recombinant vector comprising the nucleic acid molecule of claim 22 .
27. A recombinant vector comprising the nucleic acid molecule of claim 24 .
28. A recombinant vector comprising the nucleic acid molecule of claim 25 .
29. A host cell comprising the recombinant vector of claim 26 .
30. A host cell comprising the recombinant vector of claim 27 .
31. A host cell comprising the recombinant vector of claim 28 .
32. A method of manufacturing a pharmaceutical composition, the method comprising a step of:
combining a humanized or chimeric antibody or fragment thereof capable of binding to GD2, wherein the antibody or fragment thereof comprises any of the following:
(i) a variable heavy chain domain of SEQ ID NO: 1 and a variable light chain domain of SEQ ID NO:2;
(ii) a variable heavy chain domain of SEQ ID NO:3 and a variable light chain domain of SEQ ID NO:2;
(iii) a variable heavy chain domain of SEQ ID NO:4 and a variable light chain domain of SEQ ID NO:5;
(iv) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO:7;
(v) a variable heavy chain domain of SEQ ID NO:8 and a variable light chain domain of SEQ ID NO:5;
(vi) a variable heavy chain domain of SEQ ID NO:9 and a variable light chain domain of SEQ ID NO: 10; and
(vii) a variable heavy chain domain of SEQ ID NO:6 and a variable light chain domain of SEQ ID NO: 5, with
a pharmaceutically acceptable carrier or excipient to produce a pharmaceutical composition.
33. The method of claim 32 wherein the animal is a human.