Antibodies and methods of use thereof
The present disclosure provides multispecific (e.g., bispecific) antibodies that specifically bind to human GITR and/or human OX40 as well as compositions comprising such antibodies. In a specific aspect, the multispecific antibodies specifically bind to human GITR and OX40 and modulate GITR and/or OX40 activity, e.g., enhance, activate, or induce GITR and/or OX40 activity, or reduce, deactivate, or inhibit GITR and/or OX40 activity. The present disclosure also provides methods for treating disorders, such as cancer, by administering a multispecific antibody that specifically binds to human GITR and/or OX40 and modulates GITR and/or OX40 activity, e.g., enhances, activates, or induces GITR and/or OX40 activity. Also provided are methods for treating autoimmune or inflammatory diseases or disorders, by administering a multispecific antibody that specifically binds to human GITR and/or OX40 and modulates GITR and/or OX40 activity, e.g., reduces, deactivates, or inhibits GITR and/or OX40 activity.
1. A method of producing a multispecific antibody comprising a first antigen-binding region that specifically binds human OX40 and a second antigen-binding region that specifically binds human GITR, wherein:
(a) the first antigen-binding region comprises a first heavy chain comprising CDRs VH-CDR1, VH-CDR2, and VH-CDR3, and a first light chain comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein the first heavy chain comprises the VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 54, and the first light chain comprises the VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 55; and
(b) the second antigen-binding region comprises a second heavy chain comprising CDRs VH-CDR1, VH-CDR2, and VH-CDR3, and a second light chain comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein the second heavy chain comprises the VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 18, and the second light chain comprises the VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 19,
the method comprising:
(c) culturing a first host cell comprising a first polynucleotide encoding the first heavy chain and a second polynucleotide encoding the first light chain under suitable conditions such that the first heavy chain and the first light chain are expressed, and the first antigen-binding region is produced;
(d) culturing a second host cell comprising a third polynucleotide encoding the second heavy chain and a fourth polynucleotide encoding the second light chain under suitable conditions such that second heavy chain and the second light chain are expressed, and the second antigen-binding region is produced; and
(e) contacting the first and the second antigen-binding regions produced in steps (c) and (d), under suitable conditions so that the multispecific antibody is produced.
2. The method of claim 1 , wherein:
(a) the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the first antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively; and
(b) the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the second antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 7, 10, 3, 14, 5, and 16, respectively.
3. The method of claim 1 , wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 54, and/or the first light chain comprises the amino acid sequence of SEQ ID NO: 55.
4. The method of claim 1 , wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 54, and the first light chain comprises the amino acid sequence of SEQ ID NO: 55.
5. The method of claim 1 , wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and/or the second light chain comprises the amino acid sequence of SEQ ID NO: 19.
6. The method of claim 1 , wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and the second light chain comprises the amino acid sequence of SEQ ID NO: 19.
7. The method of claim 1 , wherein:
(a) the first heavy chain comprises the amino acid sequence of SEQ ID NO: 54, and the first light chain comprises the amino acid sequence of SEQ ID NO: 55; and
(b) the second heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and the second light chain comprises the amino acid sequence of SEQ ID NO: 19.
8. The method of claim 1 , wherein the first heavy chain and/or the second heavy chain comprises a heavy chain constant region selected from the group consisting of human IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
9. The method of claim 8 , wherein the first heavy chain and/or the second heavy chain comprises an IgG 1 heavy chain constant region.
10. The method of claim 9 , wherein:
(a) the first heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, and the second heavy chain comprises tryptophan at amino acid position 366; or
(b) the first heavy chain comprises tryptophan at amino acid position 366, and the second heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively,
numbered according to the EU numbering system.
11. The method of claim 10 , wherein the first heavy chain and/or the second heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
12. The method of claim 1 , wherein the first light chain and/or the second light chain comprises a human kappa light chain constant region or a human lambda light chain constant region.
13. The method of claim 9 , wherein:
(a) the first heavy chain comprises the amino acid sequence of SEQ ID NO: 54, and the first light chain comprises the amino acid sequence of SEQ ID NO: 67; and
(b) the second heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and the second light chain comprises the amino acid sequence of SEQ ID NO: 37.
14. The method of claim 13 , wherein:
(a) the first heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, and the second heavy chain comprises tryptophan at amino acid position 366; or
(b) the first heavy chain comprises tryptophan at amino acid position 366, and the second heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively,
numbered according to the EU numbering system.
15. The method of claim 14 , wherein the first heavy chain and/or the second heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
16. The method of claim 14 , wherein the first heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, and the second heavy chain comprises tryptophan at amino acid position 366, numbered according to the EU numbering system.
17. The method of claim 16 , wherein the first heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
18. The method of claim 16 , wherein the second heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
19. The method of claim 16 , wherein the first heavy chain and the second heavy chain comprise aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
20. The method of claim 14 , wherein the first heavy chain comprises tryptophan at amino acid position 366, and the second heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, numbered according to the EU numbering system.
21. The method of claim 20 , wherein the first heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
22. The method of claim 20 , wherein the second heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
23. The method of claim 20 , wherein the first heavy chain and the second heavy chain comprise aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
24. A method of producing a multispecific antibody comprising a first antigen-binding region that specifically binds human OX40 and a second antigen-binding region that specifically binds human GITR, wherein:
(a) the first antigen-binding region comprises a first heavy chain comprising CDRs VH-CDR1, VH-CDR2, and VH-CDR3, and a first light chain comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein the first heavy chain comprises the VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 54, and the first light chain comprises the VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 55; and
(b) the second antigen-binding region comprises a second heavy chain comprising CDRs VH-CDR1, VH-CDR2, and VH-CDR3, and a second light chain comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein the second heavy chain comprises the VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 18, and the second light chain comprises the VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 19,
the method comprising culturing a host cell comprising a first polynucleotide encoding the first heavy chain, a second polynucleotide encoding the first light chain, a third polynucleotide encoding the second heavy chain, and a fourth polynucleotide encoding the second light chain, under suitable conditions such that the first heavy chain, the second heavy chain, the first light chain, and the second light chain are expressed, and the multispecific antibody is produced.
25. The method of claim 24 , wherein:
(a) the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the first antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively; and
(b) the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the second antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 7, 10, 3, 14, 5, and 16, respectively.
26. The method of claim 24 , wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 54, and/or the first light chain comprises the amino acid sequence of SEQ ID NO: 55.
27. The method of claim 24 , wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 54, and the first light chain comprises the amino acid sequence of SEQ ID NO: 55.
28. The method of claim 24 , wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and/or the second light chain comprises the amino acid sequence of SEQ ID NO: 19.
29. The method of claim 24 , wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and the second light chain comprises the amino acid sequence of SEQ ID NO: 19.
30. The method of claim 24 , wherein:
(a) the first heavy chain comprises the amino acid sequence of SEQ ID NO: 54, and the first light chain comprises the amino acid sequence of SEQ ID NO: 55; and/or
(b) the second heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and the second light chain comprises the amino acid sequence of SEQ ID NO: 19.
31. The method of claim 24 , wherein the first heavy chain and/or the second heavy chain comprises a heavy chain constant region selected from the group consisting of human IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
32. The method of claim 31 , wherein the first heavy chain and/or the second heavy chain comprises an IgG 1 heavy chain constant region.
33. The method of claim 32 , wherein:
(a) the first heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, and the second heavy chain comprises tryptophan at amino acid position 366; or
(b) the first heavy chain comprises tryptophan at amino acid position 366, and the second heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively,
numbered according to the EU numbering system.
34. The method of claim 33 , wherein the first heavy chain and/or the second heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
35. The method of claim 24 , wherein the first light chain and/or the second light chain comprises a human kappa light chain constant region or a human lambda light chain constant region.
36. The method of claim 24 , wherein:
(a) the first heavy chain comprises the amino acid sequence of SEQ ID NO: 54, and the first light chain comprises the amino acid sequence of SEQ ID NO: 67; and
(b) the second heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and the second light chain comprises the amino acid sequence of SEQ ID NO: 37.
37. The method of claim 36 , wherein:
(a) the first heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, and the second heavy chain comprises tryptophan at amino acid position 366; or
(b) the first heavy chain comprises tryptophan at amino acid position 366, and the second heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively,
numbered according to the EU numbering system.
38. The method of claim 37 , wherein the first heavy chain and/or the second heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
39. The method of claim 37 , wherein the first heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, and the second heavy chain comprises tryptophan at amino acid position 366, numbered according to the EU numbering system.
40. The method of claim 39 , wherein the first heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
41. The method of claim 39 , wherein the second heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
42. The method of claim 39 , wherein the first heavy chain and the second heavy chain comprise aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
43. The method of claim 37 , wherein the first heavy chain comprises tryptophan at amino acid position 366, and the second heavy chain comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, numbered according to the EU numbering system.
44. The method of claim 43 , wherein the first heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
45. The method of claim 43 , wherein the second heavy chain comprises aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.
46. The method of claim 43 , wherein the first heavy chain and the second heavy chain comprise aspartate, leucine, and glutamate at amino acid positions 239, 330, and 332, respectively, numbered according to the EU numbering system.