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1. WO2020198683 - PROTÉINES HÉTÉROMULTIMÈRES ET LEURS MÉTHODES D'UTILISATION

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CLAIMS

What is claimed is:

1. A heteromultimeric protein comprising a first polypeptide comprising a first heavy chain constant domain 3 (CH3) domain and a second polypeptide comprising a second CH3 domain, wherein the first CH3 domain comprises a substitution relative to a wildtype CH3 domain at amino acid position 354 with a bulky hydrophobic amino acid, and/or the second CH3 domain comprises a substitution relative to a wildtype CH3 domain at amino acid position 347 with a negatively charged amino acid, and wherein the amino acid residue numbering is based on EU numbering.

2. The heteromultimeric protein of claim 1, wherein the bulky hydrophobic amino acid at amino acid position 354 forms a hydrophobic interaction with an amino acid residue in the second CH3 domain.

3. The heteromultimeric protein of claim 1 or 2, wherein the second CH3 domain comprises a tyrosine (Y) at amino acid position 349.

4. The heteromultimeric protein of any one of claims 1-3, wherein the negatively charged amino acid at amino acid position 347 forms an ionic bond with an amino acid residue in the first CH3 domain.

5. The heteromultimeric protein of any one of claims 1-4, wherein the first CH3 domain comprises a lysine (K) at amino acid position 360.

6. The heteromultimeric protein of any one of claims 1-5, wherein the first CH3 domain and the second CH3 domain are human CH3 domains.

7. The heteromultimeric protein of any one of claims 1-6, wherein the first CH3 domain comprises a substitution selected from the group consisting of S354Y, S354F and S354W. 8. The heteromultimeric protein of claim 7, wherein the first CH3 domain comprises S354Y. 9. The heteromultimeric protein of any one of claims 1-8, wherein the second CH3 domain comprises a substitution selected from the group consisting of Q347E and Q347D.

10. The heteromultimeric protein of claim 9, wherein the second CH3 domain comprises Q347E.

11. The heteromultimeric protein of any one of claims 1-10, wherein the first CH3 domain and the second CH3 domain further comprise knob-into-hole residues.

12. The heteromultimeric protein of claim 11, wherein the knob-into-hole residues are T366Y and Y407T.

13. The heteromultimeric protein of claim 12, wherein the first CH3 domain comprises T366Y and S354Y, and the second CH3 domain comprises Y407T and Q347E.

14. The heteromultimeric protein of any one of claims 1-13, wherein the first polypeptide and/or the second polypeptide comprise a heavy chain constant domain 2 (CH2).

15. The heteromultimeric protein of claim 14, wherein the heteromultimeric protein

comprises an IgG Fc region.

16. The heteromultimeric protein of claim 15, wherein the IgG Fc region is an IgG1, IgG2, IgG3, or IgG4 Fc region.

17. The heteromultimeric protein of any one of claims 1-16, wherein the first polypeptide is an antibody heavy chain, and/or the second polypeptide is an antibody heavy chain. 18. The heteromultimeric protein of claim 17, wherein the heteromultimeric protein

comprises one or more antibody light chains.

19. The heteromultimeric protein of claim 18, wherein the heteromultimeric protein is a multispecific antibody.

20. The heteromultimeric protein of claim 18 or 19, comprising:

(a) a first heavy chain comprising from the N-terminus to the C-terminus: a first heavy chain variable domain (VH1), a first heavy chain constant domain 1 (CH1), a first heavy chain constant domain 2 (CH2), and the first CH3 domain;

(b) a first light chain comprising from the N-terminus to the C-terminus: a first light chain variable domain (VL1), and a first light chain constant domain (CL);

(c) a second heavy chain comprising from the N-terminus to the C-terminus: a second heavy chain variable domain (VH2), a second CH1, a second CH2, and the second CH3 domain; and

(d) a second light chain comprising from the N-terminus to the C-terminus: a second light chain variable domain (VL2), and a second CL;

wherein VH1 and VL1 associate to form a first antigen binding site that specifically binds to a first target, and VH2 and VL2 associate to form a second antigen binding site that specifically binds to a second target.

21. The heteromultimeric protein of claim 20, wherein VL1 and VL2 have the same amino acid sequence.

22. The heteromultimeric protein of claim 20, wherein VL1 and VL2 have different amino acid sequences.

23. The heteromultimeric protein of any one of claims 20-22, wherein the first target and the second target are different epitopes of the same antigen.

24. The heteromultimeric protein of any one of claims 20-22, wherein the first target and the second target are different antigens.

25. The heteromultimeric protein of claim 24, wherein the first antigen binding site

specifically binds a tumor antigen and the second antigen binding site specifically binds CD3.

26. The heteromultimeric protein of claim 25, wherein the first antigen binding site

specifically binds CD20.

27. The heteromultimeric protein of claim 25, wherein the first antigen binding site

specifically binds HER2.

28. The heteromultimeric protein of any one of claims 20-27, comprising:

(a) a first heavy chain comprising from the N-terminus to the C-terminus: a third heavy chain variable domain (VH3), a third CH1, the VH1, the first CH1, the first CH2, and the first CH3 domain;

(b) a first light chain comprising from the N-terminus to the C-terminus: a third light chain variable domain (VL2), a third CL, the VL1, and the first CL;

wherein VH3 and VL3 associate to form a third antigen binding site that specifically binds to a third target.

29. The heteromultimeric protein of claim 28, wherein the first antigen binding site and the third antigen binding site specifically bind to the same antigen.

30. The heteromultimeric protein of claim 29, wherein the first antigen binding site and the third antigen binding site specifically bind to HER2 and the second antigen binding site specifically bind to CD3.

31. The heteromultimeric protein of claim 18 or 19, comprising:

(a) a first heavy chain comprising from the N-terminus to the C-terminus: a first VHH, a first heavy chain constant domain 2 (CH2), and the first CH3 domain;

(b) a second heavy chain comprising from the N-terminus to the C-terminus: a first heavy chain variable domain (VH1), a second CH1, a second CH2, and the second CH3 domain; and

(d) a light chain comprising from the N-terminus to the C-terminus: a first light chain variable domain (VL1), and a first CL;

wherein VH1 and VL1 associate to form a first antigen binding site that specifically binds to a first target, and the first VHH specifically binds to a second target.

32. The heteromultimeric protein of claim 31, wherein the first antigen binding site

specifically binds to CD3 and the first VHH specifically binds to a tumor antigen.

33. The heteromultimeric protein of claim 32, wherein the first VHH specifically binds to BCMA.

34. The heteromultimeric protein of any one of claims 31-33, wherein the first heavy chain comprises from the N-terminus to the C-terminus: a second VHH, the first VHH, the first CH2, and the first CH3 domain, wherein the second VHH specifically binds to a third target.

35. The heteromultimeric protein of claim 34, wherein the first VHH and the second VHH specifically bind to the same antigen.

36. The heteromultimeric protein of claim 35, wherein the first VHH and the second VHH specifically bind to BCMA.

37. The heteromultimeric protein of any one of claims 1-16, wherein the heteromultimeric protein is an immunoadhesin or an antibody-immunoadhesin chimera.

38. One or more nucleic acid(s) encoding the heteromultimeric protein of any one of claims 1-37.

39. A vector comprising the one or more nucleic acid(s) of claim 38.

40. A host cell comprising the one or more nucleic acid(s) of claim 38 or the vector of claim 39.

41. A method for preparing a multispecific antibody or a heteromultimeric protein,

comprising:

(a) culturing the host cell of claim 40 under conditions that allow expression of the one or more nucleic acid(s) or vector; and

(b) recovering the multispecific antibody or the heteromultimeric protein from the host cell culture.

42. A pharmaceutical composition comprising the heteromultimeric protein of any one of claims 1-37, and a pharmaceutically acceptable excipient.

43. A method for treating a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 42.

44. A method of generating a heteromultimeric protein that specifically binds to a first target and a second target, comprising:

(a) providing a first polypeptide comprising a first binding domain that specifically binds to the first target, and a first CH3 domain; and

(b) providing a second polypeptide comprising a second binding domain that specifically binds to the second target, and a second CH3 domain;

wherein:

(i) the first CH3 domain comprises a substitution relative to a wildtype CH3 domain at amino acid position 354 with a bulky hydrophobic amino acid, and/or the second CH3 domain comprises a substitution relative to a wildtype CH3 domain at amino acid position 347 with a negatively charged amino acid; or

(ii) the first CH3 domain comprises a substitution relative to a wildtype CH3 domain at amino acid position 347 with a negatively charged amino acid, and/or the second CH3 domain comprises a substitution relative to a wildtype CH3 domain at amino acid position 354 with a bulky hydrophobic amino acid;

and wherein the amino acid residue numbering is based on EU numbering.