Methods and means for the production of Ig-like molecules
The invention provides means and methods for producing one or more Ig-like molecules in a single host cell. Novel CH3 mutations enabling the production of monospecific and/or bispecific Ig-like molecules of interest are also provided.
1. A heterodimeric antibody comprising two heavy chains with CH3 domains, wherein the CH3 domain of the first heavy chain comprises at least one substitution of a neutral amino acid residue by a positively charged amino acid residue as compared to wildtype, and the CH3 domain of the second heavy chain comprises at least one substitution of a neutral amino acid residue by a negatively charged amino acid residue as compared to wildtype, and wherein the at least one positively charged amino acid residue substituted in the CH3 domain of said first heavy chain interacts with the at least one negatively charged amino acid residue substituted in the CH3 domain of said second heavy chain in the interface between said first and second heavy chains.
2. The heterodimeric antibody of claim 1 , wherein the at least one positively charged amino acid residue substitution in the CH3 domain of the first heavy chain is lysine (K).
3. The heterodimeric antibody of claim 1 , wherein the CH3 domain of the first heavy chain comprises a substitution of the amino acid residue at position 366 as compared to wildtype and according to the EU numbering system, by a positively charged amino acid residue, and the CH3 domain of the second heavy chain comprises a substitution of the amino acid residue at position 351 as compared to wildtype and according to the EU numbering system, by a negatively charged residue.
4. The heterodimeric antibody of claim 3 , wherein the positively charged amino acid residue substitution in the CH3 domain of the first heavy chain at position 366 is lysine (K), and the negatively charged amino acid residue substitution in the CH3 domain of the second heavy chain at position 351 is aspartic acid (D) or glutamic acid (E).
5. The heterodimeric antibody of claim 3 or 4 , wherein the CH3 domain of the first heavy chain further comprises a substitution, as compared to wildtype, of the amino acid residue at position 351 by a positively charged amino acid residue.
6. The heterodimeric antibody of claim 5 , wherein the positively charged amino acid residue substitution in the CH3 domain of the first heavy chain at position 351 is lysine (K).
7. The heterodimeric antibody of claims 1 or 3 , wherein the CH3 domain of the second heavy chain further comprises amino acid substitution(s), as compared to wildtype, selected from the group consisting of
(i) a substitution of the amino acid residue at position 349, according to the EU numbering system, by a negatively charged amino acid residue;
(ii) a substitution of the amino acid residue at position 368, according to the EU numbering system, by negatively charged amino acid residue;
(iii) a substitution of the amino acid residue at position 355, according to the EU numbering system, by a negatively charged amino acid residue;
(iv) a substitution of the amino acid residues at position 349 and 368, according to the EU numbering system, by negatively charged amino acid residues;
(v) a substitution of the amino acid residues at positions 349 and 355, according to the EU numbering system, by negatively charged amino acid residues.
8. The heterodimeric antibody of claim 7 , wherein the CH3 domain of the first heavy chain further comprises a substitution, as compared to wildtype, of the amino acid residue at position 351 by a positively charged amine acid residue.
9. The heterodimeric antibody of claim 1 , wherein the CH3 domain of the first heavy chain comprises amino acid substitutions at positions 366 and 351 as compared to wildtype and according to the EU numbering system, by positively charged amino acid residues, and the CH3 domain of the second heavy chain comprises amino acid substitutions at positions 351 and 368 as compared to wildtype and according to the EU numbering system, by negatively charged amino acid residues.
10. The heterodimeric antibody of claim 9 , wherein the positively charged amino acid residue substitutions in the CH3 domain of the first heavy chain at positions 366 and 351 are both lysine, and the negatively charged amino acid substitutions in the CH3 domain of the second heavy chain at positions 351 and 368 are each aspartic acid (D) or glutamic acid (E).
11. A heterodimeric antibody comprising two heavy chains with CH3 domains wherein, (i) the CH3 domain of the first heavy chain comprises a substitution of the amino acid residue at position 366 as compared to wildtype and according to the EU numbering system, by a positively charged amino acid residue, and the CH3 domain of the second heavy chain comprises substitutions of the amino acid residues at positions 351 and 368 as compared to wildtype and according to the EU numbering system, by negatively charged amino acid residues; or
(ii) the CH3 domain of the first heavy chain comprises a substitution of the amino acid residue at position 366 as compared to wildtype and according to the EU numbering system, by a positively charged amino acid residue, and the CH3 domain of the second heavy chain comprises substitutions of the amino acid residues at positions 349 and 351 as compared to wildtype and according to the EU numbering system, by negatively charged amino acid residues; or
(iii) the CH3 domain of the first heavy chain comprises a substitution of the amino acid residue at position 366 as compared to wildtype and according to the EU numbering system, by a positively charged amino acid residue, and the CH3 domain of the second heavy chain comprises substitutions of the amino acid residues at positions 349, 351 and 368 as compared to wildtype and according to the EU numbering system, by negatively charged amino acid residues; or
(iv) the CH3 domain of the first heavy chain comprises a substitution of the amino acid residue at position 366 as compared to wildtype and according to the EU numbering system, by a positively charged amino acid residue, and the CH3 domain of the second heavy chain comprises substitutions of the amino acid residues at positions 349 and 351 and 355 as compared to wildtype and according to the EU numbering system, by negatively charged amino acid residues; or
(v) the CH3 domain of the first heavy chain comprises substitutions of the amino acids at positions 351 and 366 as compared to wildtype and according to the EU numbering system, by positively charged residues, and the CH3 domain of the second heavy chain comprises substitutions of the amino acid residues at positions 351 and 368 as compared to wildtype and according to the EU numbering system, by negatively charged amino acid residues; or
(vi) the CH3 domain of the first heavy chain comprises substitutions of the amino acids at positions 351 and 366 as compared to wildtype and according to the EU numbering system, by positively charged residues, and the CH3 domain of the second heavy chain comprises substitutions of the amino acid residues at positions 349, 351 and 355 as compared to wildtype and according to the EU numbering system, by negatively charged residues; or
(vii) the CH3 domain of the first heavy chain comprises substitutions of the amino acids at positions 351 and 366 as compared to wildtype and according to the EU numbering system, by positively charged residues, and the CH3 domain of the second heavy chain comprises substitutions of the amino acid residues at positions 349 and 351 as compared to wildtype and according to the EU numbering system, by negatively charged amino acid residues; or
(viii) the CH3 domain of the first heavy chain comprises substitutions of the amino acids at positions 351 and 366 as compared to wildtype and according to the EU numbering system, by positively charged residues, and the CH3 domain of the second heavy chain comprises substitutions of the amino acid residues at positions 349 and 351 and 368 as compared to wildtype and according to the EU numbering system, by negatively charged amino acid residues.
12. The heterodimeric antibody of claim 11 , wherein the positively charged amino acid substitutions in the CH3 domain of the first heavy chain are lysine (K).
13. The heterodimeric antibody of claim 1 , wherein the antibody further comprises a common light chain.
14. The heterodimeric antibody of claim 13 , wherein the common light chain is a germline light chain.
15. The heterodimeric antibody of claim 13 , wherein the common light chain is a rearranged germline human kappa light chain.
16. The heterodimeric antibody of claim 15 , wherein the common light chain is the rearranged germline human kappa light chain IgVK1-39/JK or IgVK3-20/JK.
17. The heterodimeric antibody of claim 1 , wherein the heterodimeric antibody is human IgG.
18. The heterodimeric antibody of claim 17 , wherein the heterodimeric antibody is human IgG1.
19. The heterodimeric antibody of claim 1 , wherein each variable region of the first and second heavy chains binds to different epitopes.
20. The heterodimeric antibody of claim 19 , wherein the different epitopes are located on the same target molecule.
21. The heterodimeric antibody of claim 20 , wherein the target molecule is a soluble molecule.
22. The heterodimeric antibody of claim 20 , wherein the target molecule is a membrane-bound molecule.
23. The heterodimeric antibody of claim 19 , wherein the different epitopes are located on different target molecules.
24. The heterodimeric antibody of claim 23 , wherein the different target molecules are expressed on the same cells.
25. The heterodimeric antibody of claim 23 , wherein the different target molecules are expressed on different cells.
26. The heterodimeric antibody of claim 23 , wherein the different target molecules are soluble molecules.
27. The heterodimeric antibody of claim 23 , wherein one target molecule is a soluble molecule and a second target molecule is a membrane bound molecule.
28. The heterodimeric antibody of claim 19 , wherein at least one of said epitopes is located on a tumor cell.
29. The heterodimeric antibody of claim 19 , wherein at least one of said epitopes is located on an effector cell.
30. The heterodimeric antibody of claim 29 , wherein said effector cell is an NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell or a neutrophilic granulocyte.
31. The heterodimeric antibody of claim 19 , wherein at least one of said epitopes is located on a CD3, CD16, CD25, CD28, CD64, CD89, NKG2D or a NKp46 molecule.
32. A pharmaceutical composition comprising the heterodimeric antibody of claim 1 and a pharmaceutically acceptable carrier.
33. The heterodimeric antibody of claim 1 , wherein the at least one negatively charged amino acid residue substitution in the CH3 domain of the second heavy chain is aspartic acid (D) or glutamic acid (E).
34. The heterodimeric antibody of claim 3 or 4 , wherein the CH3 domain of the second heavy chain further comprises a substitution of the amino acid residue at position 368 as compared to wildtype and according to the EU numbering system, by a negatively charged amino acid residue.