Sequence asymmetric modified IgG4 bispecific antibodies
View Patent ↗The present disclosure provides an asymmetric mixed antibody comprising two heavy chains or heavy chain fragments each comprising at least a variable region, a hinge region and a CH1 domain, wherein a first heavy chain or fragment thereof is a class IgG4 and has: a the inter-chain cysteine at position 127, numbered according to the Kabat numbering system, in the CH1 domain is substituted with another amino acid; and b optionally one or more of the amino acids positioned in the upper hinge region is substituted with cysteine, and wherein the second heavy chain or fragment thereof is characterized in that part or all of the chain has a different amino acid sequence to said first heavy chain in at least the region outside the variable region (for example the constant region), formulations comprising the same, therapeutic used of both of the above, and processes for preparing the antibodies and formulation.
1. An asymmetric antibody of the class IgG4, comprising (a) a first heavy chain paired to a first light chain and (b) a second heavy chain paired to a second light chain, wherein each of the first and second heavy chains comprises a variable region, and a constant region with a hinge region, and a C H 1 domain, wherein in the first heavy chain
(a) an inter-chain cysteine at position 127 is substituted with serine, threonine, alanine, or glycine; and
(b) an amino acid at position 227, 228, 229 or 230 is substituted with cysteine, and
wherein the second heavy chain is IgG4 wild type or a mutated IgG4 wild type with an S241P mutation, and wherein amino acid numbering is according to the Kabat numbering system.
2. The antibody according to claim 1 , wherein the glycine at position 230 of the first heavy chain is substituted with cysteine.
3. The antibody according to claim 1 , wherein the tyrosine at position 229 of the first heavy chain is substituted with cysteine.
4. The antibody according to claim 1 , wherein the lysine at position 228 of the first heavy chain is substituted with cysteine.
5. The antibody according to claim 1 , wherein the serine at position 227 of the first heavy chain is substituted with cysteine.
6. The antibody according to claim 1 , wherein the cysteine at position 239 and the cysteine at position 242 of the first heavy chain are substituted with serine, threonine, alanine, or glycine.
7. The antibody according to claim 1 , wherein the cysteine at position 239 of the first heavy chain is substituted with serine, threonine, alanine, or glycine.
8. The antibody according to claim 1 , wherein the cysteine at position 242 of the first heavy chain is substituted with serine, threonine, alanine, or glycine.
9. The antibody according to claim 6 , wherein the cysteine at position 239 and/or the cysteine at position 242 of the first heavy chain is substituted with serine.
10. The antibody according to claim 1 , wherein three alanines are inserted between positions 238 and 239 of the first heavy chain.
11. The antibody according to claim 1 , wherein a threonine-histidine-threonine amino acid sequence is inserted between positions 238 and 239 of the first heavy chain.
12. The antibody according to claim 1 , wherein the second heavy chain is a mutated IgG4 wild type in which the serine at position 241 is substituted with proline.
13. The antibody according to claim 1 , wherein in the first heavy chain the glycine at position 230 is substituted with cysteine, the serine at position 227 is substituted with proline, the tyrosine at position 229 is substituted with serine, the proline at position 237 is substituted with aspartic acid, the proline at position 238 is substituted with lysine, the amino acid sequence threonine-histidine-threonine is inserted between positions 238 and 239 and the serine at position 241 is substituted with proline.
14. The antibody according to claim 1 , wherein the two variable regions of the heavy chains are identical.
15. The antibody according to claim 1 , wherein the amino acid sequences of the two heavy chain variable regions are not identical.
16. The antibody according to claim 1 , wherein the antibody is bi-specific.
17. The antibody according to claim 1 , wherein one or both heavy chains comprises an upper hinge region and a core region of 12 to 17 amino acids in length.
18. An asymmetric antibody of the class IgG4, comprising first heavy chain paired to a first light chain and a second heavy chain each paired to a second light chain, wherein each of the first and second heavy chains comprises a variable region, a constant region with a hinge region, and a C H 1 domain, wherein in the first heavy chain
(a) an inter-chain cysteine at position 127 is substituted with serine, threonine, alanine, or glycine; and
(b) the cysteine at position 239 or cysteine at position 242 is substituted with serine, threonine, alanine, or glycine;
wherein the second heavy chain is an IgG4 wild type or a mutated IgG4 wild type with an S241P mutation, and
wherein amino acid numbering is according to the Kabat numbering system.
19. An IgG4 antibody, the improvement comprising
(a) a first heavy chain paired to a first light chain and a second heavy chain paired to a second light chain, wherein the amino acid sequences of the first and second heavy chains are not identical in the regions outside the variable regions, and
(b) in the first heavy chain
(i) an inter-chain cysteine at position 127 is substituted with serine, threonine, alanine, or glycine;
(ii) an amino acid at position 227, 228, 229 or 230 is substituted with cysteine, and
(iii) the amino acid at position 242 is substituted with serine, threonine, alanine, or glycine,
wherein amino acid numbering is according to the Kabat numbering system, and the IgG4 antibody has improved stability compared to the corresponding wild-type IgG4 antibody.
20. The antibody according to claim 19 , wherein the glycine at position 230 of the first heavy chain is substituted with cysteine.
21. The antibody according to claim 19 , wherein the tyrosine at position 229 of the first heavy chain is substituted with cysteine.
22. The antibody according to claim 19 , wherein the lysine at position 228 of the first heavy chain is substituted with cysteine.
23. The antibody according to claim 19 , wherein the serine at position 227 of the first heavy chain is substituted with cysteine.
24. The antibody according to claim 19 , wherein the cysteine at position 239 of the first heavy chain is substituted with serine, threonine, alanine, or glycine.
25. The antibody according to claim 19 , wherein the cysteine at position 239 and/or the cysteine at position 242 of the first heavy chain is substituted with serine.
26. The antibody according to claim 19 , wherein three alanines are inserted between positions 238 and 239 of the first heavy chain.
27. The antibody according to claim 19 , wherein a threonine-histidine-threonine sequence is inserted between positions 238 and 239 of the first heavy chain.
28. The antibody according to claim 19 , wherein the serine at position 241 of the first heavy chain is substituted with proline.
29. The antibody according to claim 19 , wherein in the first heavy chain the glycine at position 230 is substituted with cysteine, the serine at position 227 is substituted with proline, the tyrosine at position 229 is substituted with serine, the proline at position 237 is substituted with aspartic acid, the proline at position 238 is substituted with lysine, the amino acid sequence threonine-histidine-threonine is inserted between positions 238 and 239 and the serine at position 241 is substituted with proline.
30. The antibody according to claim 19 , wherein the two variable regions of the heavy chains are identical.
31. The antibody according to claim 19 , wherein the amino acid sequences of the two variable regions of the heavy chains are not identical.
32. The antibody according to claim 19 , wherein the antibody is bi-specific.
33. The antibody according to claim 19 , wherein the hinge regions of the heavy chains are different.
34. The antibody according to claim 19 , wherein the hinge regions of the heavy chains are the same.
35. The antibody according to claim 19 , wherein one or both heavy chains comprises an upper hinge region and a core region of 12 to 17 amino acids in length.
36. An expression vector, comprising a sequence encoding an antibody of claim 1 .
37. A host cell, comprising a vector as defined in claim 36 .