NOVEL POLYPEPTIDES
20220056148 · 2022-02-24
Inventors
- Anna Sall (Lund, SE)
- Laura Von Schantz (Lund, SE)
- Anneli Nilsson (Lund, SE)
- Barnabas Nyesiga (Lund, SE)
- Jessica Petersson (Lund, SE)
- Mattias Levin (Lund, SE)
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
C07K2317/64
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K16/2878
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention provides bispecific antibodies of an antibody format, and which comprise one or more Fab fragments and an immunoglobulin molecule. The invention further provides compositions of said bispecific antibodies, as well as methods and uses of the same. The invention further provides a method of generating bispecific antibodies of the format.
Claims
1. A polypeptide comprising an antigen binding fragment, wherein the antigen binding fragment comprises an antibody VL region and an antibody VH region, wherein the antigen binding fragment comprises four or more mutations to promote association of the heavy chain with the light chain, wherein the VL region comprises a mutation at position 44 (according to IMGT numbering), and wherein the VH region comprises a mutation at position 44 (according to IMGT numbering).
2. The polypeptide according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of: an Fv fragment (such as a single chain Fv fragment, or a disulphide-bonded Fv fragment), a Fab-like fragment (such as a Fab fragment; a Fab′ fragment or an F(ab).sub.2 fragment) and domain antibodies.
3. The polypeptide according to claim 1, wherein the polypeptide is an immunoglobulin molecule.
4. The polypeptide according to claim 3, further comprising one or more mutations are in one or more of the following regions: (i) the CH1 region and/or (ii) the CKappa or CLambda region.
5. The polypeptide according to claim 3 or 4, wherein the CH1 region comprises a mutation at position 168 and/or at position 170 (according to EU numbering); and wherein the CKappa or CLambda region comprises a mutation at position 135 and/or at position 176 (according to Kabat numbering).
6. The polypeptide according to any one of the preceding claims, wherein the VL region mutation at position 44 comprises a mutation at position Q44 (according to IMGT numbering), and wherein the VH region mutation at position 44 comprises a mutation at position Q44 (according to IMGT numbering).
7. The polypeptide according to claim 6, wherein the VL region mutation at position Q44 comprises a mutation at position Q44E, Q44D, Q44R or Q44K (according to IMGT numbering), and wherein the VH region mutation at position Q44 comprises a mutation at position Q44R, Q44K, Q44E, or Q44D (according to IMGT numbering).
8. The polypeptide according to any one of the preceding claims, wherein the antigen binding fragment comprises two or more mutations at positions selected from the list consisting of: 44, 49 and 120 (according to IMGT numbering).
9. The polypeptide according to claim 8, wherein the VL region comprises one or more mutation at positions selected from the list consisting of: 44, 49 and 120 (according to IMGT numbering).
10. The polypeptide according to claim 8, wherein the VH region comprises one or more mutation at positions selected from the list consisting of: 44, 49 and 120 (according to IMGT numbering).
11. The polypeptide according to any one of claims 8 to 10, wherein the two or more mutations are positions selected from the list consisting of: Q44, G49, A49, Q120 and A120 (according to IMGT numbering).
12. The polypeptide according to claim 11, wherein the two or more mutations are positions selected from the list consisting of: Q44E, Q44R, Q44K, G49C, A49D, Q120C, A120C and Q120K (according to IMGT numbering).
13. The polypeptide according to any one of the preceding claims, wherein the mutations prevent the formation of aggregates and a Fab by-product.
14. The polypeptide according to any one of the preceding claims, wherein the mutations prevent formation of aggregates by generating steric hindrance and/or incompatibility between charges.
15. The polypeptide according to any one of the preceding claims, wherein the mutations prevent formation of a Fab by-product by generating steric hindrance and/or incompatibility between charges.
16. The polypeptide according to any one of the preceding claims wherein the polypeptide comprises one or more mutation pairs each comprising two functionally compatible mutations.
17. The polypeptide according to claim 16, wherein the functionally compatible mutations are selected from: (a) cavity and protruding surface mutations (i.e. steric mutations); and/or (b) hydrophobic swap mutations; and/or (c) charged mutations (i.e. salt mutations); and/or (d) double charged mutations; and/or (e) mutations resulting in the formation of a disulphide bridge.
18. The polypeptide according to claim 17, wherein the polypeptide comprises one or more mutation pairs in one or more of the following region groups: (a) the CH1 and CKappa or CLambda region; and/or (b) the VL and VH regions.
19. The polypeptide according to claim 18 wherein the mutation pairs are in the CH1 and CKappa or CLambda regions are selected from: (a) cavity and protruding surface mutations (i.e. steric mutations); and/or (b) hydrophobic swap mutations; and/or (c) charged mutations (i.e. salt mutations); and/or (d) mutations resulting in the formation of a disulphide bridge.
20. The polypeptide according to any one of claims 17 to 19 wherein the mutation pairs are in the VH and VL regions, and wherein the mutation pairs are selected from: (a) charged mutations (i.e. salt mutations); and/or (b) double charged mutations; and/or (c) mutations resulting in the formation of a disulphide bridge.
21. The polypeptide according to claims 18 to 20 wherein the polypeptide comprises one or more mutation pairs selected from: (a) steric mutations in the CH1 and CKappa or CLambda regions; (b) steric mutations in the CH1 and CKappa or CLambda regions and salt mutations in the VH and VL regions; (c) hydrophobic mutations in the CH1 and CKappa or CLambda regions; (d) hydrophobic mutations in the CH1 and CKappa or CLambda regions and salt mutations in the VH and VL regions; (e) salt mutations in the CH1 and CKappa or CLambda regions and salt mutations in the VH and VL regions;
22. The polypeptide according to any one of claims 4 to 21, wherein the mutations are at positions selected from the group consisting of: (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or (b) a position selected from the one or more of the following position ranges in the CKappa or CLambda domain: position 132 to 138, position 173 to 179, position 130 to 136, position 111 to 117 and position 134 to 140 (according to Kabat numbering).
23. The polypeptide according to any one of claims 4 to 22, wherein the mutations are at positions selected from the group consisting of: (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or (b) one or more of the following positions in the CKappa or CLambda domain: L135, S176, V133, S114 and N137 (according to Kabat numbering).
24. The polypeptide according to any one of claims 4 to 23, wherein the mutations are selected from the group consisting of: (a) one or more of the following mutations in the CH1 domain: H168A, F170G, L145Q, S183V and T187E (according to EU numbering); and/or (b) one or more of the following mutations in the CKappa or CLambda domain: L135Y, S176W, V133T, S176V, S114A and N137K (according to Kabat numbering).
25. The polypeptide according to any one of the preceding claims, wherein the polypeptide is a bispecific antibody.
26. The polypeptide according to claim 26, wherein the polypeptide is an immunoglobulin molecule comprising at least two antigen binding fragments, with the first antigen binding fragment having specificity for the first antigen and a second antigen binding fragment having specificity for a second antigen, preferably wherein the first and second antigen binding fragment are a first Fab and a second Fab.
27. The polypeptide according to claim 26, wherein the bispecific antibody comprises (a) an immunoglobulin molecule having specificity for a first antigen, the immunoglobulin molecule comprising a first heavy chain polypeptide and a first light chain polypeptide; and (b) at least one Fab fragment having specificity for a second antigen, the Fab fragment comprising a second heavy chain polypeptide and a second light chain polypeptide wherein the second light chain polypeptide is fused to the C-terminus of the first heavy chain polypeptide and wherein the bispecific antibody comprises one or more mutations to promote association of the first heavy chain polypeptide with the first light chain polypeptide and/or to promote association of the second heavy chain polypeptide with the second light chain polypeptide.
28. The polypeptide according to any one of claims 3 to 27, wherein the immunoglobulin molecule comprises two copies of the first heavy chain polypeptide and/or two copies of the first light chain polypeptide.
29. The polypeptide according to claim 27 or to 28, wherein the antibody comprises two Fab fragments according to (b).
30. The polypeptide according to any one of claims 27 to 29, wherein the immunoglobulin molecule comprises two copies of the first heavy chain polypeptide and two copies of the first light chain polypeptide, and the bispecific antibody further comprises two Fab fragments according to (b), and the first Fab fragment is fused to the C-terminus of the first copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment; and the second Fab fragment is fused to the C-terminus of the second copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment.
31. The polypeptide according to any one of claims 3 to 30, wherein the immunoglobulin molecule comprises a human Fc region or a variant of a said region, where the region is an IgG1, IgG2, IgG3 or IgG4 region, preferably an IgG1 or IgG4 region.
32. The polypeptide according to claim 31, wherein the Fc region is a naturally occurring (i.e. wildtype) human Fc region.
33. The polypeptide according to claim 31, wherein the Fc region is a non-naturally occurring (e.g. mutated) human Fc region.
34. The polypeptide according to any one of claims 31 to 33, wherein the Fc region has modified glycosylation, for example wherein the Fc region is afucosylated.
35. The polypeptide according to any one of claims 27 to 34, wherein the one or more mutations are in one or more of the following regions: (i) the CH1 region of the first heavy chain polypeptide and/or (ii) the CH1 region of the second heavy chain polypeptide and/or (iii) the VH region of the second heavy chain polypeptide, and/or (iv) the CKappa or CLambda region of the first light chain polypeptide and/or (v) the CKappa or CLambda region of the second light chain polypeptide, and/or (vi) the VL region of the second light chain polypeptide.
36. The polypeptide according to any one of claims 27 to 35, wherein the one or more mutations prevent the binding of the second heavy chain polypeptide to the first light chain polypeptide and/or prevent self-aggregation of the first heavy chain polypeptide fused to the second light chain polypeptide.
37. The polypeptide according to any one of claims 27 to 35, wherein the one or more mutations prevent the formation of aggregates and a Fab by-product.
38. The polypeptide according to any one of claims 27 to 37, wherein the mutations prevent formation of aggregates by generating steric hindrance and/or incompatibility between charges.
39. The polypeptide according to any one of claims 27 to 38, wherein the mutations prevent formation of a Fab by-product by generating steric hindrance and/or incompatibility between charges.
40. The polypeptide according to any one of claims 27 to 39, wherein the antibody comprises one or more mutation pairs each comprising two functionally compatible mutations.
41. The polypeptide according to claim 40, wherein the functionally compatible mutations are selected from: (a) cavity and protruding surface mutations (i.e. steric mutations); and/or (b) hydrophobic swap mutations; and/or (c) charged mutations (i.e. salt mutations); and/or (d) double charged mutations; and/or (e) mutations resulting in the formation of a disulphide bridge.
42. The polypeptide according to claim 41, wherein the bispecific antibody comprises one or more mutation pairs in one or more of the following region groups: (a) the CH1 and CKappa or CLambda region of the immunoglobulin; and/or (b) the CH1 and CKappa or CLambda region of the Fab; and/or (c) the VL and VH regions of the immunoglobulin; and/or (d) the VL and VH regions of the Fab.
43. The polypeptide according to claim 42, wherein the mutation pairs are in the CH1 and CKappa or CLambda regions of the Fab and/or the immunoglobulin, and wherein the mutation pairs are selected from: (a) cavity and protruding surface mutations (i.e. steric mutations); and/or (b) hydrophobic swap mutations; and/or (c) charged mutations (i.e. salt mutations); and/or (d) mutations resulting in the formation of a disulphide bridge.
44. The polypeptide according to any one of claims 41 to 43, wherein the mutation pairs are in the VH and VL regions of the Fab, and wherein the mutation pairs are selected from: (a) charged mutations (i.e. salt mutations); and/or (b) double charged mutations; and/or (c) mutations resulting in the formation of a disulphide bridge.
45. The polypeptide according to any one of claims 41 to 44, wherein the bispecific antibody comprises one or more mutation pairs selected from: (a) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin; (b) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, and salt mutations in the VH and VL regions of the Fab; (c) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (d) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin and salt mutations in the CH1 and CKappa or CLambda regions of the Fab; (e) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (f) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (g) disulphide bridge-forming mutations in the CH1 and CKappa or CLambda regions of the Fab; (h) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (i) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (j) salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (k) salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (l) salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (m) salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (n) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (o) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge forming mutations in the VH and VL regions of the Fab; (p) steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, hydrophobic mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; or (q) steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, hydrophobic mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab. (r) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutation in the CH1 and alanine mutation in the CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab;
46. The polypeptide according to any one of claims 27 to 45, wherein the mutations are at positions selected from the group consisting of: (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or (b) a position selected from the one or more of the following position ranges in the CKappa or CLambda domain: position 132 to 138, position 173 to 179, position 130 to 136, position 111 to 117 and position 134 to 140 (according to Kabat numbering); and/or (c) a position selected from one or more of the following position ranges in the VL: position 41 to 47, position 117 to 123 and position 46 to 52 (according to IMGT numbering); and/or (d) a position selected from one or more of the following position ranges in the VH: position 41 to 47, position 46 to 52 and position 117 to 123 (according to IMGT numbering).
47. The polypeptide according to any one of claims 27 to 46, wherein the mutations are at positions selected from the group consisting of: (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or (b) one or more of the following positions in the CKappa or CLambda domain: 135, 176, 133, 114 and 137 (according to Kabat numbering); and/or (c) one or more of the following positions in the VL: 44, 120 and 49 (according to IMGT numbering); and/or (d) one or more of the following positions in the VH: 44, 49 and 120 (according to IMGT numbering).
48. The polypeptide according to any one of claims 27 to 47, wherein the mutations are at positions selected from the group consisting of: (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or (b) one or more of the following positions in the CKappa or CLambda domain: L135, S176, V133, S114 and N137 (according to Kabat numbering); and/or (c) one or more of the following positions in the VL: Q44, Q120 or A120 and A49 (according to IMGT numbering); and/or (d) one or more of the following positions in the VH: Q44, G49 and Q120 (according to IMGT numbering).
49. The polypeptide antibody according to claim 48, wherein the mutations are selected from the group consisting of: (a) one or more of the following mutations in the CH1 domain: H168A, F170G, L145Q, S183V and T187E (according to EU numbering); and/or (b) one or more of the following mutations in the CKappa or CLambda domain: L135Y, S176W, V133T, S176V, S114A and N137K (according to Kabat numbering); and/or (c) one or more of the following mutations in the VL: Q44R, Q44K, Q44E, Q120C or A120C, Q44D and A49D (according to IMGT numbering); and/or (d) one or more of the following mutations in the VH: Q44E, Q44R, G49C, Q44K, Q44D, Q120C and Q120K (according to IMGT numbering).
50. A bispecific antibody comprising: (a) an immunoglobulin molecule having specificity for a first antigen, the immunoglobulin molecule comprising a first heavy chain polypeptide and a first light chain polypeptide; and (b) at least one Fab fragment having specificity for a second antigen, the Fab fragment comprising a second heavy chain polypeptide and a second light chain polypeptide wherein the second light chain polypeptide is fused to the C-terminus of the first heavy chain polypeptide and wherein the bispecific antibody comprises one or more mutations to promote association of the first heavy chain polypeptide with the first light chain polypeptide and/or to promote association of the second heavy chain polypeptide with the second light chain polypeptide.
51. The bispecific antibody according to claim 50, wherein (a) comprises one or more antigen binding fragment having specificity for the first antigen, wherein the antigen binding fragment comprises an antibody VL region and an antibody VH region, wherein the antigen binding fragment comprises four or more mutations, wherein the VL region comprises a mutation at position 44 (according to IMGT numbering), and wherein the VH region comprises a mutation at position 44 (according to IMGT numbering).
52. The polypeptide according to claim 50 or 51, wherein (a) comprises (i) the CH1 region and/or (ii) the CKappa or CLambda region; and wherein the CH1 region comprises a mutation at position 168 and/or at position 170 (according to EU numbering); and wherein the CKappa or CLambda region comprises a mutation at position 135 and/or at position 176 (according to Kabat numbering); wherein (b) comprises (i) the CH1 region and/or (ii) the CKappa or CLambda region; and wherein the CH1 region comprises a mutation at position 187; and wherein the CKappa or CLambda region comprises a mutation at position 114 and/or at position 137 (according to Kabat numbering).
53. The bispecific antibody according to claim 51 or 52, wherein the VL region mutation at position 44 comprises a mutation at position Q44 (according to IMGT numbering), and wherein the VH region mutation at position 44 comprises a mutation at position Q44 (according to IMGT numbering).
54. The bispecific antibody according to any one of claims 51 to 53, wherein the VL region mutation at position Q44 comprises a mutation at position Q44R, Q44K Q44E or Q44D (according to IMGT numbering), and wherein the VH region mutation at position Q44 comprises a mutation at position Q44R or Q44K (according to IMGT numbering).
55. The bispecific antibody according to any one of claims 51 to 54, wherein the antigen binding fragment comprises two or more mutations at positions selected from the list consisting of: 44, 49 and 120 (according to IMGT numbering).
56. The bispecific antibody according to claim 55, wherein the VL region comprises one or more mutation at positions selected from the list consisting of: 44, 49 and 120 (according to IMGT numbering).
57. The bispecific antibody according to claim 55, wherein the VH region comprises one or more mutation at positions selected from the list consisting of: 44, 49 and 120 (according to IMGT numbering).
58. The bispecific antibody according to any one of claims 55 to 57, wherein the two or more mutations are positions selected from the list consisting of: Q44, G49, A49 and Q120 (according to IMGT numbering).
59. The bispecific antibody according to claim 58, wherein the two or more mutations are positions selected from the list consisting of: Q44E, Q44R, Q44K, Q44D, G49C, G49D, A49D, A49C, Q120C and Q120K (according to IMGT numbering).
60. The bispecific antibody according to any one of claims 51 to 59, wherein the immunoglobulin molecule comprises two copies of the first heavy chain polypeptide and/or two copies of the first light chain polypeptide.
61. The bispecific antibody according to any one of claims 51 to 60, wherein the antibody comprises two Fab fragments according to (b).
62. The bispecific antibody according to any one of claims 51 to 61, wherein the immunoglobulin molecule comprises two copies of the first heavy chain polypeptide and two copies of the first light chain polypeptide, and the bispecific antibody further comprises two Fab fragments according to (b), and the first Fab fragment is fused to the C-terminus of the first copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment; and the second Fab fragment is fused to the C-terminus of the second copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment.
63. The bispecific antibody according to any one of claims 51 to 62, wherein the immunoglobulin molecule comprises a human Fc region or a variant of a said region, where the region is an IgG1, IgG2, IgG3 or IgG4 region, preferably an IgG1 or IgG4 region.
64. The bispecific antibody according to claim 63, wherein the Fc region is a naturally occurring (i.e. wildtype) human Fc region.
65. The bispecific antibody according to claim 63, wherein the Fc region is a non-naturally occurring (e.g. mutated) human Fc region.
66. The bispecific antibody according to any one of claims 63 to 65 wherein the Fc region has modified glycosylation, for example wherein the Fc region is afucosylated.
67. The bispecific antibody according to any one of claims 51 to 66, wherein the one or more mutations are in one or more of the following regions: (i) the CH1 region of the first heavy chain polypeptide and/or (ii) the VH region of the first heavy chain polypeptide, and/or (iii) the CH1 region of the second heavy chain polypeptide and/or (iv) the VH region of the second heavy chain polypeptide, and/or (v) the CKappa or CLambda region of the first light chain polypeptide and/or (vi) the VL region of the first light chain polypeptide, and/or (vii) the CKappa or CLambda region of the second light chain polypeptide, and/or (viii) the VL region of the second light chain polypeptide.
68. The bispecific antibody according to any one of claims 51 to 67, wherein the one or more mutations prevent the binding of the second heavy chain polypeptide to the first light chain polypeptide and/or prevent self-aggregation of the first heavy chain polypeptide fused to the second light chain polypeptide.
69. The bispecific antibody according to any one of claims 51 to 68, wherein the one or more mutations prevent the formation of aggregates and a Fab by-product.
70. The bispecific antibody according to any one of claims 51 to 69, wherein the mutations prevent formation of aggregates by generating steric hindrance and/or incompatibility between charges.
71. The bispecific antibody according to any one of claims 51 to 70, wherein the mutations prevent formation of a Fab by-product by generating steric hindrance and/or incompatibility between charges.
72. The bispecific antibody according to any one of claims 51 to 71, wherein the antibody comprises one or more mutation pairs each comprising two functionally compatible mutations.
73. The bispecific antibody according to claim 72, wherein the functionally compatible mutations are selected from: (a) cavity and protruding surface mutations (i.e. steric mutations); and/or (b) hydrophobic swap mutations; and/or (c) charged mutations (i.e. salt mutations); and/or (d) double charged mutations; and/or (e) mutations resulting in the formation of a disulphide bridge.
74. The bispecific antibody according to claim 73, wherein the bispecific antibody comprises one or more mutation pairs in one or more of the following region groups: (a) the CH1 and CKappa or CLambda region of the immunoglobulin; and/or (b) the CH1 and CKappa or CLambda region of the Fab; and/or (c) the VL and VH regions of the immunoglobulin; and/or (d) the VL and VH regions of the Fab.
75. The bispecific antibody according to claim 74 wherein the mutation pairs are in the CH1 and CKappa or CLambda regions of the Fab and/or the immunoglobulin, and wherein the mutation pairs are selected from: (a) cavity and protruding surface mutations (i.e. steric mutations); and/or (b) hydrophobic swap mutations; and/or (c) charged mutations (i.e. salt mutations); and/or (d) mutations resulting in the formation of a disulphide bridge.
76. The bispecific antibody according to any one of claims 73 to 75 wherein the mutation pairs are in the VH and VL regions of the Fab and/or the immunoglobulin, and wherein the mutation pairs are selected from: (a) charged mutations (i.e. salt mutations); and/or (b) double charged mutations; and/or (c) mutations resulting in the formation of a disulphide bridge.
77. A bispecific antibody according to any one of claims 74 to 76 wherein the bispecific antibody comprises one or more mutation pairs selected from: (a) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin; (b) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, and salt mutations in the VH and VL regions of the Fab; (c) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (d) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin and salt mutations in the CH1 and CKappa or CLambda regions of the Fab; (e) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (f) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (g) disulphide bridge-forming mutations in the CH1 and CKappa or CLambda regions of the Fab; (h) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (i) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (j) salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (k) salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (l) salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (m) salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; (n) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; (o) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge forming mutations in the VH and VL regions of the Fab; (p) steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, hydrophobic mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; or (q) steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, hydrophobic mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab. (r) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutation in the CH1 and alanine mutation in the CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab;
78. The bispecific antibody according to any one of claims 51 to 77, wherein the mutations are at positions selected from the group consisting of: (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or (b) a position selected from the one or more of the following position ranges in the CKappa or CLambda domain: position 132 to 138, position 173 to 179, position 130 to 136, position 111 to 117 and position 134 to 140 (according to Kabat numbering); and/or (c) a position selected from one or more of the following position ranges in the VL: position 41 to 47, position 117 to 123 and position 46 to 52 (according to IMGT numbering); and/or (d) a position selected from one or more of the following position ranges in the VH: position 41 to 47, position 46 to 52 and position 117 to 123 (according to IMGT numbering).
79. The bispecific antibody according to any one of claims 51 to 78, wherein the mutations are at positions selected from the group consisting of: (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or (b) one or more of the following positions in the CKappa or CLambda domain: 135, 176, 133, 114 and 137 (according to Kabat numbering); and/or (c) one or more of the following positions in the VL: 44, 120 and 49 (according to IMGT numbering); and/or (d) one or more of the following positions in the VH: 44, 49 and 120 (according to IMGT numbering).
80. The bispecific antibody according to any one of claims 51 to 79, wherein the mutations are at positions selected from the group consisting of: (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or (b) one or more of the following positions in the CKappa or CLambda domain: L135, S176, V133, S114 and N137 (according to Kabat numbering); and/or (c) one or more of the following positions in the VL: Q44, Q120 or A120 and A49 (according to IMGT numbering); and/or (d) one or more of the following positions in the VH: Q44, G49 and Q120 (according to IMGT numbering).
81. The bispecific antibody according to claim 80, wherein the mutations are selected from the group consisting of: (a) one or more of the following mutations in the CH1 domain: H168A, F170G, L145Q, S183V and T187E (according to EU numbering); and/or (b) one or more of the following mutations in the CKappa or CLambda domain: L135Y, S176W, V133T, S176V, S114A and N137K (according to Kabat numbering); and/or (c) one or more of the following mutations in the VL: Q44R, Q44E, Q120C or A120C, Q44D and A49D (according to IMGT numbering); and/or (d) one or more of the following mutations in the VH: Q44E, Q44R, G49C, Q44K and Q120K (according to IMGT numbering).
82. The polypeptide according to any one of claims 26 to 50 or the bispecific antibody according to any one of claims 51 to 81, wherein the bispecific antibody is tetravalent, with dual binding to each of the two antigens.
83. The polypeptide according to any one of claims 26 to 50 and 82 or the bispecific antibody according to any one of claims 51 to 82, wherein the Fab fragment(s) is linked to the C terminal end of the immunoglobulin via a linker.
84. The polypeptide according to claim 83 or the bispecific antibody according to claim 83, wherein the linker is a peptide with the amino acid sequence SGGGGSGGGGS (SEQ ID NO: 5), SGGGGSGGGGSAP (SEQ ID NO: 6), NFSQP (SEQ ID NO: 7), KRTVA (SEQ ID NO: 8), GGGGSGGGGSGGGGS (SEQ ID NO: 9), (SG)m, where m=1 to 7, or GGGGSGGGGS (SEQ ID NO: 34).
85. The polypeptide according to any one of claims 1 to 50 and 82 to 84 or the bispecific antibody according to any one of claims 51 to 84, wherein the first and/or second antigen is an immunomodulator.
86. The polypeptide according to claim 85 or the bispecific antibody according to claim 85, wherein the immunomodulator is a checkpoint molecule.
87. The polypeptide according to claim 86 or the bispecific antibody according to claim 86, wherein the checkpoint molecule is a stimulatory checkpoint molecule, optionally wherein the stimulatory checkpoint molecule is selected from CD40, CD137, GITR, CD27, ICOS and OX40.
88. The polypeptide according to claim 86 or the bispecific antibody according to claim 86, wherein the checkpoint molecule is an inhibitory checkpoint molecule, optionally wherein the inhibitory checkpoint molecule is selected from CTLA-4, PD-1, Tim3, Lag3, Tigit and VISTA.
89. The polypeptide according to any one of claims 1 to 50 and 82 to 88 or the bispecific antibody according to any one of claims 51 to 88, wherein the first and/or second antigen is a tumour cell-associated antigen.
90. The polypeptide according to claim 89 or the bispecific antibody according to claim 89, wherein the tumour cell-associated antigen is selected from the group consisting of: (m) products of mutated oncogenes and tumour suppressor genes; (n) overexpressed or aberrantly expressed cellular proteins; (o) tumour antigens produced by oncogenic viruses; (p) oncofetal antigens; (q) altered cell surface glycolipids and glycoproteins; (r) cell type-specific differentiation antigens; (s) hypoxia-induced antigens; (t) tumour peptides presented by MHC class I; (u) epithelial tumour antigens; (v) haematological tumour-associated antigens; (w) cancer testis antigens; and (x) melanoma antigens.
91. The polypeptide according to claim 90 or the bispecific antibody according to claim 90 wherein the tumour cell-associated antigen is selected from the group consisting of 5T4, CD20, CD19, MUC 1, CA-125, CO17-1A, EpCAM, HER2, EphA2, EphA3, DR5, FAP, OGD2, VEGFR, Her3, mesothelin and EGFR.
92. The polypeptide according to any one of claims 1 to 50 and 82 to 91 or the bispecific antibody according to any one of claims 51 to 91, wherein the first and second antigen are selected from the group consisting of: CD40, EpCAM, 5T4, CD137, OX40, CTLA-4, GITR, EGFR and HER2.
93. The polypeptide according to any one of claims 26 to 50 and 82 to 92 or the bispecific antibody according to any one of claims 51 to 92, wherein the bispecific antibody targets a pair of antigens selected from: OX40 and CTLA-4, OX40 and CD137, GITR and CTLA-4, CD137 and CTLA-4, 5T4 and CD137, and OX40 and 5T4.
94. The polypeptide according to claim 93 or the bispecific antibody according to claim 93, wherein the first and second antigen are selected from CD40 and EpCAM.
95. The polypeptide according to any one of claims 1 to 50 and 82 to 94 or the bispecific antibody according to any one of claims 51 to 94 comprising one or more CDR sequences selected from: TABLE-US-00036 a) CDRH1: (SEQ ID NO: 10) GFTFSSYA; and/or b) CDRH2: (SEQ ID NO: 11) IGSYGGGT; and/or c) CDRH3: (SEQ ID NO: 12) ARYVNFGMDY; and/or d) CDRL1: (SEQ ID NO: 13) QSISSY; and/or e) CDRL2: (SEQ ID NO: 14) AAS; and/or f) CDRL3: (SEQ ID NO: 15) QQYGRNPPT; and/or g) CDRH1: (SEQ ID NO: 18) GYAFTNYW; and/or h) CDRH2: (SEQ ID NO: 19) IFPGSGNI; and/or i) CDRH3: (SEQ ID NO: 20) ARLRNWDEPMDY; and/or j) CDRL1: (SEQ ID NO: 21) QSLLNSGNQKNY; and/or k) CDRL2: (SEQ ID NO: 22) WAS; and/or l) CDRL3: (SEQ ID NO: 23) QNDYSYPLT.
96. The polypeptide according to claim 95 or the bispecific antibody according to claim 95 comprising one or more variable region sequences selected from: TABLE-US-00037 (a) VH of SEQ ID NO: 16 (EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSGIGSYGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CARYVNFGMDYWGQGTLVTVSS); and/or (b) VL of SEQ ID NO: 17 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYGRNP PTFGQGTKLEIK); and/or (c) VH of SEQ ID NO: 24 (EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLE WIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVY FCARLRNWDEPMDYWGQGTTVTVSS); and/or (d) VL of SEQ ID NO: 25 (ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPG QPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQ NDYSYPLTFGAGTKLEIK)
97. The polypeptide according to claim 96 or the bispecific antibody according to claim 96, wherein: (a) the first heavy chain polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 16; and/or (b) the first light chain polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 17; and/or (c) the second heavy chain polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 24; and/or (d) the second light chain polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 25.
98. The polypeptide according to any one of claims 1 to 50 and 82 to 97 or the bispecific antibody according to any one of claims 51 to 97, bispecific antibody according to any one of the preceding claims, wherein the polypeptide or bispecific antibody is capable of inducing antibody dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or apoptosis.
99. The polypeptide according to any one of claims 1 to 50 or the bispecific antibody according to any one of claims 51 to 98, wherein the polypeptide or bispecific antibody is capable of inducing: (a) activation of B cells; and/or (b) activation of dendritic cells; and/or (c) activation of cytotoxic T cells, i.e. CD8+ T cells; and/or (d) activation of helper T cells, i.e. CD4.sup.+ T cells; and/or (e) improved tumour antigen cross-presentation by dendritic cells; and/or (f) expansion of tumour antigen-specific cytotoxic T cells; and/or (g) direct tumour cell killing via ADCC and/or via inhibition of tumour growth and survival signals; and/or (h) anti-angiogenic effects via interaction with endothelial and/or stromal cells; and/or (i) activation of natural killer cells; and/or (j) Treg depletion; and/or (k) reprograming of Tregs into effector T cells; and/or (l) depletion of tumour myeloid cell populations; and/or (m) reprogramming of tumour myeloid cell populations; and/or (n) internalisation of tumour debris by antigen-presenting cells; and/or (o) internalisation of tumour extracellular vesicles, e.g. exosomes, by antigen-presenting cells; and/or (p) localization to tumour tissue by binding to tumour cells.
100. The polypeptide according to any one of claims 1 to 50 and 82 to 99 or the bispecific antibody according to any one of claims 51 to 99, which induces an increase in the activity of an effector T cell, optionally wherein said increase is at least 1.5-fold, 4.5-fold or 7-fold higher than the increase in activity of an effector T cell induced by a combination of the immunoglobulin molecule and Fab fragment administered to the T cell as separate molecules.
101. The polypeptide according to claim 100 or the bispecific antibody according to claim 100, wherein said increase in T cell activity is an increase in proliferation and/or IFNγ or IL-2 production by the T cell.
102. The polypeptide according to any one of claims 1 to 50 and 82 to 101 or the bispecific antibody according to any one of claims 51 to 101, which induces an increase in the activation of an antigen-presenting cell, such as a B cell or dendritic cell.
103. The polypeptide according to claim 102 or the bispecific antibody according to claim 102, wherein said increase in activation is an increase in the expression of the co-stimulatory molecules CD80 or CD86 by the antigen-presenting cell.
104. The polypeptide according to any one of claims 1 to 50 and 82 to 103 or the bispecific antibody according to any one of claims 51 to 103, which induces an increase in the uptake of tumour debris or tumour extracellular vesicles by an antigen-presenting cell, such as a B cell or dendritic cell.
105. The polypeptide according to claim 104 or the bispecific antibody according to claim 104, wherein said increase in uptake is measured by the co-localization or internalization of the tumour debris or tumour extracellular vesicles by the antigen-presenting cell.
106. The polypeptide according to any one of claims 26 to 50 and 82 to 105 or the bispecific antibody according to any one of claims 51 to 105, wherein the bispecific antibody binds to the first and/or the second antigen with a K.sub.D of less than 100×10.sup.−9M or less than 50×10.sup.−9M or less than 25×10.sup.−9M, preferably less than 10, 9, 8, 7, or 6×10.sup.−9M, more preferably less than 5, 4, 3, 2, or 1×10.sup.−9M, most preferably less than 9×10.sup.−10M.
107. The polypeptide according to any one of claims 27 to 50 and 82 to 106 or the bispecific antibody according to any one of claims 51 to 106, wherein one or more amino acids are removed from the N-termini end of the light chain of (b).
108. The polypeptide according to claim 107 or the bispecific antibody according to claim 107, wherein: three amino acids are removed from the N-termini end of the light chain of (b); or six amino acids are removed from the N-termini end of the light chain of (b); or nine amino acids are removed from the N-termini end of the light chain of (b).
109. An isolated nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 50 and 82 to 108 or a bispecific antibody according to any one of claims 51 to 108, or a component polypeptide chain thereof.
110. The nucleic acid molecule according to claim 109 wherein the molecule is a cDNA molecule.
111. The nucleic acid molecule according to claim 109 or 110 encoding an antibody heavy chain or variable region thereof.
112. A vector comprising a nucleic acid molecule according to any one of claims 109 to 111.
113. The vector according to claim 112 wherein the vector is an expression vector.
114. A recombinant host cell comprising a nucleic acid molecule according to any one of claims 109 to 112 or a vector according to claim 112 or 113.
115. The host cell according to claim 114, wherein the host cell is a bacterial cell.
116. The host cell according to claim 114 wherein the host cell is a mammalian cell.
117. The host cell according to claim 114 wherein the host cell is a human cell.
118. A method for producing a polypeptide according to any one of claims 1 to 50 and 82 to 108 or a bispecific antibody according to any one of claims 51 to 108, the method comprising culturing a host cell as defined in any of claims 114 to 117 under conditions which permit expression of the bispecific antibody or component polypeptide chain thereof.
119. A pharmaceutical composition comprising an effective amount of a polypeptide according to any one of claims 1 to 50 and 82 to 108 or a bispecific antibody according to any one of claims 51 to 108 and a pharmaceutically-acceptable diluent, carrier or excipient.
120. The pharmaceutical composition according to claim 119 adapted for parenteral delivery.
121. The pharmaceutical composition according to claim 119 adapted for intravenous delivery.
122. A polypeptide according to any one of claims 1 to 50 and 82 to 108 or a bispecific antibody according to any one of claims 51 to 108 for use in medicine.
123. A polypeptide according to any one of claims 1 to 50 and 82 to 108 or a bispecific antibody according to any one of claims 51 to 108 for use in treating or preventing a neoplastic disorder in a subject.
124. The polypeptide or antibody for use according to claim 123, wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body.
125. The polypeptide or antibody for use according to claim 124, wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer and sarcomas.
126. The polypeptide or antibody for use according to claim 125, wherein the solid tumour is selected from the groups consisting of renal cell carcinoma, colorectal cancer, lung cancer, prostate cancer and breast cancer.
127. The polypeptide or antibody for use according to any one of claims 123 to 126 wherein the antibody is for use in combination with one or more additional therapeutic agents.
128. The polypeptide or antibody for use according to claim 127, wherein the one or more additional therapeutic agents is/are an immunotherapeutic agent that binds a target selected from the group consisting of PD-1/PD-L1, CTLA-4, CD137, CD40, GITR, LAG3, TIM3, CD27, VISTA, OX40 and KIR.
129. Use of a polypeptide according to any one of claims 1 to 50 and 82 to 108 or a bispecific antibody according to any one of claims 51 to 108 in the preparation of a medicament for treating or preventing a neoplastic disorder in a subject.
130. The use according to claim 129 wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body.
131. The use according to claim 130 wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer and sarcomas.
132. The use according to claim 131 wherein the solid tumour is selected from the groups consisting of renal cell carcinoma, colorectal cancer, lung cancer, prostate cancer and breast cancer.
133. The use according to any one of claims 129 to 132 wherein the antibody is for use in combination with one or more additional therapeutic agents.
134. The use according to claim 133 wherein the one or more additional therapeutic agents is/are an immunotherapeutic agent that binds a target selected from the group consisting of PD-1/PD-L1, CTLA-4, CD137, CD40, GITR, LAG3, TIM3, CD27, VISTA, OX40 and KIR.
135. A method for the treatment or diagnosis of a neoplastic disorder in a subject, comprising the step of administering to the subject an effective amount of a polypeptide according to any one of claims 1 to 50 and 82 to 108 or a bispecific antibody according to any one of claims 51 to 108.
136. The method according to claim 135 wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body.
137. The method according to claim 136 wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer and sarcomas.
138. The method according to claim 137 wherein the solid tumour is selected from the groups consisting of renal cell carcinoma, colorectal cancer, lung cancer, prostate cancer and breast cancer.
139. The method according to any one of claims 135 to 138 wherein the subject is human.
140. The method according to any one of claims 135 to 139 wherein the method comprises administering the bispecific antibody systemically.
141. The method according to any one of claims 135 to 140 further comprising administering to the subject one or more additional therapeutic agents.
142. The method according to claim 141 wherein the one or more additional therapeutic agents is/are an immunotherapeutic agent that binds a target selected from the group consisting of PD-1/PD-1L, CTLA-4, CD137, CD40, GITR, LAG3, TIM3, CD27, VISTA, OX40 and KIR.
143. A method of producing a polypeptide according to any one of claims 27 to 50 and 82 to 108 or a bispecific antibody according to any one of claims 51 to 108 comprising expressing three polypeptide chains in the same host cell, wherein the three polypeptide chains are: (d) an immunoglobulin heavy chain (the first heavy chain) fused via a polypeptide linker to a second light chain; (e) a first light chain; and (f) a second heavy chain
144. The method of producing a bispecific antibody according to claim 143 further comprising the step of modifying the ratios of the chains (a), (b) and (c) to optimise formation of a bispecific antibody.
145. A bispecific antibody substantially as described herein with reference to the description and figures.
146. A polynucleotide substantially as described herein with reference to the description and figures.
147. A pharmaceutical composition substantially as described herein with reference to the description and figures.
148. Use of a bispecific antibody substantially as described herein with reference to the description and figures.
149. A method of treatment substantially as described herein with reference to the description and figures.
150. A polypeptide substantially as described herein with reference to the description and figures.
Description
[1653] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:
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EXAMPLES
Example 1—Generation and Manufacturability Evaluation of 20 Variants in IgG-Fab Architecture
[1670] Material and Methods
[1671] Design
[1672] Novel bispecific constructs consisting of an immunoglobulin (IgG) coupled to two Fab fragments connected via a polypeptide linker between the C-terminal end of the IgG and the N-terminal end of the light chain of the Fab fragments (
[1673] Manufacturability
[1674] Bispecific antibodies were expressed using transient HEK293 and Expi293 HEK (Life technologies) cultures at different volumes ranging from 600 μL-2 L according to manufacturer's instructions. Purification of bispecifics from supernatants was made on protein A using the NGC system (BioRad), the ÄKTA Avant system (GE Healthcare) or Predictor MabSelectSure 50 μl 96 well plates (GE Healthcare). Cells were transfected with three different vectors encoding separately for each of the three polypeptides chains (i.e. the immunoglobulin heavy chain linked to the Fab light chain, the immunoglobulin light chain and the Fab heavy chain). Different transfection ratios of the three vectors were tested. Aggregation was measured with SE-HPLC in a 1260 Infinity II system (Agilent Technologies) using a TSK gel Super SW mAB HTP 4 μm, 4.6×150 mm column (TOSOH Bioscience) and 100 mM Sodium Phosphate, pH 6.8, 300 mM NaCl as mobile phase at ambient temperature and a flow rate of 0.35 ml/min.
[1675] Results
[1676] A total of 20 different variants were generated and evaluated. Two different antibody combinations (Combo 1 (CD137-5T4 bispecific) and Combo 2 (CD40-EpCAM bispecific)) were tested. A description of the mutational strategies used, how these differed between the two different FAbs, and which mutations that were tested, are listed in Table 1, 2 and 3.
[1677] The expression and the aggregation levels after transient small-scale production in HEK cells and purification on protein A columns varied dramatically between bsAb variants that differed only in the mutations introduced between the CH1-CKappa and VH-VL interfaces as seen in Table 4. Variant 1 construct carrying no mutations aggregated severely during expression/purification. All mutations introduced improved the purity and quality of the expressed bispecific antibodies lowering the levels of aggregation. However, some mutation combinations such as in variants 6, 9, 10, 13, 17 and 19 generated constructs, where the produced bispecific antibody displayed surprisingly low levels of aggregation. The aggregation could further be reduced by changing the transfection ratio of the three vectors used to transform HEK cells for production of the bispecific antibody. Production of bispecific antibodies with optimized transfection ratios resulted in expression of bispecific constructs with a very high purity, displaying aggregation levels at less than 5%, which suggests that the method described would result in bispecific antibodies of high quality (Table 4).
[1678] The results are particularly surprising as data showed that it was not possible to predict in beforehand which combination of mutations and an in which domain (the IgG or the Fab) that would be most beneficial.
TABLE-US-00020 TABLE 1 Mutational strategies to avoid pairing of the long heavy chain with itself and pairing of L1 with H2 short and promote correct chain pairing in the Fab domain and immunoglobulin. Immunoglobulin Fab CH1 -CK VH-VL CH1-CK VH-VL Variant 1 — — — — Variant 2 Set 1 (steric) — — — Variant 3 Set 1 (steric) Set 5b (salt) — Set 5a (salt) Variant 4 Set 1 (steric) Set 5b (salt) — Set 5a (salt) and Set 6 (SS) Variant 5 Set 2 (hydrophob) — Set 3 (salt) Variant 6 Set 2 (hydrophob) Set 5b (salt) Set 3 (salt) Set 5a (salt) Variant 7 Set 2 (hydrophob) Set 5b (salt) Set 3 (salt) Set 5a (salt) and Set 6 (SS) Variant 8 — — Set 4 (SS) — Variant 9 Set 1 (steric) Set 5b (salt) Set 3 (salt) Set 5a (salt) Variant 9′ Set 1 (steric) Set 5b (salt) Set 3′ (charge-Alanine) Set 5a (salt) Variant 10 Set 1 (steric) Set 5b (salt) Set 3 (salt) Set 5a (salt) and Set 6 (SS) Variant 11 Set 3 (salt) Set 5b (salt) Set 1 (steric) Set 5a (salt) Variant 12 Set 3 (salt) Set 5b (salt) Set 1 (steric) Set 5a (salt) and Set 6 (SS) Variant 13 — Set 7 (2 × salt) Set 1 (steric) Set 5a (salt) Variant 14 — Set 7 (2 × salt) Set 1 (steric) Set 5a (salt) and Set 6 (SS) Variant 15 Set 3 (salt) Set 7 (2 × salt) Set 1 (steric) Set 5a (salt) Variant 16 Set 3 (salt) Set 7 (2 × salt) Set 1 (steric) Set 5a (salt) and Set 6 (SS) Variant 17 Set 2 (hydrophob) Set 5b (salt) Set 1 (steric) and Set 3 (salt) Set 5a (salt) Variant 18 Set 2 (hydrophob) Set 5b (salt) Set 1 (steric) and Set 3 (salt) Set 5a (salt) and Set 6 (SS) Variant 19 Set 1 (steric) and Set 3 (salt) Set 5b (salt) Set 2 (hydrophob) Set 5a (salt) Variant 20 Set 1 (steric) and Set 3 (salt) Set 5b (salt) Set 2 (hydrophob) Set 5a (salt) and Set 6 (SS)
TABLE-US-00021 TABLE 2 Mutations introduced between CH1 and CKappa interface and VL and VH interface. Domain Mutation* Set 1 CH1 H168A, F170G CKappa L135Y, S176W Set 2 CH1 L145Q, S183V CKappa V133T, S176V Set 3 CH1 T187E CKappa S114A, N137K Set 3′ CH1 T187E CKappa S114A Set 4 CH1 F126C CKappa S121C Set 5a VL Q44R VH Q44E Set 5b VL Q44E VH Q44R Set 6 VL Q120C or A120C VH G49C Set 7 VL Q44D, A49D VH Q44K, Q120K *Mutation in CH1 and CKappa interface are in the EU numbering system, Mutations in VL (variable light) and VH (variable heavy) are in IMGT numbering system.
TABLE-US-00022 TABLE 3a Mutations‡ included in the different variants generated. H1 long L1 H2 short Variant 1 VH.sub.A - CH1.sub.A - FC - linker - VL.sub.B - CKappa1.sub.B VL.sub.A - CKappa.sub.A VH.sub.B - CH1.sub.B Variant 2 VH.sub.A - CH1.sub.A (H172A, F174G) - FC - linker - VL.sub.B - CKappa1.sub.B VL.sub.A - CKappa.sub.A (L135Y, S176W) VH.sub.B - CH1.sub.B Variant 3 VH.sub.A (Q44R) - CH1.sub.A (H172A, F174G) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W) Variant 4 VH.sub.A (Q44R) - CH1.sub.A (H172A, F174G) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E, Q120C) - CH1.sub.B VL.sub.B (Q44R, G49C) - CKappa1.sub.B (L135Y, S176W) Variant 5 VH.sub.A - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A - CKappa.sub.A VH.sub.B - CH1.sub.B VL.sub.B - CKappa1.sub.B (S114A, N137K) (V133T, S176V) (T187E) Variant 6 VH.sub.A (Q44R) - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (S114A, N137K) (V133T, S176V) (T187E) Variant 7 VH.sub.A (Q44R) - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E, Q120C) - CH1.sub.B VL.sub.B (Q44R, G49C) - CKappa1.sub.B (S114A, N137K) (V133T, S176V) (T187E) Variant 8 VH.sub.A - CH1.sub.A - FC - linker - VL.sub.B - CKappa1.sub.B (S121C) VL.sub.A - CKappa.sub.A VH.sub.B - CH1.sub.B (F126C) Variant 9 VH.sub.A (Q44R) - CH1.sub.A (H172A, F174G) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (S114A, N137K) (L135Y, S176W) (T187E) Variant 9′ VH.sub.A (Q44R) - CH1.sub.A (H168A, F170G) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (S114A) (L135Y, S176W) (T187E) Variant 10 VH.sub.A (Q44R) - CH1.sub.A (H172A, F174G) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C) - CKappa1.sub.B (S114A, N137K) (L135Y, S176W) (T187E) Variant 11 VH.sub.A (Q44R) - CH1.sub.A (T187E) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W) (S114A, N137K) (H172A, F174G) Variant 12 VH.sub.A (Q44R) - CH1.sub.A (T187E) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C) - CKAPPA1.sub.B (L135Y, S176W) (S114A, N137K) (H172A, F174G) Variant 13 VH.sub.A (Q44K, Q120K) - CH1.sub.A- FC - linker - VL.sub.A (Q44D, A49D) - CKappa.sub.A VH.sub.B (Q44E)- CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W) (H172A, F174G) Variant 14 VH.sub.A (Q44K, Q120K) - CH1.sub.A- FC - linker - VL.sub.A (Q44D, A49D) - CKappa.sub.A VH.sub.B (Q44E, G49C)- CH 1.sub.B VL.sub.B (Q44R, Q120C) - CKappa1.sub.B (L135Y, S176W) (H172A, F174G) Variant 15 VH.sub.A (Q44K, Q120K) - CH1.sub.A (T187E) - FC - linker - VL.sub.A (Q44D, A49D) - CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W) (S114A, N137K) (H172A, F174G) Variant 16 VH.sub.A (Q44K, Q120K) - CH1.sub.A (T187E) - FC - linker - VL.sub.A (Q44D, A49D) - CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C) - CKappa1.sub.B (L135Y, S176W) (S114A, N137K) (H172A, F174G) Variant 17 VH.sub.A (Q44R) - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W, S114A, N137K) (V133T, S176V) (H172A, F174G, T187E) Variant 18 VH.sub.A (Q44R) - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C) - CKappa1.sub.B (V133T, S176V) (H172A, F174G, T187E) (L135Y, S176W, S114A, N137K) Variant 19 VH.sub.A (Q44R) - CH1.sub.A (H172A, F174G, T187E) - FC - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B linker - VL.sub.B (Q44R) - CKappa1.sub.B (V133T, S176V) (L135Y, S176W, S114A, N137K) (L145Q, S183V) Variant 20 VH.sub.A (Q44R) - CH1.sub.A (H172A, F174G, T187E) - FC - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B linker - VL.sub.B (Q44R, Q120C) - CKappa1.sub.B (V133T, S176V) (L135Y, S176W, S114A, N137K) (L145Q, S183V) ‡Mutation in CH1 and CKappa interface are in the EU numbering system, except for mutations H172A and F174G that are in the Kabat numbering system. Mutations in VL (variable light) and VH (variable heavy) are in IMGT numbering system.
TABLE-US-00023 TABLE 3b Mutations‡ included in the different variants generated. H1 long L1 H2 short Variant 1 VH.sub.A - CH1.sub.A - FC - linker - VL.sub.B - CKappa1.sub.B VL.sub.A - CKappa.sub.A VH.sub.B - CH1.sub.B Variant 2 VH.sub.A - CH1.sub.A (H168A, F170G) - FC - linker - VL.sub.B - CKappa1.sub.B VL.sub.A - CKappa.sub.A (L135Y, S176W) VH.sub.B - CH1.sub.B Variant 3 VH.sub.A (Q44R) - CH1.sub.A (H168A, F170G) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W) Variant 4 VH.sub.A (Q44R) - CH1.sub.A (H168A, F170G) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E, Q120C) - CH1.sub.B VL.sub.B (Q44R, G49C) - CKappa1.sub.B (L135Y, S176W) Variant 5 VH.sub.A - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A - CKappa.sub.A VH.sub.B - CH1.sub.B VL.sub.B - CKappa1.sub.B (S114A, N137K) (V133T, S176V) (T187E) Variant 6 VH.sub.A (Q44R) - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (S114A, N137K) (V133T, S176V) (T187E) Variant 7 VH.sub.A (Q44R) - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E, Q120C) - CH1.sub.B VL.sub.B (Q44R, G49C) - CKappa1.sub.B (S114A, N137K) (V133T, S176V) (T187E) Variant 8 VH.sub.A - CH1.sub.A - FC - linker - VL.sub.B - CKappa1.sub.B (S121C) VL.sub.A - CKappa.sub.A VH.sub.B - CH1.sub.B (F126C) Variant 9 VH.sub.A (Q44R) - CH1.sub.A (H168A, F170G) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R)-CKappa1.sub.B (S114A, N137K) (L135Y, S176W) (T187E) Variant 9′ VH.sub.A (Q44R) - CH1.sub.A (H168A, F170G) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (S114A) (L135Y, S176W) (T187E) Variant 10 VH.sub.A (Q44R) - CH1.sub.A (H168A, F170G) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C) - CKappa1.sub.B (S114A, N137K) (L135Y, S176W) (T187E) Variant 11 VH.sub.A (Q44R) - CH1.sub.A (T187E) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W) (S114A, N137K) (H168A, F170G) Variant 12 VH.sub.A (Q44R) - CH1.sub.A (T187E) - FC - linker - VL.sub.A (Q44E) - CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C) - CKappa1.sub.B (L135Y, S176W) (S114A, N137K) (H168A, F170G) Variant 13 VH.sub.A (Q44K, Q120K) - CH1.sub.A- FC - linker - VL.sub.A (Q44D, A49D) - CKappa.sub.A VH.sub.B (Q44E)- CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W) (H168A, F170G) Variant 14 VH.sub.A (Q44K, Q120K) - CH1.sub.A- FC - linker - VL.sub.A (Q44D, A49D) - CKappa.sub.A VH.sub.B (Q44E, G49C)- CH 1.sub.B VL.sub.B (Q44R, Q120C) - CKappa1.sub.B (L135Y, S176W) (H168A, F170G) Variant 15 VH.sub.A (Q44K, Q120K) - CH1.sub.A (T187E) - FC - linker - VL.sub.A (Q44D, A49D) - CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W) (S114A, N137K) (H168A, F170G) Variant 16 VH.sub.A (Q44K, Q120K) - CH1.sub.A (T187E) - FC - linker - VL.sub.A (Q44D, A49D) - CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C or A120C - CKappa1.sub.B (L135Y, S176W) (S114A, N137K) (H168A, F170G) Variant 17 VH.sub.A (Q44R) - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (L135Y, S176W, S114A, N137K) (V133T, S176V) (H168A, F170G, T187E) Variant 18 VH.sub.A (Q44R) - CH1.sub.A (L145Q, S183V) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C or A120C) - CKappa1.sub.B (V133T, S176V) (H168A, F170G, T187E) (L135Y, S176W, S114A, N137K) Variant 19 VH.sub.A (Q44R) - CH1.sub.A (H168A, F170G, T187E) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E) - CH1.sub.B VL.sub.B (Q44R) - CKappa1.sub.B (V133T, S176V) (L135Y, S176W, S114A, N137K) (L145Q, S183V) Variant 20 VH.sub.A (Q44R) - CH1.sub.A (H168A, F170G, T187E) - FC - linker - VL.sub.A (Q44E)- CKappa.sub.A VH.sub.B (Q44E, G49C) - CH1.sub.B VL.sub.B (Q44R, Q120C or A120C) - CKappa1.sub.B (V133T, S176V) (L135Y, S176W, S114A, N137K) (L145Q, S183V) ‡Mutation in CH1 and CKappa interface are in the EU numbering system, Mutations in VL (variable light) and VH (variable heavy) are in IMGT numbering system.
TABLE-US-00024 TABLE 4 Aggregation after expression and protein A purification as measured with SE-HPLC % aggregates, chains % aggregates, chain transfected at ratios transfection ratio Variant 1:1:1 optimized Combo 1 v1 (no mutations) 29.8 Combo 1 v2 10.4 Combo 1 v3 5.5 1.8 Combo 1 v4 5.6 1.8 Combo 1 v5 13.5 Combo 1 v6 6.1 1.6 Combo 1 v7 7 Combo 1 v8 15.5 Combo 1 v9′ 6.2 2 Combo 1 v10 6.8 1.8 Combo 1 v11 12.6 Combo 1 v12 12.5 Combo 1 v13 13.6 Combo 1 v14 10.2 Combo 1 v15 15.2 Combo 1 v16 13.6 Combo 1 v17 6.5 1.8 Combo 1 v18 6.8 2.6 Combo 1 v19 5.9 2.3 Combo 1 v20 9.9 Combo 2 v1 (no mutations) 48.7 Combo 2 v2 35.4 Combo 2 v3 11.1 Combo 2 v4 7.9 Combo 2 v5 38.9 Combo 2 v6 16.4 Combo 2 v7 11 Combo 2 v8 28.8 Combo 2 v9 8.6 1.8 Combo 2 v10 7 2.1 Combo 2 v11 25.3 Combo 2 v12 23.5 Combo 2 v13 13.2 3.5 Combo 2 v14 16.3 Combo 2 v15 21.8 Combo 2 v16 19.8 Combo 2 v17 11.7 1.9 Combo 2 v18 7.3 Combo 2 v19 8.7 2.5 Combo 2 v20 11.8
Example 2—Stability and Solubility
[1679] Material and Methods
[1680] Thermostability
[1681] The melting temperatures were measured with the UNCle system (UNchained labs). The intrinsic fluorescence was measured during linear temperature ramping from 20° C. to 95° C. at a rate of 0.4° C./minute. The data analysis was performed with the UNcle Analysis software version 2.0 using default settings.
[1682] Stability
[1683] Samples in non-optimized buffer (PBS) at low (<1 mg/mL) or high (10 mg/mL) protein concentration were incubated at 2-8° C., room temperature and 40° C. for 1, 2 and 4 weeks or 1 and 2 weeks or subjected to 3 rounds of freeze thawing. Protein degradation was measured with SE-HPLC, SDS-PAGE, A280, dual ELISA and by visual inspection.
[1684] Shear Stress Stability
[1685] Samples in duplicates were subjected to shear stress in the form of heavy agitation at 2000 rpm on the 96-well plate shaker MixMate (Eppendorf) for at least 30 minutes. Protein precipitates were removed by centrifugation at 3000 g for 10 minutes and the absorbance at 280 nm was measured using a Clariostar.
[1686] Colloidal Stability
[1687] Samples were mixed with PBS/PEG solutions with different PEG concentrations ranging from 8%-36%, added to 96-well filter plates in triplicates and incubated over night at room temperature. Filtrates obtained by centrifugation at 12000 g for 15 minutes were collected and spun down before the absorbance at 280 nm was measured (to determine loss of protein) and compared to controls.
[1688] Low pH Stability
[1689] Stability at low pH was analysed, in conjunction with protein A purification, in two ways, by exposing bsAb samples to low pH (3.5) during 30, 60, 90 or 120 minutes, either during elution from protein A or after elution and an intermediate neutralization.
[1690] Serum Stability
[1691] The stability in serum was tested by incubating samples in 50% serum/50% PBS or PBS/0.1% bovine serum albumin at 37° C. for 2 hours, 1, 2 and 7 days before analysing binding in dual ELISA.
[1692] Solubility
[1693] The solubility in PBS or 20 mM Histidine/150 mM Arginine/pH 6.0 at 10 mg/mL was measured by concentrating the samples in ultrafiltration centrifugal units (Vivaspin 6, 10 kDa MWCO, GE Healthcare). Degradation was analysed with HPLC, SDS-PAGE, A280 and by visual inspection.
[1694] Results
[1695] All variants displayed excellent stability. Incubation at elevated temperatures in a non-optimized buffer (PBS) resulted only in minor degradation, <3% increase in aggregation and <3% increase in fragmentation as measured by SE-HPLC (Table 5), SDS-PAGE, dual ELISA, A280 and visual inspection, indicating a good storage stability. In addition, freeze/thawing treatments of 1 or 3 rounds showed minimal degradation as measured by SE-HPLC, indicating again that the stability of the construct is high. Shear stress stability studies showed low loss of protein (<20%) after severe agitation for all of the tested constructs (Table 5) indicating that the bsAb are not sensitive to shear stress. Evaluation of the colloidal stability showed to reach 50% loss of the protein concentrations >9% of PEG were required (
[1696] The melting temperatures measured with UNcle were found to be >64° C. for Combo 2 constructs (CD40-EpCAM bispecific) and >69° C. for Combo 1 constructs (CD137-5T4 bispecific) (Table 6) indicating that the thermostability of these constructs is high.
[1697] Samples were also concentrated to 10 mg/mL in PBS or Histidine buffer. No degradation, either due to change in buffer or to increase in concentration was observed, as measured with SE-HPLC, SDS-PAGE, A280 and by visual inspection, indicating that the constructs have a solubility of at least around 10 mg/mL in both tested buffers.
[1698] Binding to both targets remained unaltered even after incubation of constructs at 37° C. in human serum compared to samples incubated in PBS with BSA as measured in dual ELISA (
TABLE-US-00025 TABLE 5 Changes in degradation after different treatments to evaluate stability. Protein loss % 25 C., 4 weeks 40 C., 4 weeks Freeze/thaw, 1 round Freeze/thaw, 3 rounds after shear bsAb Δ% HMWs Δ% LMWs Δ% HMWs Δ% LMWs Δ% HMWs Δ% LMWs Δ% HMWs Δ% LMWs stress treatment Combo 1 V3 0 −1 1 2 1 −1 0 −1 2 Combo 1 V4 1 −1 2 1 1 0 1 −1 7 Combo 1 V6 0 0 0 2 0 0 0 0 10 Combo 1 V7 0 0 1 2 1 0 1 −1 4 Combo 1 V9′ 0 0 0 2 0 0 0 0 6 Combo 1 V10 1 1 1 3 0 0 1 0 7 Combo 1 V13* 0 0 −1 1 0 0 1 0 10 Combo 1 V17 0 0 0 2 1 0 1 0 9 Combo 1 V18 1 1 3 3 0 0 0 0 11 Combo 2 V3 0 0 −1 2 0 0 0 0 9 Combo 2 V4 −1 0 −1 2 −1 0 −1 0 4 Combo 2 V6 0 0 −1 2 −1 0 0 0 4 Combo 2 V7 0 0 −1 2 −1 0 0 0 0 Combo 2 V9 0 0 0 1 0 0 0 0 5 Combo 2 V10 0 0 0 1 0 0 0 0 −1 Combo 2 V13* 0 1 0 1 1 0 2 0 6 Combo 2 V17 0 0 0 1 1 0 1 0 3 Combo 2 V18 0 0 0 1 0 0 0 0 5 Combo 3 V13* 0 0 1 −2 0 0 0 0 7 Positive shear 86 stress assay control *Stability for the constructs was assessed after incubation at 25 and 40° C. for only 2 weeks.
TABLE-US-00026 TABLE 6 Melting temperatures determined with Uncle. bsAb Tm (° C.) Combo 1 v2 70 Combo 1 v3 69 Combo 1 v4 69 Combo 1 v5 72 Combo 1 v6 70 Combo 1 v7 70 Combo 1 v8 69 Combo 1 v9′ 69 Combo 1 v10 67 Combo 1 v13 68 Combo 1 v17 70 Combo 1 v18 70 Combo 2 v3 66 Combo 2 v4 68 Combo 2 v6 66 Combo 2 v7 68 Combo 2 v9 66 Combo 2 v10 68 Combo 2 v13 65 Combo 2 v17 64 Combo 2 v18 66
Example 3—Binding Studies
[1699] Material and Methods
[1700] Dual ELISA
[1701] Plates were coated with 0.5 μg/mL antigen, Ag1 or Ag2, in PBS over night at 4° C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again. Samples serially diluted in PBS/0.5% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with 0.5 μg/mL biotinylated Ag1 or Ag2, whichever of the antigens that was not used for coating, for at least 1 hour at room temperature. Dual complexed bsAb with Ag1 and Ag2 were detected with HRP-labelled streptavidin. SuperSignal Pico Luminescent was used as substrate and luminescence signals were measured using Fluostar Optima.
[1702] Octet
[1703] Kinetic measurements were performed using the Octet RED96 platform (ForteBIo). Antigens, either biotinylated or Fc labelled, were coupled to either Streptavidin or Amine reactive Second generation sensors (ForteBio) at antigen concentrations of 0.4, 1.5, 0.25 or 0.5 μg/mL. Tested bsAb and mAbs (serially diluted ½ in 1× Kinetic buffer (ForteBio) with start concentrations of 20, 15, 10 or 25 nM) were analysed for binding to antigen-coupled sensors. The association was followed for 300, 1000 or 600 seconds and the dissociation in 1× Kinetic buffer for 1000, 2000, 600 or 300 seconds. Sensor tips were regenerated using 10 mM glycine, pH 2.2. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14). The processed data were fitted using a 1:1 Langmuir binding model.
[1704] Results
[1705] Binding studies showed good binding to both targets for each combination as analysed by Octet and dual ELISA. Octet measurements showed similar KD values for the bsAb constructs in the new format compared to monoclonal or bispecific antibody controls (Table 7). In dual ELISA assays where simultaneous binding to each target combination was evaluated it was shown that indeed all novel bsAb constructs regardless of variant displayed dual binding. This was observed for bsAb constructs against different target combination as shown in representative ELISAs in
TABLE-US-00027 TABLE 7 Binding kinetics of bsAb and mAb constructs. KD kon kdis Construct Target (nM) (1/Ms) (1/s) Combo 1 v6 Combo 1 Ag1 0.02 4E+6 6E−5 Combo 1 v9′ Combo 1 Ag1 0.02 3E+6 6E−5 mAb Combo 1 Ag1 Combo 1 Ag1 0.03 4E+6 1E−4 bsAb control Combo 1 Combo 1 Ag1 0.03 3E+6 7E−5 Combo 1 V6 Combo 1 Ag2 0.2 1E+5 3E−5 Combo 1 V9′ Combo 1 Ag2 1.0 7E+4 7E−5 mAb Combo 1 Ag 2 Combo 1 Ag2 0.2 5E+5 8E−5 Combo 2 V9 Combo 2 Ag1 1 1E+6 1E−3 Combo 2 V10 Combo 2 Ag1 1 1E+6 1E−3 Combo 2 V19 Combo 2 Ag1 2 7E+5 1E−3 mAb Combo 2 Ag1 Combo 2 Ag1 1 1E+6 1E−3 bsAB control Combo 2 Combo 2 Ag1 1 9E+5 7E−4 Combo 2 V9 Combo 2 Ag2 8 1E+5 1E−3 Combo 2 V10 Combo 2 Ag2 9 2E+5 2E−3
Example 4—Functional Assay T Cell Activation
[1706] Material and Methods
[1707] T Cell Activation Assay
[1708] The functional activity of the Combo 1 bsAbs (CD137-5T4 bispecific) was evaluated in a CD8+ T cell assay, where cells were cultured in microtiter plates coated with Combo 1 Ag2-Fc and CD3 antibody. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll-Paque (p 1.077 g/ml) (GE Healthcare #17-1440-02) from leucocyte concentrates obtained from healthy donors (Clinical Immunology and Transfusion Medicine, Labmedicin Region Skåne, Lund Sweden). CD8+ T cells were enriched by negative selection using the CD8+ T cell isolation kit (Miltenyi 130-096-495). Plates were coated overnight at 4° C. with 3 μg/ml αCD3, clone OKT3 (Affymetrix eBioscience #16-0037-85), washed and coated with 5 μg/ml 5T4-Fc for 2 h at 37° C. After the Ag2-Fc coating, plates were washed and blocked for a minimum of 30 minutes with RPMI (Gibco #61870010) containing 10% FCS (Heat inactivated, Gibco #10270-106 lot 41Q9248K) and 10 mM HEPES (Gibco #15630056). Combo 1 bsAbs (CD137-5T4 bispecific) bsAbs were diluted in RPMI containing 10% FCS and 10 mM HEPES and added to the plates 30 minutes before addition of CD8+ T cells (0.07×10.sup.6 cells/well). Assay plates were incubated for 68 h at 37° C., and culture supernatant harvested. IFN-γ levels in the supernatants were measured by ELISA (ED OptiEIA #555142).
[1709] Results
[1710] Constructs were shown to have good T cell activation function. The functional activity of Combo 1 constructs (CD137-5T4 bispecific antibodies) was determined using human CD8+ T cells cultured in 5T4-Fc coated plates and was based on sets of experiments with a total of 4 donors for each set. CD8+ T cell activation of the bispecific antibodies was assessed both in the presence and absence of 5T4-Fc, to verify a 5T4 crosslinking dependency of CD137 activation. As shown in
Example 5—B Cell Activation Function
[1711] Material and Methods
[1712] B Cell Activation Assay
[1713] Transfected CHO cells expressing Combo 2 (CD40-EpCAM bispecific) Ag2 and CHO cells transfected with an empty vector were UV irradiated, seeded in 96-well flat-bottom tissue culture-treated plates and incubated at 37° C. overnight. PBMCs were isolated from healthy donors as described above. B cells were enriched using human B cell isolation kit II (Miltenyi Biotec) and cultured with the irradiated CHO-Ag2 or CHO-ctrl cells and Combo 2 bsAbs in RPMI 1640 (Gibco) supplemented with 10% FCS, 10 mM HEPES (Gibco) and 10 ng/ml IL-4 (Gibco). After 48h, cells were harvested and stained with CD19-PE-Cy7, CD86-APC and Fixable Viability Stain 450 (all ED Biosciences). Expression of CD86 on CD19+ B cells was analyzed by flow cytometry.
[1714] Results
[1715] As shown in
Example 6—Dendritic Cell Activation Function
[1716] Material and Methods
[1717] DC Activation Assay
[1718] PBMCs were isolated as described above. Monocytes were enriched by positive selection using CD14 microbeads (Miltenyi Biotec) and cultured in the presence of GM-CSF (150 ng/ml, Gibco) and IL-4 (50 ng/ml, Gibco) for 7 days to generate monocyte-derived DCs. Transfected CHO cells expressing Combo 2 (CD40-EpCAM bispecific) Ag2 and CHO cells transfected with an empty vector were UV irradiated, seeded in 96-well flat-bottom tissue culture-treated plates and incubated at 37° C. overnight. DCs were cocultured with CHO-Ag2 or CHO-ctrl cells and Combo 2 bsAbs in medium supplemented with GM-CSF and IL-4 for 48h, followed by flow cytometry analysis of CD86 and HLA-DR expression on DCs.
[1719] Results
[1720] Both Combo 2 constructs (CD40-EpCAM bispecific) variant 9 and variant 10 (v9 and v10 respectively) induced dose-dependent DC activation in the presence but not absence of Combo 2 Ag2 (EpCAM), measured as % of CD86+ HLA-DR+ cells among CD1a+ CD14-DCs (
Example 7—Internalization Function
[1721] Material and Methods
[1722] A tumor cell line expressing Combo 2 (CD40-EpCAM bispecific) Ag2 was stained with the fluorescent membrane dye PKH26 (Sigma-Aldrich) followed by heat shock at 45° C. for 10 min to induce cell death. Heat-shocked tumor cells were incubated at 37° C. overnight, spun down and supernatant containing tumor cell debris was collected. Raji cells were labelled with the nuclear stain Hoechst 33342 (0.045 μg/ml, Thermo Fisher) and seeded in 96-well flat-bottom plates (Costar). Tumor cell debris and Combo 2 bsAbs or control mAb were added, and cells were imaged using Cytation5 (BioTek) every hour. Gen5 software was used to analyse the number of colocalized tumor debris and Raji cells.
[1723] Results
[1724] Combo 2 constructs (CD40-EpCAM bispecific) were shown to induce internalization of tumour cell debris. A CD40-expressing cell line was incubated with fluorescently cell debris from a necrotic tumour cell line expressing Combo 2 Ag2. Different Combo 2 bsAbs or a control mAb were added, and the localization of tumour cell debris in CD40+ cells was analysed using a live cell imaging system. All bispecific constructs enhanced internalization of tumour debris in a dose-dependent manner (
Example 8—Fc Gamma Receptor Interaction Measured by Octet
[1725] Material and Methods
[1726] Binding towards soluble human and mouse Fcγ receptors (FcγR) of either novel IgG-Fab bsAb constructs with different Fc isotype variants or monoclonal isotype controls were evaluated using the Octet RED96 platform (ForteBIo). Samples consisting of variant 9 IgG-Fab bsAb constructs carrying IgG1, IgG1 LALA, IgG2 or IgG4 S228P Fc domains were diluted in 200 nM 1× Kinetic buffer and captured on 8 parallel sensors for 300 seconds. After setting a new baseline, the captured antibodies were assayed against any of the FcγR (hFcγRI (1257-FC-050, R&D Systems), hFcγRIIa (1330-CD-050, R&D Systems), hFcγRIIb (1875-CD-050, R&D Systems), hFcγRIIIa (4325-FC-050, R&D Systems), hFcγRIIIa V176F (8894-FC-050, R&D Systems), mFcγRI (2074-FC-050, R&D Systems), mFcγRIIb (1460-CD-050, R&D Systems), mFcγRIII (1960-FC-050, R&D Systems) mFcγRIV (50036-M27H-50, Sino Biologicals) for 60 seconds followed by dissociation for 60 seconds in Kinetic buffer. The FcgRs were ran in seven 1:2 dilutions starting at 100 nM. Sensor regeneration using 10 mM Glycine pH 1.7 was performed before capturing of the next bsAb/mAb. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14). The processed data were fitted using a 1:1 Langmuir binding model.
[1727] Results
[1728] Octet measurement show similar Fcγ receptor interaction for novel bsAb constructs compared to mAb isotype controls regardless of Fc isotype as demonstrated by similar measured affinities as shown in
TABLE-US-00028 TABLE 8 Octet measurements of Fcgamma receptor interaction with novel constructs compared to monoclonal antibodies hFcgRI hFcgRIIa hFcγRIIIa 176V Construct K.sub.D (M) k.sub.on (1/Ms) k.sub.dis (1/s) K.sub.D (M) k.sub.on (1/Ms) k.sub.dis (1/s) K.sub.D (M) k.sub.on (1/Ms) k.sub.dis (1/s) IgG1 bsAb <1E−12 5E+05 <1E−07 2E−07 2E+06 4E−01 3E−08 1E+06 3E−02 IgG1 mAb <1E−12 3E+05 <1E−07 3E−07 1E+06 4E−01 5E−08 6E+05 3E−02 IgG1 LALA bsAb 4E−08 7E+05 3E−02 <LOD <LOD <LOD <LOD <LOD <LOD IgG1 LALA mAb 5E−08 5E+05 2E−02 <LOD <LOD <LOD <LOD <LOD <LOD IgG4 bsAb 3E−09 8E+05 2E−03 <LOD <LOD <LOD <LOD <LOD <LOD IgG4 mAb 2E−09 7E+05 1E−03 <LOD <LOD <LOD <LOD <LOD <LOD IgG2 bsAb <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD IgG2 mAb <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD
Example 9—Interaction to Fc Gamma Receptors Expressed on Cells Measured by FACS
[1729] Material and Methods
[1730] CHO K1 cells (ATCC) transfected with FcγRI, FcγRIIa (R131), FcγRIIa (H131) and FcγRIIb were used, as a pool or single cell sorted, for the analysis of binding of novel bispecific antibody constructs with different Fc isotypes to the above mentioned Fcγ receptors using flow cytometry. Cells were incubated with variant 9 IgG-Fab bsAb constructs carrying IgG1, IgG1 LALA or IgG2 Fc domains or a monoclonal isotype controls (at 50, 10, 2 or 0.4 μg/mL), followed by secondary staining with PE-conjugated goat anti-human IgG (F(ab′)2) antibody (Jackson ImmunoResearch Inc. PA, US). Cells were analyzed using a FACSVerse™ (BD Biosciences, US), and data was analyzed using the FlowJo software (BD Bioscience).
[1731] Results
[1732] Results from repeated studies show that novel bsAb constructs of different isotypes bind to Fc gamma receptors expressed on cells in a similar manner as is observed for monoclonal antibodies. Experiments with hFcγRI expressing cells shows dose dependent response with IgG1 RUBYs and IgG1 mAb. Neither RUBYs with IgG1 LALA or IgG2 nor monoclonal antibodies with these isotypes appear to interact with these cells as expected and shown in
Example 10—IgG Like bsAb
[1733] Material and Methods
[1734] Design
[1735] To evaluate if the combination of mutations found to be beneficial for the generation of functional and developable IgG-Fab bispecific antibody constructs could also be used to promote correct chain pairing in other bispecific antibody architectures, constructs containing the combination of mutations in variants 9 (or 9′), 13, and 19 were generated in an IgG like architecture. These were compared to variant 1 IgG like bsAbs. Using Knob-in-Hole mutations, IgG like bsAbs of variants 1, 9′, 13, and 19 were generated for Combo 1 constructs (CD137-5T4 bispecific) and variants 1, 9, 13, and 19 for Combo 2 constructs (CD40-EpCAM bispecific).
[1736] Production of IgG like bsAbs. Bispecific antibodies were expressed using transient Expi293 HEK (Life technologies) cultures at 30 mL according to manufacturer's instructions. Purification of bispecifics from supernatants was made on protein A using the NGC system (BioRad). Cells were transfected with four different vectors encoding separately for each of the four polypeptides chains (i.e. the immunoglobulin heavy chain of a first antibody carrying also the Knob mutations, the immunoglobulin heavy chain of a second antibody, the immunoglobulin light chain the first antibody and the immunoglobulin light chain of the second antibody). The same transfection ratio (non-optimized) was used for all constructs.
[1737] ELISA
[1738] IgG like bsAb were analyzed in dual ELISA to investigate binding differences that can indicate differences in amount of light chain mispairing. Plates were coated with 0.5 μg/mL antigen Ag1 in PBS over night at 4° C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again. Samples serially diluted in PBS/0.5% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with 0.5 μg/mL biotinylated Ag2 for at least 1 hour at room temperature. Dual complexed bsAb with Ag1 and Ag2 were detected with HRP-labelled streptavidin. SuperSignal Pico Luminescent was used as substrate and luminescence signals were measured using Fluostar Optima.
[1739] Generation of Fab Fragments
[1740] To be able to analyse amount of light chain mispairing with ESI-LC-MS purified Fab fragments from IgG like bsAbs were generated using the GingisKHAN Fab kit (Genovis, B0-GFK-020) under manufacturer's recommended conditions. The kit mainly involves two steps, GingisKHAN digestion of IgG and purification of the fragments on an IgG-CH1 specific affinity spin column. Briefly, 2000 U GingisKHAN was reconstituted in 200 μL ddH.sub.2O, and the 10× reducing agent (supplied by the vendor) was freshly prepared in 50 μL ddH2O before each digestion. 150 μg antibody in 100 mM Tris, pH 8.0 were digested with the reconstituted GingisKHAN and freshly prepared 10× reducing agent at 37° C. for 1 hour. The digests were analyzed by SE-HPLC and LC-ESI-MS.
[1741] Molecular Weight Determination with LC-ESI-MS
[1742] Determination of the Molecular Weight (MW) of intact proteins was performed by Liquid Chromatography coupled to Electrospray Ionization Mass Spectrometry (LC-ESI-MS). LC-measurement were made with an Agilent 1200 system (Agilent, Santa Clara, Calif.). A; Poroshell 300SB-C8 2.1×75 mm, 5 μm column (Agilent) was used for chromatographic separation. Mobile phases A and B were 0.1% TFA in water and acetonitrile, respectively. Mass spectra were collected with a maXis Impact mass spectrometer (Bruker Daltonics, Germany) at a mass-to-charge ratio range of 500-4500. Molecular mass was determined by deconvolution of the mass spectra data using the MaxEnt I software package. The determined MW was compared to the theoretical MW calculated from the protein amino acid sequence.
[1743] Results
[1744] Dual ELISA demonstrates higher binding for constructs carrying combination of mutations compare to constructs without mutations to promote correct light chain pairing. As shown in
[1745] Further, mass spectrometry analysis was used to detect the amount of correctly paired light chains. Samples of Fab fragments generated by enzymatic cleavage from IgG-like bsAb constructs and purified on CH1 columns were tested on LC-ESI-MS. Determined masses were compared with theoretical molecular weight values. Results show the mutations can greatly improve correct pairing of heavy and light chains. As observed in Table 10 the amount of mispaired Fab species in the samples from IgG-like bsAb with no mutations (variant 1 constructs) is as expected very high (close to 50% for Combo 2 construct). The variant 19 mutations decrease the amount of mispaired Fab species substantially to as little as 6% (for the Combo 1 construct). In all, this demonstrates that the combination of mutations discovered for IgG-Fab bispecific may be beneficial in the generation of other bispecific antibody of other architectures as well, as observed for IgG-like bsAb in this example.
TABLE-US-00029 TABLE 10 Relative amount of correctly paired and mispaired Fab fragment species in Combo 1 and Combo 2 variants 1 and 19. Relative amount (%) Relative amount (%) of correctly paired of mispaired Fab species Fab species Combo 1 variant 1 63 38 Combo 1 variant 19 94 6 Combo 2 variant 1 53 47 Combo 2 variant 19 92 8
Example 11—BsAbs with Truncated Light Chain
[1746] Material and Methods
[1747] Design
[1748] To evaluate if the linker position could be moved in the IgG-Fab constructs bispecific antibodies with truncated Fab fragments fused to the C terminal end of IgGs were also engineered. Variants with differently truncated Fabs were included in the study. The truncation was made in the N-terminal end of the Fab fragment by deleting nucleotides coding for the first 3 (variant 21), 6 (variant 22) or 9 (variant 23) amino acids of the light chain of variant 9 IgG-Fab bsAb constructs.
[1749] Thermostability
[1750] The melting temperatures were measured with the UNcle system (UNchained labs). The intrinsic fluorescence was measured during linear temperature ramping from 20° C. to 95° C. at a rate of 0.4° C./minute. The data analysis was performed with the UNcle Analysis software version 2.0 using default settings.
[1751] Stability
[1752] Samples in non-optimized buffer (PBS) at low (<1 mg/mL) were incubated at 2-8° C., room temperature and 40° C. 2 weeks or subjected to 3 rounds of freeze thawing. Protein degradation was measured with SE-HPLC, SDS-PAGE, A280, dual ELISA and by visual inspection.
[1753] Shear Stress Stability
[1754] Samples in duplicates were subjected to shear stress in the form of heavy agitation at 2000 rpm on the 96-well plate shaker MixMate (Eppendorf) for at least 30 minutes. Protein precipitates were removed by centrifugation at 3000 g for 10 minutes and the absorbance at 280 nm was measured using Big Lunatic.
[1755] Colloidal Stability
[1756] Samples were mixed with PBS/PEG solutions with different PEG concentrations ranging from 8%-36%, added to 96-well filter plates in triplicates and incubated over night at room temperature. Filtrates obtained by centrifugation at 12000 g for 15 minutes were collected and spun down before the absorbance at 280 nm was measured (to determine loss of protein) and compared to controls.
[1757] Production and Manufacturing
[1758] Bispecific antibodies were expressed using transient Expi293 HEK (Life technologies) cultures at different volumes ranging from high through put to 30 mL according to manufacturer's instructions. Purification of bispecifics from supernatants was made on protein A using the NGC system (BioRad), the ÄKTA Avant system (GE Healthcare) or Predictor MabSelectSure 50 μl 96 well plates (GE Healthcare). Cells were transfected with three different vectors encoding separately for each of the three polypeptides chains (i.e. the immunoglobulin heavy chain linked to the Fab light chain, the immunoglobulin light chain and the Fab heavy chain). Aggregation was measured with SE-HPLC in a 1260 Infinity II system (Agilent Technologies) using a TSK gel Super SW mAB HTP 4 μm, 4.6×150 mm column (TOSOH Bioscience) and 100 mM Sodium Phosphate, pH 6.8, 300 mM NaCl as mobile phase at ambient temperature and a flow rate of 0.35 ml/min.
[1759] Dual ELISA
[1760] Plates were coated with 0.5 μg/mL antigen Ag1 in PBS over night at 4° C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again. Samples serially diluted in PBS/0.5% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with 0.5 μg/mL biotinylated Ag2 for at least 1 hour at room temperature. Dual complexed bsAb with Ag1 and Ag2 were detected with HRP-labelled streptavidin. SuperSignal Pico Luminescent was used as substrate and luminescence signals were measured using Fluostar Optima.
[1761] Results
[1762] The expression yields of constructs varied substantially depending of the combination of IgG and Fab domains. Combo 1 constructs could be expressed regardless of truncation variant.
[1763] Binding tested in dual ELISA with variant 21 and 22 constructs showed that variant 21 constructs display higher target interaction as observed in
[1764] Further, the developability of variant 21 constructs was evaluated. All variants displayed high stability. Incubation at elevated temperatures in a non-optimized buffer (PBS) resulted only in low degradation, <10% increase in aggregation and <5% increase in fragmentation as measured by SE-HPLC (Table 12), SDS-PAGE, dual ELISA, A280 and visual inspection, indicating a good storage stability. In addition, freeze/thawing treatments of 1 or 3 rounds showed minimal degradation <3% degradation as measured by SE-HPLC, indicating again that the stability of the constructs is high.
[1765] Shear stress stability studies showed low loss of protein (<25%) after severe agitation for all of the tested constructs (Table 11 indicating that the bsAb are not sensitive to shear stress. Evaluation of the colloidal stability showed to reach 50% loss of the protein concentrations >9% of PEG were required (
[1766] The melting temperatures measured with UNcle were found to be >65° C. for the variant 21 constructs as shown in Table 13 indicating that the thermostability of these constructs is high.
[1767] In all the experiments with truncated light chains show that removal of some amino acids, in particular 3 as in variant 21, results in constructs that are both stable and display excellent binding. Furthermore, this strategy was shown to be compatible with variant 9 combination of mutations and will likely work with other IgG-Fab bsAb variants.
TABLE-US-00030 TABLE 11 Expression yields for Combo 1 (5T4-CD137 bispecific) variants 21, 22 and 23 Combo ID Yield(mg/L) Combo 1 v21 127 Combo 1 v22 85 Combo 1 v23 144
TABLE-US-00031 TABLE 12 Changes in degradation after different treatments to evaluate stability. Protein loss % 25 C., 2 weeks 40 C., 2 weeks Freeze/thaw, 1 round Freeze/thaw, 3 rounds after shear bsAb Δ% HMWs Δ% LMWs Δ% HMWs Δ% LMWs Δ% HMWs Δ% LMWs Δ% HMWs Δ% LMWs stress treatment Combo 1 V21 1.7 −0.1 9.5 0.7 0.1 0.2 0.8 −0.1 22.2
TABLE-US-00032 TABLE 13 Melting temperatures determined with Uncle. Construct Tm (° C.) Combo 1 v21 67
Example 12—Strategy to Remove Protein a Site in Unwanted Fab By-Products
[1768] Material and Methods
[1769] Design
[1770] To investigate if the protein A purification of unwanted Fab by-product, could be decreased, mutations at position 65, which is close to the protein A site for variable heavy chains of framework group IGHV3, were introduced. Constructs with mutations T65E or T65A in the variable FAb heavy chain were compared with constructs without mutations in this position
[1771] Production
[1772] Bispecific antibodies were expressed using transient Expi293 HEK (Life technologies) cultures in 700 μl according to manufacturer's instructions. Purification of bispecifics from supernatants was made on protein A Predictor MabSelectSure 50 μl 96 well plates (GE Healthcare). Cells were transfected with three different vectors encoding separately for each of the three polypeptides chains (i.e. the immunoglobulin heavy chain linked to the Fab light chain, the immunoglobulin light chain and the Fab heavy chain).
[1773] HPLC Analysis
[1774] Aggregation was measured with SE-HPLC in a 1260 Infinity II system (Agilent Technologies) using a TSK gel Super SW mAB HTP 4 μm, 4.6×150 mm column (TOSOH Bioscience) and 100 mM Sodium Phosphate, pH 6.8, 300 mM NaCl as mobile phase at ambient temperature and a flow rate of 0.35 ml/min.
[1775] Results
[1776] Mutations in position 65 showed improved purity after protein A purification as observed from HPLC measurements. The investigated mutations were shown to decrease the amount of purified Fab substantially with close to 100% reduction of amount of purified Fab in the majority of tested constructs (Table 14)
TABLE-US-00033 TABLE 14 Relative reduction of amount (%) of by-product FAb after protein A purification compared to constructs with no mutation in position 65. Reduction of amount of by-product FAb after protein A purification compared to constructs BsAb construct with no mutation in position 65 (%) Combo 4, V9 T65E 88 Combo 4, V9 T65A 96 Combo 5, V9 T65E 98 Combo 5, V9 T65A 98
Example 13: Anti-Tumour Effect of the Combo 2 Variant 9 (CD40-EpCAM) Bispecific Antibody (Also Known as 1132-3174.R in RUBY™ Format)
[1777] Background and Aim
[1778] 1132-3174.R is a CD40-EpCAM bispecific antibody in the novel IgG-Fab bispecific format (as described herein) wherein 1132 refers to its CD40 agonist domain and 3174 to its EpCAM-binding, tumour-targeting domain. The antibody has been LALA-mutated to silence Fcγ receptor binding.
[1779] The aim of this study was to evaluate the anti-tumour effect of 1132-3174.R in human CD40 transgenic (hCD40tg) mice inoculated with murine MB49 tumours transfected with human EpCAM (MB49-hEpCAM) or MB49-wt (hEpCAM negative) tumours.
[1780] Materials and Methods
[1781] Female hCD40tg mice of 13-16 weeks of age were inoculated with either 2.5×10.sup.5 MB49-wt or MB49-hEpCAM cells s.c. in the right flank. On days 10, 13 and 16 after inoculation, the mice were administered i.p. with 100 μg of wildtype CD40 monospecific antibody, 1132, or 250 μg of the LALA-mutated equivalent, 1132.m2. Alternatively, the mice received 417 μg of 1132-3174.R. A group of vehicle-treated mice was also included. The tumours were frequently measured with a caliper in width (w), length (l) and height (h) and the tumour volume was calculated using the formula: (w/2×l/2×h/2×π×(4/3)).
[1782] In an alternative experimental set-up, hCD40tg mice were inoculated with MB49-wt or MB49-hEpCAM cells s.c. as previously and, instead, mice were administered i.p. with 100 μg 1132, 100 μg 1132.m2 or 167 μg (dose of molecular mass equivalence to the monospecific antibodies) or 417 μg (dose 2.5 fold higher in terms of molecular mass, compared to monospecific antibodies) 1132-3174.R on days 10, 13 and 16 after inoculation. A group of vehicle-treated mice was also included. Tumours were frequently measured as previously.
[1783] Results
[1784] The data (shown in
Example 14: Anti-Tumour Effect of Combo 2 Variant (CD40-EpCAM) Bispecific Antibody (Also Known as 1132-3174.R)
[1785] Background and Aim
[1786] 1132-3174.R is a CD40-EpCAM bispecific antibody in the novel IgG-Fab bispecific format (as described herein) wherein 1132 refers to its CD40 agonist domain and 3174 to its EpCAM-binding, tumour-targeting domain. The antibody has been LALA-mutated to silence Fcγ receptor binding.
[1787] The aim of this study was to evaluate the anti-tumour effect of 1132-3174.R in human CD40 transgenic (hCD40tg) mice inoculated with murine MB49 tumours transfected with human EpCAM (MB49-hEpCAM) or MB49-wt (hEpCAM negative) tumours.
[1788] Materials and Methods
[1789] Female hCD40tg mice of 13-16 weeks of age were inoculated with either 2.5×105 MB49-wt or MB49-hEpCAM cells s.c. in the right flank. On days 10, 13 and 16 after inoculation, the mice were administered i.p. with 100 μg of wildtype CD40 monospecific antibody 1132 or 100 μg of the LALA-mutated equivalent 1132.m2. Alternatively, the mice received 167 μg 1132-3174.R (dose of molecular mass equivalence to the monospecific antibodies) or 417 μg 1132-3174.R (dose 2.5 fold higher in terms of molecular mass, compared to monospecific antibodies). A group of vehicle-treated mice was also included. The mice were kept in the study until the individual tumour volume reached the ethical limit of 2000 mm3, at which point the mice were sacrificed.
[1790] Results and Conclusions
[1791] The data (shown in
[1792] The invention is further defined by the following numbered paragraphs: [1793] 1. A bispecific antibody comprising: (a) an immunoglobulin molecule having specificity for a first antigen, the immunoglobulin molecule comprising a first heavy chain polypeptide and a first light chain polypeptide; and (b) at least one Fab fragment having specificity for a second antigen, the Fab fragment comprising a second heavy chain polypeptide and a second light chain polypeptide wherein the second light chain polypeptide is fused to the C-terminus of the first heavy chain polypeptide and wherein the bispecific antibody comprises one or more mutations to promote association of the first heavy chain polypeptide with the first light chain polypeptide and/or to promote association of the second heavy chain polypeptide with the second light chain polypeptide. [1794] 2. A bispecific antibody according to paragraph 1, wherein the immunoglobulin molecule comprises two copies of the first heavy chain polypeptide and/or two copies of the first light chain polypeptide. [1795] 3. A bispecific antibody according to paragraph 1 or 2, wherein the antibody comprises two Fab fragments according to (b). [1796] 4. A bispecific antibody according to any one of the preceding paragraphs, wherein the immunoglobulin molecule comprises two copies of the first heavy chain polypeptide and two copies of the first light chain polypeptide, and the bispecific antibody further comprises two Fab fragments according to (b), and the first Fab fragment is fused to the C-terminus of the first copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment; and the second Fab fragment is fused to the C-terminus of the second copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment. [1797] 5. A bispecific antibody according to any one of the preceding paragraphs, wherein the immunoglobulin molecule comprises a human Fc region or a variant of a said region, where the region is an IgG1, IgG2, IgG3 or IgG4 region, preferably an IgG1 or IgG4 region. [1798] 6. A bispecific antibody according to paragraph 5 wherein the Fc region is a naturally occurring (i.e. wildtype) human Fc region. [1799] 7. A bispecific antibody according to paragraph 5 wherein the Fc region is a non-naturally occurring (e.g. mutated) human Fc region. [1800] 8. A bispecific antibody according to any one of paragraphs 5 to 7 wherein the Fc region has modified glycosylation, for example wherein the Fc region is afucosylated. [1801] 9. A bispecific antibody according to any one of the preceding paragraphs wherein the one or more mutations are in one or more of the following regions: [1802] (i) the CH1 region of the first heavy chain polypeptide and/or [1803] (ii) the VH region of the first heavy chain polypeptide, and/or [1804] (iii) the CH1 region of the second heavy chain polypeptide and/or [1805] (iv) the VH region of the second heavy chain polypeptide, and/or [1806] (v) the CKappa or CLambda region of the first light chain polypeptide and/or [1807] (vi) the VL region of the first light chain polypeptide, and/or [1808] (vii) the CKappa or CLambda region of the second light chain polypeptide, and/or [1809] (viii) the VL region of the second light chain polypeptide. [1810] 10. A bispecific antibody according to any one of the preceding paragraphs wherein the one or more mutations prevent the binding of the second heavy chain polypeptide to the first light chain polypeptide and/or prevent self-aggregation of the first heavy chain polypeptide fused to the second light chain polypeptide. [1811] 11. A bispecific antibody according to any one of the preceding paragraphs wherein the one or more mutations prevent the formation of aggregates and a Fab by-product. [1812] 12. A bispecific antibody according to any one of the preceding paragraphs, wherein the mutations prevent formation of aggregates by generating steric hindrance and/or incompatibility between charges. [1813] 13. A bispecific antibody according to any one of the preceding paragraphs, wherein the mutations prevent formation of a Fab by-product by generating steric hindrance and/or incompatibility between charges. [1814] 14. A bispecific antibody according to any one of the preceding paragraphs wherein the antibody comprises one or more mutation pairs each comprising two functionally compatible mutations. [1815] 15. A bispecific antibody according to paragraph 14, wherein the functionally compatible mutations are selected from: [1816] (a) cavity and protruding surface mutations (i.e. steric mutations); and/or [1817] (b) hydrophobic swap mutations; and/or [1818] (c) charged mutations (i.e. salt mutations); and/or [1819] (d) double charged mutations; and/or [1820] (e) mutations resulting in the formation of a disulphide bridge. [1821] 16. A bispecific antibody according to paragraph 15, wherein the bispecific antibody comprises one or more mutation pairs in one or more of the following region groups: [1822] (a) the CH1 and CKappa or CLambda region of the immunoglobulin; and/or [1823] (b) the CH1 and CKappa or CLambda region of the Fab; and/or [1824] (c) the VL and VH regions of the immunoglobulin; and/or [1825] (d) the VL and VH regions of the Fab. [1826] 17. A bispecific antibody according to paragraph 16 wherein the mutation pairs are in the CH1 and CKappa or CLambda regions of the Fab and/or the immunoglobulin, and wherein the mutation pairs are selected from: [1827] (a) cavity and protruding surface mutations (i.e. steric mutations); and/or [1828] (b) hydrophobic swap mutations; and/or [1829] (c) charged mutations (i.e. salt mutations); and/or [1830] (d) mutations resulting in the formation of a disulphide bridge. [1831] 18. A bispecific antibody according to any one of paragraphs 15 to 17 wherein the mutation pairs are in the VH and VL regions of the Fab and/or the immunoglobulin, and wherein the mutation pairs are selected from: [1832] (a) charged mutations (i.e. salt mutations); and/or [1833] (b) double charged mutations; and/or [1834] (c) mutations resulting in the formation of a disulphide bridge. [1835] 19. A bispecific antibody according to any one of paragraphs 16 to 18 wherein the bispecific antibody comprises one or more mutation pairs selected from: [1836] (a) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin; [1837] (b) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, and salt mutations in the VH and VL regions of the Fab; [1838] (c) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; [1839] (d) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin and salt mutations in the CH1 and CKappa or CLambda regions of the Fab; [1840] (e) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; [1841] (f) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; [1842] (g) disulphide bridge-forming mutations in the CH1 and CKappa or CLambda regions of the Fab; [1843] (h) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; [1844] (i) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; [1845] (j) salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; [1846] (k) salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; [1847] (l) salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; [1848] (m) salt mutations in the VH and VL regions of the immunoglobulin, steric mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab; [1849] (n) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; [1850] (o) hydrophobic mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge forming mutations in the VH and VL regions of the Fab; [1851] (p) steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, hydrophobic mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; or [1852] (q) steric mutations and salt mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, hydrophobic mutations in the CH1 and CKappa or CLambda regions of the Fab, and salt mutations and disulphide bridge-forming mutations in the VH and VL regions of the Fab. [1853] (r) steric mutations in the CH1 and CKappa or CLambda regions of the immunoglobulin, salt mutations in the VH and VL regions of the immunoglobulin, salt mutation in the CH1 and alanine mutation in the CKappa or CLambda regions of the Fab, and salt mutations in the VH and VL regions of the Fab; [1854] 20. A bispecific antibody according to any one of the preceding paragraphs, wherein the mutations are at positions selected from the group consisting of: [1855] (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or [1856] (b) a position selected from the one or more of the following position ranges in the CKappa or CLambda domain: position 132 to 138, position 173 to 179, position 130 to 136, position 111 to 117 and position 134 to 140 (according to Kabat numbering); and/or [1857] (c) a position selected from one or more of the following position ranges in the VL: position 41 to 47, position 117 to 123 and position 46 to 52 (according to IMGT numbering); and/or [1858] (d) a position selected from one or more of the following position ranges in the VH: position 41 to 47, position 46 to 52 and position 117 to 123 (according to IMGT numbering). [1859] 21. A bispecific antibody according to any one of the preceding paragraphs, wherein the mutations are at positions selected from the group consisting of: [1860] (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or [1861] (b) one or more of the following positions in the CKappa or CLambda domain: 135, 176, 133, 114 and 137 (according to Kabat numbering); and/or [1862] (c) one or more of the following positions in the VL: 44, 120 and 49 (according to IMGT numbering); and/or [1863] (d) one or more of the following positions in the VH: 44, 49 and 120 (according to IMGT numbering). [1864] 22. A bispecific antibody according to any one of the preceding paragraphs, wherein the mutations are at positions selected from the group consisting of: [1865] (a) one or more of the following positions in the CH1 domain: H168, F170, L145, S183 and T187 (according to EU numbering); and/or [1866] (b) one or more of the following positions in the CKappa or CLambda domain: L135, S176, V133, S114 and N137 (according to Kabat numbering); and/or [1867] (c) one or more of the following positions in the VL: Q44, Q120 or A120 and A49 (according to IMGT numbering); and/or [1868] (d) one or more of the following positions in the VH: Q44, G49 and Q120 (according to IMGT numbering). [1869] 23. A bispecific antibody according to paragraph 22, wherein the mutations are selected from the group consisting of: [1870] (a) one or more of the following mutations in the CH1 domain: H168A, F170G, L145Q, S183V and T187E (according to EU numbering); and/or [1871] (b) one or more of the following mutations in the CKappa or CLambda domain: L135Y, S176W, V133T, S176V, S114A and N137K (according to Kabat numbering); and/or [1872] (c) one or more of the following mutations in the VL: Q44R, Q44E, Q120C or A120C, Q44D and A49D (according to IMGT numbering); and/or [1873] (d) one or more of the following mutations in the VH: Q44E, Q44R, G49C, Q44K and Q120K (according to IMGT numbering). [1874] 24. A bispecific antibody according to any one of the preceding paragraphs, wherein the bispecific antibody is tetravalent, with dual binding to each of the two antigens. [1875] 25. A bispecific antibody according to any one of the preceding paragraphs, wherein the Fab fragment(s) is linked to the C terminal end of the immunoglobulin via a linker. [1876] 26. A bispecific antibody according to paragraph 25, wherein the linker is a peptide with the amino acid sequence SGGGGSGGGGS (SEQ ID NO: 5), SGGGGSGGGGSAP (SEQ ID NO: 6), NFSQP (SEQ ID NO: 7), KRTVA (SEQ ID NO: 8), GGGGSGGGGSGGGGS (SEQ ID NO: 9), (SG)m, where m=1 to 7 or GGGGSGGGGS (SEQ ID NO: 34). [1877] 27. A bispecific antibody according to any one of the preceding paragraphs, wherein the first and/or second antigen is an immunomodulator. [1878] 28. A bispecific antibody according to paragraph 27, wherein the immunomodulator is a checkpoint molecule. [1879] 29. A bispecific antibody according to paragraph 28, wherein the checkpoint molecule is a stimulatory checkpoint molecule, optionally wherein the stimulatory checkpoint molecule is selected from CD40, CD137, GITR, CD27, ICOS and OX40. [1880] 30. A bispecific antibody according to paragraph 28, wherein the checkpoint molecule is an inhibitory checkpoint molecule, optionally wherein the inhibitory checkpoint molecule is selected from CTLA-4, PD-1, Tim3, Lag3, Tigit and VISTA. [1881] 31. A bispecific antibody according to any one of the preceding paragraphs, wherein the first and/or second antigen is a tumour cell-associated antigen. [1882] 32. A bispecific antibody according to paragraph 31 wherein the tumour cell-associated antigen is selected from the group consisting of: [1883] (m) products of mutated oncogenes and tumour suppressor genes; [1884] (n) overexpressed or aberrantly expressed cellular proteins; [1885] (o) tumour antigens produced by oncogenic viruses; [1886] (p) oncofetal antigens; [1887] (q) altered cell surface glycolipids and glycoproteins; [1888] (r) cell type-specific differentiation antigens; [1889] (s) hypoxia-induced antigens; [1890] (t) tumour peptides presented by MHC class I; [1891] (u) epithelial tumour antigens; [1892] (v) haematological tumour-associated antigens; [1893] (w) cancer testis antigens; and [1894] (x) melanoma antigens. [1895] 33. A bispecific antibody according to paragraph 32 wherein the tumour cell-associated antigen is selected from the group consisting of 5T4, CD20, CD19, MUC 1, CA-125, CO17-1A, EpCAM, HER2, EphA2, EphA3, DR5, FAP, OGD2, VEGFR, Her3, mesothelin and EGFR. [1896] 34. A bispecific antibody according to any one of the preceding paragraphs wherein the first and second antigen are selected from the group consisting of: CD40, EpCAM, 5T4, CD137, OX40, CTLA-4, GITR, EGFR and HER2. [1897] 35. A bispecific antibody according to any one of the preceding paragraphs wherein the bispecific antibody targets a pair of antigens selected from: OX40 and CTLA-4, OX40 and CD137, GITR and CTLA-4, CD137 and CTLA-4, OX40 and 5T4. [1898] 36. A bispecific antibody according to paragraph 35, wherein the first and second antigen are selected from CD40 and EpCAM. [1899] 37. A bispecific antibody according to paragraph 36 comprising one or more CDR sequences selected from:
TABLE-US-00034 a) CDRH1: (SEQ ID NO: 10) GFTFSSYA; and/or b) CDRH2: (SEQ ID NO: 11) IGSYGGGT; and/or c) CDRH3: (SEQ ID NO: 12) ARYVNFGMDY; and/or d) CDRL1: (SEQ ID NO: 13) QSISSY; and/or e) CDRL2: (SEQ ID NO: 14) AAS; and/or f) CDRL3: (SEQ ID NO: 15) QQYGRNPPT; and/or g) CDRH1: (SEQ ID NO: 18) GYAFTNYW; and/or h) CDRH2: (SEQ ID NO: 19) IFPGSGNI; and/or i) CDRH3: (SEQ ID NO: 20) ARLRNWDEPMDY; and/or j) CDRL1: (SEQ ID NO: 21) QSLLNSGNQKNY; and/or k) CDRL2: (SEQ ID NO: 22) WAS; and/or l) CDRL3: (SEQ ID NO: 23) QNDYSYPLT. [1900] 38. A bispecific antibody according to paragraph 37 comprising one or more variable region sequences selected from:
TABLE-US-00035 (a) VH of SEQ ID NO: 16 (EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSGIGSYGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CARYVNFGMDYWGQGTLVTVSS); and/or (b) VL of SEQ ID NO: 17 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYGRNP PTFGQGTKLEIK); and/or (c) VH of SEQ ID NO: 24 (EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLE WIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVY FCARLRNWDEPMDYWGQGTTVTVSS); and/or (d) VL of SEQ ID NO: 25 (ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPG QPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQ NDYSYPLTFGAGTKLEIK) [1901] 39. A bispecific antibody according to paragraph 38, wherein: [1902] (a) the first heavy chain polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 16; and/or [1903] (b) the first light chain polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 17; and/or [1904] (c) the second heavy chain polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 24; and/or [1905] (d) the second light chain polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 25. [1906] 40. A bispecific antibody according to any one of the preceding paragraphs, wherein the bispecific antibody is capable of inducing antibody dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or apoptosis. [1907] 41. A bispecific antibody according to any one of the preceding paragraphs, wherein the bispecific antibody is capable of inducing: [1908] (a) activation of B cells; and/or [1909] (b) activation of dendritic cells; and/or [1910] (c) activation of cytotoxic T cells, i.e. CD8+ T cells; and/or [1911] (d) activation of helper T cells, i.e. CD4.sup.+ T cells; and/or [1912] (e) improved tumour antigen cross-presentation by dendritic cells; and/or [1913] (f) expansion of tumour antigen-specific cytotoxic T cells; and/or [1914] (g) direct tumour cell killing via ADCC and/or via inhibition of tumour growth and survival signals; and/or [1915] (h) anti-angiogenic effects via interaction with endothelial and/or stromal cells; and/or [1916] (i) activation of natural killer cells; and/or [1917] (j) Treg depletion; and/or [1918] (k) reprograming of Tregs into effector T cells; and/or [1919] (l) depletion of tumour myeloid cell populations; and/or [1920] (m) reprogramming of tumour myeloid cell populations; and/or [1921] (n) internalisation of tumour debris by antigen-presenting cells; and/or [1922] (o) internalisation of tumour extracellular vesicles, e.g. exosomes, by antigen-presenting cells; and/or [1923] (p) localization to tumour tissue by binding to tumour cells. [1924] 42. A bispecific antibody according to any one of the preceding paragraphs, which induces an increase in the activity of an effector T cell, optionally wherein said increase is at least 1.5-fold, 4.5-fold or 7-fold higher than the increase in activity of an effector T cell induced by a combination of the immunoglobulin molecule and Fab fragment administered to the T cell as separate molecules. [1925] 43. A bispecific antibody according to paragraph 42, wherein said increase in T cell activity is an increase in proliferation and/or IFNγ or IL-2 production by the T cell. [1926] 44. A bispecific antibody according to any one of the preceding paragraphs, which induces an increase in the activation of an antigen-presenting cell, such as a B cell or dendritic cell. [1927] 45. A bispecific antibody according to paragraph 44, wherein said increase in activation is an increase in the expression of the co-stimulatory molecules CD80 or CD86 by the antigen-presenting cell. [1928] 46. A bispecific antibody according to any one of the preceding paragraphs, which induces an increase in the uptake of tumour debris or tumour extracellular vesicles by an antigen-presenting cell, such as a B cell or dendritic cell. [1929] 47. A bispecific antibody according to paragraph 46, wherein said increase in uptake is measured by the co-localization or internalization of the tumour debris or tumour extracellular vesicles by the antigen-presenting cell. [1930] 48. A bispecific antibody according to any one of the preceding paragraphs, wherein the bispecific antibody binds to the first and/or the second antigen with a K.sub.D of less than 100×10.sup.−9M or less than 50×10.sup.−9M or less than 25×10.sup.−9M, preferably less than 10, 9, 8, 7, or 6×10.sup.−9M, more preferably less than 5, 4, 3, 2, or 1×10.sup.−9M, most preferably less than 9×10.sup.−10M. [1931] 49. An isolated nucleic acid molecule encoding a bispecific antibody according to any one of the preceding paragraphs, or a component polypeptide chain thereof. [1932] 50. A nucleic acid molecule according to paragraph 49 wherein the molecule is a cDNA molecule. [1933] 51. A nucleic acid molecule according to paragraph 49 or 50 encoding an antibody heavy chain or variable region thereof. [1934] 52. A nucleic acid molecule according to paragraph 49 or 50 encoding an antibody light chain or variable region thereof. [1935] 53. A vector comprising a nucleic acid molecule according to any one of paragraphs 49 to 52. [1936] 54. A vector according to paragraph 53 wherein the vector is an expression vector. [1937] 55. A recombinant host cell comprising a nucleic acid molecule according to any one of paragraphs 49 to 52 or a vector according to paragraph 53 or 54. [1938] 56. A host cell according to paragraph 55 wherein the host cell is a bacterial cell. [1939] 57. A host cell according to paragraph 55 wherein the host cell is a mammalian cell. [1940] 58. A host cell according to paragraph 55 wherein the host cell is a human cell. [1941] 59. A method for producing bispecific antibody according to any one of paragraphs 1 to 48, the method comprising culturing a host cell as defined in any of paragraphs 55 to 58 under conditions which permit expression of the bispecific antibody or component polypeptide chain thereof. [1942] 60. A pharmaceutical composition comprising an effective amount of a bispecific antibody according to any one of the paragraphs 1 to 48 and a pharmaceutically-acceptable diluent, carrier or excipient. [1943] 61. A pharmaceutical composition according to paragraph 60 adapted for parenteral delivery. [1944] 62. A pharmaceutical composition according to paragraph 60 adapted for intravenous delivery. [1945] 63. A bispecific antibody according to any one of the paragraphs 1 to 48 for use in medicine. [1946] 64. A bispecific antibody according to any one of the paragraphs 1 to 48 for use in treating or preventing a neoplastic disorder in a subject. [1947] 65. An antibody for use according to paragraph 64 wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body. [1948] 66. An antibody for use according to paragraph 65 wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer and sarcomas. [1949] 67. An antibody for use according to paragraph 66 wherein the solid tumour is selected from the groups consisting of renal cell carcinoma, colorectal cancer, lung cancer, prostate cancer and breast cancer. [1950] 68. An antibody for use according to any one of paragraphs 64 to 67 wherein the antibody is for use in combination with one or more additional therapeutic agents. [1951] 69. An antibody for use according to paragraph 68 wherein the one or more additional therapeutic agents is/are an immunotherapeutic agent that binds a target selected from the group consisting of PD-1/PD-L1, CTLA-4, CD137, CD40, GITR, LAG3, TIM3, CD27, VISTA, OX40 and KIR. [1952] 70. Use of a bispecific antibody according to any one of paragraphs 1 to 48 in the preparation of a medicament for treating or preventing a neoplastic disorder in a subject. [1953] 71. A use according to paragraph 70 wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body. [1954] 72. A use according to paragraph 71 wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer and sarcomas. [1955] 73. A use according to paragraph 72 wherein the solid tumour is selected from the groups consisting of renal cell carcinoma, colorectal cancer, lung cancer, prostate cancer and breast cancer. [1956] 74. A use according to any one of paragraphs 70 to 73 wherein the antibody is for use in combination with one or more additional therapeutic agents. [1957] 75. An antibody for use according to paragraph 74 wherein the one or more additional therapeutic agents is/are an immunotherapeutic agent that binds a target selected from the group consisting of PD-1/PD-L1, CTLA-4, CD137, CD40, GITR, LAG3, TIM3, CD27, VISTA, OX40 and KIR. [1958] 76. A method for the treatment or diagnosis of a neoplastic disorder in a subject, comprising the step of administering to the subject an effective amount of a bispecific antibody according to any one of the paragraphs 1 to 48. [1959] 77. A method according to paragraph 76 wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body. [1960] 78. A method according to paragraph 77 wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer and sarcomas. [1961] 79. A method according to paragraph 78 wherein the solid tumour is selected from the groups consisting of renal cell carcinoma, colorectal cancer, lung cancer, prostate cancer and breast cancer. [1962] 80. A method according to any one of paragraphs 76 to 79 wherein the subject is human. [1963] 81. A method according to any one of paragraphs 76 to 80 wherein the method comprises administering the bispecific antibody systemically. [1964] 82. A method according to any one of paragraphs 76 to 81 further comprising administering to the subject one or more additional therapeutic agents. [1965] 83. A method according to paragraph 82 wherein the one or more additional therapeutic agents is/are an immunotherapeutic agent that binds a target selected from the group consisting of PD-1/PD-1L, CTLA-4, CD137, CD40, GITR, LAG3, TIM3, CD27, VISTA, OX40 and KIR. [1966] 84. A method of producing a bispecific antibody according to any one of paragraphs 1 to 48 comprising expressing three polypeptide chains in the same host cell, wherein the three polypeptide chains are: [1967] (d) an immunoglobulin heavy chain (the first heavy chain) fused via a polypeptide linker to a second light chain; [1968] (e) a first light chain; and [1969] (f) a second heavy chain [1970] 85. A method of producing a bispecific antibody according to paragraph 84 further comprising the step of modifying the ratios of the chains (a), (b) and (c) to optimise formation of a bispecific antibody.