Immunotherapeutic proteins

Abstract

An bispecific immunotherapeutic protein and methods of use and production thereof are disclosed, wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptide comprising an Fc region component comprising at least a constant heavy chain domain 3 (CH3) (or at least a constant heavy domain 4 (CH4) domain), wherein said one or more polypeptide includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1 heavy chain (Eu numbering).

Description

Production thereof are disclosed, wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptides comprising an Fc region component comprising at least a constant heavy chain domain 3 (CH3) (or at least a constant heavy domain 4 (CH4) domain), wherein said one or more polypeptides includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1 heavy chain (Eu numbering).

Claims

CLAIMS1. A bispecific immunotherapeutic protein comprising two antigen recognition structures directed at two different epitopes and comprising one or more immunoglobulin heavy chain polypeptide comprising an Fc region component comprising at least a constant heavy chain domain 3 (CH3) (or at least a constant heavy domain 4 (CH4) domain), wherein said one or more polypeptide includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgGl, IgG2, IgG3 or IgG4 heavy chain (Eu numbering).

2. The bispecific immunotherapeutic protein of claim 1, wherein the Fc region component comprises a constant heavy domain 3 (CH3) and a constant heavy domain 2 (CH2) of an immunoglobulin heavy chain, optionally in combination with a lower, core and / or upper hinge sequence.

3. The bispecific immunotherapeutic protein of claim 1, wherein the Fc region component comprises a constant heavy domain 3 (CH3) only.

4. The bispecific immunotherapeutic protein of any one of claims 1 to 3, wherein the Fc region component is derived from an IgG or IgA heavy chain polypeptide.

5. The bispecific immunotherapeutic protein of any one of claims 1 to 4, wherein the bispecific immunotherapeutic protein is an immunoglobulin molecule comprising first and second immunoglobulin heavy chain polypeptides comprising an Fc region component comprising CH2 and CH3 domains so that the first and second polypeptides form, by dimerisation, an Fc fragment or Fc- like fragment.

6. The bispecific immunotherapeutic protein of claim 4, wherein the Fc region component is derived from an IgGl heavy chain polypeptide.

7. The bispecific immunotherapeutic protein of claim 4, wherein the Fc region component is derived from an IgG2 heavy chain polypeptide, an IgG3 heavy chain polypeptide, an IgG4 heavy chain polypeptide or an IgA heavy chain polypeptide.

8. The bispecific immunotherapeutic protein of any one of claims 1 to 7, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgGl, IgG2, IgG3 or IgG4 heavy chain polypeptide is a H to an aromatic amino acid or cyclic amino acid.

9. The bispecific immunotherapeutic protein of any one of claims 1 to 7, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgGl, IgG2, IgG3 or IgG4 heavy chain polypeptide is a H to a hydrophobic amino acid.

10. The bispecific immunotherapeutic protein of any one of claims 1 to 9, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgGl, IgG2, IgG3 or IgG4 heavy chain polypeptide is an H— >Y substitution.

11. The bispecific immunotherapeutic protein of any one of claims 1 to 9, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgGl, IgG2, IgG3 or IgG4 heavy chain polypeptide is an H— >F substitution.

12. The bispecific immunotherapeutic protein of any one of claims 1 to 11, wherein the bispecific immunotherapeutic protein is a cell bridging bispecific immunotherapeutic protein.

13. The bispecific immunotherapeutic protein of any one of claims 1 to 12, wherein the bispecific immunotherapeutic protein is an antigen-crosslinking bispecific immunotherapeutic protein.

14. The bispecific immunotherapeutic protein of any one of claims 1 to 13, wherein the two epitopes are on different cells.

15. The bispecific immunotherapeutic protein of any one of claims 1 to 14, wherein one epitope is on a cancer cell and one epitope is on an immune cell.

16. The bispecific immunotherapeutic protein of any one of claims 1 to 15, wherein the two epitopes are on the same cell.

17. The bispecific immunotherapeutic protein of any one of claims 1 to 16, wherein the two epitopes are on the same molecule.

18. The bispecific immunotherapeutic protein of any one of claims 1 to 17, wherein one or both of the epitopes is on a molecule selected from: a cancer-associated antigen, an autoantigen, an allergen, an antigen associated with an inflammatory disease, an antigen from a transplanted tissue or organ or an antigen of an infectious agent such as an antigen of a eukaryotic, a soluble molecule, bacterial or viral pathogen.

19. The bispecific immunotherapeutic protein of any one of claims 1 to 18, wherein one or both of the epitopes is on a molecule selected from: cluster of differentiation 37 (CD37), programmed death-ligand 1 (PD-L1), cluster of differentiation 3 (CD3), cluster of differentiation 20 (CD20), cluster of differentiation 127 (4-1BB), human epidermal growth factor receptor-2 (HER2), death receptor 5 (DR5)DR5 B-cell maturation antigen (BCMA), FcyRIII, FcyRIIb, FcyRIIa, FcalphaRI, FcyRI, natural cytotoxicity triggering receptor 1 (NKp46), killer cell lectin like receptor KI (NKG2D), natural cytotoxicity triggering receptor (3NKp30), death receptor 5 (DR5), TNP conjugates (2, 4, 6 trinitrophenyl conjugates), biotin-conjugates, hapten-conjugates, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 10 (IL-10), interleukin- 12 (IL-12), interleukin- 13 (IL-13), interleukin- 17 (IL-17), tumor necrosis factor (TNF), tumor necrosis factor alpha (TNFa), cluster of differentiation 19 (CD19), cluster of differentiation 22 (CD22), cluster of differentiation 27 (CD27), cluster of differentiation 28 (CD28), cluster of differentiation 30 (CD30), cluster of differentiation 33 (CD33), cluster of differentiation 38 (CD38), cluster of differentiation 39 (CD39), cluster of differentiation 47 (CD47), cluster of differentiation 52 (CD52), cluster of differentiation 79 (CD79), cluster of differentiation 123 (CD123), C-type lectin-like molecule-1 (CLL- 1), C-type lectin domain family 12 member A (CLEC12A), delta-like 4 (DLL4), cluster of differentiation antigen 135 (FLT-3), Fc receptor-like protein 5 (FcRH5), G protein-coupled receptor class C group 5 member D (GPRC5D), carcinoembryonic antigen (CEA), epithelial cellular adhesion molecule (EpCAM), prostate-specific membrane antigen (PSMA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene-3 (LAG-3), epidermal growth factor receptor (EGFR), tyrosine-protein kinase Met (cMet), glycoprotein 100 (GP100), angiopoietin-2 (Ang-2), vascular endothelial growth factor A (VEGF-A), tumor-associated glycoprotein 72 (Tag72), claudin 18 (CLDN18.2), cluster of differentiation (CD66), CD300 molecule like family member F (CD300f), glypican 2 (GPC2), fms related receptor tyrosine kinase 3 (FLT3), CD276 molecule (B7-H3), cell adhesion molecule (CEA), delta like canonical notch ligand 3 (DLL3), disialoganglioside (GD2), glycoprotein A33 (gpA33), glypican 3 (GPC3), guanylate cyclase 2C (GUCY2C), major histocompatibility complex, class I A (HLA-A), mage family member A4 (MAGE- A4), mucin 1 cell surface associated (MUC1), mesothelin (MSLN), mucin 16 cell surface associated (MUC16), mucin 17 cell surface associated (MUC17), new york esophageal squamous cell carcinoma 1 (NY-ESO1), PRAME nuclear receptor transcriptional regulator (PRAME), prostate stem cell antigen (PSCA), somatostatin receptor 2 (SSTR2), STEAP Family Member 1 (STEAP1), cluster of differentiation 127 (4-1BB), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), survivin and SARS-CoV-2 S protein.

20. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein the molecule is a soluble molecule.

21. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a CD37 epitope.

22. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a DR5 epitope.

23. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a HER2 epitope.

24. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a CD3 epitope.

25. The bispecific immunotherapeutic protein of any one of clams 1 to 19, wherein one or both of the epitope / s is a CD20 epitope.

26. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a CD 19 epitope.

27. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a CD28 epitope.

28. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a PD-L1 epitope.

29. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a 4- IBB epitope.

30. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a BCM A epitope.

31. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a FcyRIII epitope.

32. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a FcyRIIb epitope.

33. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a FcalphaRl epitope.

34. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a FcyRl epitope.

35. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein one or both of the epitope / s is a C5 epitope.

36. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein the epitopes are on a pair of proteins selected from: PDL-1 and 4- IBB; HER2 and 4- IBB; HER2 and FcyRIII; BCMA and FcyRIII; CD3 and BCMA; CD3 and CD20; CD3 and CD 19; FcyRIIb and TNP-BSA; CD3 and CD28; CD3 and CD123; CD3 and CD33; CD3 and CD37; CD3 and CD38; CD3 and CLEC12A; CD3 and FLT-3; CD3 and Survivin; CD20 and CD47; CTLA-4 and PD-1; LAG-3 and PD-1; PD-1 and PD-L1; and, CD30 and FcyRIII.

37. The bispecific immunotherapeutic protein of any one of claims 1 to 19, wherein the bispecific immunotherapeutic protein is selected from: blinatumomab; emicizumab; amivantamab-vmjw; tebentafusp-tebn; faricimab-svoa; cadonilimab; mosunetuzumab; teclistamab, catumaxomab, odronextamab and ozoralizumab.

38. The bispecific immunotherapeutic protein of any one of claims 1 to 37, wherein the bispecific immunotherapeutic protein is produced from a platform selected from: DEKK, ART-Ig, CrossMab, DuoBody, Ortho-Fab, strand-exchange engineered domain (SEED), Knob-into-hole, DuetMab, K' / . body, YBODY, bispecific engagement by antibodies based on the T cell receptor (BEAT), crossover dual variable (CODV), Wuxibody, DVD-Ig, Adaptir, two-in-one and dual-action antigen-binding fragment (Fab) (DAF), FIT-Ig, TsBsIgG, Triomab, and XmAb.

39. The bispecific immunotherapeutic protein of any one of claims 1 to 38, wherein the bispecific immunotherapeutic protein is a mini-body.

40. The bispecific immunotherapeutic protein of any one of claims 1 to 39, wherein the bispecific immunotherapeutic protein forms oligomers through self-association in solution at physiological pH.

41. The bispecific immunotherapeutic protein of claim 40, wherein the bispecific immunotherapeutic protein is a hexamer.

42. The bispecific immunotherapeutic protein of any one of claims 1 to 39, wherein the bispecific immunotherapeutic protein forms oligomers upon binding to a relevant target through "on target" oligomerisation.

43. The bispecific immunotherapeutic protein of claim 42, wherein the bispecific immunotherapeutic protein forms a hexamer upon binding to a relevant target through "on target" oligomerisation.

44. The bispecific immunotherapeutic protein of any one of claims 1 to 38, wherein the antibody is produced by a quadroma.

45. The bispecific immunotherapeutic protein of any one of claims 1 to 44, wherein the polypeptide comprises a further mutation selected from one or more of: i) an amino acid substitution at a position corresponding to 274, of the amino acid sequence of human IgGl, IgG2, IgG3 or IgG4 heavy chain (Eu numbering); ii) an amino acid substitution at a position corresponding to 219 of the amino acid sequence of human IgGl, IgG2, IgG3 or IgG4 heavy chain (Eu numbering); iii) an amino acid substitution at a position corresponding to 220 of the amino acid sequence of human IgGl, IgG2, IgG3 or IgG4 heavy chain (Eu numbering); and iv) an amino acid substitution at a position corresponding to 219 and 220 of the amino acid sequence of human IgGl, IgG2, IgG3 or IgG4 heavy chain (Eu numbering).

46. The bispecific immunotherapeutic protein of claim 45, wherein the further mutation is a substitution at position corresponding to C219 or a substitution at position corresponding to C220 of an amino acid sequence of human IgGl, IgG2, IgG3 or IgG4 heavy chain (Eu numbering).

47. The bispecific immunotherapeutic protein of claim 45, wherein the further mutation is a substitution selected from K274 with glutamine, C219 with serine, S219 with cysteine, and C220 with serine.

48. An oligomer comprising the bispecific immunotherapeutic protein of any one of claims 1 to 47.

49. A nucleic acid encoding the bispecific immunotherapeutic protein of any one of claims 1 to 47.

50. The nucleic acid of claim 49, wherein the nucleic acid is selected from an RNA, DNA or combination thereof.

51. The use of the bispecific immunotherapeutic protein, oligomer, or nucleic acid of any one of claims 1 to 50 for treating or preventing a disease or condition in a subject, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases, and proliferative diseases.

52. The use of the bispecific immunotherapeutic protein, oligomer, or nucleic acid of any one of claims 1 to 50 in the manufacture of a medicament for treating or preventing a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

53. A method for treating or preventing a disease or condition, comprising administering to the subject an effective amount of the bispecific immunotherapeutic protein, oligomer, or nucleic acid of any one of claims 1 to 50, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

54. The use of claim 51 or 52 or method of claim 53, wherein the bispecific immunotherapeutic protein or oligomer removes soluble molecules and / or immune complexes from circulation.

55. The use of claim 51 or 52 or method of claim 53, wherein the bispecific immunotherapeutic protein enhances death receptor signalling responses.

56. The use of claim 51 or 52 or method of claim 53, wherein the bispecific immunotherapeutic protein enhances complement-based lysis.

57. A pharmaceutical composition or medicament comprising the bispecific immunotherapeutic protein, oligomer, or nucleic acid of any one of claims 1 to 50, and a pharmaceutically acceptable carrier, diluent and / or excipient.

58. The pharmaceutical composition or medicament of claim 57, wherein the carrier is saline at physiological pH.

59. The pharmaceutical composition or medicament of claim 57, wherein the carrier is saline at pH less than 6.5.

60. A kit comprising at least one bispecific immunotherapeutic protein, oligomer, or nucleic acid of any one of claims 1 to 50.

61. A method of producing a bispecific immunotherapeutic protein of claim 1 to 50, comprising culturing a host cell comprising a construct encoding said protein under conditions suitable for the expression of said protein, and recovering the protein from culture supernatant under conditions of:(i) mildly acidic pH to recover bispecific immunotherapeutic protein in a monomeric form; or(ii) substantially neutral pH to recover bispecific immunotherapeutic protein in an oligomeric form.

62. A method of producing a bispecific immunotherapeutic protein of any one of claims 1 to 50, comprising culturing a host cell comprising a construct encoding said protein under conditions suitable for the expression of said protein, and recovering the protein from culture supernatant using a method comprising affinity chromatography using an elution buffer comprising a concentration of arginine of less than 130 mM and at less than or equal to pH 5.0.

63. A method of producing / modifying a bispecific immunotherapeutic protein comprising substituting an amino acid at a position corresponding to H429 of the amino acid sequence of human IgGl, IgG2, IgG3 or IgG4 heavy chain (Eu numbering), wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptide comprising an Fc region component comprising at least a constant heavy chain domain 3 (CH3) (or at least a constant heavy domain 4 (CH4) domain).

64. A mini-body comprising one or more immunoglobulin heavy chain polypeptide comprising an Fc region component comprising at least a constant heavy chain domain 3 (CH3) (or at least a constant heavy domain 4 (CH4) domain), wherein said one or more polypeptide includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgGl, IgG2, IgG3 or IgG4 heavy chain (Eu numbering).

65. The bispecific immunotherapeutic protein of claim 63 or the mini-body of claim 64, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgGl, IgG2, IgG3 or IgG4 heavy chain polypeptide is an H— >Y substitution.

66. The bispecific immunotherapeutic protein of claim 63 or the mini-body of claim 64, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgGl, IgG2, IgG3 or IgG4 heavy chain polypeptide is an H— >F substitution.

67. An oligomer comprising the mini -body of any one of claims 64 to 66.

68. A nucleic acid encoding the mini-body of any one of claims 64 to 66.

69. The nucleic acid of claim 68, wherein the nucleic acid is selected from an RNA, DNA or combination thereof.

70. The use of the mini-body, oligomer or nucleic acid of any one of claims 64 to 69 for treating or preventing a disease or condition in a subject, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

71. The use of the mini -body, oligomer or nucleic acid of any one of claims 64 to 69 in the manufacture of a medicament for treating or preventing a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

72. A method for treating or preventing a disease or condition, comprising administering to the subject an effective amount of the mini-body, oligomer or nucleic acid of any one of claims 64 to 69, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

73. The use of claim 70 or 71 or method of claim 72, wherein the mini-body or oligomer removes soluble molecules and / or immune complexes from circulation.

74. The use of claim 70 or 71 or method of claim 72, wherein the mini-body or oligomer enhances death receptor signalling responses.

75. The use of claim 70 or 71 or method of claim 72, wherein the mini-body or oligomer enhances complement-based lysis.

76. The use of claim 70 or 71 or method of claim 72, wherein the mini-body or oligomer enhances T cell or NK cell-based lysis.

77. A pharmaceutical composition or medicament comprising the mini-body, oligomer or nucleic acid of any one of claims 64 to 69, and a pharmaceutically acceptable carrier, diluent and / or excipient.

78. A kit comprising at least one mini-body, oligomer or nucleic acid of any one of claims 64 to 69.

79. A method of producing a mini-body of any one of claims 64 to 69, comprising culturing a host cell comprising a construct encoding said protein under conditions suitable for the expression of saidprotein, and recovering the protein from culture supernatant under conditions of:(i) mildly acidic pH to recover bispecific immunotherapeutic protein in a monomeric form; or(ii) substantially neutral pH to recover bispecific immunotherapeutic protein in an oligomeric form.

80. A method of producing a mini-body of any one of claims 64 to 69, comprising culturing a host cell comprising a construct encoding said protein under conditions suitable for the expression of said protein, and recovering the protein from culture supernatant using a method comprising affinity chromatography using an elution buffer comprising a concentration of arginine of less than 130 mM and at less than or equal to pH 5.0.

81. A method of producing / modifying a mini-body comprising substituting an amino acid at a position corresponding to H429 of the amino acid sequence of human IgGl heavy chain (Eu numbering), wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptide comprising an Fc region component comprising at least a constant heavy chain domain 3 (CH3) (or at least a constant heavy domain 4 (CH4) domain).

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