Improved bispecific FOLR1 protease-activated T-cell antibodies

Claims

1. A protease-activated bispecific T-cell activating molecule comprising: (a) a first antigen-binding fragment capable of binding to CD3; (b) a second antigen-binding fragment capable of binding to an antigen of a target cell; and (c) a masking moiety covalently attached to the bispecific T cell activating molecule via a peptide linker, wherein the masking moiety is capable of binding to an idiotype of the first or second antigen-binding moiety, thereby reversibly masking the first or second antigen-binding moiety, wherein the linker comprises the protease recognition sequence XQARK (SEQ ID NO: 39), where X is histidine (H) or proline (P).

2. The protease-activated bispecific T cell activating molecule of claim 1, wherein the masking moiety is covalently attached to the first antigen-binding moiety and reversibly masks the first antigen-binding moiety.

3. The protease-activated bispecific T-cell activating molecule of claim 1 or 2, wherein the masking moiety is covalently attached to the heavy chain variable region of the first antigen-binding moiety.

4. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 3, wherein the masking fragment is an scFv.

5. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 4, wherein (i) the second antigen-binding fragment is a standard Fab molecule or (ii) the second antigen-binding fragment is a crossover Fab molecule in which the variable or constant regions of a Fab light chain and a Fab heavy chain are exchanged.

6. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 5, wherein the first antigen-binding fragment is a standard Fab molecule.

7. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 6, comprising a third antigen-binding fragment which is a Fab molecule capable of binding to an antigen of a target cell.

8. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 7, wherein the third antigen-binding fragment is identical to the second antigen-binding fragment.

9. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 8, wherein the target cell antigen is FolR1.

10. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 9, wherein the first and second antigen-binding fragments are fused to each other, optionally via a peptide linker.

11. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 10, wherein the second antigen-binding fragment is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding fragment.

12. A protease-activated bispecific T cell activating molecule according to any one of claims 1-11, further comprising an Fc domain consisting of a first and a second subunit capable of stable association.

13. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 12, wherein the Fc domain is an Fc domain of IgG, in particular IgG1 or IgG4.

14. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 13, wherein the Fc domain exhibits reduced binding affinity for the Fc receptor and / or reduced effector function compared to the native IgG1 Fc domain.

15. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 14, wherein the antigen-binding fragment capable of binding to CD3 comprises a heavy chain variable region (VH): (a) the amino acid sequence of the heavy chain complementarity determining region (HCDR)1 of SYAMN (SEQ ID NO: 1); (b) the amino acid sequence of HCDR2 RIRSKYNNYATYYADSVKG (SEQ ID NO: 2); (c) the amino acid sequence HCDR3 ASNFPASYVSYFAY (SEQ ID NO: 3); and a light chain variable region (VL) containing: (d) the amino acid sequence of the light chain complementarity determining region (LCDR)1 GSSTGAVTTSNYAN (SEQ ID NO: 7); (d) the amino acid sequence of LCDR2 GTNKRAP (SEQ ID NO: 8); and (e) an LCDR3 amino acid sequence selected from ALWYSNLWV (SEQ ID NO: 9).

16. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 15, wherein the antigen-binding fragment capable of binding to CD3 comprises a VH region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, and / or a VL region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

10.

17. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 14, wherein the antigen-binding fragment capable of binding to CD3 comprises a heavy chain variable region (VH) comprising: (a) the amino acid sequence of the heavy chain complementarity determining region (HCDR)1 of SYAMN (SEQ ID NO: 1); (b) the amino acid sequence of HCDR2 RIRSKYNNYATYYADSVKG (SEQ ID NO: 2); (c) the amino acid sequence of HCDR3 HTTFPSSYVSYYGY (SEQ ID NO: 4); and a light chain variable region (VL) containing: (d) the amino acid sequence of the light chain complementarity determining region (LCDR)1 GSSTGAVTTSNYAN (SEQ ID NO: 7); (d) the amino acid sequence of LCDR2 GTNKRAP (SEQ ID NO: 8); and (e) an LCDR3 amino acid sequence selected from ALWYSNLWV (SEQ ID NO: 9).

18. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 15, wherein the antigen-binding fragment capable of binding to CD3 comprises a VH region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6, and / or a VL region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

10.

19. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 18, wherein the masking moiety comprises a VH region comprising: (a) the amino acid sequence of HCDR1 DYSMN (SEQ ID NO: 15), (b) an HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 16), WINTETGEPRYTDDFTG (SEQ ID NO: 17) and WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) the amino acid sequence of HCDR3 EGDYDVFDY (SEQ ID NO: 19); and the VL region containing: (d) the LCDR1 amino acid sequence RASKSVSTSSYSYMH (SEQ ID NO: 25) or KSSKSVSTSSYSYMH (SEQ ID NO: 26); (d) the amino acid sequence of LCDR2 YVSYLES (SEQ ID NO: 27); and (e) the amino acid sequence of LCDR3 QHSREFPYT (SEQ ID NO: 28) or QQSREFPYT (SEQ ID NO: 29).

20. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 19, wherein the masking moiety comprises a VH region comprising: (a) the amino acid sequence of HCDR1 DYSMN (SEQ ID NO: 15); (b) the amino acid sequence HCDR2 WINTETGEPRYTDDFKG (SEQ ID NO: 16); (c) the amino acid sequence of HCDR3 EGDYDVFDY (SEQ ID NO: 19); and the VL region containing: (d) the amino acid sequence of LCDR1 RASKSVSTSSYSYMH (SEQ ID NO: 25); (d) the amino acid sequence of LCDR2 YVSYLES (SEQ ID NO: 27); and (e) the amino acid sequence of LCDR3 QHSREFPYT (SEQ ID NO: 28).

21. A protease-activated bispecific T cell activating molecule according to any one of claims 1 to 19, wherein the masking moiety comprises a VH region comprising: (a) the amino acid sequence of HCDR1 DYSMN (SEQ ID NO: 15); (b) the amino acid sequence HCDR2 WINTETGEPRYTDDFKG (SEQ ID NO: 16); (c) the amino acid sequence of HCDR3 EGDYDVFDY (SEQ ID NO: 19); and the VL region containing: (d) the amino acid sequence of LCDR1 KSSKSVSTSSYSYMH (SEQ ID NO: 26); (d) the amino acid sequence of LCDR2 YVSYLES (SEQ ID NO: 27); and (e) the amino acid sequence of LCDR3 QHSREFPYT (SEQ ID NO: 28).

22. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 19, wherein the masking moiety comprises a VH region comprising: (a) the amino acid sequence of HCDR1 DYSMN (SEQ ID NO: 15); (b) the amino acid sequence of HCDR2 WINTETGEPRYTDDFTG (SEQ ID NO: 17); (c) the amino acid sequence of HCDR3 EGDYDVFDY (SEQ ID NO: 19); and the VL region containing: (d) the amino acid sequence of LCDR1 KSSKSVSTSSYSYMH (SEQ ID NO: 26); (d) the amino acid sequence of LCDR2 YVSYLES (SEQ ID NO: 27); and (e) the amino acid sequence of LCDR3 QHSREFPYT (SEQ ID NO: 28).

23. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 19, wherein the masking moiety comprises a VH region comprising: (a) the amino acid sequence of HCDR1 DYSMN (SEQ ID NO: 15); (b) the amino acid sequence of HCDR2 WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) the amino acid sequence of HCDR3 EGDYDVFDY (SEQ ID NO: 19); and the VL region containing: (d) the amino acid sequence of LCDR1 KSSKSVSTSSYSYMH (SEQ ID NO: 26); (d) the amino acid sequence of LCDR2 YVSYLES (SEQ ID NO: 27); and (e) the amino acid sequence of LCDR3 QHSREFPYT (SEQ ID NO: 28).

24. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 19, wherein the masking moiety comprises a VH region comprising: (a) the amino acid sequence of HCDR1 DYSMN (SEQ ID NO: 15); (b) the amino acid sequence of HCDR2 WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) the amino acid sequence of HCDR3 EGDYDVFDY (SEQ ID NO: 19); and the VL region containing: (d) the amino acid sequence of LCDR1 RASKSVSTSSYSYMH (SEQ ID NO: 25); (d) the amino acid sequence of LCDR2 YVSYLES (SEQ ID NO: 27); and (e) the amino acid sequence of LCDR3 QQSREFPYT (SEQ ID NO: 29).

25. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 24, wherein the second antigen-binding fragment is capable of binding to FolR1 and comprises a VH region comprising: (a) the amino acid sequence of HCDR1 NAWMS (SEQ ID NO: 11); (b) the amino acid sequence of HCDR2 RIKSKTDGGTTDYAAPVKG (SEQ ID NO: 12); and (c) the amino acid sequence of HCDR3 PWEWSWYDY (SEQ ID NO: 13); and the VL region containing: (d) LCDR1 GSSTGAVTTSNYAN (SEQ ID NO: 7); (d) the amino acid sequence of LCDR2 GTNKRAP (SEQ ID NO: 8); and (e) the amino acid sequence of LCDR3 ALWYSNLWV (SEQ ID NO: 9).

26. The protease-activated bispecific T cell activating molecule of any one of claims 1 to 25, wherein the antigen-binding fragment capable of binding to FolR1 comprises a VH region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14, and / or a VL region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

10.

27. The protease-activatable bispecific T cell activating molecule of any one of claims 1 to 26, wherein the protease-cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41).

28. An idiotype-specific polypeptide for reversibly obscuring the CD3 antigen binding site of a molecule, wherein the idiotype-specific polypeptide is covalently attached to the molecule via a peptide linker, wherein the linker comprises the protease recognition sequence XQARK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P).

29. The idiotype-specific polypeptide of claim 28, wherein the idiotype-specific polypeptide is an anti-idiotypic scFv.

30. The idiotype-specific polypeptide of claim 29, wherein the molecule is a bispecific T-cell activating molecule.

31. The idiotype-specific polypeptide of claim 30, wherein the linker comprises a PQARK protease recognition sequence (SEQ ID NO: 41).

32. A pharmaceutical composition comprising a protease-activated bispecific T-cell activating molecule according to any one of claims 1-27 and a pharmaceutically acceptable carrier.

33. A pharmaceutical composition comprising an idiotype-specific polypeptide according to any one of claims 28-31 and a pharmaceutically acceptable carrier.

34. An isolated polynucleotide encoding a protease-activated bispecific antigen-binding T-cell activating molecule according to any one of claims 1-27.

35. An isolated polynucleotide encoding an idiotype-specific polypeptide according to any one of claims 28-31.

36. A vector, in particular an expression vector, containing the polynucleotide according to claim 34 or 35.

37. A host cell containing the vector according to claim 36.

38. A method for producing a protease-activated bispecific T-cell activating molecule, comprising the steps of a) culturing a host cell according to claim 37 under conditions suitable for expressing the protease-activated bispecific T-cell activating molecule, and b) isolating the protease-activated bispecific T-cell activating molecule.

39. A protease-activated bispecific T-cell activating molecule according to any one of claims 1-27 for use as a medicinal product.

40. A protease-activated bispecific T-cell activating molecule for use according to claim 39, wherein the medicament is for treating or slowing the progression of cancer, treating or slowing the progression of an immune-related disease, or enhancing or stimulating an immune response or immune function in an individual.

41. Use of a protease-activated bispecific T-cell activating molecule according to any one of claims 1-27 for the manufacture of a medicament for the treatment of a disease.

42. The use of a protease-activated bispecific T-cell activating molecule according to claim 41, wherein the disease is cancer.

43. A method of treating a disease in an individual comprising administering to said individual a therapeutically effective amount of a composition comprising a protease-activated bispecific T-cell activating molecule according to any one of claims 1-27.

44. The method of claim 43 for treating or slowing the progression of cancer.