Immunoconjugates comprising anti-HER2 antibodies and pyrrolobenzodiazepines

1. An immunoconjugate comprising an antibody and a cytotoxic agent, wherein the immunoconjugate has the formula Ab-(L-D)p, wherein:

(a) Ab is the antibody;

(b) L is a linker;

(c) D is a cytotoxic agent; and

(d) p ranges from 1-8;

and wherein D is a pyrrolobenzodiazepine and L-D is of Formula A:

wherein:

R 2 is

where R 36a and R 36b are independently selected from H, F, C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C 1-4 alkyl amido and C 1-4 alkyl ester; or, when one of R 36a and R 36b is H, the other is selected from nitrile and a C 1-4 alkyl ester;

R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, NO 2 , Me 3 Sn and halo;

R 7 is independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, NO 2 , Me 3 Sn and halo;

Y has the formula:

G is a linker connected to the antibody;

n is an integer selected in the range of 0 to 48;

either:

(a) R 10 is H, and R 11 is OH, OR A , where R A is C 1-4 alkyl; or

(b) R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or

(c) R 10 is H and R 11 is OSO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;

R and R′ are each independently selected from optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups, and optionally in relation to the group NRR′, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

R 16 , R 17 , R 19 , R 20 , R 21 and R 22 are as defined for R 6 , R 7 , R 9 , R 10 , R 11 and R 2 respectively;

Z is CH or N;

T and T″ are independently selected from a single bond or a C 1-9 alkylene, which chain may be interrupted by one or more heteroatoms, e.g., O, S, N(H), NMe, provided that the number of atoms in the shortest chain of atoms between X and X′ is 3 to 12 atoms; and

X and X′ are independently selected from O, S and N(H);

and wherein the antibody is an antibody that binds HER2 comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:15; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:16; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:13; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

2. The immunoconjugate of claim 1 , wherein the antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 11 and a light chain variable region comprising the sequence of SEQ ID NO: 10.

3. The immunoconjugate of claim 1 , wherein the antibody is a monoclonal antibody.

4. The immunoconjugate of claim 3 , wherein the antibody is a humanized or chimeric antibody.

5. The immunoconjugate of claim 1 , wherein the antibody is an antibody fragment that binds HER2.

6. The immunoconjugate of claim 1 , wherein HER2 is human HER2 comprising amino acids 23 to 1255 of SEQ ID NO: 1.

7. The immunoconjugate of claim 1 , wherein the antibody binds to extracellular domain I of HER2.

8. The immunoconjugate of claim 7 , wherein extracellular domain I of HER2 has the sequence of SEQ ID NO: 35.

9. The immunoconjugate of claim 4 , wherein the antibody is an IgG1, IgG2a or IgG2b antibody.

10. The immunoconjugate of claim 1 , wherein the antibody comprises one or more engineered free cysteine amino acids residues.

11. The immunoconjugate of claim 10 , wherein the one or more engineered free cysteine amino acids residues are located in the heavy chain.

12. The immunoconjugate of claim 10 , wherein the one or more engineered free cysteine amino acids residues are located in the light chain.

13. The immunoconjugate of claim 11 , wherein the antibody comprises at least one mutation in the heavy chain constant region selected from A118C and S400C.

14. The immunoconjugate of claim 12 , wherein the antibody comprises at least one mutation in the light chain constant region selected from K149C and V205C.

15. The immunoconjugate of claim 1 , wherein the antibody comprises:

a) a heavy chain comprising the sequence of SEQ ID NO: 19 and a light chain comprising the sequence of SEQ ID NO: 18; or

b) a heavy chain comprising the sequence of SEQ ID NO: 19 and a light chain comprising the sequence of SEQ ID NO: 23; or

c) a heavy chain comprising the sequence of SEQ ID NO: 24 and a light chain comprising the sequence of SEQ ID NO: 18.

16. The immunoconjugate of claim 1 , wherein the antibody comprises the heavy chain constant region of SEQ ID NO: 28.

17. The immunoconjugate of claim 1 , wherein the antibody comprises the light chain constant region of SEQ ID NO: 25.

18. The immunoconjugate of claim 1 , wherein R 9 is H and R 19 is H.

19. The immunoconjugate of claim 1 , wherein R 6 is H and R 16 is H.

20. The immunoconjugate of claim 1 , wherein R 7 is OR and R 17 is OR, where each R is optionally substituted C 1-4 alkyl.

21. The immunoconjugate of claim 20 , wherein each R is Me.

22. The immunoconjugate of claim 1 , wherein X is O and X′ is O.

23. The immunoconjugate of claim 1 , wherein T is a single bond or is selected from a C 1 alkylene group and a C 2 alkylene group and wherein T′ is a single bond or is selected from a C 1 alkylene group and a C 2 alkylene group.

24. The immunoconjugate of claim 1 , wherein T is a C 1 alkylene group and T′ is a C 1 alkylene group.

25. The immunoconjugate of claim 1 , wherein R 36a and R 36b are both H.

26. The immunoconjugate of claim 1 , wherein R 36a and R 36b are both methyl.

27. The immunoconjugate of claim 1 , wherein one of R 36a and R 36b is H, and the other is selected from C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.

28. The immunoconjugate of claim 1 , wherein one of R 36a and R 36b is H, and the other is selected from methyl and ethyl.

29. The immunoconjugate of claim 1 , wherein R 10 is H, R 11 is OH, R 20 is H, and R 21 is OH.

30. The immunoconjugate of claim 1 , wherein R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, and wherein R 20 and R 21 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.

31. The immunoconjugate of claim 1 , wherein R 16 , R 17 , R 19 , R 20 , R 21 , R 22 , X′ and T′ are the same as R 6 , R 7 , R 9 , R 10 , R 11 , R 2 , X and T, respectively.

32. The immunoconjugate of claim 1 , comprising the structure:

33. The immunoconjugate of claim 32 , comprising the structure:

34. The immunoconjugate of claim 1 , wherein p ranges from 1.3-2 or from 2-5.

35. A pharmaceutical formulation comprising the immunoconjugate of claim 1 and a pharmaceutically acceptable carrier.

36. The pharmaceutical formulation of claim 35 , further comprising an additional therapeutic agent.

37. The pharmaceutical formulation of claim 36 , wherein the additional therapeutic agent is an antibody or immunoconjugate that binds HER2.

38. The pharmaceutical formulation of claim 37 , wherein the additional therapeutic agent is (i) an antibody or immunoconjugate that binds to domain II of HER2, and/or (ii) an antibody or immunoconjugate that binds to domain IV or HER2.

39. The pharmaceutical formulation of claim 38 , wherein the additional therapeutic agent is (i) an antibody or immunoconjugate that binds to epitope 2C4, and/or (ii) an antibody or immunoconjugate that binds to epitope 4D5.

40. The pharmaceutical formulation of claim 36 , wherein the additional therapeutic agent is trastuzumab, trastuzumab-MCC-DM1 (T-DM1), and/or pertuzumab.

41. The pharmaceutical formulation of claim 36 , further comprising (1) trastuzumab or T-DM1, and (2) pertuzumab.

42. A method of treating an individual having a HER2-positive cancer, the method comprising administering to the individual an effective amount of the immunoconjugate of claim 1 .

43. The method of claim 42 , wherein the HER2-positive cancer is breast cancer or gastric cancer.

44. The method of claim 43 , wherein the HER2-positive breast cancer is early-stage breast cancer.

45. The method of claim 43 , wherein the HER2-positive breast cancer is metastatic breast cancer.

46. The method of claim 42 , further comprising administering an additional therapeutic agent to the individual.

47. A method of treating an individual having a HER2-positive cancer, the method comprising administering to the individual an effective amount of the immunoconjugate of claim 1 and at least one additional therapeutic agent to the individual.

48. The method of claim 47 , wherein the additional therapeutic agent is an antibody or immunoconjugate that binds HER2.

49. The method of claim 48 , wherein the additional therapeutic agent is (i) an antibody or immunoconjugate that binds to domain II of HER2, and/or (ii) an antibody or immunoconjugate that binds to domain IV or HER2.

50. The method of claim 49 , wherein the additional therapeutic agent is (i) an antibody or immunoconjugate that binds to epitope 2C4, and/or (ii) an antibody or immunoconjugate that binds to epitope 4D5.

51. The method of claim 47 , wherein the additional therapeutic agent is selected from trastuzumab, trastuzumab-MCC-DM1 (T-DM1), and pertuzumab.

52. The method of claim 47 , wherein the additional therapeutic agents are (1) trastuzumab or T-DM1, and (2) pertuzumab.

53. The method of claim 47 , wherein the HER2-positive cancer is breast cancer or gastric cancer.

54. The method of claim 53 , wherein the HER2-positive breast cancer is metastatic breast cancer.

55. The method of claim 53 , wherein the HER2-positive breast cancer is early-stage breast cancer.

56. The method of claim 42 , wherein the HER2-positive cancer is recurrent cancer.

57. The method of claim 56 , wherein the recurrent cancer is locally recurrent cancer.

58. The method of claim 42 , wherein the HER2-positive cancer is advanced cancer.

59. The method of claim 42 , wherein the HER2-positive cancer is non-resectable.

60. A method of treating an individual having a HER2-positive cancer, comprising:

a) subjecting the individual to neoadjuvant treatment with the immunoconjugate claim 1 ,

b) removing the cancer by definitive surgery, and

c) subjecting the individual to adjuvant treatment with the immunoconjugate of claim 1 .

61. The method of claim 60 , wherein the HER2-positive cancer is breast cancer or gastric cancer.

62. The method of claim 61 , wherein the HER2-positive cancer is breast cancer.

63. A method of inhibiting proliferation of a HER2-positive cell, the method comprising exposing the cell to the immunoconjugate of claim 1 under conditions permissive for binding of the immunoconjugate to HER2 on the surface of the cell, thereby inhibiting proliferation of the cell.

64. The method of claim 63 , wherein the cell is a breast cancer cell or a gastric cancer cell.