Il-2 conjugates and methods of use to treat autoimmune diseases

Abstract

This article discloses compositions, kits, and methods comprising interleukin (IL) conjugates (e.g., IL-2 conjugates) for the treatment of one or more indications. Pharmaceutical compositions and kits comprising one or more of these interleukin conjugates (e.g., IL-2 conjugates) are also described herein.

Description

Technical field

Prior technology

Content of invention

Implementation

Claims

1. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structure of formula (I): (I) wherein: Z is CH2 and Y is; Y is CH2 and Z is; Z is CH2 and Y is; or Y is CH2 and Z is; W is a PEG group with a molecular weight of about 50 kDa; X has the following structure: ; X-1 refers to the point connected to the preceding amino acid residue; X+1 refers to the point connected to the following amino acid residue; and the at least one amino acid residue substituted by the structure of formula (I) is located in the IL-2 polypeptide at a position selected from K8, H15, L18, D19, N25, N87 and N118, wherein the residue position corresponds to positions 8, 15, 18, 19, 25, 87 and 118 as described in SEQ ID NO:

3.

2. The IL-2 conjugate of claim 1, wherein the IL-2 polypeptide is composed of the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue of formula (I) is located in the IL-2 polypeptide at a position selected from K8, H15, L18, D19, N25, N87 and N118.

3. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structural substitution of formula (I): (I) Wherein: Z is CH2 and Y is; Y is CH2 and Z is; Z is CH2 and Y is; or Y is CH2 and Z is; W is a PEG group with a molecular weight of about 50 kDa; X has the following structure: ; X-1 refers to the point connected to the preceding amino acid residue; X+1 refers to the point connected to the following amino acid residue; and the at least one amino acid residue that is substituted by the structure of formula (I) is located in the IL-2 polypeptide at a position selected from K9, H16, L19, D20, N26, N88 and N119, wherein the residue position corresponds to positions 9, 16, 19, 20, 26, 88 and 119 as described in SEQ ID NO:

4.

4. The IL-2 conjugate of claim 3, wherein the IL-2 polypeptide is composed of the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid residue of formula (I) is located in the IL-2 polypeptide at a position selected from K9, H16, L19, D20, N26, N88 and N119.

5. An IL-2 conjugate as claimed in any of claims 1 to 4, wherein Z is CH2 and Y is CH2.

6. An IL-2 conjugate as claimed in any of claims 1 to 4, wherein Y is CH2 and Z is CH2.

7. An IL-2 conjugate as claimed in any of claims 1 to 4, wherein Z is CH2 and Y is CH2.

8. An IL-2 conjugate as claimed in any of claims 1 to 4, wherein Y is CH2 and Z is CH2.

9. The IL-2 junction as requested in item 3 or 4, wherein the position is K9.

10. The IL-2 junction as claimed in claim 3 or 4, wherein the position is H16.

11. The IL-2 junction as requested in item 3 or 4, wherein the position is L19.

12. The IL-2 junction as requested in item 3 or 4, wherein the position is D20.

13. The IL-2 junction as claimed in claim 3 or 4, wherein the position is N26.

14. The IL-2 junction as requested in item 3 or 4, wherein the position is N88.

15. The IL-2 junction as claimed in claim 3 or 4, wherein the position is N119.

16. The IL-2 junction of request item 1 or 2, wherein the position is K8.

17. The IL-2 junction of claim 1 or 2, wherein the position is H15.

18. The IL-2 junction of claim 1 or 2, wherein the position is L18.

19. The IL-2 junction of claim 1 or 2, wherein the position is D19.

20. The IL-2 junction as claimed in claim 1 or 2, wherein the position is N25.

21. The IL-2 junction of claim 1 or 2, wherein the position is N87.

22. The IL-2 junction of claim 1 or 2, wherein the position is N118.

23. An IL-2 conjugate comprising an amino acid sequence comprising any one of SEQ ID NOS: 49-52, 54, 55, 57, 214-217, 219, 220, and 222, wherein [AzK_PEG50kDa] has the structure of formula (II) or formula (III): (II) (III) Wherein: W represents a PEG group with a molecular weight of approximately 50 kDa; X represents the following structure: ; X-1 refers to the point where it is attached to the preceding amino acid residue; and X+1 refers to the point where it is attached to the following amino acid residue.

24. A pharmaceutical composition comprising a mixture of IL-2 conjugates, wherein the mixture comprises (i) an IL-2 conjugate having an amino acid sequence of any one of SEQ ID NOS: 49-52, 54, 55, 57, 214-217, 219, 220 and 222 having the structure of formula (II), and (ii) an IL-2 conjugate having an amino acid sequence of any one of SEQ ID NOS: 49-52, 54, 55, 57, 214-217, 219, 220 and 222 having the structure of formula (III): (II) (III) Wherein: W represents a PEG group with a molecular weight of approximately 50 kDa; X represents the following structure: ; X-1 refers to the point where it is attached to the preceding amino acid residue; and X+1 refers to the point where it is attached to the following amino acid residue.

25. An IL-2 conjugate comprising an amino acid sequence comprising any one of SEQ ID NOS: 124-127, 129, 130, 132, 169-172, 174, 175 and 177, wherein [AzK_L1_PEG50kDa] has the structure of formula (IV) or formula (V): (IV) (V) Wherein: W represents a PEG group with a molecular weight of approximately 50 kDa; X represents the following structure: ; X-1 refers to the point where it is attached to the preceding amino acid residue; and X+1 refers to the point where it is attached to the following amino acid residue.

26. The IL-2 conjugate of claim 25, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO:

170.

27. The IL-2 conjugate of claim 25, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO:

125.

28. A pharmaceutical composition comprising a mixture of IL-2 conjugates, wherein the mixture comprises (i) an IL-2 conjugate having an amino acid sequence of any one of SEQ ID NOS: 124-127, 129, 130, 132, 169-172, 174, 175 and 177 having the structure of formula (IV), and (ii) an IL-2 conjugate having an amino acid sequence of any one of SEQ ID NOS: 124-127, 129, 130, 132, 169-172, 174, 175 and 177 having the structure of formula (V): (IV) (V) Wherein: W represents a PEG group with a molecular weight of approximately 50 kDa; X represents the following structure: ; X-1 refers to the point where it is attached to the preceding amino acid residue; and X+1 refers to the point where it is attached to the following amino acid residue.

29. The pharmaceutical composition of claim 28, wherein each of the IL-2 conjugates comprises the amino acid sequence of SEQ ID NO:

170.

30. The pharmaceutical composition of claim 28, wherein each of the IL-2 conjugates comprises the amino acid sequence of SEQ ID NO:

125.

31. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structural substitution of formula (VI) or formula (VII): (VI) (VII) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of about 50 kDa; X has the following structure: X-1 refers to the point connected to the preceding amino acid residue; X+1 refers to the point connected to the following amino acid residue; and the at least one amino acid residue substituted by the structure of formula (VI) or formula (VII) is located in the IL-2 polypeptide at a position selected from K8, H15, L18, D19, N25, N87 and N118, wherein the residue position corresponds to positions 8, 15, 18, 19, 25, 87 and 118 as described in SEQ ID NO:

3.

32. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structure of formula (VIII) or formula (IX): (VIII) (IX) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of about 50 kDa; X has the following structure: X-1 refers to the point connected to the preceding amino acid residue; X+1 refers to the point connected to the following amino acid residue; and the at least one amino acid residue substituted by the structure of formula (VIII) or formula (IX) is located in the IL-2 polypeptide at a position selected from K8, H15, L18, D19, N25, N87 and N118, wherein the residue position corresponds to positions 8, 15, 18, 19, 25, 87 and 118 as described in SEQ ID NO:

3.

33. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structural substitution of formula (X) or formula (XI): (X) (XI) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of about 50 kDa; the wavy line refers to the covalent bond connecting the first amino acid residue before the at least one amino acid residue in SEQ ID NO:3 and the first amino acid residue after the at least one amino acid residue; and the at least one amino acid residue substituted by the structure of formula (X) or formula (XI) is located in the IL-2 polypeptide at a position selected from K8, H15, L18, D19, N25, N87 and N118, wherein the residue position corresponds to positions 8, 15, 18, 19, 25, 87 and 118 as described in SEQ ID NO:

3.

34. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structural substitution of formula (XII) or formula (XIII): (XII) (XIII) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of approximately 50 kDa; the wavy lines refer to the covalent bonds respectively connected to the first amino acid residue before and the first amino acid residue after the at least one amino acid residue in SEQ ID NO:3; and the at least one amino acid residue substituted in the structure of formula (XII) or formula (XIII) is located in the IL-2 polypeptide at positions selected from K8, H15, L18, D19, N25, N87 and N118, wherein the residue positions correspond to positions 8, 15, 18, 19, 25, 87 and 118 as described in SEQ ID NO:

3.

35. An IL-2 conjugate of any one of claims 31 to 34, wherein the IL-2 polypeptide is composed of the amino acid sequence of SEQ ID NO: 3, wherein the position is selected from K8, H15, L18, D19, N25, N87 and N118.

36. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structural substitution of formula (VI) or formula (VII): (VI) (VII) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of about 50 kDa; X has the following structure: X-1 refers to the point connected to the preceding amino acid residue; X+1 refers to the point connected to the following amino acid residue; and the at least one amino acid residue substituted by the structure of formula (VI) or formula (VII) is located in the IL-2 polypeptide at a position selected from K9, H16, L19, D20, N26, N88 and N119, wherein the residue position corresponds to positions 9, 16, 19, 20, 26, 88 and 119 as described in SEQ ID NO:

4.

37. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structure of formula (VIII) or formula (IX): (VIII) (IX) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of about 50 kDa; X has the following structure: X-1 refers to the point connected to the preceding amino acid residue; X+1 refers to the point connected to the following amino acid residue; and the at least one amino acid residue substituted by the structure of formula (VIII) or formula (IX) is located in the IL-2 polypeptide at a position selected from K9, H16, L19, D20, N26, N88 and N119, wherein the residue position corresponds to positions 9, 16, 19, 20, 26, 88 and 119 as described in SEQ ID NO:

4.

38. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structural substitution of formula (X) or formula (XI): (X) (XI) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of approximately 50 kDa; the wavy lines refer to the covalent bonds connecting the first amino acid residue before and the first amino acid residue after the at least one amino acid residue in SEQ ID NO: 4; and the at least one amino acid residue substituted with the structure of formula (X) or formula (XI) is located in the IL-2 polypeptide at a position selected from K9, H16, L19, D20, N26, N88 and N119, wherein the residue positions correspond to positions 9, 16, 19, 20, 26, 88 and 119 as described in SEQ ID NO:

4.

39. An IL-2 conjugate comprising an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 polypeptide is substituted by a structural substitution of formula (XII) or formula (XIII): (XII) (XIII) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of approximately 50 kDa; the wavy lines refer to the covalent bonds respectively connected to the first amino acid residue before and the first amino acid residue after the at least one amino acid residue in SEQ ID NO: 4; and the at least one amino acid residue substituted in the structure of formula (XII) or formula (XIII) is located in the IL-2 polypeptide at a position selected from K9, H16, L19, D20, N26, N88 and N119, wherein the residue positions correspond to positions 9, 16, 19, 20, 26, 88 and 119 as described in SEQ ID NO:

4.

40. The IL-2 conjugate of any one of claims 36 to 39, wherein the IL-2 polypeptide is composed of the amino acid sequence of SEQ ID NO: 4, wherein the position is selected from K9, H16, L19, D20, N26, N88, E100 and N119.

41. Use of an IL-2 conjugate as claimed in any one of claims 1 to 23, 25 to 27 and 31 to 40 or a pharmaceutical composition as claimed in any one of claims 24 and 28 to 30 for the preparation of a medicament for treating autoimmune diseases in subjects in need.

42. As claimed in claim 41, wherein the autoimmune disease is selected from the following: graft-versus-host disease (GVHD), atopic dermatitis, Crohn's disease, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes mellitus, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cholangitis, primary biliary cirrhosis, non-alcoholic fatty liver disease (NASH), psoriasis, rheumatoid arthritis, scleroderma, CREST syndrome, Sjögren's syndrome. Syndrome), systemic lupus erythematosus, thyroiditis, uveitis, leukoplakia, Wergener's granulomatosis, Addison's disease (adrenal insufficiency), Hashimoto's thyroiditis, autoimmune hepatitis, infertility, ANCA-associated vasculitis, psoriatic arthritis, celiac disease, ulcerative colitis, lichen sclerosus, and Behcet's disease.

43. As claimed in claim 42, wherein the autoimmune disease is juvenile / childhood type 1 diabetes.

44. As claimed in claim 41 or 42, wherein the subject exhibits an increased concentration of rheumatoid factor in the subject's blood sample prior to administration of a therapeutically effective amount of the IL-2 conjugate or pharmaceutical composition to the subject.

45. Use of an IL-2 conjugate as claimed in any one of claims 1 to 23, 25 to 27 and 31 to 40, or a pharmaceutical composition as claimed in any one of claims 24 and 28 to 30, for the preparation of a medicament for treating rheumatoid arthritis in a subject with this need, wherein the treatment comprises: (a) Determine the concentration of rheumatoid factor in the subject's blood sample; (b) If the concentration of rheumatoid factor in the blood sample of the subject is greater than about 14 IU / mL, the subject shall be given a therapeutically effective amount of the IL-2 conjugate or the pharmaceutical composition.

46. ​​As requested in claim 41 or 42, wherein, prior to administering a therapeutically effective amount of the IL-2 conjugate or the pharmaceutical composition to the subject, the subject exhibits an abnormal erythrocyte sedimentation rate (ESR) as determined by the Westergren rate method or the Wintrobe rate method, if necessary.

47. Use of an IL-2 conjugate as claimed in any one of claims 1 to 23, 25 to 27 and 31 to 40, or a pharmaceutical composition as claimed in any one of claims 24 and 28 to 30, for the preparation of a medicament for treating an autoimmune disease in a subject with the need, wherein the treatment comprises: (a) Measure the erythrocyte sedimentation rate (ESR) in the blood sample of the subject; and (b) if the ESR in the blood sample of the subject is measured to be abnormal, administer a therapeutically effective amount of the IL-2 conjugate or the pharmaceutical composition to the subject.

48. Use of an IL-2 conjugate as claimed in any one of claims 1 to 23, 25 to 27 and 31 to 40, or a pharmaceutical composition as claimed in any one of claims 24 and 28 to 30, for the preparation of a medicament for treating an autoimmune disease in a subject with the need, wherein the treatment comprises: (a) Determine the concentration of C-reactive protein (CRP) in the subject's blood sample; (b) If the concentration of C-reactive protein (CRP) in the subject's blood sample is found to be abnormal, the subject shall be given a therapeutically effective amount of the IL-2 conjugate or the pharmaceutical composition.

49. As claimed in claim 48, wherein prior to administering a therapeutically effective amount of the IL-2 conjugate or the pharmaceutical composition to the subject, the subject exhibits a C-reactive protein (CRP) concentration greater than 10 mg / L in a blood sample.

50. Use of an IL-2 conjugate as claimed in any one of claims 1 to 23, 25 to 27 and 31 to 40, or a pharmaceutical composition as claimed in any one of claims 24 and 28 to 30, for the preparation of a medicament for treating rheumatoid arthritis in a subject with this need, wherein the treatment comprises: If the concentration of anti-cyclic citrullinated peptide (anti-CCP) in the subject's blood sample is found to be abnormal, the subject shall be administered a therapeutically effective amount of the IL-2 conjugate or the pharmaceutical composition.

51. Use of an IL-2 conjugate as claimed in any one of claims 1 to 23, 25 to 27 and 31 to 40, or a pharmaceutical composition as claimed in any one of claims 24 and 28 to 30, for the preparation of a medicament for treating rheumatoid arthritis in a subject with this need, wherein the treatment comprises: If the concentration of anti-cyclic amino peptide (anti-CCP) in the subject's blood sample is determined to be greater than about 20 IU / mL, the subject shall be given a therapeutically effective amount of the IL-2 conjugate or the pharmaceutical composition.

52. A method for manufacturing an IL-2 conjugate, the method comprising: An IL-2 polypeptide having the following structure of non-natural amino acids: , wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or 4, wherein at least one amino acid residue at position X is replaced by a non-natural amino acid, wherein position X is selected from amino acid positions K8, H15, L18, D19, N25, N87 and N118 in SEQ ID NO: 3, or position X is selected from amino acid positions K9, H16, L19, D20, N26, N88 and N119 in SEQ ID NO: 4, position X-1 refers to the position connected to the preceding amino acid residue, and position X+1 refers to the position connected to the following amino acid residue, and the wavy line refers to the covalent bond connected to the amino acid residues at positions X+1 and X-1 in SEQ ID NO: 3 or 4, respectively, reacts with mPEG-DBCO of the following formula or wherein n such that mPEG-DBCO comprises approximately 50 The IL-2 conjugate is generated by using mPEG with a molecular weight of kDa.

53. The method of claim 52, wherein position X is an amino acid position K8, L18, D19, N25, N87 or N118 within SEQ ID NO:

3.

54. The method of claim 52, wherein position X is an amino acid position K9, H16, L19, D20, N26, N88 or N119 within SEQ ID NO:

4.

55. The method of claim 52, wherein the IL-2 junction is an IL-2 junction of any one of claims 1 to 23, 25 to 27 and 31 to 40.

56. The IL-2 junction as claimed in claim 10, wherein: Z is CH2 and Y is CH2; or Y is CH2 and Z is CH2.

57. An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein a modified amino acid having the structure of formula (XII) or (XIII) is present at position 15 of SEQ ID NO: 3, substituted with histidine: (XII) (XIII) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of about 50 kDa; and the wavy lines refer to the covalent bonds respectively connected to the amino acid residues at position 14 and position 16 in SEQ ID NO:

3.

58. An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 80, wherein X in SEQ ID NO: 80 is N6-((2-azidoethoxy)-carbonyl)-L-lysine (N6-(2-azidoethoxy)-carbonyl-L-lysine, AzK), which is covalently linked to a PEG group having a molecular weight of about 50 kDa via a conjugate group comprising a dibenzocyclooctylene group.

59. The IL-2 conjugate of claim 58, wherein the AzK system covalently linked to the PEG group via the conjugating group is configured as formula (XII) or formula (XIII): (XII) (XIII) Wherein: n is an integer such that the PEG group having the structure -(OCH2CH2)n-OCH3 has a molecular weight of about 50 kDa; and the wavy lines refer to the covalent bonds respectively connected to the first amino acid residue before X and the first amino acid residue after X in SEQ ID NO:

80.

60. A pharmaceutical composition comprising a mixture of IL-2 conjugates as claimed in claim 59, wherein the mixture comprises (i) an IL-2 conjugate having the structure of formula (XII); and (ii) an IL-2 conjugate having the structure of formula (XIII).

61. The pharmaceutical composition of claim 60 further includes diluents, buffers, stabilizers, surfactants, or any combination thereof.

62. The pharmaceutical composition of claim 60 further includes sorbitol.

63. The pharmaceutical composition of claim 60 further comprises polysorbate-20.

64. A dispenser device comprising a pharmaceutical composition as claimed in claim 60.

65. A syringe comprising the pharmaceutical composition as claimed in claim 60.

66. A vial containing the pharmaceutical composition as claimed in claim 60.

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