ADAMTS-bound immunoglobulin

Abstract

To provide polypeptides for osteoarthritis (OA) that have improved prophylactic, therapeutic, and / or pharmacological properties. [Solution] A polypeptide comprising A disintegrin having a thrombospondin motif and at least one immunoglobulin monovariate domain (ISVD) that binds to metalloproteinase (ADAMTS) 5, wherein the ISVD that binds to ADAMTS 5 comprises three complementarity-determining regions (CDR1 to CDR3, respectively).

Claims

1. A polypeptide comprising A disintegrin having a thrombospongin motif and at least one immunoglobulin monovariate domain (ISVD) that binds to metalloproteinase (ADAMTS).

2. The polypeptide according to claim 1, wherein ADAMTS is selected from the group consisting of ADAMTS1 to ADAMTS19, preferably ADAMTS5, ADAMTS4, ADAMTS1, ADAMTS8, ADAMTS9, ADAMTS15, and ADAMTS20, and most preferably ADAMTS5.

3. The polypeptide according to claim 2, wherein the ISVD that binds to ADAMTS, preferably ADAMTS5, does not bind to ADAMTS4, MMP1, or MMP14.

4. The polypeptide according to claim 3, wherein the ISVD bound to ADAMTS5 includes three complementarity-determining regions (CDR1 to CDR3, respectively), wherein (i) CDR1 is sequence numbers 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; and Selected from the group consisting of amino acid sequences having one, two, or three amino acid differences from sequence numbers 21, 35, 20, 22, 25, 33, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32, and 34; (ii) CDR2 is sequence numbers 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and Selected from the group consisting of amino acid sequences having one, two, or three amino acid differences from sequence numbers 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49, and 52; and (iii) CDR3 is sequence numbers 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70; and The polypeptide is selected from the group consisting of amino acid sequences having one, two, three or four amino acid differences from sequence numbers 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67, and 70.

5. The polypeptide according to claim 3, wherein the ISVD that specifically binds to ADAMTS5 comprises three complementarity-determining regions (CDR1 to CDR3, respectively), (i) CDR1 is selected from the following group: (a) Sequence ID No. 22; and (b) an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid differences from sequence number 22, where amino acid differences are defined as follows: - At position 2, S is changed to R; and / or - At position 3, A is changed to T; and / or - At position 4, V is changed to F; and / or - At position 6, V is changed to S; and / or - At position 7, N is changed to Y; and / or -At position 10, A is changed to G; (ii) CDR2 is sequence number 36; and (iii) CDR3 is sequence number 54. The aforementioned polypeptide.

6. The polypeptide according to claim 3, wherein the ISVD that specifically binds to ADAMTS5 comprises three complementarity-determining regions (CDR1 to CDR3, respectively), (i) CDR1 is sequence number 33; (ii) CDR2 is selected from the following group: (c) Sequence ID 50; and (d) an amino acid sequence having one, two, or three amino acid differences from sequence number 50, where the amino acid differences are defined as follows: -At position 8, M is changed to I; and / or -At position 9, P is changed to T; and / or -At position 10, Y is changed to F; and (iii) CDR3 is selected from the following group: (e) Sequence ID 68; and (f) An amino acid sequence having one or two amino acid differences from sequence number 68, where the amino acid difference is defined as follows: - At position 5, F is changed to L; and / or - At position 11, D is changed to E; The aforementioned polypeptide.

7. The polypeptide according to claim 3, wherein the ISVD that specifically binds to ADAMTS5 comprises three complementarity-determining regions (CDR1 to CDR3, respectively), (i) CDR1 is sequence number 28; (ii) CDR2 is selected from the following group: (c) Sequence ID 44; and (d) an amino acid sequence having one, two, or three amino acid differences from sequence number 44, where the amino acid differences are defined as follows: -At position 3, S is changed to T; and / or - At position 4, R is changed to W; and / or -At position 8, T is changed to I; and / or -At position 9, T is changed to L; and (iii) CDR3 is selected from the following group: (e) Sequence ID 62; and (f) An amino acid sequence having one or two amino acid differences from sequence number 62, where the amino acid difference is defined as follows: - At position 1, G is changed to S; and / or - At position 14, D is changed to E; The aforementioned polypeptide.

8. A polypeptide according to any one of claims 1 to 7, -CDR1 is selected from the group consisting of sequence numbers 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32, and 34; -CDR2 is selected from the group consisting of sequence numbers 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and - CDR3 is selected from the group consisting of sequence numbers 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67, and 70; The aforementioned polypeptide.

9. A polypeptide according to any one of claims 1 to 7, wherein the ISVD comprises a combination of CDR1, CDR2, and CDR3 selected from the group consisting of: CDR1 is sequence number 21, CDR2 is sequence number 37, and CDR3 is sequence number 55; CDR1 is sequence number 35, CDR2 is sequence number 53, and CDR3 is sequence number 118; CDR1 is sequence number 35, CDR2 is sequence number 53, and CDR3 is sequence number 71; CDR1 is sequence number 20, CDR2 is sequence number 36, and CDR3 is sequence number 54; CDR1 is sequence number 22, CDR2 is sequence number 36, and CDR3 is sequence number 54; CDR1 is sequence number 25, CDR2 is sequence number 40, and CDR3 is sequence number 58; CDR1 is sequence number 33, CDR2 is sequence number 50, and CDR3 is sequence number 68; CDR1 is sequence number 33, CDR2 is sequence number 51, and CDR3 is sequence number 69; CDR1 is sequence number 28, CDR2 is sequence number 44, and CDR3 is sequence number 62; CDR1 is sequence number 28, CDR2 is sequence number 45, and CDR3 is sequence number 63; CDR1 is sequence number 28, CDR2 is sequence number 43, and CDR3 is sequence number 61; CDR1 is sequence number 24, CDR2 is sequence number 39, and CDR3 is sequence number 57; CDR1 is sequence number 23, CDR2 is sequence number 38, and CDR3 is sequence number 56; CDR1 is sequence number 26, CDR2 is sequence number 41, and CDR3 is sequence number 59; CDR1 is sequence number 27, CDR2 is sequence number 119, and CDR3 is sequence number 60; CDR1 is sequence number 27, CDR2 is sequence number 42, and CDR3 is sequence number 60; CDR1 is sequence number 29, CDR2 is sequence number 46, and CDR3 is sequence number 64; CDR1 is sequence number 30, CDR2 is sequence number 47, and CDR3 is sequence number 65; CDR1 is sequence number 31, CDR2 is sequence number 48, and CDR3 is sequence number 66; CDR1 is sequence number 32, CDR2 is sequence number 49, CDR3 is sequence number 67; and CDR1 is sequence number 34, CDR2 is sequence number 52, and CDR3 is sequence number 70; The aforementioned polypeptide.

10. The polypeptide according to any one of claims 1 to 9, wherein the ISVD comprises four framework regions (FR1 to FR4, respectively) and three complementarity determination regions CDR1, CDR2, and CDR3, or is essentially composed of these.

11. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide is sequence number 129 or 130, or a polypeptide having at least 95% sequence identity with sequence number 129 or 130.

12. The polypeptide according to any one of claims 4, 8, and 9, wherein CDR1 is sequence number 21, CDR2 is sequence number 37, and CDR3 is sequence number 55.

13. The polypeptide according to any one of claims 4 to 12, wherein ISVD is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15, and 18.

14. When the polypeptide is determined, for example, by KinExA or alternatively by Gyrolab, K Ｄ is 1E －０７ M to 1E －１３ M, for example 1E －０８ M to 1E －１２ M, preferably at most 1E －０７ M, preferably 1E －０８ M or 1E －０９ M less than, or 1E －１０ M less than, for example 5E －１１ M, 4E －１１ M, 3E －１１ M, 2E －１１ M, 1.7E －１１ M, 1E －１１ M etc., or 5E －１２ M, 4E －１２ M, 3E －１２ M, 1E －１２ M and binds to ADAMTS5, the polypeptide according to any one of claims 1 to 13.

15. If the polypeptide is determined by, for example, a human FRET assay or human AlphaLISA, IC ５０ ga 1E －０７ M-1E －１２ During M, for example, 1E －０８ M-1E －１１ A polypeptide according to any one of claims 1 to 14, which inhibits the activity of ADAMTS5 between M.

16. Polypeptides, IC ５０ up to 1E －０７ M, preferably 1E －０８ M, 5E －０９ M, or 4E －９ M, 3E －９ M, 2E －９ M, for example, 1E －９ The polypeptide according to claim 15, which inhibits the (enzymatic) activity of ADAMTS5, such as M.

17. When the polypeptide is determined, for example by binding ELISA, EC ５０ ga 1E －０７ M-1E －１２ During M, for example, 1E －０８ M-1E －１１ A polypeptide according to any one of claims 1 to 16, which adjusts ADAMTS5 between M.

18. If the polypeptide is determined, for example, by SPR, then the off-rate is 1E －０４ s －１ A polypeptide according to any one of claims 1 to 14, which binds to ADAMTS5 at a rate less than [value missing].

19. The polypeptide according to any one of claims 1 to 18, wherein ADAMTS5 is human ADAMTS5 (SEQ ID NO: 149), bovine ADAMTS5 (SEQ ID NO: 150), rat ADAMTS5 (SEQ ID NO: 151), guinea pig ADAMTS5 (SEQ ID NO: 152), mouse ADAMTS5 (SEQ ID NO: 153), or cynomolgus monkey ADAMTS5 (SEQ ID NO: 154), preferably human ADAMTS5, most preferably SEQ ID NO:

149.

20. The polypeptide according to any one of claims 1 to 19, wherein the polypeptide antagonizes the activity of ADAMTS5, for example, protease activity, for example, cleavage of aggrecan, versican, brevican, neurocan, decorin, and / or biglycan, preferably cleavage of aggrecan; preferably antagonizes the aggrecanase activity of ADAMTS5.

21. The polypeptide according to any one of claims 1 to 19, wherein the polypeptide blocks the binding of ADAMTS5 to aggrecan by at least 20%, for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, when determined, for example, by FRET, AlphaLISA, or ELISA.

22. The polypeptide according to any one of claims 1 to 20, wherein the polypeptide inhibits the protease activity of ADAMTS5, inhibiting the proteolysis of a substrate, preferably aggrecan, such as aggrecan, versican, brevican, neurocan, decorin, and / or biglycan.

23. The polypeptide according to any one of claims 1 to 22, comprising at least two ISVDs, wherein at least one ISVD is specifically bound to ADAMTS, preferably ADAMTS5, and preferably selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18.

24. The polypeptide according to claim 23, wherein at least two ISVDs specifically bind to ADAMTS, preferably to ADAMTS5.

25. A polypeptide comprising two or more ISVDs, each individually specifically bound to ADAMTS5, wherein a) At least the "first" ISVD specifically binds to the first antigenic determinant, epitope, portion, domain, subunit, or conformation of ADAMTS5; and here, b) At least the “second” ISVD specifically binds to a second antigenic determinant, epitope, partial, domain, subunit, or conformation of ADAMTS5, which is distinct from the first antigenic determinant, epitope, partial, domain, subunit, or conformation; The aforementioned polypeptide.

26. The polypeptide according to claim 25, wherein the "first" ISVD that specifically binds to ADAMTS5 is selected from the group consisting of SEQ ID NOs: 2, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15, and 18.

27. The polypeptide according to claim 25 or 26, wherein the "second" ISVD that specifically binds to ADAMTS5 is sequence number 118 or 19.

28. The polypeptide according to any one of claims 25 to 27, wherein the polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb), and SEQ ID NO: 128 (clone 131 9D3-093-Alb), or a polypeptide having at least 95% amino acid sequence identity with SEQ ID NO: 126, 127, 128, 130, or 131.

29. The polypeptide according to any one of claims 1 to 28, further comprising ISVD that binds to serum albumin.

30. The polypeptide according to claim 29, wherein the ISVD that binds to serum albumin essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), where CDR1 is sequence number 146, CDR2 is sequence number 147, and CDR3 is sequence number 148.

31. The polypeptide according to claim 30, wherein the ISVD that binds to serum albumin is selected from the group consisting of ALB8 (SEQ ID NO: 131), ALB23 (SEQ ID NO: 132), ALB129 (SEQ ID NO: 133), ALB132 (SEQ ID NO: 134), ALB11 (SEQ ID NO: 135), ALB11(S112K)-A (SEQ ID NO: 136), ALB82 (SEQ ID NO: 137), ALB82-A (SEQ ID NO: 138), ALB82-AA (SEQ ID NO: 139), ALB82-AAA (SEQ ID NO: 140), ALB82-G (SEQ ID NO: 141), ALB82-GG (SEQ ID NO: 142), ALB82-GGG (SEQ ID NO: 143), ALB92 (SEQ ID NO: 144), and ALB223 (SEQ ID NO: 145).

32. The polypeptide according to claim 31, wherein: Sequence ID 129 (Clone 577 2F3) ＳＯ -Alb), Sequence ID 130 (Clone 579 2F3) ＳＯ -093-Alb), Sequence ID 120 (Clone 4 2A12-Alb), Sequence ID 121 (Clone 5 2D7-Alb), Sequence ID 122 (Clone 6 2F3-Alb) Sequence ID 123 (Clone 69049-Alb), Sequence ID 124 (Clone 70 9D3-Alb), Sequence ID 125 (Clone 71 3B2-Alb), Sequence ID 126 (Clone 129 2F3-093-Alb), Sequence ID 127 (clone 130 049-093-Alb), and Sequence ID 128 (Clone 131 9D3-093-Alb) The polypeptide selected from the group consisting of the following:

33. The polypeptide according to any one of claims 1 to 32, further comprising at least one ISVD that specifically binds to aggrecan, preferably selected from the group consisting of SEQ ID NO: 156 (nanobody 00745 PEA114F08) and SEQ ID NO: 157 (nanobody 00747 PEA604F02).

34. The polypeptide according to any one of claims 1 to 32, comprising at least two ISVDs that specifically bind to aggrecan.

35. The polypeptide according to claim 34, wherein at least two ISVDs that specifically bind to aggrecan may be the same or different.

36. The polypeptide according to claim 34 or 35, wherein at least two ISVDs that specifically bind to aggrecan are independently selected from the group consisting of SEQ ID NOs: 156 and 157.

37. The polypeptide according to any one of claims 33 to 36, wherein the ISVD that specifically binds to aggrecan specifically binds to human aggrecan [SEQ ID NO: 155].

38. The polypeptide according to any one of claims 33 to 37, wherein the ISVD that specifically binds to aggrecan specifically binds to canine aggrecan, bovine aggrecan, rat aggrecan; porcine aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus monkey aggrecan and / or rhesus monkey aggrecan.

39. The polypeptide according to any one of claims 33 to 38, wherein ISVD, which specifically binds to aggrecan, preferably binds to cartilage and / or cartilaginous tissue such as the meniscus.

40. The polypeptide according to any one of claims 1 to 39, wherein the polypeptide is stable in synovial fluid (SF) at 37°C for at least 7 days, for example, 14 days, 21 days, 1 month, 2 months, or 3 months.

41. The polypeptide according to any one of claims 23 to 40, wherein at least two ISVDs are linked to each other directly or via a linker.

42. The polypeptide according to claim 41, wherein the linker is selected from the group consisting of SEQ ID NOs: 158 to 174, preferably SEQ ID NO:

169.

43. A polypeptide according to any one of claims 1 to 42, further comprising C-terminal elongation.

44. The polypeptide according to claim 43, wherein the C-terminal extension is C-terminal extension (X)n, where n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4 or 5 (and preferably 1 or 2, for example 1); and each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

45. The polypeptide according to any one of claims 1 to 44, wherein the polypeptide has at least 80%, 90%, 95%, or 100% sequence identity with any of sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 116, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130.

46. For example, a method for treating and / or preventing a disease or disorder of an individual in which ADAMTS5 activity is involved, the method comprising administering to the individual a polypeptide according to any one of claims 1 to 45 in an amount effective to treat or prevent the symptoms of the disease or disorder.

47. The method according to claim 46, wherein the disease or disorder is selected from the group consisting of arthropathy and chondrodysplasia, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment, chondrodysplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and agricanopathies.

48. A polypeptide according to any one of claims 1 to 45, for use as a pharmaceutical.

49. A polypeptide according to any one of claims 1 to 45 for use in the treatment or prevention of symptoms of ADAMTS5-related diseases, such as arthropathy and chondrodysplasia, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment, chondrodysplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and agricanopathies.

50. A polypeptide according to any one of claims 1 to 45, wherein the polypeptide cross-blocks the binding to ADAMTS5 of at least one polypeptide represented by any one of sequence numbers 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18, and / or the binding to ADAMTS5 is cross-blocked by at least one polypeptide represented by any one of sequence numbers 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18.

51. A polypeptide that cross-blocks the binding of a polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18 to ADAMTS5, and / or whose binding to ADAMTS5 is cross-blocked by at least one polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18, wherein the polypeptide comprises at least one VH, VL, dAb, immunoglobulin single variable domain (ISVD) that specifically binds to ADAMTS5, wherein the binding to ADAMTS5 modulates the activity of ADAMTS5.

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