Matrix metalloprotease-cleavable and serine protease-cleavable substrates and methods of use thereof
The invention relates generally to polypeptides that include at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP), to activatable antibodies and other larger molecules that include these polypeptides that include at least a CM1 that is a substrate for at least one MMP protease and at least a CM2 that is a substrate for at least one SP protease, and to methods of making and using these polypeptides that include at least a CM1 that is a substrate for at least one MMP protease and at least a CM2 that is a substrate for at least one SP protease in a variety of therapeutic, diagnostic and prophylactic indications.
1. An isolated polypeptide comprising a CM1-CM2 substrate comprising at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP), wherein the CM1-CM2 substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 22.
2. The isolated polypeptide of claim 1 , wherein the MMP is MMP2, MMP9, or MMP14.
3. The isolated polypeptide of claim 1 , wherein the SP is uPA or matriptase.
4. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 83, 449, 451, 472, and 474 and a heavy chain amino acid sequence comprising SEQ ID NO: 108.
5. The isolated polypeptide of claim 1 , wherein the CM1-CM2 substrate comprises the amino acid sequence of SEQ ID NO: 1.
6. The isolated polypeptide of claim 1 , wherein the CM1-CM2 substrate comprises the amino acid sequence of SEQ ID NO: 22.
7. An isolated polypeptide comprising an antibody or antigen binding fragment thereof (AB) that binds a target, and a CM1-CM2 substrate comprising at least a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and at least a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP), wherein the CM1-CM2 substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 22.
8. The isolated polypeptide of claim 7 , wherein the MMP, the SP, or both the MMP and the SP are co-localized in a tissue with the target.
9. The isolated polypeptide of claim 7 , wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab′) 2 fragment, a scFv, and a scAb.
10. The isolated polypeptide of claim 7 , wherein the AB is linked to the CM1.
11. The isolated polypeptide of claim 10 , wherein the AB is linked directly to the CM1.
12. The isolated polypeptide of claim 10 , wherein the AB is linked to the CM1 via a linking peptide.
13. The isolated polypeptide of claim 7 , wherein the AB is linked to CM2.
14. The isolated polypeptide of claim 13 , wherein the AB is linked directly to CM2.
15. The isolated polypeptide of claim 13 , wherein the AB is linked to the CM2 via a linking peptide.
16. The isolated polypeptide of claim 7 , wherein the MMP is MMP2, MMP9, or MMP14.
17. The isolated polypeptide of claim 7 , wherein the SP is uPA or matriptase.
18. The isolated polypeptide of claim 7 , wherein the isolated polypeptide comprises a masking moiety (MM).
19. The isolated polypeptide of claim 18 , wherein the MM has a dissociation constant for binding to the AB that is greater than the dissociation constant of the AB for binding to the target.
20. The isolated polypeptide of claim 18 , wherein the MM is a polypeptide of no more than 40 amino acids in length.
21. The isolated polypeptide of claim 18 , wherein the MM is linked to the CM1 such that the isolated polypeptide in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM-CM1-CM2-AB or AB-CM2-CM1-MM.
22. The isolated polypeptide of claim 21 , wherein the isolated polypeptide comprises a linking peptide between the MM and the CM1.
23. The isolated polypeptide of claim 21 , wherein the isolated polypeptide comprises a linking peptide between CM2 and the AB.
24. The isolated polypeptide of claim 18 , wherein the MM is linked to CM2 such that the isolated polypeptide in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM-CM2-CM1-AB or AB-CM1-CM2-MM.
25. The isolated polypeptide of claim 24 , wherein the isolated polypeptide comprises a linking peptide between the MM and CM2.
26. The isolated polypeptide of claim 24 , wherein the isolated polypeptide comprises a linking peptide between CM1 and the AB.
27. The isolated polypeptide of claim 18 , wherein the isolated polypeptide comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the isolated polypeptide has the structural arrangement from N-terminus to C-terminus as follows in the uncleaved state: MM-LP1-CM1-CM2-LP2-AB, AB-LP2-CM2-CM1-LP1-MM, MM-LP1-CM2-CM1-LP2-AB, or AB-LP2-CM1-CM2-LP1-MM.
28. The isolated polypeptide of claim 27 , wherein the two linking peptides need not be identical to each other.
29. The isolated polypeptide of claim 27 , wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.
30. The isolated polypeptide of claim 18 , wherein the amino acid sequence of the MM is different from that of the target and is no more than 10% identical to the amino acid sequence of a natural binding partner of the AB.
31. The isolated polypeptide of claim 18 , wherein the MM does not interfere or compete with the AB for binding to the target in a cleaved state.
32. The isolated polypeptide of claim 18 , wherein the SP is uPA or matriptase.
33. The isolated polypeptide of claim 18 , wherein the MMP is MMP2, MMP9, or MMP14.
34. The isolated polypeptide of claim 18 , wherein the CM1-CM2 substrate comprises the amino acid sequence of SEQ ID NO: 1.
35. The isolated polypeptide of claim 18 , wherein the CM1-CM2 substrate comprises the amino acid sequence of SEQ ID NO: 22.
36. The isolated polypeptide of claim 7 , wherein the isolated polypeptide comprises the light chain amino acid sequence of SEQ ID NO: 420, and a heavy chain amino acid sequence comprising SEQ ID NO: 67.
37. The isolated polypeptide of claim 7 , wherein the isolated polypeptide comprises a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 83, 449, 451, 472, and 474, and a heavy chain amino acid sequence comprising SEQ ID NO: 108.
38. A conjugated activatable antibody comprising the isolated polypeptide of claim 7 conjugated to an agent.
39. The conjugated activatable antibody of claim 38 , wherein the agent is conjugated to the AB via a linker.
40. The conjugated activatable antibody of claim 39 , wherein the linker is a cleavable linker.
41. The conjugated activatable antibody of claim 39 , wherein the linker is a non-cleavable linker.
42. The conjugated activatable antibody of claim 38 , wherein the agent is a toxin or fragment thereof.
43. The conjugated activatable antibody of claim 38 , wherein the agent is a microtubule inhibitor.
44. The conjugated activatable antibody of claim 38 , wherein the agent is a nucleic acid damaging agent.
45. The conjugated activatable antibody of claim 38 , wherein the agent is selected from the group consisting of a dolastatin, an auristatin, a maytansinoid, a duocarmycin, and a calicheamicin.
46. The conjugated activatable antibody of claim 45 , wherein the agent is auristatin E.
47. The conjugated activatable antibody of claim 45 , wherein the agent is monomethyl auristatin E (MMAE).
48. The conjugated activatable antibody of claim 45 , wherein the agent is monomethyl auristatin D (MMAD).
49. The conjugated activatable antibody of claim 45 , wherein the agent is a maytansinoid selected from the group consisting of DM1 and DM4.
50. The conjugated activatable antibody of claim 38 , wherein the agent is a detectable moiety.
51. The conjugated activatable antibody of claim 50 , wherein the detectable moiety is a diagnostic agent.
52. The isolated polypeptide of claim 38 , wherein the CM1-CM2 substrate comprises the amino acid sequence of SEQ ID NO: 1.
53. The isolated polypeptide of claim 38 , wherein the CM1-CM2 substrate comprises the amino acid sequence of SEQ ID NO: 22.
54. A pharmaceutical composition comprising the isolated polypeptide of claim 7 and a carrier.
55. The pharmaceutical composition of claim 54 comprising an additional agent.
56. The pharmaceutical composition of claim 55 , wherein the additional agent is a therapeutic agent.
57. The isolated polypeptide of claim 7 , wherein the CM1-CM2 substrate comprises the amino acid sequence of SEQ ID NO: 1.
58. The isolated polypeptide of claim 7 , wherein the CM1-CM2 substrate comprises the amino acid sequence of SEQ ID NO: 22.