Activatable binding polypeptides and methods of identification and use thereof
Activatable binding polypeptides (ABPs), which contain a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM) are provided. Activatable antibody compositions, which contain a TBM containing an antigen binding domain (ABD), a MM and a CM are provided. Furthermore, ABPs which contain a first TBM, a second TBM and a CM are provided. The ABPs exhibit an “activatable” conformation such that at least one of the TBMs is less accessible to target when uncleaved than after cleavage of the CM in the presence of a cleaving agent capable of cleaving the CM. Further provided are libraries of candidate ABPs, methods of screening to identify such ABPs, and methods of use. Further provided are ABPs having TBMs that bind VEGF, CTLA-4, or VCAM, ABPs having a first TBM that binds VEGF and a second TBM that binds FGF, as well as compositions and methods of use.
1. A method of manufacturing an enzyme activatable binding polypeptide (ABP), the method comprising:
(a) culturing a cell comprising a nucleic acid construct that encodes the ABP under conditions that lead to expression of the ABP, wherein the ABP comprises a masking moiety (MM), a cleavable moiety (CM), and an antigen binding domain (ABD) that specifically binds Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4),
(i) wherein the ABP in an uncleaved state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-ABD or ABD-CM-MM;
(ii) wherein the MM is a peptide that inhibits binding of the ABD to the target, and wherein the MM comprises an amino acid sequence selected from the group consisting of MILLCAAGRTWVEACANGR (SEQ ID NO:63), AERLCAWAGRFCGS (SEQ ID NO:65), WADVMPGSGVLPWTS (SEQ ID NO:67) and SDGRMGSLELCALWGRFCGS (SEQ ID NO:69); and
(iii) wherein, the CM is positioned in the ABP such that, in an uncleaved state, the MM interferes with specific binding of the ABD to CTLA-4, and in a cleaved state the MM does not interfere or compete with specific binding of the ABD to CTLA-4; and
(b) recovering the ABP.
2. The method of claim 1 , further comprising:
(c) testing the ABP for the ability to maintain an activatable phenotype while in soluble form.
3. The method of claim 1 , wherein the MM is a peptide of no more than about 40 amino acids in length.
4. The method of claim 1 , wherein the ABD comprises a Fab fragment, a scFv or a single chain antibody (SCAB).
5. The method of claim 1 , wherein the CM is a polypeptide that functions as a substrate for a protease that is co-localized in a tissue with the target, wherein the protease cleaves the CM in the ABP when the ABP is exposed to the protease.
6. The method of claim 1 , wherein the CM is a polypeptide of up to 15 amino acids in length.
7. The method of claim 1 , wherein the CM of the ABP in an uncleaved state is coupled to the N-terminus of the ABD.
8. The method of claim 7 , wherein the CM of the ABP in an uncleaved state is coupled to the N-terminus of a V L chain of the ABD.
9. The method of claim 1 , wherein the CM of the ABP in an uncleaved state is coupled to the C-terminus of the ABD.
10. The method of claim 1 , wherein the ABD is from ipilimumab or tremelimumab.
11. The method of claim 1 , wherein the CM is a substrate for an enzyme selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.
12. The method of claim 1 , wherein the CM is a substrate for an enzyme selected from the group consisting of an MMP and a CATHEPSIN.
13. The method of claim 1 , wherein the ABP comprises a linker peptide, wherein the linker peptide is positioned between the MM and the CM.
14. The method of claim 1 , wherein the ABP comprises a linker peptide, wherein the linker peptide is positioned between the ABD and the CM.
15. The method of claim 1 , wherein the ABP comprises a first linker peptide (L 1 ) and a second linker peptide (L 2 ), wherein the first linker peptide is positioned between the MM and the CM and the second linker peptide is positioned between the ABD and the CM.
16. The method of claim 15 , wherein each of L 1 and L 2 is a peptide of about 1 to 20 amino acids in length, and wherein each of L 1 and L 2 need not be the same linker.
17. The method of claim 15 , wherein one or both of L 1 and L 2 comprises a glycine-serine polymer.
18. The method of claim 15 , wherein at least one of L 1 and L 2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GSGGS) n (SEQ ID NO:1) and (GGGS) n (SEQ ID NO:2), where n is an integer of at least one.
19. The method of claim 15 , wherein at least one of L 1 and L 2 comprises an amino acid sequence having the formula (GGS) n , where n is an integer of at least one.
20. The method of claim 15 , wherein at least one of L 1 and L 2 comprises an amino acid sequence selected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO:3), Gly-Gly-Ser-Gly-Gly (SEQ ID NO:4), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:5), Gly-Ser-Gly-Gly-Gly (SEQ ID NO:6), Gly-Gly-Gly-Ser-Gly (SEQ ID NO:7), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO:8).
21. A method of manufacturing an enzyme activatable binding polypeptide (ABP), the method comprising:
(a) providing a masking moiety (MM), a cleavable moiety (CM), and an antibody or an antigen binding fragment thereof (ABD) that specifically binds Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), wherein the MM is a peptide that inhibits binding of the ABD to CTLA-4, and wherein the MM comprises an amino acid sequence selected from the group consisting of MILLCAAGRTWVEACANGR (SEQ ID NO:63), AERLCAWAGRFCGS (SEQ ID NO:65), WADVMPGSGVLPWTS (SEQ ID NO:67) and SDGRMGSLELCALWGRFCGS (SEQ ID NO:69); and
(b) coupling the MM to the CM and coupling the ABD to the CM to produce an ABP, wherein:
(i) the ABP in an uncleaved state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-ABD or ABD-CM-MM; and
(ii) the CM is positioned in the ABP such that, in an uncleaved state, the MM interferes with specific binding of the ABD to CTLA-4 and in a cleaved state the MM does not interfere or compete with specific binding of the ABD to CTLA-4.
22. The method of claim 21 , further comprising:
(c) testing the ABP for the ability to maintain an activatable phenotype while in soluble form.
23. The method of claim 21 , wherein the MM is a peptide of no more than about 40 amino acids in length.
24. The method of claim 21 , wherein the ABP is manufactured by culturing a cell comprising a nucleic acid construct that encodes the ABP under conditions that lead to expression of the ABP.
25. The method of claim 21 , wherein the ABD comprises a Fab fragment, a scFv or a single chain antibody (SCAB).
26. The method of claim 21 , wherein the CM is a polypeptide that functions as a substrate for a protease that is co-localized in a tissue with the target, wherein the protease cleaves the CM in the ABP when the ABP is exposed to the protease.
27. The method of claim 21 , wherein the CM is a polypeptide of up to 15 amino acids in length.
28. The method of claim 21 , wherein the CM of the ABP in an uncleaved state is coupled to the N-terminus of the ABD.
29. The method of claim 28 , wherein the CM of the ABP in an uncleaved state is coupled to the N-terminus of a V L chain of the ABD.
30. The method of claim 21 , wherein the CM of the ABP in an uncleaved state is coupled to the C-terminus of the ABD.
31. The method of claim 21 , wherein the ABD is from ipilimumab or tremelimumab.
32. The method of claim 21 , wherein the CM is a substrate for an enzyme selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.
33. The method of claim 21 , wherein the CM is a substrate for an enzyme selected from the group consisting of an MMP and a CATHEPSIN.
34. The method of claim 21 , wherein the ABP comprises a linker peptide, wherein the linker peptide is positioned between the MM and the CM.
35. The method of claim 21 , wherein the ABP comprises a linker peptide, wherein the linker peptide is positioned between the ABD and the CM.
36. The method of claim 21 , wherein the ABP comprises a first linker peptide (L 1 ) and a second linker peptide (L 2 ), wherein the first linker peptide is positioned between the MM and the CM and the second linker peptide is positioned between the ABD and the CM.
37. The method of claim 36 , wherein each of L 1 and L 2 is a peptide of about 1 to 20 amino acids in length, and wherein each of L 1 and L 2 need not be the same linker.
38. The method of claim 36 , wherein one or both of L 1 and L 2 comprises a glycine-serine polymer.
39. The method of claim 36 , wherein at least one of Ll and L2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GSGGS) n (SEQ ID NO:1) and (GGGS) n (SEQ ID NO:2), where n is an integer of at least one.
40. The method of claim 36 , wherein at least one of L 1 and L 2 comprises an amino acid sequence having the formula (GGS) n , where n is an integer of at least one.
41. The method of claim 36 , wherein at least one of L 1 and L 2 comprises an amino acid sequence selected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO:3), Gly-Gly-Ser-Gly-Gly (SEQ ID NO:4), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:5), Gly-Ser-Gly-Gly-Gly (SEQ ID NO:6), Gly-Gly-Gly-Ser-Gly (SEQ ID NO:7), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO:8).