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. An isolated polypeptide comprising
an antigen binding domain of an antibody (ABD) that binds a target, wherein the target is VEGF or CTLA-4; and
a cleavable moiety (CM) comprising the amino acid sequence VHMPLGFLGP (SEQ ID NO:142).
2. The isolated polypeptide of claim 1 , wherein the antigen binding domain is selected from the group consisting of a Fab fragment, a F(ab′) 2 fragment, a scFv, and a single chain antibody (scAb).
3. The isolated polypeptide of claim 1 , wherein the CM is a substrate for a protease that is co-localized in a tissue with the target.
4. The isolated polypeptide of claim 1 , wherein when the polypeptide is in an uncleaved state, the CM is positioned in the polypeptide N-terminal to the ABD.
5. The isolated polypeptide of claim 1 , wherein when the polypeptide is in an uncleaved state, the CM is positioned in the polypeptide C-terminal to the ABD.
6. The isolated polypeptide of claim 1 , wherein when the polypeptide is in an uncleaved state, the CM is positioned in the polypeptide N-terminal to a variable light (V L ) chain of the ABD.
7. The isolated polypeptide of claim 1 , wherein the ABD is positioned at the N-terminus of the isolated polypeptide.
8. The isolated polypeptide of claim 1 , wherein the ABD is positioned at the C-terminus of the isolated polypeptide.
9. The isolated polypeptide of claim 1 , wherein the ABD is linked to the CM.
10. The isolated polypeptide of claim 9 , wherein the ABD is linked to the CM via a linker peptide.
11. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises a masking moiety (MM), and wherein when the isolated polypeptide is in an uncleaved state, the MM interferes with specific binding of the ABD to its target.
12. The isolated polypeptide of claim 11 , wherein the MM is a polypeptide of no more than 40 amino acids in length.
13. The isolated polypeptide of claim 11 , wherein MM does not have an amino acid sequence of a naturally occurring binding partner of the ABD.
14. The isolated polypeptide of claim 11 , wherein the MM does not interfere or compete with the ABD for binding to the target when the polypeptide is in a cleaved state.
15. The isolated polypeptide of claim 11 , wherein the MM is linked to the CM such that the isolated polypeptide in an uncleaved state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-ABD or ABD-CM-MM.
16. The isolated polypeptide of claim 15 , wherein the isolated polypeptide comprises a linker peptide between the MM and the CM.
17. The isolated polypeptide of claim 15 , wherein the isolated polypeptide comprises a linker peptide between the CM and the ABD.
18. The isolated polypeptide of claim 15 , wherein the isolated polypeptide comprises a first linker peptide (L 1 ) and a second linker peptide (L 2 ), and wherein when the isolated polypeptide is in an uncleaved state, the isolated polypeptide comprises a structural arrangement from N-terminus to C-terminus as follows: MM-L 1 -CM- L 2 -ABD or ABD- L 2 -CM- L 1 -MM.
19. The isolated polypeptide of claim 18 , wherein the two linker peptides are not the same.
20. The isolated polypeptide of claim 18 , wherein each of L 1 and L 2 is a peptide of about 1 to 20 amino acids in length.
21. The isolated polypeptide of claim 18 , wherein one or both of L 1 and L 2 comprise a glycine-serine copolymer.
22. The isolated polypeptide of claim 18 , wherein at least one of L 1 and L 2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GGS) n , GSGGS (SEQ ID NO: 1) and (GGGS) n (SEQ ID NO: 2), where n is an integer of at least one.
23. The isolated polypeptide of claim 18 , wherein at least one of L 1 and L 2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).
24. The isolated polypeptide of claim 9 , wherein the isolated polypeptide comprises a masking moiety (MM), and wherein when the isolated polypeptide is in an uncleaved state, the MM interferes with specific binding of the ABD to its target.
25. The isolated polypeptide of claim 24 , wherein the MM is a polypeptide of no more than 40 amino acids in length.
26. The isolated polypeptide of claim 24 , wherein MM does not have an amino acid sequence of a naturally occurring binding partner of the ABD.
27. The isolated polypeptide of claim 24 , wherein the MM does not interfere or compete with the ABD for binding to the target when the polypeptide is in a cleaved state.
28. The isolated polypeptide of claim 24 , wherein the MM is linked to the CM such that the isolated polypeptide in an uncleaved state comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM-ABD or ABD-CM-MM.
29. The isolated polypeptide of claim 28 , wherein the isolated polypeptide comprises a linker peptide between the MM and the CM.
30. The isolated polypeptide of claim 28 , wherein the isolated polypeptide comprises a linker peptide between the CM and the ABD.
31. The isolated polypeptide of claim 28 , wherein the isolated polypeptide comprises a first linker peptide (L 1 ) and a second linker peptide (L 2 ), and wherein when the isolated polypeptide is in an uncleaved state, the isolated polypeptide comprises a structural arrangement from N-terminus to C-terminus as follows: MM-L 1 -CM- L 2 -ABD or ABD- L 2 -CM- L 1 -MM.
32. The isolated polypeptide of claim 31 , wherein the two linker peptides are not the same.
33. The isolated polypeptide of claim 31 , wherein each of L 1 and L 2 is a peptide of about 1 to 20 amino acids in length.
34. The isolated polypeptide of claim 31 , wherein one or both of L 1 and L 2 comprise a glycine-serine copolymer.
35. The isolated polypeptide of claim 31 , wherein at least one of L 1 and L 2 comprises an amino acid sequence selected from the group consisting of (GS) n , (GGS) n , GSGGS (SEQ ID NO: 1) and (GGGS) n (SEQ ID NO: 2), where n is an integer of at least one.
36. The isolated polypeptide of claim 31 , wherein at least one of L 1 and L 2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).