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 activatable binding polypeptide (ABP) that in an activated state binds vascular endothelial growth factor (VEGF), the ABP comprising, in an N-to C-terminal direction or in a C- to N-terminal direction:
a masking moiety (MM), wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37 and 38;
a cleavable moiety (CM); and
an antigen binding domain (ABD) that specifically binds to VEGF.
2. The ABP of claim 1 , wherein the MM comprises a polypeptide of no more than 40 amino acids in length.
3. The ABP of claim 1 , wherein the ABP comprises a linker peptide between the MM and the CM.
4. The ABP of claim 1 , wherein the ABP comprises a linker peptide between the CM and the ABD.
5. The ABP of claim 1 , wherein the ABP comprises a Fab fragment, ScFv, or single chain antibody (SCAB) comprising the ABD.
6. The ABP 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 VEGF, wherein the protease cleaves the CM in the ABP when the ABP is exposed to the protease.
7. The ABP of claim 1 , wherein the CM is a polypeptide of up to 15 amino acids in length.
8. The ABP of claim 1 , wherein the CM is positioned in the ABP in an uncleaved state N-terminal to the ABD.
9. The ABP of claim 1 , wherein the CM is positioned in the ABP in an uncleaved state C-terminal to the ABD.
10. The ABP of claim 8 , wherein the CM is positioned in the ABP in an uncleaved state N-terminal to a variable light (VL) chain of the ABD.
11. The ABP of claim 1 , wherein the ABD is positioned at the N-terminus of the ABP.
12. The ABP of claim 1 , wherein the ABD is positioned at the C-terminus of the ABP.
13. The ABP of claim 1 , wherein the MM, CM, and ABD are positioned in an N- to C-terminal direction of the ABP in an uncleaved state.
14. The ABP of claim 1 , wherein the ABD is from an antibody selected from the group consisting of bevacizumab and ranibizumab.
15. The ABP 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, Caspase10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.
16. The ABP of claim 1 , wherein the CM is a substrate for an enzyme selected from the group consisting of an MMP and a CATHEPSIN.
17. The ABP of claim 1 , comprising a detectable moiety.
18. The ABP of claim 17 , wherein the detectable moiety is a diagnostic agent.
19. The ABP 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.
20. The ABP of claim 19 , 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.
21. The ABP of claim 19 , wherein one or both of L 1 and L 2 comprise a glycine-serine copolymer.
22. The ABP of claim 21 , wherein both of L 1 and L 2 comprise a glycine-serine copolymer.
23. The ABP of claim 19 , wherein at least one of L 1 and L 2 comprises an amino acid sequence selected from the group consisting of (GS) n , GSGGS (SEQ ID NO: 1) and (GGGS) n (SEQ ID NO: 2), where n is an integer of at least one.
24. The ABP of claim 19 , 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.
25. The ABP of claim 19 , 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).
26. The ABP of claim 19 , wherein at least one of L 1 and L 2 comprises an amino acid sequence of (GSGGS: SEQ ID NO: 1) n , wherein n is an integer of at least 1.
27. The ABP of claim 19 , wherein at least one of L 1 and L 2 comprises an amino acid sequence of GGSG (SEQ ID NO: 3).
28. The ABP of claim 19 , wherein at least one of L 1 and L 2 comprises an amino acid sequence of GGSGG (SEQ ID NO: 4).
29. A method of inhibiting activity of a vascular endothelial growth factor (VEGF) or a VEGF receptor in a subject, comprising:
administering to the subject a therapeutically effective amount of a composition comprising an ABP according to claim 1 and a pharmaceutically acceptable excipient.