Methods for manufacturing activatable binding polypeptides comprising matrix metalloprotease cleavable moieties

A method for manufacturing an activatable binding polypeptide (ABP), wherein the ABP comprises a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM), and wherein the TBM specifically binds to a target protein, wherein the target protein is VEGF-A, the method comprising:

• a) selecting a MM from a plurality of candidate polypeptides, wherein the selected MM inhibits binding of the TBM to its target protein, and wherein the selected MM does not have an amino acid sequence of a naturally occurring binding partner of the TBM;
• and
• b) constructing the ABP comprising the following structure MM-CM-TBM or TBM-CM-MM, comprising synthesizing a polypeptide construct that includes the ABP,
• wherein the VL of the TBM comprises the complementarity determining regions (CDRs) of the VL domain of SEQ ID NO:
• 30 and the VH of the TBM comprises the complementarity determining regions (CDRs) of the VH domain of SEQ ID NO:
• 30,
• wherein the MM is positioned in the ABP such that it is capable of binding the TBM and is capable of inhibiting binding of the TBM to the target protein when the ABP is in an uncleaved state, and
• wherein the CM is a substrate for a protease, wherein the CM is a matrix metalloprotease (MMP) substrate, wherein the MMP substrate is a matrix metalloprotease 1 (MMP-1) substrate, a matrix metalloprotease 2 (MMP-2) substrate, a matrix metalloprotease 9 (MMP-9) substrate, or a matrix metalloprotease 14 (MMP-14) substrate, and wherein said CM is positioned in the ABP such that in a cleaved state in the presence of the target protein, the TBM binds the target protein, and in an uncleaved state in the presence of the target protein, binding of the TBM to the target protein is inhibited by the MM.