Patent ID: 8563269

Claim:
A method of manufacturing an activatable antibody, the method comprising: (a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 218-220, 236-265 and 266, a first peptide linker (LP1), a cleavable moiety (CM), a second peptide linker (LP2), and an antibody or an antigen binding fragment thereof (AB) that specifically binds Epidermal Growth Factor Receptor (EGFR), (i) wherein: the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB for binding to EGFR; (ii) wherein the CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of urokinase type plasminogen activator (uPA), legumain and matriptase (MT-SP1), and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease; (iii) wherein the AB has an equilibrium dissociation constant of at most 100 nM for binding to EGFR; (iv) wherein each of LP1 and LP2 is a peptide, and wherein the two LPs need not be identical to each other; (v) wherein the activatable antibody in an uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM; (vi) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to EGFR and wherein the MM of the activatable antibody in a cleaved state does not interfere or compete with specific binding of the AB to EGFR; and (vii) wherein the MM of the activatable antibody in an uncleaved state inhibits the binding of the AB to EGFR such that the dissociation constant (K d ) of the AB of the activatable antibody in an uncleaved state towards EGFR is at least 100 times greater than the K d of the AB of the activatable antibody in a cleaved state towards EGFR; and (b) recovering the activatable antibody.