Compositions and methods for conjugating activatable antibodies

A method of partially reducing an activatable antibody and conjugating an agent thereto resulting in selectivity in the placement of the agent, the method comprising:

• reducing at least one disulfide bond in the activatable antibody with a reducing agent without reducing any intrachain disulfide bonds in the activatable antibody, thereby partially reducing the activatable antibody,
• wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP),
• wherein the ratio of the reducing agent to the activatable antibody is less than 5:1, and
• wherein the reduction time is 30 minutes to 2 hours;
• and
• conjugating an agent to at least one thiol of the partially reduced activatable antibody,
• wherein the activatable antibody comprises an antibody or an antigen binding fragment thereof (AB) that specifically binds to a target, a masking moiety (MM) coupled to the AB that inhibits the binding of the AB to the target when the activatable antibody is in an uncleaved state, and a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease,
• wherein at least one intrachain disulfide bond in the activatable antibody is within the MM, and
• wherein the partial reduction of the activatable antibody and conjugation of the agent thereto does not disturb or negatively affect activation and/or efficacy of the activatable antibody, wherein the activity and/or efficacy of the activatable antibody is selected from the group consisting of:
• the masking activity of the MM to the AB when the activatable antibody is in an uncleaved state, the activation of the uncleaved activatable antibody by the protease, and the binding activity of the activated activatable antibody to the target of the AB.