Compositions and methods for detecting protease activity in biological systems

A method of treating a patient with an activatable antibody, the method comprising:

• I. detecting presence or absence of a co-localized cleaving agent and a target in a tissue sample bound to a support matrix by
• (a) contacting the tissue sample with an uncleaved first activatable antibody,
• wherein the tissue sample is from the patient,
• wherein the uncleaved first activatable antibody comprises:
• (i) an antibody or an antigen binding fragment thereof (AB) that specifically binds to a target,
• (ii) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease, whereby cleavage of the uncleaved activatable antibody in the CM results in an activated activatable antibody;
• (iii) a masking moiety (MM) coupled to the AB via the CM, wherein the MM inhibits the binding of the AB of the activatable antibody in an uncleaved state to the target, wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target;
• (b) washing the tissue sample bound to the support matrix;
• and
• (c) measuring in situ the presence or absence of a detectable level of activated first activatable antibody in the tissue sample;
• wherein presence of a detectable level of activated first activatable antibody in the tissue sample from the patient indicates that the patient is positive for the presence of both the target and the protease, and
• wherein absence of a detectable level of activated first activatable antibody in the tissue sample from the patient indicates that the patient is negative for the presence of either or both the target and the protease;
• and
• II. administering to the patient identified in step (I) as positive for the presence of both the target and the protease a therapeutically effective amount of a second activatable antibody comprising an AB that specifically binds to the same target, and a CM that functions as a substrate for the same protease, as the first activatable antibody in (a)(I) and (a) (ii), and a MM, wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to the target.