Abstract: . There is great interest in developing strategies to identify proteolytic substrates. Synthetic peptide libraries are among the most widely used to screen substrates-based activity, however, this technique is limited by the size of libraries and is time-consuming. Several combinatorial library-based display technologies such as phage, bacteria and yeast display, can identify better substrates for protease activity with higher-throughput sequencing and enrichment of specific substrates over multiple rounds of selection. Even though these profiling techniques provide greater coverage of protease cleavable substrates, such cells are less capable of accessing difficult to deliver sites, pose immunogenicity concerns, and have not been applied to discriminate on-from off-target activity in vivo. Disclosed herein are in vitro and in vivo methods for screening protease substrates.

Abstract: Compositions and methods for pharmacodynamic monitoring of immunotherapy are provided herein. The compositions include an immunotherapeutic agent linked to protease substrates. Upon administration, the compositions target to sites of disease where proteases are upregulated during responsive immunotherapy and subsequently cleave the attached substrates. Cleavage fragments are detected in a sample from the body and detection of the fragments is indicative of an effect of the immunotherapeutic agent.

Abstract: An activity-based nanosensor composition for detecting protease activity comprising a cleavable detectable substrate and methods of use are disclosed.

Abstract: The disclosure provides methods and compositions to digitally profile biological activity by detecting the activity of at least two biomarkers using protease activity sensors or biocomparators.

Abstract: An activity-based nanosensor composition for detecting protease activity comprising a cleavable detectable substrate and methods of use are disclosed.