ABSTRACT
Proteases have been implicated in the development of many pathological conditions, including cancer. Detection of protease activity in diseased tissues could therefore be useful for diagnosis, prognosis, and the development of novel therapeutic approaches. Due to tight post-translational regulation, determination of the expression level of proteases alone may not be indicative of protease activities, and new methods for measuring protease activity in biological samples such as tumor biopsies are needed. Here we report a novel zymography-based technique, called the IHZTM assay, for the detection of specific protease activities in situ. The IHZ assay involves imaging the binding of a protease-activated monoclonal antibody prodrug, called a Probody® therapeutic, to tissue. Probody therapeutics are fully recombinant, masked antibodies that can only bind target antigen after removal of the mask by a selected protease. A fluorescently labeled Probody molecule is incubated with a biological tissue, thereby enabling its activation by tissue endogenous proteases. Protease activity is measured by imaging the activated Probody molecule binding to antigen present in the sample. The method was evaluated in xenograft tumor samples using protease specific substrates and inhibitors, and the measurements correlated with efficacy of the respective Probody therapeutics. Using this technique, a diverse profile of MMP and serine protease activities was characterized in breast cancer patient tumor samples. The IHZ assay represents a new type of in situ zymography technique that can be used for the screening of disease-associated proteases in patient samples from multiple pathological conditions.
Subject(s)
Molecular Imaging/methods , Molecular Probes/metabolism , Neoplasms/diagnosis , Peptide Hydrolases/analysis , Prodrugs/metabolism , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Female , Humans , Mice , Microscopy, Fluorescence , Molecular Probes/genetics , Molecular Probes/pharmacology , Neoplasms/pathology , Peptide Hydrolases/metabolism , Prodrugs/pharmacology , Protein Processing, Post-Translational , Proteolysis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
PROBODY therapeutics (Pb-Tx) are protease-activatable prodrugs of monoclonal antibodies (mAbs) designed to target tumors where protease activity is elevated while avoiding normal tissue. They are composed of a parental mAb, a mask that inhibits antibody binding to target, and a protease-cleavable substrate between the mask and the mAb. We report a quantitative systems pharmacology model for the rational design and clinical translation of Pb-Tx. The model adequately described monkey pharmacokinetic data following the administration of six anti-CD166 Pb-Tx of varying mask strength and substrate cleavability and captured the trend of decreasing Pb-Tx systemic clearance with increasing mask strength. Projections to humans suggested both higher levels of Pb-Tx in tumor relative to parental mAb and an optimal mask strength for maximizing tumor receptor-mediated uptake. Simulations further suggested the majority of circulating species in humans would be intact/masked Pb-Tx, with no significant flux of cleaved/activated species from tumor to the systemic compartment.
