Associate Professor Georgia Institute of Technology, United States
Introduction: Protease dysregulation is a major hallmark of cancer, yet the substrate repertoire of the tumor microenvironment remains largely undefined. Here we design masked protease-activatable receptors (MAPRs) that display degenerate peptide libraries for deep profiling of the substrate repertoire in cancer. MAPRs comprise synthetic Notch receptors blocked from signaling by a peptide mimotope until its substrate linker is cleaved by an extracellular protease. By genetically encoding T cells to express a MAPR library displaying ~160,000 4-mer amino acid linkers, we cataloged the substrate repertoire of 25 proteases across 5 catalytic classes by sequencing reporter-positive cells. The adoptive transfer of a HER2-targeted MAPR T cell library in mice bearing HER2-positive breast cancer xenografts revealed multiple peptides selectively activated in the tumor, enabling development of a bespoke imaging probe from an isolated substrate. MAPR T cell display may be useful for drug and biosensor design by identifying substrates based on their tumor-selective activity.
Materials and
Methods: MAPR T cell library was generated by co-transducing 10 million CD3+ T cells with a HER2 MAPR lentiviral library encoding 160,000 substrates sequences (MOI 1) and a lentivirus encoding the UAS-activated BFP reporter (MOI 5). Ten million FACS-sorted T cells expressing the MAPR library were intravenously injected into naïve mice or mice bearing HER2-negative or HER2-positive MDA-MB-468 tumors (~100 mm3 in size).
Results, Conclusions, and Discussions: We demonstrate that T cell display of a MAPR library comprising ~160,000 substrate linkers of randomized 4-mers can map the substrate repertoire of recombinant proteases in vitro by next-generation sequencing of reporter-positive T cells. In vivo, the adoptive transfer of an αHER2 MAPR T cell library to mice bearing HER2-positive xenograft tumors identifies hit sequences that exhibit tumor-selective activity in both discovery and independent validation studies with monoclonal MAPR T cells. One hit sequence was further developed into a bespoke fluorogenic peptide for cancer imaging that detected tumors without loss in selectivity, demonstrating cross-platform usefulness. Our results support MAPR T cell display for deep profiling of the protease-substrate repertoire in cancer.
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