Abstract B047: Next-generation gene expression enables tumor-focused immuno-oncology therapies
Authors: Stern Patrick 
Published: February 2019

Abstract
To move cancer immuno-therapies forward, the tumor must participate in treatment. The goal is treatments that provoke tumors to activate cancer-killing immunity, but the basic research technologies to develop these therapies were sorely lacking until now. Our next-generation gene expression system can mimic pharmacologic intervention for any target in growing tumors in mice, enabling high-confidence target assessments before committing to compound development. We have used this technology to understand the mechanisms controlling CTL-activating apoptosis—Immunizing Cell Death (ICD)—and have identified drug-able pathways that will synergize with existing clinical cytotoxic drugs and cause tumors to provoke antitumor immunity. Developed over 8 years at the MIT Koch Institute, our patented Switch expression system is the only “translation control” system and is 1000-times faster than existing “transcription-based” technologies. This speed enables gene expression with drug-like kinetics. More importantly, our novel control enables unmatched drug target assessments by mimicking targeted therapy in growing tumors. Switch is designed to express a first gene and then “switch” for a second gene (e.g., express a kinase and replace with kinase-dead to mimic kinase inhibitors) in growing tumors or in vitro. Switch has been used in 20+ cell lines and transplantable tumor models and is the follow-on to CRISPR-based efforts where the endogenous gene is deleted. Delivered into cancer cells with optimized lentiviral vectors, Switch can replace the CRISPRed genomic target and then cells may be transplanted into mice or grown/ differentiated in vitro. When engineered cells reach the desired point (e.g., a mature tumor), Tamoxifen-CreER recombination causes deletion of the first gene and expression of the second gene. This strategy recapitulates the “logic” of drug treatment—acute functional perturbation of a target protein—and elevates preclinical drug R&D to the next level beyond the “deletion phenotypes” of CRISPR and RNAi. We have identified the critical molecular control mechanisms regulating the immunogenicity of normal cells dying by apoptosis. Normal cells, killed by apoptosis in the appropriate way, can elicit potent tumor-suppressive CD8+ immunity. We developed a cell-based prophylactic melanoma vaccine model to identify critical regulators of this process. Normal mouse fibroblasts (expressing melanoma antigens) were engineered with Switch-regulated apoptosis-inducing and stress-modulating constructs and transplanted into mouse mammary fat pads. Tamoxifen treatment caused expression of apoptosis-inducing and stress-modulating factors in transplanted cells—an “in vivo immunization” with dying cells. One week later mice were challenged with B16 melanoma and tumor growth monitored. Through 485 mice and 25+ experiments, we identified initiators and modifiers of normal cell apoptosis that activate cell-killing immunity with as few as 50K cells and confer protection (or not) from melanoma challenge. We confirmed 12 targets as core regulators of immunizing cell death and developed a therapeutic strategy for converting cytotoxic clinical assets into immune-provoking therapies. Switch can be used in combination with existing clinical assets to identify synergistic targets in vivo to beat drug resistance before committing to compound development. Switch provides unmatched precision and control of pharmacologically relevant targets and presents a critical bridge between in vivo validation of target efficacy and in vitro profiling for functional protein biomarkers. Switch technology enabled development of a therapeutic strategy for converting existing clinical oncology assets into first-in-class tumor-focused immuno-oncology therapies.
Citation Format: Patrick Stern. Next-generation gene expression enables tumor-focused immuno-oncology therapies [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B047.