Project Grant R43CA254820

SIMULTANEOUS TARGETING OF TUMOR AND STROMA CELLS TO ENHANCE SOLID TUMOR CAR-T CELL THERAPY - ABSTRACT THE USE OF T CELLS ENGINEERED TO EXPRESS SPECIFIC CHIMERIC ANTIGEN RECEPTORS (CARS) TO TREAT CANCER HAS GENERATED DURABLE CURES FOR MANY TYPES OF CANCER AND RESULTED IN THE FIRST FDA APPROVED CAR-T CELL THERAPY TO TREAT CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA IN 2017. DESPITE THIS SUCCESS, CAR-T IMMUNOTHERAPIES HAVE BEEN MUCH LESS EFFECTIVE AT TARGETING SOLID TUMORS. PART OF THIS LIMITED SUCCESS STEMS FROM THE SOLID TUMOR MICROENVIRONMENT, WHICH FORMS A PHYSICAL BARRIER TO IMMUNE CELL INFILTRATION AND PRODUCES SOLUBLE FACTORS THAT DOWNREGULATE T CELL ACTIVITY AND ACCELERATE T CELL EXHAUSTION. WHILE IMMUNOTHERAPIES TARGETING SOLID TUMORS ARE INITIALLY EFFECTIVE, THE TUMOR MICROENVIRONMENT'S INHIBITION OF T CELLS PREVENTS THESE TREATMENTS FROM PRODUCING DURABLE RESPONSES. IN THIS APPLICATION, WE PROPOSE A NOVEL CAR-T CELL THERAPY AIMED TO IMPROVE OUTCOMES FOR PATIENTS WITH ADVANCED STAGE PANCREATIC CANCER BY OVERCOMING THE DEFICIENCIES THAT PLAGUE CURRENT CAR-T CELL THERAPIES. TO THIS END, WE WILL ENGINEER T CELLS TO EXPRESS MULTIPLE CARS, ENABLING THESE CELLS TO TARGET TUMOR CELLS AND CELLS IN THE IMMUNE-SUPPRESSIVE TUMOR MICROENVIRONMENT. SPECIFICALLY, WE WILL LEVERAGE THE NON-VIRAL, TC BUSTER DNA TRANSPOSON SYSTEM TO INSERT A LARGE MULTICISTRONIC GENETIC CONSTRUCT CONTAINING MULTIPLE CARS AND A SELECTION MARKER INTO T CELLS. USING THIS PLATFORM, WE WILL GENERATE T CELLS WITH CARS TARGETING MESOTHELIN (MSLN), A PROTEIN EXPRESSED BY 80-85% OF PANCREATIC CANCER TUMORS, AND FIBROBLAST ACTIVATION PROTEIN (FAP), A MARKER OF CANCER ASSOCIATED FIBROBLASTS IN THE TUMOR MICROENVIRONMENT. WE WILL THEN SELECT A PURE POPULATION OF T CELLS EXPRESSING MSLN- AND FAP-CARS AND DETERMINE THE ACTIVITY AND SPECIFICITY OF THESE CELLS IN VITRO. WE EXPECT THAT ENGINEERED T CELLS WILL GENERATE A SPECIFIC AND ROBUST RESPONSE, ELICITING CYTOTOXIC FUNCTIONS ONLY AGAINST CELLS EXPRESSING THEIR TARGET ANTIGEN. WE WILL THEN DETERMINE THE EFFICACY OF ENGINEERED T CELLS IN VIVO USING A XENOGRAFT MOUSE MODEL TO GENERATE MSLN AND FAP POSITIVE PANCREATIC CARCINOMAS FOLLOWED BY ADOPTIVE TRANSFER OF T CELLS. WE EXPECT IMMUNOTHERAPEUTIC DELIVERY OF BISPECIFIC T CELLS EXPRESSING FAP-CARS AND MSLN- CARS WILL ELICIT A ROBUST AND LONG-LASTING T CELL RESPONSE AGAINST MSLN+/FAP+ SOLID TUMORS RESULTING IN TUMOR SHRINKAGE AND INCREASED SURVIVAL. FURTHERMORE, WE EXPECT THE DEVELOPMENT OF A FLEXIBLE, EFFICIENT, AND RELIABLE PROCESS TO GENERATE BISPECIFIC T CELLS WITH A SINGLE, NON-VIRAL GENE DELIVERY APPROACH WILL FACILITATE THE EMERGENCE OF NOVEL THERAPIES TO OVERCOME MANY ISSUES FACING ENGINEERED T CELL THERAPY TODAY, INCLUDING ANTIGEN ESCAPE AND TARGET SPECIFICITY.