Project Grant F31CA310226

ELUCIDATING THE ROLE OF CD8+ TCR ATTRIBUTES IN CANCER IMMUNOTHERAPY - PROJECT ABSTRACT PANCREATIC DUCTAL ADENOCARCINOMA (PDA) IS A HIGHLY LETHAL MALIGNANCY WITH THE LOWEST 5-YEAR SURVIVAL RATE AMONG ALL SOLID TUMORS. ADOPTIVE CELL TRANSFER (ACT) OF TUMOR NEOEPITOPE SPECIFIC CD8+ T CELLS HAS SHOWN THERAPEUTIC PROMISE, BUT CURRENT APPROACHES RELY ON PEPTIDE-MHC TETRAMERS TO SELECT HIGH-AFFINITY T CELL RECEPTORS (TCRS), POTENTIALLY OVERLOOKING FUNCTIONALLY SUPERIOR CLONES. OUR PRELIMINARY DATA REVEAL THAT LOW- AFFINITY TCRS-PARTICULARLY THOSE UNDETECTABLE BY TETRAMERS-CAN MEDIATE POTENT ANTITUMOR RESPONSES, SUGGESTING THAT TCR BOND LIFETIME UNDER MECHANICAL FORCE, RATHER THAN AFFINITY ALONE, MAY DICTATE THERAPEUTIC EFFICACY. TO ADDRESS THIS, I DEVELOPED A NOVEL PANEL OF T CELL RECEPTOR EXCHANGE (TREX) MICE EXPRESSING PDA- SPECIFIC TCRS SPANNING A SPECTRUM OF AFFINITIES FOR THE SAME TUMOR NEOANTIGEN (CB101-109:H2-DB). STRIKINGLY, THE LOWEST-AFFINITY TCR (TCR5.1) DEMONSTRATED ENHANCED TUMOR CONTROL COMPARED TO HIGHER-AFFINITY COUNTERPARTS, CHALLENGING CONVENTIONAL TCR SELECTION PARADIGMS. THIS PROPOSAL WILL DEFINE HOW TCR BIOPHYSICAL PROPERTIES INFLUENCE CD8+ T CELL FATE AND FUNCTION IN PDA THROUGH TWO SPECIFIC AIMS. AIM 1, WILL EMPLOY A BIOMEMBRANE FORCE PROBE (BFP) TO QUANTIFY TCR-PMHC BOND LIFETIMES AND CORRELATE THESE WITH ACT OUTCOMES IN ORTHOTOPIC PDA MODELS, USING HIGH-RESOLUTION ULTRASOUND AND MULTIMODAL T CELL PROFILING. IN COLLABORATION WITH BRIAN EVAVOLD'S LAB AT THE UNIVERSITY OF UTAH, I WILL USE BFP ANALYSIS TO QUANTIFY EACH TCRS AFFINITY, BOND LIFETIME, AND IDEAL FORCE CONDITIONS. I WILL THEN EVALUATE THE EFFECTOR FUNCTIONS OF EACH CLONOTYPE EX VIVO USING TITRATED PEPTIDE STIMULATION ASSAYS AND ASSESS TUMOR CLEARANCE IN VIVO THROUGH ACT IN IMMUNOCOMPETENT ORTHOTOPIC PDA MODELS. AIM 2 WILL DISSECT COMPETITIVE FITNESS OF TCRS DURING IMMUNOTHERAPY BY CO-TRANSFERRING T CELL CLONES INTO IMMUNOCOMPETENT HOSTS, COMBINING SCRNASEQ AND SPATIAL ANALYSES TO UNCOVER MECHANISMS DRIVING TCR-DEPENDENT PERSISTENCE. BY "PARKING" NAIVE TREX CD8+ T CELLS IN MICE PRIOR TO ORTHOTOPIC TUMOR IMPLANTATION, I WILL IDENTIFY THE IMPACT OF COMPETITION BETWEEN TCRS OF VARYING AFFINITIES ON THE ANTI-TUMOR RESPONSE DURING IMMUNE CHECKPOINT BLOCKADE OR CD40 AGONIST THERAPIES. TRACKING THESE CLONAL POPULATIONS THROUGH SINGLE-CELL RNA SEQUENCING AND SPATIAL ANALYSES WILL ALLOW FOR THE IDENTIFICATION OF THE MOLECULAR AND CELLULAR MECHANISMS THAT DRIVE TCR-DEPENDENT PERSISTENCE AND STEM-LIKE MAINTENANCE. TOGETHER, THESE STUDIES WILL TEST THE CENTRAL HYPOTHESIS THAT LOW-AFFINITY, LONG-LIVED TCR INTERACTIONS PROMOTE THE DEVELOPMENT OF DURABLE, STEM-LIKE T CELL POPULATIONS THAT ARE RESISTANT TO EXHAUSTION - A POTENTIAL KEY TO ACHIEVING SUSTAINED ANTITUMOR RESPONSES. THE SUCCESSFUL COMPLETION OF THIS RESEARCH WILL PROVIDE TRANSFORMATIVE INSIGHTS INTO TCR- INTRINSIC DETERMINANTS OF THERAPEUTIC EFFICACY, WITH DIRECT IMPLICATIONS FOR THE DESIGN OF IMPROVED ACT PROTOCOLS FOR PDA AND OTHER SOLID TUMORS.