Project Grant R01CA292680

INCREASING THE EFFICACY OF NON-ACTIVATED CAR T CELLS BY MODULATING IFN1 SIGNALING - CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS HAVE DEMONSTRATED THEIR EFFICACY IN TREATING BLOOD-BASED CANCERS. HOWEVER, THE DURABILITY OF RESPONSES IS OFTEN HINDERED BY CHALLENGES RELATED TO LONG-TERM T CELL PERSISTENCE AND ENGRAFTMENT. THE SUCCESS OF CAR T CELL IMMUNOTHERAPY RELIES ON THE DIFFERENTIATION STATUS AND OVERALL FITNESS OF THE CAR T CELL PRODUCT. CURRENT PROTOCOLS INVOLVE THE ACTIVATION AND EX VIVO EXPANSION OF PATIENT T CELLS, HOWEVER, ACTIVATION LEADS TO IRREVERSIBLE DIFFERENTIATION, COMPROMISING THEIR THERAPEUTIC POTENCY. OUR RECENT WORK SHOWED THAT A MANUFACTURING PROTOCOL UTILIZING NON-ACTIVATED T CELLS RESULTS IN SUPERIOR DIFFERENTIATION CHARACTERISTICS AND REDUCED EXHAUSTION, WITH CONCORDANT BENEFITS IN LONG-TERM TUMOR CONTROL. NEVERTHELESS, AS QUIESCENT T CELLS ARE HIGHLY RESISTANT TO LENTIVIRAL INFECTION, CAR T MANUFACTURING YIELD IS A SIGNIFICANT LIMITATION WITH NON-ACTIVATED T CELLS. THE GOAL OF THIS STUDY IS TO HARNESS THE INTRINSIC STEMNESS QUALITIES OF NON-ACTIVATED CAR T CELLS AND IMPROVE THEIR TRANSDUCTION EFFICIENCY AND EFFECTOR FUNCTION, THEREBY ENHANCING THEIR DURABLE EFFICACY FOLLOWING INFUSION. QUIESCENT T CELLS INITIATE A TYPE I INTERFERON (IFN1)-MEDIATED INNATE RESPONSE UPON LENTIVIRAL VECTOR TRANSDUCTION, WHICH LIMITS CAR T CELL TRANSDUCTION EFFICIENCY. OUR PRELIMINARY DATA INDICATE THAT PRE-TREATMENT OF NON-ACTIVATED T CELLS WITH AN IFN1-BINDING PROTEIN ENHANCES CAR T CELL TRANSDUCTION EFFICIENCY, AND PROMOTES A MORE NAIVE AND CENTRAL MEMORY PHENOTYPE. THIS RESEARCH WILL DELVE INTO THE IMPACT OF IFN1 BLOCKADE ON CAR LENTIVIRUS TRANSDUCTION AND FUNCTION OF NON-ACTIVATED T CELLS, BOTH IN VITRO AND IN XENOGRAFT MODELS IN VIVO. GIVEN THAT SUSTAINED TYPE I IFN SIGNALING FACILITATES TUMOR IMMUNE ESCAPE AND RESISTANCE TO THERAPIES, WE HYPOTHESIZE THAT CONTINUOUS IFN1 BLOCKADE NOT ONLY ENHANCES T CELL FITNESS BY INHIBITING THE INNATE RESPONSE TO THE LENTIVIRAL VECTOR BUT ALSO AMPLIFIES THE THERAPEUTIC EFFICACY OF T CELLS IN TUMORS RELIANT ON IFN-MEDIATED IMMUNE EVASION. TO EXPLORE THIS FURTHER, WE WILL EVALUATE THE EFFECT OF SUSTAINED IFN1 BLOCKADE BY CONSTRUCTING LENTIVIRAL TRANSFER PLASMIDS ENCODING BOTH A CAR AND A SECRETED ANTI-IFN1 BINDING PROTEIN IN VARIOUS XENOGRAFT MODELS OF CANCER. ANOTHER KEY ASPECT OF OUR INVESTIGATION IS THE INTERPLAY BETWEEN SAMHD1 AND IFN1 SIGNALING PATHWAYS IN QUIESCENT T CELLS. SAMHD1 RESTRICTS NUCLEOTIDE AVAILABILITY FOR REVERSE TRANSCRIPTION AND IS UPREGULATED BY IFN1. GIVEN THAT VPX, A COMPONENT OF NATURAL HIV, DEGRADES SAMHD1, WE WILL ASSESS THE IMPACT OF VPX INCORPORATION ON THE EFFICIENCY OF REVERSE TRANSCRIPTION AND VECTOR INTEGRATION OF CAR LENTIVIRUS IN NON-ACTIVATED T CELLS. OUR HYPOTHESIS IS THAT RESTORING VPX, WHICH TARGETS RATE-LIMITING STEPS OF THE VIRAL TRANSDUCTION PATHWAY, WILL SYNERGIZE WITH IFN1 INHIBITION IN QUIESCENT T CELLS, ULTIMATELY ENHANCING LENTIVIRAL TRANSDUCTION EFFICIENCY AND BOLSTERING THE FUNCTION IN NON-ACTIVATED CAR T CELLS. THESE STUDIES REPRESENT A SIGNIFICANT STEP TOWARD ENHANCING T CELL FITNESS BY COUNTERING ANTI-VIRAL DEFENSES TRIGGERED DURING MANUFACTURING. WE ARE DEDICATED TO ADVANCING THE NON-ACTIVATED CAR T CELL PLATFORM, WITH THE ULTIMATE GOAL OF IMPROVING THE CLINICAL POTENTIAL OF NON-ACTIVATED CAR T CELLS AS A VIABLE CAR THERAPY. GIVEN OUR WELL-ESTABLISHED TRANSLATIONAL INFRASTRUCTURE, OUR FINDINGS HOLD IMMEDIATE CLINICAL RELEVANCE.