Utilizing distinct CAR and TCR signaling to generate enhanced cellular immunotherapy

Abstract CAR-T cells utilize TCR signaling cascades and the recognition functions of antibodies. CAR-T technology has achieved significant success in treatment of certain, primarily liquid, cancers. Nonetheless, many challenges hinder the development of this therapy, for example the efficacy for solid tumors. These challenges show our inadequate understanding of this technology, particularly regarding CAR signaling, and how it may differ from TCR signaling. To dissect CAR signaling, CAR and TCR targeting the same antigen were compared directly. This comparison revealed that CAR was sufficient to bind monomeric antigens due to its high affinity but required oligomeric antigens for its activation. CAR sustained the transduced signal significantly longer than did TCR. CD8 coreceptor was recruited to the CAR synapse but played a negligible role in signaling, unlike for TCR signaling. Surprisingly, we identified a non-canonical CAR signaling triggered in the absence of SRC family kinase (SFK) LCK, which is essential for TCR signaling. We show that LCK-deficient CAR-T cells are strongly signaled through CAR and have a better in vivo efficacy because of reduced exhaustion phenotype and enhanced induction of memory. This non-canonical signaling of CAR-T cells provides new insight into the initiation of TCR and CAR signaling as well as important clinical implications for improvement of CAR function. Supported by grants from Singapore Ministry of Health’s National Medical Research Council: OFIRG19nov-0066; and Ministry of Education, NUHSRO/2020/110/T1/SEED-MAR/06, and NUS ILO TAP Grant: TAP2002019-04-25. LW, QW and JL were supported by research scholarships from Yong Loo Lin School of Medicine.