Allogeneic CAR-T activity enabled by knockout of LCK 2267386

Abstract Introduction CAR-T technology has achieved great success in treatment of liquid cancers, but poor quality and quantity of the patients’ T cells, and the time taken to produce sufficient CAR-T cells for therapy are major bottlenecks. Allogeneic CAR-T could solve these problems, but can cause Graft versus Host Disease (GVHD). We recently showed that CD28-CAR signaling still works after deletion of kinase LCK, essential for TCR signaling. LCK-KO CAR-T cells signal strongly through CAR and have better in vivo efficacy in both liquid and solid tumor models. They show enhanced persistence, induction of memory, and reduced exhaustion phenotype. The ability to block TCR signals while leaving CAR signaling intact suggested a method to enable allo-CAR-T therapy. The current method for stopping GVHD in allo-CAR-T is to block the TCR itself, usually by KO of the TRAC locus. Methods We knocked CAR constructs into TRAC or LCK loci in human or mouse T cells, then tested these for their ability to cause GVHD in mice. We tested their ability to protect immunodeficient NSG mice from leukemia. Results We find that LCK-knockout CAR-T blocked activation through endogenous TCR equally well as the TRAC knockout, and that both methods block development of GVHD. LCK-KO CAR-T also show superior in vivo persistence compared to TCR KO T cells. CAR constructs using the interdomain B and kinase domains of ZAP70 (“zCAR”) were independent of LCK, and were superior to the standard CAR constructs, particularly in vivo. Conclusion The enhanced persistence of LCK-deficient CAR-T cells is explained by FYN-dependent tonic signaling. Exchanging the CD3ζ element of the CAR construct for the IDB+kinase domains of ZAP70, combined with LCK-knockout, provides enhanced CAR-T activity while inhibiting any TCR-dependent signaling. This blocks any development of GVHD. The ability to separate TCR and CAR/zCAR signaling through knockout of LCK has important clinical implications for improvement of allogeneic CAR-T cells. Funding Source A*STAR Singapore Therapeutics Development Review (STDR) (H24H9a0005) Singapore Ministry of Health’s National Medical Research Council (MOH-000523) Augusta University Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)