Optimization of CAR T cell signaling by incorporation of null mutations

An obstacle with continued clinical development of CAR T cells is the limited understanding of CAR T cell biology and its mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP) we could optimize CAR T cell signaling and reduce exhaustion. In vivo we found that mice given CAR T cells with a PYAP CD28 endodomain had a significant survival advantage. We also observed that mutant CAR T cells had significantly less expression of PD1 and were increased in the bone marrow and spleen. In addition, these mutant CAR T cells had optimized signaling resulting in a reduction of exhaustion related transcription factors and genes. Our results demonstrate that CAR T cells with a mutant CD28 endodomain have better survival and decreased exhaustion and is the result of decreased CAR dependent NFAT/NR4A1 transcription factors. This work allows for development of enhanced CAR T cell therapies by optimizing CAR T cell signaling. For both ATAC-seq and RNA-seq m1928z, m19hBBz, and mut06 CAR T cells were produced from C57BL6 mice. CAR T cells were simulated with 3T3-mCD19 target cells at a 10:1 E:T ratio for 24 hours. After stimulation CAR positive cells were sorted using a BD ARIA FACS sorter. Samples were pelleted and snap frozen in liquid nitrogen. All samples were done in duplicate.