An introduction to chimeric antigen receptor (CAR) T‐cell immunotherapy for human cancer

Chimeric antigen receptors (CARs) redirect T cell cytotoxicity against cancer cells, providing a promising approach to cancer immunotherapy. Despite extensive clinical use, the attributes of CAR co-stimulatory domains that impact persistence and resistance to exhaustion of CAR-T cells remain largely undefined. Here, we report the influence of signaling domains of coreceptors CD28 and 4-1BB on the metabolic characteristics of human CAR T cells. Inclusion of 4-1BB in the CAR architecture promoted the outgrowth of CD8(+) central memory T cells that had significantly enhanced respiratory capacity, increased fatty acid oxidation and enhanced mitochondrial biogenesis. In contrast, CAR T cells with CD28 domains yielded effector memory cells with a genetic signature consistent with enhanced glycolysis. These results provide, at least in part, a mechanistic insight into the differential persistence of CAR-T cells expressing 4-1BB or CD28 signaling domains in clinical trials and inform the design of future CAR T cell therapies.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes 1 using recombinant viral vectors, which do not target transgenes to specific genomic sites 2,3 . In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR) 4,5 . Here, we developed a CRISPR-Cas9 genome targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (> 1kb) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we apply this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, demonstrating improved signaling function. Second, we replace the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognize tumour antigen and mount productive anti-tumour cell responses in vitro and in vivo. Taken together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

The success of adoptive T cell gene transfer for treatment of cancer and HIV is predicated on generating a response that is both durable and safe. We report long-term results from three clinical trials to evaluate gammaretroviral vector-engineered T cells for HIV. The vector encoded a chimeric antigen receptor (CAR) composed of CD4 linked to the CD3ζ signaling chain (CD4ζ). CAR T cells were detected in 98% of samples tested for at least 11 years after infusion at frequencies that exceeded average T cell levels after most vaccine approaches. The CD4ζ transgene retained expression and function. There was no evidence of vector-induced immortalization of cells; integration site distributions showed no evidence of persistent clonal expansion or enrichment for integration sites near genes implicated in growth control or transformation. The CD4ζ T cells had stable levels of engraftment, with decay half-lives that exceeded 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression before T cell transfer is not required to achieve long-term persistence of gene-modified T cells. Further, our results emphasize the safety of T cells modified by retroviral gene transfer in clinical application, as measured in >500 patient-years of follow-up. Thus, previous safety issues with integrating viral vectors are hematopoietic stem cell or transgene intrinsic, and not a general feature of retroviral vectors. Engineered T cells are a promising form of synthetic biology for long-term delivery of protein-based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases.