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.

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. Here 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) comprised of CD4 linked to the CD3-ζ signaling chain (CD4ζ). CAR T-cells were detected in 98% of samples tested for at least 11 years post-infusion at frequencies that exceed 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 as 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 have stable levels of engraftment, with decay half-lives that exceed 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression prior to T cell transfer is not required in order 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.

The majority of chimeric antigen receptor (CAR) T cell research has focused on attacking cancer cells. Here we show that targeting the tumor-promoting, non-transformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single chain Fv FAP (mAb 73.3) with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFNγ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAPhi stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8+ T cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective suggesting that further clinical development of anti-human FAP-CAR is warranted.