Vasco Dos Reis Gonçalves

The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods. While viral vectors offer high transfection efficiencies, concerns regarding potential malignant transformation coupled with costly and time-consuming vector manufacturing are constant drivers in the search for cheaper, easier-to-use, safer, and more efficient alternatives. In this review, we examine different non-viral gene transfer methods as alternatives for CAR-T cell production, their advantages and disadvantages, and examples of their applications. Transposon-based gene transfer methods lead to stable but non-targeted gene integration, are easy to handle, and achieve high gene transfer rates. Programmable endonucleases allow targeted integration, reducing the potential risk of integration-mediated malignant transformation of CAR-T cells. Non-integrating CAR-encoding vectors avoid this risk completely and achieve only transient CAR expression. With these promising alternative techniques for gene transfer, all avenues are open to fully exploiting the potential of next-generation CAR-T cell therapy and applying it in a wide range of applications.

Low antigen sensitivity and a gradual loss of effector functions limit the clinical applicability of chimeric antigen receptor (CAR)–modified T cells and call for alternative antigen receptor designs for effective T cell–based cancer immunotherapy. Here, we applied advanced microscopy to demonstrate that TCR/CD3-based synthetic constructs (TCC) outperform second-generation CAR formats with regard to conveyed antigen sensitivities by up to a thousandfold. TCC-based antigen recognition occurred without adverse nonspecific signaling, which is typically observed in CAR–T cells, and did not depend—unlike sensitized peptide/MHC detection by conventional T cells—on CD4 or CD8 coreceptor engagement. TCC-endowed signaling properties may prove critical when targeting antigens in low abundance and aiming for a durable anticancer response.

Rational design of chimeric antigen receptors (CARs) with optimized anticancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative, single-molecule, live-cell imaging and found the sensitivity of CAR T cells toward antigen approximately 1,000-times reduced as compared to T cell antigen-receptor-mediated recognition of nominal peptide–major histocompatibility complexes. While CARs outperformed T cell antigen receptors with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-protein kinase ZAP-70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR designs, which presently limit the efficacy of CAR T cell therapies to target tumors with diminished antigen expression.