Nat Biomed Eng | 基于亮氨酸拉链技术进行多重受体CAR T细胞的免疫磁珠分离（纪念斯隆凯特琳癌症中心）

   Despite the success of CAR T cell therapies in refractory B cell malignancies and myeloma, the efficacy is limited by a number of challenges, including CAR target-antigen heterogeneity on tumor cells and associated antigen-loss escape, acquired CAR T cell dysfunction and tumor-induced immune suppression. Dual-specificity CAR T cells have been developed to address antigen heterogeneity and antigen-loss escape. However, clinical trials have revealed that B cell leukemia and lymphoma cells with low antigen expression or negative for multiple antigens can evade the effects of tandem-CAR and dual-CAR T cell therapies, as well as sequential treatments with CAR T cells targeting different single antigens. Meanwhile, strategies have been developed to overcome inhibitory receptor-associated immune suppression and improve T cell persistence, including the coexpression of dominant-negative or switch receptors that attenuate PD-1, CD200R and Fas signaling or reprogramme these inhibitory receptors to transmit activating rather than inhibitory costimulatory signals. In addition, approaches have been used to mitigate CAR T cell dysfunction and lack of persistence due to tonic or antigen-driven CAR signaling, including temporal modulation of CAR expression or activation, optimization of CAR expression density, reduction of CAR CD3ζ signal intensity and utilization of 4-1BB instead of CD28 costimulation.

    Considering the diverse mechanisms of immune evasion utilized by cancer cells, combinations of multiple synthetic biology interventions are crucial for optimizing the efficacy of CAR T cells. However, engineering multiple features into a single T cell product is challenging due to the transgene-packaging constraints of current gene-delivery vectors. To address these challenges, here the authors developed a leucine zipper-based cell-sorting system that enables a single-step immunomagnetic enrichment of cells dual-transduced with distinct vectors, aiming to produce homogeneous and purified cellular products with a doubled transgene integration capacity. The 'Zip sorting' approach enabled the generation of T cells capable of expressing a maximum of four different CARs concurrently, as well as the simultaneous expression of a maximum of three 'switch' receptors. Moreover, this platform facilitated the production of highly purified complex cellular therapeutics, utilizing clinically applicable immunomagnetic separation techniques, beginning with heterogeneous cell populations characterized by low initial co-transduction rates. Importantly, they demonstrated that T cells with multiple CARs and multiple switch receptors eliminated antigenically heterogeneous populations of leukaemia cells coexpressing multiple inhibitory ligands in syngeneic mouse models. Collectively, Zip-sorted multi-CAR multi-switch-receptor T cells can overcome multiple mechanisms of CAR T cell resistance by integrating diverse therapeutic strategies.