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Chimeric antigen receptor (CAR)-modified T and NK cell immunotherapy is a promising approach for cancer treatment. Due to the lack of tunability in anti-tumor activity, conventional CAR therapies have limited efficacy at low tumor antigen densities. To tune the CAR response to tumor cell surface antigens, we have developed a split CAR using the SpyCatcher-SpyTag system. The SpyCatcher serves as the ectodomain to constitute a SpyCatcher-CAR (SpyCAR), while SpyTag is attached to the antibodies that recognize tumor antigens. With dimerization mediated by SpyCatcher and SpyTag, the number and activation level of SpyCARs recruited by tumor antigens depends on the SpyTag number in the "antibody-SpyTag" fusion protein. The results demonstrated that the increasing number of SpyTags effectively enhanced the cytotoxicity of SpyCAR-NK92 cells against target cells. The development of SpyCAR with tunable cytotoxicity provides a novel strategy for CAR-based tumor immunotherapies. • The SpyCAR system was developed based on non-covalent SpyCatcher-SpyTag. • The SpyCatcher-SpyTag interaction mediates enhanced cytotoxicity of SpyCAR-NK92 cells. • The cytotoxicity of SpyCAR-NK92 cells is in a SpyTag number-dependent manner. [ABSTRACT FROM AUTHOR]

Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer NK cells for the efficient expression of CARs. This study reported the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. We constructed a third-generation NKG2D-based CAR that kills cancer cells by targeting up to eight stress-induced ligands (NKG2DLs). Our results demonstrated that the CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered NK cells. This research helps to optimize the production of CAR-NK cells and enhance their functional activity, to provide CAR-NK cell products with better and more uniform quality. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)-modified immune cells have emerged as a promising approach for cancer treatment, but single-target CAR therapy in solid tumors is limited by immune escape caused by tumor antigen heterogeneity and shedding. Natural killer group 2D (NKG2D) is an activating receptor expressed in human NK cells, and its ligands, such as MICA and MICB (MICA/B), are widely expressed in malignant cells and typically absent from healthy tissue. NKG2D plays an important role in anti-tumor immunity, recognizing tumor cells and initiating an anti-tumor response. Therefore, NKG2D-based CAR is a promising CAR candidate. Nevertheless, the shedding of MICA/B hinders the therapeutic efficacy of NKG2D-CARs. Here, we designed a novel CAR by engineering an anti-MICA/B shedding antibody 1D5 into the CAR construct. The engineered NK cells exhibited significantly enhanced cytotoxicity against various MICA/B-expressing tumor cells and were not inhibited by NKG2D antibody or NKG2D-Fc fusion protein, indicating no interference with NKG2D-MICA/B binding. Therefore, the developed 1D5-CAR could be combined with NKG2D-CAR to further improve the obstacles caused by MICA/B shedding. • 1D5-CAR targeting MICA/B α3 was designed based on structure. • 1D5-CAR-modified NK cells exhibited potent cytotoxicity. • 1D5-CAR didn't disturb NKG2D-MICA/B interaction. • 1D5-CAR could be combined with NKG2D-CAR. [ABSTRACT FROM AUTHOR]