Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma.

Glioblastoma (GB) remains refractory to chimeric antigen receptor (CAR)-T cell therapy, mainly attributed to tumor heterogeneity and antigen escape. CAR-T cells utilizing monomeric streptavidin-2 (mSA2) instead of a traditional target binding domain, bind biotinylated antibodies and can be directed to variable targets to mediate anti-tumor effects. Although such an approach might circumvent the aforementioned challenges, the potential of mSA2 CAR-T cells for brain tumor treatment remains unexplored. In this study, we generated mSA2 CAR-T cells and tested their efficacy against GB by tailoring their specificity toward GB-associated markers CD276, EPHA2, CD70 and IL13Ra2. In vitro, mSA2 CAR-T cells specifically recognized multiple primary GB cell lines in a target- and biotinylated antibody-dependent manner. Moreover, in heterogenous tumor environments, mSA2 CAR-T cells simultaneously targeted multiple subpopulations, guided by combinations of biotinylated antibodies, indicating their potential to address tumor heterogeneity. Finally, the mSA2 CAR-T cell-mediated anti-tumor functions were demonstrated in vivo. Immunocompromised mice orthotopically implanted with CD70⁺ or CD276⁺ GB cells and treated with mSA2 CAR-T cells pre-armed with antibodies against these two antigens exhibited control of tumor growth and induction of GB cell apoptosis after therapy. Taken together, our study suggests that antibody-guided mSA2 CAR-T cells can target potentially any surface GB-related antigen both in vitro and in vivo, either univalently or multivalently, with underlined clinical implications.