Generation of a Generic Nanobody-based CAR-T Cell Platform

Abstract

Recent advances in cancer therapy, particularly immune checkpoint blockade (ICB), have significantly improved immune responses against tumors. However, many patients do not respond to ICB, and some experience disease recurrence after initial success.<br /> Chimeric antigen receptor (CAR) T cell therapy has emerged as an alternative to ICB, but its effectiveness in solid tumors is limited by tumor heterogeneity, poor trafficking, CAR-T cell exhaustion, and antigen loss. Nanobody-based CAR-T cells offer a promising alternative to these challenges and represent an emerging therapeutic strategy.<br /> Here, we developed a novel nanobody-based CAR-T cell platform using the previously described ALFA system. To generate ALFA-tag expressing cancer cell lines, we have used classical overexpression and cutting-edge CRISPR-based approaches. The latter approach enabled the precise integration of the ALFA epitope into the genomic sequences of two promising surface targets: Mesothelin and Nectin-2. In addition, we have successfully engineered murine CAR-T cells containing the nanobody-ALFA as the antigen recognition domain ("ALFA-CAR"), and demonstrated their specific activation and cytotoxicity when co-cultured with ALFA-tagged cancer cells in vitro.<br /> To further explore the potential of the ALFA-CAR platform, we generated nanobody-based CAR-T cells targeting the native Mesothelin and Nectin-2 proteins. By comparing the activation profiles and cytotoxic capacity of ALFA-CAR cells with Mesothelin- and Nectin-2-specific CARs, we observed that ALFA-CARs resembles the killing dynamics of their native counterparts. Moreover, we have designed specific ALFA-tag mutant variants to study the impact of epitope-nanobody affinities for the functionality of CAR-T cells.<br /> In conclusion, we developed a versatile nanobody-based CAR-T cell platform (ALFA-CAR) that can be used for both the identification of new tumor antigens and the refinement of existing targets. This approach allows for direct, side-by-side comparisons in a highly controlled setting, offering a promising avenue for advancing CAR-T cell therapies.