Dual Targeting of BCMA and SLAMF7 with the CARtein System: Chimeric Antigen Receptors with Intein-mediated Splicing Elicit Specific T Cell Activation against Multiple Myeloma

Noelia Moares^1,2Pablo Gonzalez-Garcia^1,2Juan P Muñoz-Miranda^1,2Antonio Gabucio^1,2Rosa Luna-Espejo¹Javier Ocaña Cuesta¹Wenjie Yi-He^1,2Ricardo Fernandez-Cisnal¹Cecilia Fernandez-Ponce^1,2Francisco Garcia-Cozar^1,2*

• ¹Institute for Biomedical Research and Innovation of Cádiz, University of Cádiz, Cádiz, Spain
• ²Department of Biomedicine, Biotechnology and Public Health, Faculty of Medicine, University of Cádiz, Cádiz, Spain

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy against multiple myeloma (MM). Despite these advances, several barriers continue to limit the overall effectiveness of this approach, including high production costs, extended manufacturing timelines, safety concerns, and the risk of tumor antigen escape due to selective therapeutic pressure. To address these limitations, innovative CAR T strategies are being developed. One promising approach involves the engineering of modular CAR systems, which leverage adaptor molecules to enable multi-antigen targeting, thus enhancing specificity, safety, and overall efficiency of CAR T cell therapy. CAR T-cells directed against BCMA and SLAMF7 antigens have elicited strong and robust antitumor responses in MM therapy. Hence, we developed a novel modular CAR platform directed against BCMA and SLAMF7 by exploiting split intein-mediated protein splicing mechanism. This approach enables the formation of specific covalent peptide bonds between CAR modules, while maintaining an almost seamless CAR structure, preserving its integrity and functionality. The rational design of the intein-spliced CAR system (CARtein) was optimized through the use of advanced protein structure prediction software. Moreover, we show that cells expressing the spliced CARtein constructs, designed to target BCMA, SLAMF7, or both antigens simultaneously, induce a robust and highly specific activation. Our results suggest that the CARtein platform may serve as a promising, versatile, and highly specific tool for the modular design and engineering of CARs.