Rational engineering of sdAb-based CAR T cells targeting BCMA enhances antitumor efficacy and persistence in Multiple Myeloma

Ibarra-Garcia, V.; Calleja-Cervantes, M. E.; Rochira, F.; Azagra, I.; San Martin-Uriz, P.; Aguirre-Ruiz, P.; Rodriguez-Diaz, S.; Martinez-Turrillas, R.; Iglesias, E.; Ibanez-Sala, I.; Molina, E.; Bernasconi-Bisio, F.; Rodriguez-Marquez, P.; Puchades-Carrasco, L.; Gomez-Cebrian, N.; Lasarte, J. J.; Hernaez, M.; Alfonso-Pierola, A.; Tamariz, E.; Rodriguez-Otero, P.; San Miguel, J.; Rodriguez-Madoz, J. R.; Pineda-Lucena, A.; Prosper, F.

doi:10.64898/2026.06.02.729637

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bioRxiv · 10.64898/2026.06.02.729637

Rational engineering of sdAb-based CAR T cells targeting BCMA enhances antitumor efficacy and persistence in Multiple Myeloma

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Abstract

BCMA-directed CAR T therapies have transformed the treatment of relapsed/refractory multiple myeloma (MM), yet durable responses remain limited by insufficient persistence and functional exhaustion. The contribution of antigen-binding domain properties to CAR T cell performance is not fully defined. Here, we report the rational discovery and engineering of BCMA-targeting single-domain antibodies (sdAbs) and their incorporation into second-generation CAR T cells. We generated sdAbs recognizing distinct BCMA epitopes with diverse binding kinetics. Comprehensive biophysical and functional characterization identified sdAb5 as a lead candidate with balanced binding kinetics and a non-overlapping epitope relative to clinically approved constructs. sdAb-based CAR T cells exhibited potent antigen-specific cytotoxicity, minimal tonic signaling, and preserved IL-2 production without excessive inflammatory activation. In xenograft models, sdAb5-based CAR T cells induced durable tumor control, enhanced persistence, and improved responses to tumor rechallenge compared with ide-cel and cilta-cel constructs. Notably, this superior functionality correlated with balanced binding kinetics rather than maximal affinity. Single-cell transcriptomic analyses corroborated these findings, revealing a restrained inflammatory activation in sdAb5-based CAR T cells with preservation of transcriptional plasticity. Collectively, these findings support antigen-binding optimization as a key determinant of CAR T-cell durability and identify sdAb5 as a candidate for next-generation BCMA-directed therapies in MM.

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Ibarra-Garcia, V., Calleja-Cervantes, M. E., Rochira, F., Azagra, I., San Martin-Uriz, P., Aguirre-Ruiz, P., Rodriguez-Diaz, S., Martinez-Turrillas, R., Iglesias, E., Ibanez-Sala, I., Molina, E., Bernasconi-Bisio, F., Rodriguez-Marquez, P., Puchades-Carrasco, L., Gomez-Cebrian, N., Lasarte, J. J., Hernaez, M., Alfonso-Pierola, A., Tamariz, E., Rodriguez-Otero, P., San Miguel, J., Rodriguez-Madoz, J. R., Pineda-Lucena, A., Prosper, F.. 2026-06-05. Rational engineering of sdAb-based CAR T cells targeting BCMA enhances antitumor efficacy and persistence in Multiple Myeloma. https://doi.org/10.64898/2026.06.02.729637

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