Innovative sdab-based CAR-T cells targeting BCMA outperform current CAR-T therapies for multiple myeloma

Abstract

Abstract Introduction: Current chimeric antigen receptor (CAR) T cell therapies offer a valuable treatment option for patients with relapsed or refractory multiple myeloma (R/R MM). While BCMA-directed products, such as idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel), have demonstrated high initial remission rates, their long-term efficacy remains limited, particularly for the first one, with median progression-free survival of 13.8 and 34.9 months, respectively. These limitations underscore the pressing need for next-generation CAR-T strategies capable of achieving durable responses and extending clinical benefit. Methods In this study, we have developed and evaluated innovative strategies to enhance the therapeutic efficacy of BCMA CAR-T cells by leveraging single-domain antibodies (sdAbs) as recognition moieties. Using phage display libraries from immunized llamas, we identified multiple families of novel BCMA-specific sdAbs. A representative sdAb from each family was selected for further characterization evaluating their affinity, binding kinetics and epitope competition via ELISA, surface plasmon resonance (SPR) and biolayer interferometry (BLI). Results We observd that sdAbs exhibited a broad affinity range (KD values from 1.16×10⁻⁷ to 8.62×10⁻¹⁰ M) and recognized multiple distinct epitopes on BCMA. Then, selected sdAbs were incorporated into 4-1BB second-generation CAR constructs, replacing the scFv with the sequence of each sdAb. Functional assessment using Jurkat triple reporter system showed reduced tonic signaling while maintaining robust activation, compared to scFv-based CAR. Based on performance, four sdAb-based CAR constructs were selected for further evaluation. Thus, sdAb-based CAR-T cells generated from healthy donors were phenotypically and functionally compared to ide-cel (scFv-based) and cilta-cel (biparatopic sdAb-based) reference products. Flow cytometry analysis demonstrated that our sdAb-based CAR-T cells were enriched in stem-cell-like memory and central memory cells. Functionally, sdAb-based CAR-T cells displayed potent and specific cytotoxicity against BCMA-expressing MM cell lines, with increased IL-2 production. Moreover, in vivo antitumoral efficacy analyses, evaluated in xenograft models of MM in NSG mice, revealed that two of the selected sdAb-based CAR-T cells (sdAb5 and sdAb10) presented enhanced antitumoral efficacy with improved survival rate compared to ide-cel. Interestingly, although sdAb5 presented a similar antitumoral efficacy than cilta-cel after CAR-T cell administration, overperformed cilta-cel in tumor rechallenge experiments designed to mimic clinical relapse, conferring a sustained protection evidenced by a greater CAR-T cell persistence in spleen and bone marrow, a significantly reduced tumor burden and a prolonged survival. Affinity studies revealed that sdAb5 displayed a KD similar to one of the sdAb comprising cilta-cel, providing mechanistic insights into their comparable initial therapeutic efficacy. To further investigate the underlying molecular mechanisms driving sdAb5 function in vivo, we performed single-cell transcriptomic profiling and flow cytometry analysis on CAR-T cells isolated at different time-points after CAR-T cell administration. Unlike cilta-cel, presenting high levels of terminally differentiated CD8⁺ effector cells, sdAb5 exhibited a Th2 polarization that transitioned into CD8⁺ effector and memory-like cells that prolonged sdAb5 persistence, being detectable even at day 78 after administration. More importantly, upon tumor rechallenge, sdAb5 underwent robust re-expansion exhibiting a specific effector-memory transcriptional program, that correlated with the already mentioned improved efficacy. These data clearly reveal that sdAb5 can confer durable tumor control through enhanced persistence, adaptability, and immunologic memory. Conclusion In this work we successfully engineered and characterized a panel of sdAb-based BCMA CAR-T cells with diverse biophysical and functional properties. Among them, sdAb5 emerged as a lead candidate, offering enhanced persistence, memory potential, and long-term efficacy exceeding that of approved CAR-T therapies in preclinical models. These findings highlight the promise of sdAb-based CAR-T cells, and our novel sdAb5 in particular, as a promising next-generation CAR-T therapies, paving the way for further clinical development and potential therapeutic application in R/R MM patients.