Rational design of BCMA-Targeted CAR-T cells using single-domain antibodies:: from binder discovery to preclinical validation

• Autores: Vianca Lizeth Ibarra García
• Directores de la Tesis: Juan Roberto Rodríguez Madoz (dir. tes.), Antonio Pineda (dir. tes.)
• Lectura: En la Universidad de Navarra ( España ) en 2025
• Idioma: inglés
• Tribunal Calificador de la Tesis: Juan Antonio Bueren Roncero (presid.), Juan José Lasarte Sagastibelza (secret.), Fermín Sánchez-Guijo Martín (voc.)
• Programa de doctorado: Programa de Doctorado en Medicina Aplicada y Biomedicina por la Universidad de Navarra
• Calificación: Sobresaliente Cum Laude
• Materias:
  • Ciencias de la vida
    • Biología celular
    • Inmunología
  • Ciencias médicas
    • Ciencias clínicas
      • Genética clínica
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• Resumen

  • Current chimeric antigen receptor (CAR) T cell therapies offer a valuable treatment option for patients with relapsed or refractory multiple myeloma (R/R MM). BCMA-directed products such as ide-cel and cilta-cel have demonstrated high initial remission rates; however, long-term disease control remains limited, with median progression-free survival (PFS) of 13.8 and 34.9 months, respectively. These outcomes underscore the pressing need for next-generation CAR-T strategies capable of improving response durability and extending clinical benefit. In this study, we developed and evaluated innovative strategies to enhance the therapeutic efficacy of BCMA-targeted CAR-T cells by leveraging single-domain antibodies (sdAbs) as antigen recognition domains. Using phage display libraries derived from immunized llamas, we identified multiple families of novel sdAbs specific for BCMA. A representative sdAb from each family was selected for further characterization, including affinity measurements, binding kinetics, and epitope competition assessed via ELISA, surface plasmon resonance (SPR), and biolayer interferometry (BLI). Subsequently, we generated sdAb-based CAR constructs by replacing the scFv of a second-generation 4-1BB CAR with the selected sdAb sequences. These constructs were functionally screened using the Jurkat triple reporter (TPR) system, which revealed reduced tonic signaling while preserving antigen-induced activation compared to scFv-based CARs. Based on these results, four sdAb-based CAR constructs were selected for further investigation. CAR-T cells were generated from healthy donor T cells and compared to ide-cel (scFv-based CAR-T) and cilta-cel (biparatopic sdAb-based CAR-T) in terms of phenotype and function. Flow cytometry analysis showed that sdAb-based CAR-T cells were enriched for stem cell memory and central memory subsets, while expressing similar levels of exhaustion markers (PD-1, LAG-3, TIM-3). Functionally, sdAb-CAR-T cells exhibited specific cytotoxicity against BCMA-expressing MM cell lines and increased IL-2 production. In vivo efficacy was evaluated in NSG mouse xenograft models. Two candidates, sdAb5 and sdAb10, demonstrated superior antitumor activity and improved survival compared to ide-celh. Notably, sdAb5 exhibited antitumor efficacy comparable to cilta-cel. In a tumor rechallenge model mimicking clinical relapse, sdAb5 conferred sustained protection, as evidenced by reduced tumor burden, prolonged survival, and enhanced CAR-T cell persistence in both spleen and bone marrow. Affinity measurements revealed that sdAb5 exhibited a KD similar to that of cilta-cels biparatopic construct, providing a mechanistic rationale for their comparable in vivo performance. Interestingly, combining sdAb5 and sdAb10 into a biparatopic CAR, mimicking the cilta-cel design, resulted in markedly reduced efficacy. This was likely due to a substantial increase in the KD, impairing CAR-T cell dissociation following antigen engagement. In conclusion, we identified and characterized multiple sdAb-based CAR-T cell constructs with distinct binding properties, some of which outperformed or matched the efficacy of clinically approved therapies. These results underscore the potential of sdAb-based CAR-T cells as promising next-generation therapeutics for patients with relapsed or refractory MM and support their further clinical development.