Identification and Evaluation of Novel Sdab-Based CAR-T Cells Against BCMA with Improved Therapeutic Efficacy for Multiple Myeloma
Authors: Ibarra-Garcia Vianca , Molina Eva , Bernasconi-Bisio Franco , Rochira Federica , Rodríguez Diaz Saray , Martínez Turrillas Rebeca , Rodriguez-Marquez Paula , Jauregui Patricia , Vanrell Lucia , Villar Sara , Rodríguez-Otero Paula , Lasarte Juan J , Rodriguez-Madoz Juan Roberto , Prosper Felipe , Pineda-Lucena Antonio 
Published: December 2024

The latest advances in cancer immunotherapy for Multiple Myeloma (MM) based on Chimeric Antigen Receptors (CAR) T cells, represent a real option for relapsed/refractory patients (1). However, despite high remission rates observed after B-Cell Maturation Antigen (BCMA) CAR-T therapy, there is a lack of long-term responses (median PFS of 13.8 months for ide-cel and 34.9 months for cilta-cel) (2), highlighting the need for improved therapeutic strategies. In this work we developed and evaluated innovative approaches to enhance the therapeutic efficacy of BCMA CAR-T cells, based on the benefits of utilizing single-domain antibodies (sdAbs) as recognition moieties of CAR-T cells.

Analysis of phage display libraries, generated from immunized llamas, allowed the identification of several families of novel SdAbs specific for BCMA, that were subsequently sequenced and subcloned for SdAb purification. A representative SdAb from each family was selected and fully characterized in terms of affinity, binding kinetics, and epitope competition by ELISA, surface plasmon resonance (SPR) and biolayer interferometry (BLI). SdAb-based CAR constructs were generated replacing the scFv sequence of a 2nd generation 4-1BB CAR with the selected SdAb sequences, and their activation profile and tonic signal were evaluated in Jurkat triple reporter system (TPR). Interestingly, in comparison to several scFv-based CAR constructs (based on ARI0002h and a humanized version of ide-cel), our selected SdAb-based CARs presented a similar activation capacity with lower tonic signal. Finally, four different SdAb-based CAR constructs were selected for further analysis, based on their affinity, binding kinetics, absence of tonic signaling, and specific activation in response to BCMA+ cells.

SdAb-based CAR-T cells were produced via lentiviral transduction of activated T cells from healthy donors and expanded in the presence of IL7 and IL15. Then, SdAb-based CAR-T cells were phenotypically and functionally characterized in a side-by-side comparison to scFv-based CAR-T cells. FACS analysis of the final CAR-T products revealed that both, SdAb-based and scFv-based CAR-T cells, presented similar phenotypic profiles, enriched in stem-cell-like memory and central memory T cells, with reduced levels of exhaustion makers (PD1, LAG-3 and TIM-3). Moreover, selected sdAb-based CAR-T cells exhibited a similar specific cytotoxic activity, measured using the Bright-GloTM Luciferase Assay System, compared to scFv-based CAR-T cells. Interestingly, sdAb-based CAR-T cells produced lower levels of IFNγ and IL-2 in response to MM cell lines expressing BCMA. Finally, antitumoral efficacy of SdAb-based CAR-T cells was evaluated in vivo, in xenograft models of MM using immunodeficient NSG mice. Thus, animals were treated with a sub-optimal dose of 105 CAR-T cells/mouse, 14 days after administration of 3x106 MM1S tumoral cells. Two of the selected sdAb-based CAR-T cells presented enhanced antitumoral efficacy, as evidenced by luciferase imaging, with improved survival rate, compared to scFv-based CAR-T cells. However, combination of both sdAb-based CAR-T cells did not improved antitumoral efficacy. The development and evaluation of a single biparatopic SdAb-based CAR construct combining both SdAbs presenting improved properties is ongoing.

In conclusion, we have successfully identified and characterized multiple sdAb-based CAR-T cells with different affinities, demonstrating their potential to surpass the antitumoral efficacy of traditional scFv-based CAR-T cells. These findings underscore the promise of sdAb-based CAR-T cell therapy as a novel and effective treatment for BCMA-positive MM and potentially other hematological malignancies. The enhanced cytotoxicity and antitumoral efficacy observed in preclinical models pave the way for further clinical development and potential therapeutic application, offering new hope for patients with relapsed or refractory multiple myeloma.

References: Berdeja J., et al. The Lancet (2021). Cancanelli L., et al. Hematology Reports (2023).