CAR-T therapies have revolutionized cancer immunotherapy, representing a promising advanced therapy for relapsed/refractory (R/R) multiple myeloma (MM) patients. Despite the high remission rates observed with BCMA CAR-T cells, a significant number of patients still relapse. This lack of long-term response could be related to CAR-T cells fitness. We hypothesized that MM disease stage has an impact on CAR-T cell phenotype and functionality.

Second generation CAR-T cells targeting BCMA were generated from senior healthy donors (SHD), MM patients at diagnosis (MMd) and after >2 lines of treatment (MM>2Lines). Phenotypic analyses were performed by flow cytometry. Cytotoxic activity was measured by luciferase-based procedures and cytokine production was quantified by Luminex. In vivo antitumoral efficacy was evaluated in xenogeneic tumor models in NSG mice. RNA-seq was performed following stablished protocols.

Phenotypic analysis revealed decreasing memory phenotype and significant increase in effector cells during disease progression, with no significant differences in activation and exhaustion markers. In vitro characterization of CAR-T cells revealed reduced cytotoxicity and cytokine production against MM cell lines once MM is diagnosed. Moreover, mice treated with CAR-T cells from MM>2Lines patients presented reduced survival. Transcriptomic analyses revealed major differences between MM>2Lines and MMd/SHD. CAR-T cells from MM>2Lines patients showed decreased score of proliferation and polyfunctional gene sets, with enrichment in genes related to terminally effector cells, corroborating previous functional results.

Our results indicate that CAR-T generated from patients at later stages of the disease present phenotypic, transcriptomic, and functional features that compromise antitumor efficacy.