Assessing CAR-T Cell Product Quality: The Crucial Role of Metabolic and Persistence Analysis after Antigen Stimulation

• Published in: Blood 2024-11, Vol.144, p.2096-2096
• Main Authors: Guijarro-Albaladejo, Beatriz, Sierro, Belén, Escamilla Gómez, Virginia, Hernández-Díaz, Paola, de la Rosa-Garrido, María, Carrasco Brocal, Inmaculada, Bejarano Garcia, Jose Antonio, Alcalde Mellado, Patricia, Rodriguez Gil, Alfonso, Lara-Chica, Maribel, Pérez-Simón, José Antonio, Garcia-Guerrero, Estefania
• Format: Article
• Language: English
• ISSN: 0006-4971
• DOI: 10.1182/blood-2024-204757

Introduction The quality of the leukapheresis product greatly influences the characteristics of the CAR-T cell product and the efficacy of the procedure. However, there is a controversy regarding the characteristics of the CAR-T cell product and its correlation with the efficacy of the therapy (Finney et al., 2019; Carniti et al., 2024). Therefore, it would be desirable to stablish which assays may help to identify the optimal CAR-T cell product. In this study, we evaluated two BCMA-CAR T cell products with different costimulatory domains, which exhibit different characteristics in vitro and in vivo, and we have identified which technics are optimal for assessing the quality of the product. Methods BCMA-CAR T lymphocytes were generated with 4-1BB (CARTemis-1-BB) and CD28 (CARTemis-1-28) costimulatory domains. Differences in expansion, CAR percentage, activation, exhaustion, and subpopulations were analyzed before and at days 3 and 5 after antigen encounter by flow cytometry. Persistence was assessed with cytotoxicity assays after repeated antigen stimulation. Mitochondrial and glycolytic metabolism were also analyzed before and after antigen stimulation, and gene expression profiling was conducted at day 5 following antigen stimulation. Results The manufacturing process of both products involved 8 days of activation and expansion using anti-CD3/CD28 beads and IL-7/IL-15. Following the 8-day manufacturing period, we performed a series of analyses that were repeated 3 and 5 days after antigen stimulation with irradiated K562-BCMA expressing cell line at 1:4 E:T ratio. Regarding expansion, although we observed a similar fold expansion between the two products, the absolute number of CAR-T cells in CARTemis-1-BB was higher than in CARTemis-1-28 (2.6x106 versus 1x106, p=0,02). Next, we examined the immunophenotype differences between the CARTemis-1-BB and CARTemis-1-28 products before and after antigen stimulation. No significant differences were observed in terms of activation or exhaustion markers between the two products. To evaluate the persistence of the cytotoxicity, we conducted repeated antigen stimulation assays with multiple myeloma cell lines every 72 hours. Both CAR-T cell products performed equally after a single antigen stimulation. However, repeated antigen stimulation assays demonstrated that CARTemis-1-BB exhibited greater persistence than CARTemis-1-28, maintaining its cytotoxic effect over multiple encounters. The metabolism of CARTemis-1-BB