Assessing CAR-T Cell Product Quality: The Crucial Role of Metabolic and Persistence Analysis after Antigen Stimulation
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 
Published: December 2024

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 and CARTemis-1-28 was also analyzed. Before antigen encounter, no metabolic differences were observed between the two products. However, after antigen encounter, CARTemis-1-BB showed greater maximal respiration (78,34 pmol/min vs 23,71 pmol/min, p=0,02) and spare respiratory capacity (60,7 pmol/min vs 15,71 pmol/min, p=0.039) both of which are widely recognized as predictors of the persistence of the cytotoxic capacity of a CAR-T cell product (Pearce et al., 2009).
Finally, we analyzed the gene expression profiles of our products following antigen stimulation. Gene expression analysis revealed overexpression of genes involved in lymphocyte activation in CARTemis-1-BB, whereas CARTemis-1-28 showed overexpression of genes associated with the TGF-β signaling pathway, which is known to have an immunosuppressive effect on CAR-T cells (Ledergor et al., 2024) and may explain the decreased mitochondrial respiration observed in CARTemis-1-28 (Dimeloe et al., 2019).
Conclusions
Flow cytometry and cytotoxicity analyses performed before and after a single antigen stimulation do not demonstrate significant differences between two different CAR-T cell products. By contrast, the determination of persistence in repeated antigen stimulation assays and the metabolic analysis following one antigen stimulation clearly showed significant functional differences which might allow to select the most optimized CAR-T cell product for clinical therapy.