J Transl Med

Background: BCMA-directed CAR-T cell therapy has demonstrated remarkable clinical efficacy in multiple myeloma, but robust methods to evaluate product quality and predict long-term functionality remain underdeveloped. While immunophenotypic features of leukapheresis products correlate with clinical outcomes, the characterization of infused CAR-T cell products remains challenging and inconsistent across studies.

Methods: We compared two versions of our academic BCMA-CAR product, CARTemis-1, differing only in their costimulatory domains (4-1BB vs. CD28), and performed a detailed functional and molecular characterization before and after antigen stimulation to identify which analytical approaches best capture functionally relevant differences between CAR-T cell products. Analyses included conventional flow cytometry, short-term cytotoxicity assays, repetitive antigen stimulation, metabolic profiling, and transcriptomic evaluation. Finally, metabolic profile of patient derived CAR-T cells products (n = 15) was also analyzed.

Results: Although previous studies have reported functional differences between CAR-T cell products incorporating distinct costimulatory domains, conventional assays in our system revealed no significant differences in viability, expansion dynamics, immunophenotype, or activation/exhaustion marker expression. In contrast, more functionally demanding assays revealed clear differences between constructs. Repetitive antigen stimulation revealed superior long-term cytotoxicity in CARTemis-1-BB, while metabolic profiling demonstrated enhanced spare respiratory capacity and maximal respiration after antigen exposure. Transcriptomic analysis further showed distinct pathway regulation, with enrichment of T cell activation signatures in CARTemis-1-BB and Wnt/TGFβ-related responses in CARTemis-1-28. Importantly, metabolic profiling of infused CAR-T cell products also identified distinct signatures associated with clinical outcome, distinguishing products administered to long-term responders from those infused into short-term or non-responding patients.

Conclusions: Persistence and metabolic analyses represent key tools for optimizing CAR-T cell construct selection during preclinical development, providing a more accurate assessment of CAR-T cell product quality than standard immunophenotyping and enabling the identification of constructs with superior therapeutic potential.