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Background <p>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.</p> Methods <p>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 (<i>n</i> = 15) was also analyzed.</p> Results <p>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.</p> Conclusions <p>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.</p>

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Metabolic and functional analysis including repetitive antigen stimulation provides a more accurate assessment for CAR-T cell product quality and long-term functionality

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月11日

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  • Beatriz Guijarro-Albaladejo,
  • Belén Sierro-Martínez,
  • Inmaculada Carrasco-Brocal,
  • Paola Hernández-Díaz,
  • María de la Rosa-Garrido,
  • Raquel Muñoz-García,
  • Jose Antonio Bejarano-García,
  • Joan Mañé-Pujol,
  • Patricia Alcalde-Mellado,
  • Alfonso Rodríguez-Gil,
  • Virginia Escamilla-Gómez,
  • Maribel Lara-Chica,
  • Cristina Aguirre-Portolés,
  • Victor Galán-Gómez,
  • Laura Escribà-García,
  • Marta Español-Rego,
  • Luis Gerardo Rodríguez-Lobato,
  • Javier Briones,
  • Carlos Fernández-de Larrea,
  • Antonio Pérez-Martínez,
  • Ángela Bella,
  • Jose Antonio Pérez-Simón,
  • Estefanía García-Guerrero

摘要

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.