Activation Testing of CAR T Cells: Methods, Biomarkers, Functional Assays and Clinical Relevance
DOI:
https://doi.org/10.63282/3050-9262.IJAIDSML-V7I3P108Keywords:
CAR T Cells, Activation Testing, Biomarkers, Cytokines, Potency Assays, Flow Cytometry, Immunotherapy, Functional AssaysAbstract
Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the management of relapsed and refractory hematologic malignancies [1,7,16,17]. Despite remarkable clinical success, substantial variability exists in manufacturing quality, in vivo expansion, persistence, toxicity, and therapeutic efficacy [2,3,5,9]. Activation testing represents a critical component of CAR T-cell characterization, enabling assessment of potency, functionality, safety, and clinical performance. Modern activation testing integrates phenotypic biomarkers, cytokine profiling, cytotoxicity measurements, proliferation assays, molecular analyses, and emerging multi-omics approaches [2,7,13,20]. Recent studies emphasize the importance of memory T-cell subsets, cytokine polyfunctionality, metabolic fitness, CAR T-cell persistence, and immune microenvironmental factors as predictors of clinical outcomes. Furthermore, increasing standardization efforts seek to harmonize analytical methods used from manufacturing through post-infusion monitoring. This review summarizes current methodologies for CAR T-cell activation testing, discusses key biomarkers and functional assays, examines clinical relevance, and highlights future opportunities for integrating advanced immunological and computational approaches into CAR T-cell quality assessment.
References
[1] June CH, Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med. 2018.
[2] Mamo T, Dreyzin A, Stroncek D, McKenna DH. Emerging Biomarkers for Monitoring Chimeric Antigen Receptor T-Cell Therapy. Clin Chem. 2024.
[3] Levstek L, Janžič L, Ihan A, Kopitar AN. Biomarkers for Prediction of CAR T Therapy Outcomes: Current and Future Perspectives. Front Immunol. 2024.
[4] Maus MV, Levine BL. Chimeric Antigen Receptor T-Cell Therapy for the Community Oncologist. Oncologist. 2016.
[5] Yoo KH, et al. Predictive Markers for the Efficacy of CAR T-Cell Therapy. Blood Advances. 2025.
[6] Rafiq S, Hackett CS, Brentjens RJ. Engineering Strategies to Overcome the Current Roadblocks in CAR T Cell Therapy. Nat Rev Clin Oncol. 2020.
[7] Neelapu SS, et al. CAR T-cell Therapy Assessment and Monitoring. Nat Rev Clin Oncol. 2018.
[8] Brudno JN, Kochenderfer JN. Toxicities of CAR T Cells. Blood. 2016.
[9] Locke FL, et al. Long-Term Outcomes Following CAR T-Cell Therapy. Lancet Oncol. 2019.
[10] Fraietta JA, et al. Determinants of Response and Resistance to CD19 CAR T Cell Therapy. Nat Med. 2018.
[11] Deng Q, et al. Characteristics of Favorable CAR T-cell Products. Nat Med. 2020.
[12] Fesnak AD, June CH, Levine BL. Engineered T Cells. Nat Rev Cancer. 2016.
[13] Roddie C, et al. Biomarkers and Monitoring Strategies for CAR T Therapy. Blood. 2019.
[14] Gauthier J, Turtle CJ. Insights into Biomarkers of CAR T Cell Response. Nat Rev Clin Oncol. 2022.
[15] Guedan S, et al. State of the Art CAR T Engineering. Mol Ther. 2019.
[16] Schuster SJ, et al. Clinical Outcomes Following CAR T Therapy. N Engl J Med. 2019.
[17] Munshi NC, et al. CAR T Cells in Multiple Myeloma. N Engl J Med. 2021.
[18] Cherkassky L, et al. Functional Assessment of CAR T Cells. Cancer Immunol Res. 2016.
[19] Sterner RC, Sterner RM. CAR-T Cell Technology. Blood Cancer Journal. 2021.
[20] De Angelis B, et al. European Survey on CAR T-Cell Analytical Methods from Apheresis to Post-Infusion Immunomonitoring. Front Immunol. 2025.
