Abstract
Multiparametric flow cytometry is a powerful cellular analysis tool used in various stages of drug development. In adoptive cell therapies, the flow cytometry methods are used for the evaluation of advanced cellular products during manufacturing and to monitor cellular kinetics after infusion. In this report, we discussed the bioanalytical method development challenges to monitor cellular kinetics in CAR-T cell therapies. These method development challenges include procuring positive control samples for the development of the method, flow cytometry panel design, LLOQ, prestain sample stability, staining reagents and data analysis.
References
- 1. . Adoptive cellular therapies: the current landscape. Virchows Arch. 474, 449–461 (2019).
- 2. . Current status and perspective of CAR-T and CAR-NK cell therapy trials in Germany. Gene Ther.
doi:10.1038/s41434-021-00246-w (2021). - 3. Validation of a flow cytometry-based method to quantify viable lymphocyte subtypes in fresh and cryopreserved hematopoietic cellular products. Cytotherapy 23, 77–87 (2021).
- 4. . Different subsets of T cells, memory, effector functions, and CAR-T immunotherapy. Cancers 8, 36, 1–12 (2016).
- 5. 1. Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia. Blood 130(21), 2317–2325 (2017).
- 6. Monitoring CAR-T cell kinetics in clinical trials by multiparametric flow cytometry: benefits and challenges. Cytometry 100, 72–78 (2021).
- 7. Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 371, 1507–1517 (2014).
- 8. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat. Med. 24, 563–571 (2018).
- 9. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J. Clin. Invest. 129, 2210–2221 (2019).
- 10. Model-based cellular kinetic analysis of chimeric antigen receptor-t cells in humans. Clin. Pharmacol. Ther. 109, 716–727 (2021).
- 11. . Understanding bioanalysis regulations. In: Regulated Bioanalysis: Fundamentals and Practice. , Lowes S (Eds). AAPS Advances in the Pharmaceutical Sciences Series, Springer International Publishing AG Cham, Switzerland, 26 (2017).
- 12. Convergence of cell pharmacology and drug delivery. Stem Cells Transl. Med. 8, 874–879 (2019).
- 13. . Recommendations for the validation of flow cytometric testing during drug development: II assays. J. Immunol. Methods 363(2), 120–134 (2011).
- 14. Flow cytometry method validation protocols. Curr. Protoc. Cytom. 87, e53 (2019).
- 15. Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses. Cytometry 100, 79–91 (2021).
- 16. CLSI. Validation of assays performed by flow cytometry; draft guidance. CLSI Document H62. Clinical and Laboratory Standards Institute, PA, USA (2021).
- 17. . Flow Cytometry in the drug development process. In: Flow Cytometry in Drug Discovery and Development. John Wiley & Sons, Inc, NJ, USA, 69–240 (2011).
- 18. . Flow cytometry: an overview. Curr. Protoc. Immunol. 120, 5.1.1–5.1.11 (2018).
- 19. High-sensitivity flow cytometric assays: considerations for design control and analytical validation for identification of rare events. Cytometry 100, 42–51 (2021).
- 20. 2019 White Paper on recent issues in bioanalysis: FDA immunogenicity guidance, gene therapy, critical reagents, biomarkers and flow cytometry validation (Part 3 – recommendations on 2019 FDA immunogenicity guidance, gene therapy bioanalytical challenges, strategies for critical reagent management, biomarker assay validation, flow cytometry validation & CLSI H62). Bioanalysis 11, 2207–2244 (2019).
- 21. New directions in chimeric antigen receptor T cell [CAR-T] therapy and related flow cytometry. Cytometry B Clin. Cytom. 98, 299–327 (2020).
- 22. Improving efficiency and sensitivity: European research initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL. Leukemia 27, 142–149 (2013).
- 23. Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow.
https://doi.org/doi:10.1002/cyto.b.20118Citations 112 (2006). - 24. Flow cytometric characterization of cerebrospinal fluid cells. Cytometry Part B 80B, 271–281 (2011).
- 25. Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials. J. Immunol. Methods 385, 79–89 (2012).
- 26. . Evaluation of sample stability for cellular kinetics and pharmacodynamic flow cytometry methods. Bioanalysis 11, 1881–1184 (2019).
- 27. . Optimizing a multicolor immunophenotyping assay. Clin. Lab. Med. 27, 469–485 (2007).
- 28. . High-dimensional immunophenotyping with fluorescence-based cytometry: a practical guidebook. Methods Mol. Biol. 2032, 1–29 (2019).