학술논문
In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation.
Document Type
Academic Journal
Author
Jiang G; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Neuber B; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Hückelhoven-Krauss A; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Höpken UE; Department of Translational Tumor Immunology, Max-Delbrück Center for Molecular Medicine (MDC), 13125 Berlin, Germany.; Ding Y; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Sedloev D; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Wang L; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Reichman A; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Eberhardt F; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Wermke M; Early Clinical Trial Unit (ECTU), Medical Clinic and Poliklinik I, Carl Gustav Carus University, 01307 Dresden, Germany.; Rehm A; Department of Translational Tumor Immunology, Max-Delbrück Center for Molecular Medicine (MDC), 13125 Berlin, Germany.; Müller-Tidow C; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Schmitt A; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.; Schmitt M; Department of Internal Medicine V, University Clinic Heidelberg, 69120 Heidelberg, Germany.
Source
Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE
Subject
Language
English
Abstract
The search for target antigens for CAR-T cell therapy against multiple myeloma defined the B-cell maturation antigen (BCMA) as an interesting candidate. Several studies with BCMA-directed CAR-T cell therapy showed promising results. Second-generation point-of-care BCMA.CAR-T cells were manufactured to be of a GMP (good manufacturing practice) standard using the CliniMACS Prodigy ® device. Cytokine release in BCMA.CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was assessed via intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at effector/target cell ratios of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed via flow cytometry. Stability was tested through a comparison of these evaluations at different timepoints: d0 as well as d + 14, d + 90 and d + 365 of cryopreservation. As results, (1) Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4 h chromium-release assay. There was no significant difference after cryopreservation on different timepoints. (2) In terms of endurance of BCMA.CAR-T cell function, BCMA.CAR-T cells kept their ability to kill all tumor cells over six rounds of co-culture. (3) BCMA.CAR-T cells released high amounts of cytokines upon stimulation with tumor cells. There was no significant difference in cytokine release after cryopreservation. According to the results, BCMA.CAR-T cells manufactured under GMP conditions exerted robust and specific killing of target tumor cells with a high release of cytokines. Even after 1 year of cryopreservation, cytotoxic functions were maintained at the same level. This gives clinicians sufficient time to adjust the timepoint of BCMA.CAR-T cell application to the patient's course of the underlying disease.