In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation.

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.

Impact of serum­free media on the expansion and functionality of CD19.CAR T­cells.

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T­cells. In order to overcome a lot­to­lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum­free media [Prime­XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T­Cell Expansion Xeno­Free Medium, Takara Bio™ (TB) and TCM GMP­Prototype, CellGenix™ (CG)] to the standard CAR T­cell medium containing FBS (RCF). After 12 days of CD19.CAR T­cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T­cells was evaluated using intracellular staining, a chromium release assay and a long­term co­culture assay. Expansion and viability did not differ between the CAR T­cells generated in serum­free media compared to the standard FBS­containing medium. The CG CAR T­cells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNF­α+ CAR T­cells than the CAR T­cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T­cells generated with serum­free media exhibited a higher cytotoxicity than the CAR T­cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR T­cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte­activation gene 3 and T­cell immunoglobulin and mucin­domain containing­3. The CAR T­cells cultured in FF had a higher percentage of central memory CAR T­cells (40.0 vs. 14.3%, P=0.0470) than the CAR T­cells cultured with FBS, whereas the CAR T­cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T­cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum­free media. Given the advantages of freedom from bovine material and consistent quality, serum­free media hold promise for the future development of the field of GMP manufacturing of CAR T­cells.

Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells.

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02-0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.