Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand

Abstract

Anti-CD19 chimeric antigen receptor (CAR)-T cell therapy has evolved as a standard of care for various forms of relapsed/refractory B cell malignancies in major developed countries. However, access to industry-driven CAR-T cell therapy is limited in developing countries, partly due to the centralized manufacturing system. Here, we demonstrated the feasibility of the point-of-care (POC) manufacturing of anti-CD19 CAR-T cells from heavily pretreated patients and healthy graft donors at an academic medical center in Thailand using a closed semi-automated production platform, CliniMACS Prodigy, and established in-process quality control and release testing to ensure their identity, purity, sterility, safety, and potency. Nine out of the nine products manufactured were used in a pilot study (ISRCTN17901467). However, we did observe that starting T cells with CD4/CD8 ratios of less than one-third had a high chance of manufacturing failure, which could be minimized by serum supplementation. Further analysis of T cell phenotypes in the infused versus circulating CAR-T cells revealed the differentiation from early memory subtypes toward effector cells in vivo. The POC manufacturing and quality control settings herein could be applied to other CAR-T cell products and may benefit other academics, especially those in developing countries, making CAR-T cells more accessible.

Keywords: B cell malignancies; CAR; CAR-T cells; CD19; MT: Regular Issue; POC; Thailand; automated; chimeric antigen receptor; manufacturing; point-of-care.

Graphical abstract

Figure 1

A schematic overview outlining the workflow of CAR-T cell manufacturing using a closed semi-automated production platform Leukapheresis product was transferred to the CliniMACS Prodigy device with TS520 tubing set and TCT program on day 0, and T cells were enriched and activated through CD3 and CD28 co-stimulation. LVV transduction was performed on day 1, after which T cells were expanded in culture media, typically for 12 days, until the final CAR-T cell product was harvested. The IPC and QC samples were analyzed at the indicated time points. Q3d, every 3 days.

Figure 2

T cell composition of leukapheresis products and recovery post-enrichment Leukapheresis product was collected from an enrolled patient or a healthy donor (case IDs H3 and H7) and processed within 24 h without cryopreservation. (A) Viability of CD45⁺ leukocytes in the leukapheresis products (pre-enrichment) and the enriched CD4⁺/CD8⁺ T cells after magnetic separation (post-enrichment). (B) Representative flow cytometric plots showing the purity of CD3⁺ T cells in viable CD45⁺ cells pre- and post-enrichment. (C) Percentages and numbers of CD4⁺ and CD8⁺ T cells among viable CD45⁺ cells (upper and center) and the calculated CD4/CD8 ratio (lower) pre- and post-enrichment. M, mean value; NS, not significant.

Figure 3

T cell composition at the start and end of culture (A) Number of total CD3⁺ T cells loaded into the culture chamber at the start and at the end of culture on day 12 or as indicated (i.e., day 11 for case IDs H7 and 8, day 14 for case 5, and day 18 for case 6 [upper]). Numbers of CD4⁺ and CD8⁺ T cells are also shown (lower). Cultured cells were classified according to the culture conditions into three groups: no HS (nos. 1−4), rescued (nos. 5 and 6), and 3% HS (nos. 7−9). M, mean value. (B and C) Cells were grouped according to CD4/CD8 ratios at the start of culture (cutoff at one-third, dashed line) (B), and total CD3⁺ T cell numbers were compared between groups with relatively lower (<1/3) and higher (>1/3) ratios on day 5 of culture (C). ∗∗p < 0.01; two-sided Student’s t test. The culture conditions are also labeled. (D) Viability of CD3⁺ T cells at the start and end of culture. NS, not significant (p > 0.05); two-sided Student’s t test. (E) CD4/CD8 ratio at the start and end of culture.

Figure 4

T cell transduction efficiency and CAR-T cell yield (A) Flow cytometric gating strategy for characterization of CAR⁺ and CAR⁻ T cells among viable CD45⁺ cells. (B) Percentage of CAR⁺ rate among viable CD3⁺ T cells on day 5 and at the end of culture. NS, not significant (p > 0.05); two-sided Student’s t test. The culture conditions are also labeled. (C) Percentages of CAR⁺ CD4⁺ and CAR⁺ CD8⁺ T cells among viable CAR⁺ CD3⁺ T cells. (D) Total number of manufactured CAR⁺ CD3⁺ T (CAR-T) cells in the final cell product, SiCF-019 cells. (E) The dosing of CAR-T cells in the enrolled patients. Pt., patient. An asterisk indicates a value below the minimum target dose of 1 × 10⁶ cells per kilogram. (F) Cellular composition reported as percentages of the defined population in viable CD45⁺ cells in the final cell product (see Table S3 for all values).

Figure 5

Immunophenotypic characterization and QC release testing of the final cell product (A) A schematic diagram of different T cell subsets and their combinatorial expression of multiple markers. (B) Flow cytometric gating strategy for characterization of T cell subsets in the final cell product. (C) Percentages of T_N, T_SCM, T_CM, T_EM, and T_EFF cells among viable CD4⁺ and CD8⁺ T cells. (D) Results of QC release testing with respect to safety and sterility. (E) Stability of the final cell product in its primary package at 4°C based on viability of CD3⁺ T cells. NS, not significant (p > 0.05) versus control at 0 h; two-sided Student’s t test. T_EM and T_EFF, effector memory and effector T cells, respectively.

Figure 6

Functionality of the final cell product (A) Cytotoxicity of the manufactured SiCF-019 (effector) cells against CD19⁺ and CD19⁻ tumor (target) cells. (Top left) Flow cytometric gating strategy for specifically detecting the cell death of PKH67-labeled tumor cells after incubation with SiCF-019 cells by annexin V/7-AAD assay. Percentages of total cell death of K562, Z-138, and REH cells, comprising annexin V⁺ and/or 7-AAD⁺ cells, at various E:T ratios at 4 h are plotted. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus basal control (without SiCF-019); ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 versus indicated groups; one-way ANOVA with Tukey’s multiple comparison test. The culture conditions are also labeled. (B) Quantitative measurement of TNF-⍺ and IFN-γ by ELISA in cell-free supernatant collected from the coexposure of untransduced T cells or SiCF-019 cells with tumor cells at various E:T ratios at 4 h. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus indicated groups; one-way ANOVA with Tukey’s multiple comparison test. NS, not significant.

Figure 7

Expression of T cell exhaustion markers in culture with poor expansion (A) Expansion kinetics of total CD3⁺ T cells and CAR-T cells on various days of culture in rescued cases 5 and 6, which required additional serum supplementation (i.e., 5% HSA or 3% HS). (B) Flow cytometric analysis of the exhaustion markers TIM3 and PD-1 in cultured CD3⁺ T cells at various time points.

Figure 8

Detection of circulating CAR-T cells in the peripheral blood of patients and their differentiation status (A) Flow cytometric gating strategy for detection of viable circulating CAR-T cells in blood samples. (B) Absolute number of viable CAR-T cells in patients at various time points for up to 6 months after cell infusion. (C) Flow cytometric gating strategy for characterization of T cell subsets in blood samples. (D) Percentages of T_N/SCM, T_CM, T_EFF, and T_EM cells among viable CAR⁺ CD4⁺ and CAR⁺ CD8⁺ cells at the peak of CAR-T persistence.