Impact of serum‑free media on the expansion and functionality of CD19.CAR T‑cells

Abstract

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T‑cells. In order to overcome a lot‑to‑lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum‑free media [Prime‑XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T‑Cell Expansion Xeno‑Free Medium, Takara Bio™ (TB) and TCM GMP‑Prototype, CellGenix™ (CG)] to the standard CAR T‑cell medium containing FBS (RCF). After 12 days of CD19.CAR T‑cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T‑cells was evaluated using intracellular staining, a chromium release assay and a long‑term co‑culture assay. Expansion and viability did not differ between the CAR T‑cells generated in serum‑free media compared to the standard FBS‑containing medium. The CG CAR T‑cells had a statistically significant higher frequency of IFNγ⁺ and IFNγ⁺TNF‑α⁺ CAR T‑cells than the CAR T‑cells 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 T‑cells generated with serum‑free media exhibited a higher cytotoxicity than the CAR T‑cells 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 T‑cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte‑activation gene 3 and T‑cell immunoglobulin and mucin‑domain containing‑3. The CAR T‑cells cultured in FF had a higher percentage of central memory CAR T‑cells (40.0 vs. 14.3%, P=0.0470) than the CAR T‑cells cultured with FBS, whereas the CAR T‑cells 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 T‑cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum‑free media. Given the advantages of freedom from bovine material and consistent quality, serum‑free media hold promise for the future development of the field of GMP manufacturing of CAR T‑cells.

Keywords: chemically defined medium; chimeric antigen receptor T‑cells; fetal bovine serum; serum‑containing medium; serum‑free medium.

Figure 1

Expansion, viability, transduction rate, CD4⁺/CD8⁺ ratio and vector copy number of CD19.CAR T-cell culture. (A) Expansion on day 12 of T-cells cultured in five different media. (B) Viability of T-cells cultured in four different media assessed on day 12. (C) Transduction efficiency of CD19.CAR T-cell culture on day 12 of expansion in four different media. (D) Vector copy numbers of CD19.CAR T-cells cultured in four different media are shown. (E) CD4⁺/CD8⁺ ratio of 7AAD-CD19.CAR⁺ T cells cultured in four different media. (A-E) Each symbol represents a separate donor (n=5). Short horizontal lines indicate the mean of data in each experimental group. Data were analyzed by one-way ANOVA (represented by a longer horizontal line above the data) with Dunnett's multiple comparison test (represented as brackets). ^*P<0.05 and ^**P<0.01. CAR, chimeric antigen receptor.

Figure 2

Intracellular staining of CD19.CAR T-cells cultured in four different media. (A-C) Cells expressing IFNγ, TNF-α or IFNγ and TNF-α are shown as a percentage of all CAR T-cells, CD4⁺ CAR T-cells or CD8⁺ CAR T-cells. Box and whisker plots represent median, upper and lower quartiles and minimum and maximum (n=5). The line indicates a comparison using one-way ANOVA, whereas the brackets indicate comparisons using Dunnett's test. ^*P<0.05 and ^**P<0.01. (D) Representative data of one healthy donor from five independent experiments are shown. CAR T-cells were assessed using anti-TNF-α and anti-IFNγ antibodies by flow cytometry. Plots are gated on live CAR T-cells. CAR T-cells stimulated with CD19⁺ tumor cells are shown in blue, whereas non-transduced T-cells are visualized as a control in orange. CAR, chimeric antigen receptor.

Figure 3

Chromium release assay. (A-D) Data of five unique donors of chromium release assay are shown. Each color/symbol represents a separate donor. Experiments were performed in triplicate and are represented as the mean ± SD. The mean value of all five donors is shown as a connecting line. The ratio of effector to target cells was 30:1, 10:1, 3:1 and 1:1 and non-transduced cells (NT) were used as a control. The results of chromium release assays for each respective medium are shown. (E) The mean value is shown as a connecting line for each medium separately. Data were analyzed using one-way ANOVA (represented by a longer vertical line) with Dunnett's multiple comparison test (represented as brackets). ^*P<0.05 and ^**P<0.01.

Figure 4

Cytotoxicity of CAR T-cells in a long-term co-culture. (A and B) Absolute cell counts of (A) CAR T-cells and (B) Daudi cells following co-culture in the corresponding medium are shown. Before adding new tumor cells to the CAR T-cells every 5 days, the co-culture was measured using flow cytometry. The mean ± SEM values are displayed. (C) The ratio of CAR T-cells per µl/Daudi cells per µl at different time points of co-culture are shown in the heatmap. Blue symbolizes a ratio of >1, whereas red symbolizes a ratio of <1. Therefore, blue indicates the overgrowth of CAR T-cells and the lysis of tumor cells, while red indicates an insufficient killing of tumor cells. Data are displayed separately for every donor. CAR, chimeric antigen receptor.

Figure 5

Phenotyping of CAR T-cells. (A) Phenotyping of CAR T-cells on day 12 of production was assessed using flow cytometry. Each donor is visualized separately. (B) Phenotyping of CAR T-cells on day 12 visualized separately for EMRA, EM, naïve and CM CAR T-cells. Data were analyzed using one-way ANOVA of all five donors followed by Dunnett's multiple comparisons test (represented as brackets). ^*P<0.05 and ^**P<0.01. Boxes show the mean and error bars indicate SEM. (C-F) Phenotyping of CAR T-cells co-cultured with tumor cells. Absolute cell count of CAR T-cells divided to the four different CAR T-cell types, EMRA, EM, naïve and CM. Boxes show the mean and error bars indicate SEM. CAR, chimeric antigen receptor; EMRA, effector memory cells re-expressing RA; EM, effector memory cells; naïve, naïve cells; CM, central memory cells.

Figure 6

Exhaustion markers of CAR T-cells. The levels of exhaustion markers (A) LAG-3, (B) PD-1 and (C) TIM-3 in CAR T-cells co-cultured with tumor cells are shown. Graphs show the mean values. Data were analyzed using a two-way ANOVA followed by Dunnett's multiple comparison test. P-values of Dunnett's multiple comparison test at day 30 are represented as brackets. ^*P<0.05 and ^**P<0.01. CAR, chimeric antigen receptor; LAG-3, lymphocyte-activation gene 3; PD-1, programmed cell death protein 1; TIM-3, T-cell immunoglobulin and mucin-domain containing-3.