A simple and effective method to purify and activate T cells for successful generation of chimeric antigen receptor T (CAR-T) cells from patients with high monocyte count

Abstract

Background: Chimeric antigen receptor T (CAR-T) cells are genetically modified T cells with redirected specificity and potent T-cell-mediated cytotoxicity toward malignant cells. Despite several CAR-T products being approved and commercialized in the USA, Europe, and China, CAR-T products still require additional optimization to ensure reproducible and cost-effective manufacture. Here, we investigated the critical parameters in the CD3⁺ T-cell isolation process that significantly impacted CAR-T manufacturing's success.

Methods: CAR-T cells were prepared from cryopreserved peripheral blood mononuclear cells (PBMC). The thawed PBMC was rested overnight before the CD3⁺ T cell isolation process using CTS^™ Dynabeads^™ CD3/CD28. Different isolation media, cell-bead co-incubation time, and cell density were examined in this study. Activated CD3⁺ T cells were transduced with a gamma retroviral vector carrying the CD19 or BCMA CAR sequence. The CAR-T cells proliferated in a culture medium supplemented with interleukin 2 (IL-2).

Results: CD14⁺ monocytes hindered T-cell isolation when X-VIVO 15 basic medium was used as the selection buffer. The activation of T cells was blocked because monocytes actively engulfed CD3/28 beads. In contrast, when DPBS was the selection medium, the T-cell isolation and activation were no longer blocked, even in patients whose PBMC contained abnormally high CD14⁺ monocytes and a low level of CD3⁺ T cells.

Conclusions: In this study, we discovered that selecting CD3⁺ T-cell isolation media is critical for improving T-cell activation, transduction, and CAR-T proliferation. Using DPBS as a CD3⁺ T cell isolation buffer significantly improved the success rate and shortened the duration of CAR-T production. The optimized process has been successfully applied in our ongoing clinical trials. Trial registration NCT03798509: Human CD19 Targeted T Cells Injection Therapy for Relapsed and Refractory CD19-positive Leukemia. Date of registration: January 10, 2019. NCT03720457: Human CD19 Targeted T Cells Injection (CD19 CAR-T) Therapy for Relapsed and Refractory CD19-positive Lymphoma. Date of registration: October 25, 2018. NCT04003168: Human BCMA Targeted T Cells Injection Therapy for BCMA-positive Relapsed/Refractory Multiple Myeloma. Date of registration: July 1, 2019.

Keywords: Chimeric antigen receptor T (CAR-T) cells; Design of experiments (DoE); Monocyte; T-cell activation; T-cell purification.

Fig. 1

CAR-T cell characteristics when using CAR-T culturing medium as the T cell selection buffer. This figure showed the outcome of CAR-T cell manufacture when CAR-T culturing medium was used as the T cell bead-based selection buffer. a The percentage of CD3 positive cells in PBMC before positive selection with anti-CD3/28 Dynabeads (BF) and positively selected cells after 44–52-h activation (AF). b The percentage of CD3⁺ cell population that express surface CD25 marker (b) or CD69 (c) before and after activation. d Cell diameter measurement results throughout the CAR-T production process. RV, the day of retroviral transduction and just before cell selection (AF). Day 1–7 indicates days after retroviral transduction. e Fold expansion of CAR-T cells after retroviral transduction. f Percentage of CD3⁺ cells express CAR at days 4 and 7 after retroviral transduction. g The percentage of live cells that were positive for CD14 surface marker before selection and after activation

Fig. 2

CAR-T manufacturing data of patients participating in phase I clinical trials. The figure shows the PBMC composition, CAR expression, and CAR-T cell expansion data of 26 patients who participated in CD19 and BCMA CAR-T phase I clinical trials. a Heatmap analysis of CD14 percentage in patients’ PBMC and CAR-T expansion days required to reach the requested dosage. The left column shows the CD14 percentage (1.04–56.29%) in PBMC from high to low before cryopreservation, and the right shows CAR-T expansion days (4–11) of the corresponding patient. b CAR-T cell expansion duration of each patient was plotted against CD14 expression levels in PBMC. Among all patients, CAR-T cells (01-001-CHFY, 02-01-004-LUYN, and 03-002-009-GAXF) took the longest to expand. Expansion duration: the days between the completion of retroviral transduction and the filling of the CAR-T product. c Individual expansion rate of CAR-T cells of all patients. The expansion curve of T cells from patients (01-001-CHFY, 02-01-004-LUYN, and 03-002-009-GAXF) are marked with black, blue, and purple, respectively. d CAR-T cell expansion duration divided by different CD3⁺ T cell isolation buffers. e CAR expression percentage in CD3⁺ cells of each patient was plotted against CD14 expression levels in PBMC. f CAR expression percentage in CD3⁺ cells was compared between groups of CAR-T cells selected with two isolation buffers

Fig. 3

Manufacturing CAR-T cells using X-VIVO15 basic medium as T cell selection buffer. Using X-VIVO15 basic medium as the CD3⁺ T cell isolation buffer hampered the CAR-T cells manufacturing, and expansion deficiency was related to CD14⁺ cells. a Microscopic images showed numerous adherent cells with engulfed magnetic beads after 48-h T-cell activation inside the cell culture bag. b CD14 expression in live cells before CD3⁺ T cell selection and after 48-h T cell activation. The percentage of CD3⁺ cells express surface CD25 (c) or CD69 (d) before CD3⁺ T cell selection or after 48-h T cell activation. e Fluctuations of cell diameter values throughout the CAR-T manufacturing process. f Expansion fold of CAR-T cells after retroviral transduction. g This experiment compares the expansion fold on day seven after retroviral transduction and the average expansion fold on day 6 in the phase I clinical trial (6 patients with day six cell-counting data recorded). h CAR expression percentage in CD3⁺ cells at the end of CAR-T manufacture (the day of product filling) between this experiment and the phase I clinical trial (all 26 patients)

Fig. 4

Significant differences were observed in CAR-T manufacture using different T cell selection buffers. This figure compares DPBS or X-VIVO15 basic medium as the CAR-T manufacturing process’s CD3 + T cell selection buffer. The DoE was repeated three times, and three PBMC samples were used in each repeat. Groups and factor levels in the DoE are shown in Table 1. a and b show the percentage of CD3⁺ cells that express CD25 (a) or CD69 (b) surface markers. In c, the cell diameter was measured before CD3⁺ T cell selection and after 48-h T-cell activation. In a, b, and c, an unpaired t-test was used, and a two-tailed P value was calculated; ns, P > 0.9999; ****, P < 0.0001. d Changes in CD25 expression percentage in CD3⁺ cells, e CD69 expression percentage in CD3⁺ cells, and f live-cell diameter in the CAR-T manufacturing process. g Expansion fold of CAR-T cells after retroviral transduction. h CAR expression percentage in CD3⁺ cells at days six and nine after retroviral transduction

Fig. 5

DoE analysis of T cell isolation parameters and the validation through CAR-T manufacture. This figure shows the results of DoE analysis for the three above parameters and the validation of the optimized T cell selection process with a new round of CAR-T manufacture. a DoE distribution analysis of input factors and output responses. The diagonal pattern shows the desired responses and their distribution in research factors. CD25 (b) and CD69 (c) expression percentage in CD3⁺ cells before CD3⁺ T cell selection and after 48-h T cell activation, live cell diameter changes (d), expansion fold of CAR-T cells after retroviral transduction (e), and CAR expression percentage in CD3⁺ cells at day 3, 6, 9 after retroviral transduction of CAR-T manufacturing process for DoE validation (f)

Fig. 6

Results of BCMA CAR-T manufacture with optimized T cell selection process in the clinical trial. This figure shows that DPBS was successfully used as the T cell isolation buffer to manufacture anti-BCMA CAR-T cells for 13 patients in phase I clinical trial. a CD14 expression and CD3 expression in patients’ PBMC. b CAR-T cell expansion rate after retroviral transduction. The three patients with excessively low CD3⁺% in their PBMC are marked red, yellow, and green. CAR-T cell expansion duration of each patient was plotted against the percentage of CD14⁺ cells (c) or CD3⁺ cells (d) in PBMC before selection. CAR expression percentage in CD3⁺ cells of each patient plotted against the percentage of CD14⁺ (e) or CD3⁺ (f) cells in their PBMC