CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies

Abstract

The development of genetic modification techniques has led to a new era in cancer treatments that have been limited to conventional treatments such as chemotherapy. intensive efforts are being performed to develop cancer-targeted therapies to avoid the elimination of non-cancerous cells. One of the most promising approaches is genetically modified CAR-T cell therapy. The high central memory T cell (Tcm) and stem cell-like memory T cell (Tscm) ratios in the CAR-T cell population increase the effectiveness of immunotherapy. Therefore, it is important to increase the populations of CAR-expressing Tcm and Tscm cells to ensure that CAR-T cells remain long-term and have cytotoxic (anti-tumor) efficacy. In this study, we aimed to improve CAR-T cell therapy's time-dependent efficacy and stability, increasing the survival time and reducing the probability of cancer cell growth. To increase the sub-population of Tcm and Tscm in CAR-T cells, we investigated the production of a long-term stable and efficient cytotoxic CAR-T cell by modifications in the cell activation-dependent production using Phytohemagglutinin (PHA). PHA, a lectin that binds to the membranes of T cells and increases metabolic activity and cell division, is studied to increase the Tcm and Tscm population. Although it is known that PHA significantly increases Tcm cells, B-lymphocyte antigen CD19-specific CAR-T cell expansion, its anti-cancer and memory capacity has not yet been tested compared with aCD3/aCD28-amplified CAR-T cells. Two different types of CARs (aCD19 scFv CD8-(CD28 or 4-1BB)-CD3z-EGFRt)-expressing T cells were generated and their immunogenic phenotype, exhausted phenotype, Tcm-Tscm populations, and cytotoxic activities were determined in this study. The proportion of T cell memory phenotype in the CAR-T cell populations generated by PHA was observed to be higher than that of aCD3/aCD28-amplified CAR-T cells with similar and higher proliferation capacity. Here, we show that PHA provides long-term and efficient CAR-T cell production, suggesting a potential alternative to aCD3/aCD28-amplified CAR-T cells.

Keywords: CAR-T; immunologic memory; immunotherapy; leukemia; phytohemagglutinin.

Figure 1

(A) The schematic model of the experimental design aims to determine Tcm and Tscm sub-population, proliferation, and cytotoxicity of either CAR1928-T cells (turquoise) or CAR19BB-T cells (red) with CD28 or 4-1BB-based CAR construct upon activation of T cells with Phytohemagglutinin/ aCD3 and aCD28. (B) The flow cytometry plots of CD3+ T cell isolation from three different donors. (C) The bar graph showing CAR+ T cell frequency with CAR19BB or CAR1928 constructs after activation with PHA or anti-CD3/anti-CD28 by expression of EGFRt. (D) The bar graph showing CAR+ T cell frequency with CAR19BB or CAR1928 constructs after second activation and re-transduction with PHA or anti-CD3/anti-CD28 by expression of EGFRt. (E) The bar graph shows division numbers of the CAR-expressing total T cells during expansion for 14 days upon stimulation with T cell complete media including either with PHA or aCD3/aCD28. The two-tailed t-test statistical significance was represented by * p < 0.05 and ns: non-significant, and the bars in the graph are the mean +/− standard deviation of the groups.

Figure 2

The flow cytometry plots showing a representative set of the T cells sub-population plots from a healthy adult donor in CD4⁺ and CD8⁺ including Tn + Tscm (CD3⁺, CD45RA⁺, CD62L⁺), Tcm (CD3⁺, CD45RA⁻, CD62L⁺), TemEarly (CD3⁺, CD45RO⁺, CD27⁺, CD45RA⁻, CD62L⁻) T cells, TemLate (CD3⁺, CD45RO⁺, CD27⁻, CD45RA⁻, CD62L⁻), and Temra (CD3⁺, CD45RA⁺, CD62L⁻) in healthy donors. The two-tailed t-test statistical analysis was performed. Legend: TemEARLY: effector memory early T cells; Tcm: central memory T cells; Tn + Tscm: naïve T cells and stem cell-like memory T cells; Temra: terminally differentiated effector memory T cells; TemLATE: effector memory late T cells.

Figure 3

The bar graphs show the quantitated distribution of Tn + Tscm, Tcm, TemEARLY, TemLATE, and Temra sub-populations of total CD4+ or CD8+ T cells in PHA-activated or aCD3/aCD28-activated CD3+ T cells from three healthy donors on day 7, day 14, and day 21. The distribution of the T cell sub-types of (A) total CD4+ T cells in PHA-activated CD3+ T cells, (B) total CD8+ T cells in PHA-activated CD3+ T cells, (C) total CD8+ T cells in aCD3/aCD28-activated CD3+ T cells, and (D) total CD4+ T cells in aCD3/aCD28-activated CD3+ T cells. The two-tailed t-test statistical significance was represented by * p < 0.05 and ns: non-significant, and the bars in the graph are the mean +/− standard deviation of the corresponding groups. Legend: TemEARLY: effector memory early T cells; Tcm: central memory T cells; Tn + Tscm: naïve T cells and stem cell-like memory T cells; Temra: terminally differentiated effector memory T cells; TemLATE: effector memory late T cells.

Figure 4

(A) The bar graph shows the comparison of Tn + Tscm, Tcm, TemEARLY, TemLATE, and Temra sub-populations of (A) total CD4+ T cells and (B) total CD8+ T cells either activated with anti-CD3/anti-CD28 or PHA on day 14. The bar graphs show the distribution of T cell sub-populations of (C) total CD4+ T cells and (D) total CD8+ T cells activated either with anti-CD3/anti-CD28 or PHA on day 21. The two-tailed t-test statistical significance was represented by * p < 0.05 and ns: non-significant, and the bars in the graph are the mean +/− standard deviation of the corresponding groups. Legend: TemEARLY: effector memory early T cells; Tcm: central memory T cells; Tn + Tscm: naïve T cells and stem cell-like memory T cells; Temra: terminally differentiated effector memory T cells; TemLATE: effector memory late T cells.

Figure 5

The bar graphs show percentages of T cell sub-populations expressing CAR(CD28) or CAR(4-1BB) in (A) the CD4+ or (B) the CD8+ T cell population after the first and second activations on days 7, 14, and 21. The two-tailed t-test statistical significance, and the bars in the graph are the mean +/− standard deviation of the corresponding groups. Legend: TemEARLY (orange): effector memory early T cells; Tcm (blue): central memory T cells; Tn + Tscm (purple): naïve T cells and stem cell-like memory T cells; Temra (green): terminally differentiated effector memory T cells; TemLATE (yellow): effector memory late T cells.

Figure 6

Exhaustion profile of PHA- or aCD3/aCD28-activated CAR-T cells. (A) Histograms showing representative plots of LAG3, TIM3, and PD1 expressions of day 7 CAR-T cells and untransduced T cell control in the same sample activated with PHA after gating on CD3+, CD4+, CD8+, and EGFRt+. (B) Comparison of MFI values of exhaustion biomarkers (LAG3, TIM3, PD1) of CD4+ CAR1928 or CAR19BB T cells at day 14 in CD3+ T cells activated with anti-CD3/anti-CD28 and PHA. (C) Comparison of MFI values at Day 14 of exhaustion biomarkers (LAG3, TIM3, PD1) of CD8+ CAR1928 or CAR19BB T cells in CD3+ T cells activated with anti-CD3/anti-CD28 and PHA. (D) Comparison of MFI values on day 14 of exhaustion biomarkers (LAG3, TIM3, PD1) of total CD4+ T cells in CD3+ T cells activated with anti-CD3/anti-CD28 and PHA. (E) Comparison of MFI values on day 14 of exhaustion biomarkers (LAG3, TIM3, PD1) of total CD8+ T cells in anti-CD3/anti-CD28 and PHA-activated CD3+ T cells. (F) Comparison of MFI values on day 21 of exhaustion biomarkers (LAG3, TIM3, PD1) of total CD4+ CAR-T cells in CD3+ T cells activated with anti-CD3/anti-CD28 and PHA. (G) Comparison of exhaustion biomarkers (LAG3, TIM3, PD1) 21st day MFI values of total CD8+ CAR-T cells in CD3+ T cells activated with anti-CD3/anti-CD28 and PHA. (H) Comparison of the MFI values of the exhaustion biomarkers (LAG3, TIM3, PD1) of total CD4+ T cells (LAG3, TIM3, PD1) on day 21 among CD3+ T cells activated with anti-CD3/anti-CD28 and PHA. (I) Comparison of exhaustion biomarkers of total CD8+ T cells (LAG3, TIM3, PD1) on day 21, among CD3+ T cells activated with anti-CD3/anti-CD28 and PHA. The two-tailed t-test statistical significance was represented by * p < 0.05 and ns: non-significant, and the bars in the graph are the mean +/− standard deviation of the corresponding groups. Legend: MFI: mean fluorescence intensity; LAG3: lymphocyte-activation gene 3; TIM3: T cell immunoglobulin and mucin-domain containing-3; PD1: Programmed cell death protein 1.

Figure 7

Cytotoxicity capacity of the CAR-T cells. (A) Analysis of CD25 activation and CD107a cytotoxic de-granulation biomarkers of CAR-T cells and untransduced T cell control in CD3+ T cells in the sample co-culture by flow cytometry and death of CD19+ RAJI cells after 48 h in CAR-T RAJI co-culture experiments. (B) CD19+ RAJI Killing Assay Mortality Rates of CAR19BB and CAR1928 CAR-Ts produced by activating PHA and anti-CD3/anti-CD28 at day 2, 7, and 14. (C) Bar graph showing the percentage of living CD19+ RAJI Cells, the anti-cancer activity with CAR-T cells produced by the second activation at day 2 and 7. The two-tailed t-test statistical significance was represented by * p < 0.05, and the bars in the graph are the mean +/− standard deviation of the corresponding groups. Legend: 1:1, 5:1, or 10:1; Effector CAR-T: Target RAJI cell co-incubated for one-week.

Figure 8

Activation of CAR-T cells with CD25 and CD107a upregulation. Bar graph of (A) CD25 activation and (B) CD107a cytotoxic degranulation biomarkers of CAR-T cells and control T cells in CD3+ T cells after 48 h in 1:1 CAR-T: RAJI co-culture experiments. Bar graph of (C) CD25 activation and (D) CD107a cytotoxic de-granulation biomarkers of CAR-T cells and control T cells in CD3+ T cells after 48 h in 5:1 CAR-T: RAJI co-culture experiments. Bar graph of (E) CD25 activation and (F) CD107a cytotoxic de-granulation biomarkers of CAR-T cells and control T cells in CD3+ T cells after 48 h in 10:1 CAR-T: RAJI co-culture experiment. The two-tailed t-test statistical significance was represented by * p < 0.05, and the bars in the graph are the mean +/− standard deviation of the groups including CAR19BB aCD3/aCD28 (blue), CAR1928 aCD3/aCD28 (orange), CAR19BB PHA (gray), and CAR1928 PHA (green).