Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

Abstract

CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

Fig 1. Transduced T cells express CAR.

CAR expression on 2G (A, B) and 3G (C, D) T cells was evaluated by flow cytometry before and after co-culture. T cells were gated as viable singlet cells expressing CD3. Statistical differences were calculated using Wilcoxon matched pairs signed rank test (start vs B/IL-2 3G; p = 0.0313, 2G; p = 0.0313). Healthy donors are shown as grey circles and CLL patients as black squares.

Fig 2. The majority of CAR T cells are CD8+ effector or effector memory cells.

Lineage (A; CD3+ CD8+), exhaustion phenotype (B; PD-1+ Tim-3+) and memory/effector status (C) were evaluated by flow cytometry at start and after co-culture with autologous B cells and IL-2 or IL-2 alone. Graphs are showing CAR+ T cells, gated as viable singlet cells expressing CD3 and CAR. For memory/effector status the cells were divided into the following subtypes: naïve: CD45RA+ CCR7+, effector: CD45RA+ CCR7-, central memory (CM): CD45RA- CCR7+ and effector memory (EM): CD45RA- CCR7-. There was no statistical difference in expression of lineage or exhaustion markers between the different time-points or between groups. T cells of effector memory phenotype increased (IL-2 vs B/IL-2 3G: p = 0.0836, 2G: p = 0.1232), while effector cells decreased (start vs B/IL-2 3G: p = 0.0625, 2G: p = 0.0625). There was also a tendency to increased number of central memory cells (start vs B/IL-2 3G: p = 0.0625, 2G: p = 0.0625). Error bars represent SEM. Statistical differences were calculated with unpaired t-test with Welch correction and Wilcoxon matched-pairs signed rank test.

Fig 3. Tyrosine kinase phosphorylation downstream CAR.

Tyrosine kinase phosphorylation in mock, 2G or 3G transduced T cells, 1 and 3 hours post stimulation with autologous B cells. Darker color indicates increased signal intensity. Data was normalized for CAR expression. A selection of the most interesting phosphorylation targets in the assay is shown. Complete array data can be found in S1 Table.

Fig 4. Adaptor proteins involved in TCR signaling are phosphorylated downstream CAR.

Phosphorylation of CD3ε (A), LAT (B), ZAP-70 (C), LCK (D), SYK (E), Erk (F) and CREB (G) at start and after stimulation with B cells and IL-2 is demonstrated using MILLIPLEX® MAP T-Cell Receptor Signaling kit. The mean value is indicated in the Figure and the dotted line indicates background levels. Data was normalized for CAR expression. Statistical differences were calculated by unpaired t-test with Welch correction and Wilcoxon matched-pairs signed rank test (* = p<0.05).

Fig 5. The presence of antigen stimulates and maintains the cytotoxic capacity of CAR T cells.

The Figure demonstrates cytotoxicity of CAR T cells generated from CLL patients (n = 3) against CD19+ Daudi before (A) and after co-culture with IL-2 (B) and autologous B cells (C). The cytotoxicity was normalized to CAR expression due to variation of expression. Current CAR expression at each specific time-point was used for normalization. Error bars represent SEM.

Fig 6. 3G CAR T cells show increased proliferation compared to 2G CAR.

Proliferation was determined each week through cell count prior to addition of B cells. The Figure shows T cell expansion from start to stimulation with IL-2 (A) or B cells and IL-2 (B). Data was normalized for CAR expression. (C) Remaining viable B cells in co-culture at endpoint (after 2–4 B cell stimulations, 4:1 ratio) analyzed by flow cytometry. The mean is indicated in the Figure. Statistical differences were calculated by unpaired t-test with Welch correction (* = p<0.05, ** = p<0.01, *** = p<0.001).

Fig 7. CAR T cells release IFNγ and Granzyme B in response to antigen.

Culture supernatants were collected 24h after stimulation at day 1 (start) and at endpoint (end). CAR T cells responded to antigen stimulation through secretion of IFNγ (A, B) and Granzyme B (C, D). IFNγ levels were higher in in the groups stimulated with both B cells and IL-2 compared to B cells alone, both at the beginning (3G: p = 0.0418, 2G: p = 0.1389) and at the end of co-culture (3G: p = 0.0228 p = 2G: p = 0.1446). Data was normalized for CAR expression. Error bars represent SEM. Statistical differences were calculated by unpaired t-test with Welch correction.

Fig 8. CAR T cells eliminate tumor cells and expand in vivo.

Mice (Nu/Nu) were injected s.c. with Daudi cells and treated with vehicle, mock, 2G or 3G CAR T cells (6 mice per group). Tumor growth is shown in A. 2G and 3G CAR T cells were infused in NSG mice engrafted with human CD34+ cells (10 mice per group). Successful engraftment is shown as detectable CD45 and CD19 cells in two representative mice (B). Migration and persistence in vivo was monitored by GFP-FireFly(FF)-luciferase. The bioluminescence signal from mice infused with 2G (red line) or 3G (black line) T cells is shown in C. At day 30, peripheral blood was collected and the presence of CAR+ T cells and B cells (CD19+) were detected with flow cytometry. T cells and B cells were gated from the CD45+ population. A representative sample is shown in D.