Inhibition of MEK pathway enhances the antitumor efficacy of chimeric antigen receptor T cells against neuroblastoma

Abstract

Disialoganglioside (GD2)-specific chimeric antigen receptor (CAR)-T cells (GD2-CAR-T cells) have been developed and tested in early clinical trials in patients with relapsed/refractory neuroblastoma. However, the effectiveness of immunotherapy using these cells is limited, and requires improvement. Combined therapy with CAR-T cells and molecular targeted drugs could be a promising strategy to enhance the antitumor efficacy of CAR T cell immunotherapy. Here, we generated GD2-CAR-T cells through piggyBac transposon (PB)-based gene transfer (PB-GD2-CAR-T cells), and analyzed the combined effect of these cells and a MEK inhibitor in vitro and in vivo on neuroblastoma. Trametinib, a MEK inhibitor, ameliorated the killing efficacy of PB-GD2-CAR-T cells in vitro, whereas a combined treatment of the two showed superior antitumor efficacy in a murine xenograft model compared to that of PB-GD2-CAR-T cell monotherapy, regardless of the mutation status of the MAPK pathway in tumor cells. The results presented here provide new insights into the feasibility of combined treatment with CAR-T cells and MEK inhibitors in patients with neuroblastoma.

Keywords: CAR-T cell therapy; GD2; MEK inhibitor; chimeric antigen receptor; neuroblastoma; trametinib.

FIGURE 1

Generation of piggyBac‐mediated disialoganglioside GD2‐specific chimeric antigen receptor‐T (PB‐GD2‐CAR‐T) cells and their characteristics. A, Schematics of transposon vector expressing the GD2‐CAR construct. IR, inverted repeat; TM, trans membranous domain. B, Representative flow cytometry dot plots of the characteristics of PB‐GD2‐CAR‐T cells regarding CAR expression, programmed cell death‐1 (PD‐1) expression, and differentiation profiles in CAR‐T cells on day 14. APC, allophycocyanin. C, Coculture assay. Neuroblastoma and PB‐GD2‐CAR‐T cells were cocultured for 4 days at an effector : target (E:T) ratio of 2:1. The number of live tumor cells (GD2⁺, CD3⁻ cell fraction) were measured by flow cytometry using counting beads (mean ± SD, n = 3). D, Sequential coculture assay. In the first round, SH‐SY5Y and PB‐GD2‐CAR‐T cells were cocultured for 3 days at an E:T ratio of 2:1. For the second and third cocultures, the same number of fresh SH‐SY5Y and PB‐GD2‐CAR‐T cells as used in the previous round, were used at the same E:T ratio

FIGURE 2

Trametinib inhibits the activity of piggyBac‐mediated disialoganglioside GD2‐specific chimeric antigen receptor‐T (PB‐GD2‐CAR‐T) cells in vitro. A, An impedance‐based tumor cell killing assay. GD2‐positive neuroblastoma cell lines were cocultured with PB‐GD2‐CAR‐T cells and/or trametinib (Tram; 10 nM) at an effector : target (E:T) ratio of 2:1 in triplicate. Real‐time impedance traces of tumor cells without treatment (Tumor alone; red), and tumor cells cocultured with Tram (light green), PB‐GD2‐CAR‐T cells (CAR‐T; blue), or PB‐GD2‐CAR‐T cells and Tram (CAR‐T+Tram; pink) were acquired for 72 h. B, Additional effects of trametinib on the killing activity of PB‐GD2‐CAR‐T cells against GD2‐high SK‐N‐AS cells (AS‐High). Real‐time impedance traces of tumor cells without treatment (red), and tumor cells cocultured with PB‐GD2‐CAR‐T cells (blue), PB‐GD2‐CAR‐T cells to which Tram was added at the same time (pink), after 2 h (green), or after 6 h (light blue) were acquired for 72 h. C, ERK 1/2 phosphorylation in CD3⁺ T cells after coculture with Tram (100 nM). PB‐GD2‐CAR‐T cells were cultured in the presence of Tram or DMSO (control) for 48 h with plate‐bound CD3/CD28 Ab. D, Cytokine release assay. Production of inflammatory cytokines, γ‐interferon (IFN‐γ) and tumor necrosis factor (TNF), in PB‐GD2‐CAR‐T cells after 48 h coculture with SH‐SY5Y. E, Expression of programmed cell death‐1 (PD‐1), Tim3, and LAG3 in PB‐GD2‐CAR‐T cells. PB‐GD2‐CAR‐T cells were cultured for 48 h without SH‐SY5Y (unstimulated), or with SH‐SY5Y in the presence or absence of Tram. F, Additional effects of Tram on the proliferation of PB‐GD2‐CAR‐T cells. PB‐GD2‐CAR‐T cells on day 14 were cultured for 96 h in the presence of Tram or DMSO (control) in triplicate. The number of CAR‐T cells on day 18 was measured by Cell Counter model R1 (Olympus)

FIGURE 3

Combined therapy of piggyBac‐mediated disialoganglioside GD2‐specific chimeric antigen receptor‐T (PB‐GD2‐CAR‐T) cells with trametinib (Tram) delays tumor growth in vivo. A, Schematics of tumor challenge experiments in the xenograft model. After AS‐High‐FFluc tumor engraftment, mice were divided into four experimental groups (n = 5), as indicated. B, Tumor growth was measured as bioluminescence signal intensity and expressed as total flux (p/s). The combination treatment group showed a statistically significant reduction in tumor growth, measured as the mean total flux at day 43 (*) relative to the PB‐GD2‐CAR‐T cell group. C, Survival curves for each treatment group (n = 5). The experiment was carried out twice, and representative results are shown here. *P < .05

FIGURE 4

Pretreatment with trametinib (Tram) synergizes the antitumor effect of piggyBac‐mediated disialoganglioside GD2‐specific chimeric antigen receptor‐T (PB‐GD2‐CAR‐T) cells. A, Schematics of tumor challenge experiments in the xenograft model. After AS‐High‐FFluc tumor engraftment, mice were divided into five experimental groups (n = 4), as indicated. B, Survival curves for simultaneous treatment and pretreatment groups (n = 4). C, Schematics of tumor challenge experiments in the xenograft model. After AS‐High‐FFluc tumor engraftment and tumor growth, mice were divided into two experimental groups (n = 4), as indicated. D, Total flux change after the treatment with or without the injection of PB‐GD2‐CAR‐T cells following Tram treatment for 7 days (n = 4). The experiment was undertaken twice, and representative results are shown here. *P < .05. NS, not significant (P > .05)

FIGURE 5

Combined therapy with piggyBac‐mediated disialoganglioside GD2‐specific chimeric antigen receptor‐T (PB‐GD2‐CAR‐T) cells and trametinib delays tumor growth even in MAPK pathway nonmutated tumor mouse. A, Schematics of tumor challenge experiments in the xenograft model. After SY5Y‐FFluc tumor engraftment, mice were divided into four experimental groups (n = 3), as indicated. B, Tumor growth was measured as bioluminescence signal intensity and expressed as total flux (p/s). The combination treatment group showed a statistically significant reduction in tumor growth measured as the mean total flux at day 34 (*) relative to the PB‐GD2‐CAR‐T cell group. C, Survival curves for each treatment group (n = 3). *P < .05