IL-7 and CCR2b Co-Expression-Mediated Enhanced CAR-T Survival and Infiltration in Solid Tumors

Abstract

Chimeric antigen receptor T (CAR-T) cells are not effective in solid tumor treatment due to reduced invasion and expansion, and short survival time. This study aimed to explore whether interleukin (IL)-7 and CCR2b expression could improve GD2-CAR-T cell survival and infiltration in neuroblastoma and melanoma treatment. IL-7 and CCR2b were inserted into the classical second-generation CAR structure to construct 7×2b CAR. The 7×2b CAR-T cell phenotypes were evaluated by flow cytometry and the chemokine levels by ELISA. The 7×2b CAR-T cell migration and anti-tumor abilities were detected by Transwell assay and animal experiments in vivo. We report that compared with that of CAR-T cells, 7×2b CAR-T cell IL-7 secretion and CCR2b expression did not affect the T cell surface expression of CAR or CAR-T specificity and efficacy against tumor cells. The 7×2b CAR-T cells could induce IFN-γ secretion in GD2-positive tumor cells, killing them as well as conventional CAR-T cells. Moreover, IL-7 and CCR2b co-expression enhanced the 7×2b CAR-T cell survival and migration. Similar to conventional CAR-T, 7×2b CAR-T cells could also inhibit tumor growth and increase IFN-γ, Gzms-B, and IL-2 expression. Finally, unlike in mice injected with CAR-T cells, CD3 expression was the most abundant in the spleen and tumor tissues in mice injected with 7×2b CAR-T cells. Our study demonstrates that IL-7 and CCR2b co-expression in GD2-CAR-T cells exhibit stronger anti-tumor activity than classical second-generation CAR-T cells, shedding light on the potential novel GD2-positive neuroblastoma and melanoma treatment approach.

Keywords: CAR-T cells; CCR2; IL-7; immune cell therapy; melanoma; neuroblastoma.

Figure 1

IL-7 and CCR2 co-expression in anti-GD2 CAR-T cells. (A) The mRNA expression of CCL2 was analyzed in GSE96631 from the GEO database. (B) Structure diagram of 7×2b CAR. (C) The expression of CAR and CCR2b in CAR-T cells. (D) The T-mock and CAR-T cells were cultured in a H3 medium without IL-7. IL-7 levels in the supernatant on day 5 were detected by ELISA.

Figure 2

The effect of 7×2b CAR-T cells on tumor cells. (A) The expression of GD2 in melanoma and neuroblastoma cell lines. (B) After co-incubation for 12 h at a ratio of 10:1, the supernatant was removed to detect the IFN-γ secretion level by ELISA. (C) Luciferase-based reporter assays were used to detect the lysis (%) of 7×2b CAR-T cells against three melanoma and neuroblastoma cells with different E:T ratios.

Figure 3

The IL-7 and CCR2b co-expression enhanced 7×2b CAR-T cell survival and migration. (A) The proliferation ability of 7×2b CAR-T cells. We cultured one million conventional CAR-T and 7×2b cells (all with IL-2), and the cell number was calculated in the presence or absence of IL-7. (B) The effect of 7×2b CAR-T cell supernatant on T cell amplification. Supernatants of CAR-T were collected and mixed with H3 medium supplemented with 2% of autologous serum and 300 U/mL of IL-2 at a 1:1 ratio, which was then used to culture 1×106 CD3+ T cells for 7 days to detect the amplification ratio of CD3, CD4, and CD8 subsets. (C) The CAR-T cells were prepared independently 8 times, and the TSCM subset proportions were analyzed. (D) The CCL2 level in the supernatant was detected by ELISA after 48 h of culture of 5×106 tumor cells. (E) The migration ability of CAR-T cells induced by HEK293T cells, SK-NS-AS cells, and CCL2 (100 ng/mL). (F) Representation of the lower Transwell compartment, showing T cells in each lower compartment. N = 3, p < 0.05, p < 0.01.

Figure 4

Anti-tumor activity of the 7×2b CAR-T cells in vivo. (A) The level of CCL2 secretion in IMR-32-CCL2 cells was detected by ELISA. (B) The antitumor activity of CAR-T cells was evaluated by in vivo imaging in mice bearing IMR-32-CCL2 xenografts. (C) Fluorescence evaluation of the in vivo imaging of the mice in (B). (D) The levels of IFN-γ, IL-2, and GZMS-B in the sera of the mice were detected by ELISA. (E) IL-7 and CCL2 were also measured. (F) The human CD3 and Ki-67 positive cells in the spleen and tumor of the mice were detected by immunohistochemistry after CAR-T infusion. N = 5.