Anti-breast cancer activity of GPC3-specific CAR-T cells inducing interleukin-12 expression in immunocompetent mice / 肿瘤

ABSTRACT

Objective: To construct the murine chimeric antigen receptor-redirected T lymphocyte (CAR-T) that can target glypican 3 (GPC3) and express interleukin-12 (IL-12) in an activation-dependent manner, and to explore the anti-cancer efficacy of CAR-T cells in immunocompetent mice bearing GPC3 positive (GPC3+) breast cancer. Methods: Gene recombination technology was used to construct the retroviral vector encoding GPC3-specific CAR (m9.28z) or encoding both GPC3-specific CAR and nuclear factor of activated T cells (NFAT)-IL-12 expression system (m9.28z/IL-12). Murine T cells were retrovirally transduced to develop m9.28z CAR-T cells and m9.28z/IL-12 CAR-T cells. In vitro, the cytotoxicity assay and ELISA assay were used to assess the biological functions of m9.28z/IL-12 CAR-T cells. In vivo, the GPC3+ breast cancer E0771-GPC3 cell subcutaneously transplanted model was established in C57BL/6 mice, and the tumor-bearing mice were injected with 5×106 murine untransduced T cells (mUTD, which served as the negative control), 5×106 m9.28z CAR-T cells, 2×106 m9.28z CAR-T cells, 5×106 m9.28z/IL-12 CAR-T cells and 2×106 m9.28z/IL-12 CAR-T cells, respectively. The growth of xenografts in nude mice was observed, and the expression levels of CD8α and forkhead box protein P3 (FOXP3) in tumor tissues were detected by immunohistochemistry. Results: The retroviral vector encoding m9.28z or m9.28z/IL-12 was constructed, and the m9.28z CAR-T cells and m9.28z/IL-12 CAR-T cells were developed subsequently. In vitro, the m9.28z/IL-12 CAR-T cells killed GPC3+ breast cancer cells specifically and effectively, and secreted more tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) than m9.28z CAR-T cells (both P < 0.05). In vivo, as compared with m9.28z CAR-T cells, the m9.28z/ IL-12 CAR-T cells suppressed the growth of xenografts significantly (P < 0.001), and there were more CD8+ T cells and less regulatory T cells (Treg) in tumor tissues treated with m9.28z/IL-12 CAR-T cells (both P < 0.01). Conclusion: The m9.28z/IL-12 CAR-T cells, which can express IL-12 inducibly, can display more potent anti-cancer activity against GPC3+ tumor than m9.28z CAR-T cells in vitro and in immunocompetent breast cancer transplanted mice.