Cholesterol Esterification Enzyme Inhibition Enhances the Potency of Human Chimeric Antigen Receptor T Cells Against Pancreatic Carcinoma

Abstract

Background: The aim of this study was to assess the effectiveness of inhibiting cholesterol acyltransferase 1 (ACAT-1) in chimeric antigen receptor T (CAR-T) cells on potentiating the antitumor response against mesothelin (MSLN)-expressing pancreatic carcinoma (PC) cells. Methods: We engineered ACAT-1-inhibited CAR-T cells (CAR-T-1847 and CAR-T-1848) using the anti-MSLN CAR lentiviral vector and siRNA targeting the conserved region of the ACAT-1 gene and characterized the efficacy of these modified CAR-T cells in terms of the cytotoxicity and cytokine release of both MSLN-positive and MSLN-negative PC cells using in vitro methods and in vivo mouse xenografts. Engineered CAR-T-2598 cells were used as the negative control group. Findings: Western blot and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assays revealed significantly lower ACAT-1 protein and mRNA levels in CAR-T-1847 and CAR-T-1848 cells, compared to CAR-T-2598 cells, indicating successful ACAT-1 inhibition. However, the siRNA knockdown efficiency was only approximately 20%. The ACAT-1-inhibited CAR-T-1847 and CAR-T-1848 cells showed a higher cytotoxicity at effector-to-target cell (E/T) ratios of 8:1 and 10:1, respectively, and induced a higher secretion of proinflammatory cytokines interleukin-2 (IL-2) and interferon-gamma (IFNγ), compared with the control group, in vitro. In addition, bioluminescence imaging of tumor xenografts of ACAT-1-inhibited anti-MSLN CAR-T cells in MSLN-positive PC mice in vivo showed significant tumor regression, which is consistent with the in vitro observations. Interpretation: Our findings demonstrate a novel immunotherapeutic strategy involving the transplantation of ACAT-1-inhibited anti-MSLN CAR-T cells to target PC cells and the feasibility of enhancing the antitumor potency of CAR-T cells through the unconventional strategy of metabolic enzyme inhibition. Funding Statement: The Fund of the Translational Medical Center of HMU (grant no. 201809), the China Postdoctoral Science Foundation (grant no. 2018M641863), the Foundation of the Health Commission of Heilongjiang Province of China (grant no. 2018349), a Grant from the First Hospital of HMU (grant no. 2019B22). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All experiments involving the use of human specimens were approved by the Ethics Committee of First Hospital of Harbin Medical University and were conducted according to the principles outlined in the Declaration of Helsinki. All animal procedures were performed with the approval from the Institutional Animal Care and Use Committee.