Background Hepatocellular carcinoma (HCC) is the leading cause of cancer mortality worldwide. While HBV/HCV infection is the primary cause of HCC, overexpression of MET, the receptor of hepatocyte growth factor (HGF), occurs in 50% HCC patients, and is an indicator of poor prognosis. Although the multi-target MET tyrosine kinase inhibitor cabozantinib is FDA approved for treating advanced HCC, the long-term efficacy versus toxicity remains unknown. Our study is to develop specific MET-targeting chimeric antigen receptor T (CAR-T) cells for treating HCC with MET overexpression.

Methods Based on a well-established anti-MET monoclonal antibody, we synthesized and cloned the single-chain variable fragment (ScFv) sequence into two retroviral based 2nd generation CAR vectors (MET-CAR.CD28.ζ. and MET-CAR.4-1BB.ζ.). A MET-CAR without CD3ζ domain (MET-CARΔ) served as a negative control. To produce MET-CAR-T cells, healthy PBMCs were stimulated with anti-CD3/CD28 antibodies in the presence of IL-7/IL-15 followed by transduction with MET-CAR viral particles. T cell transduction efficacy was determined using flow cytometry. HCC cell lines with variable MET expression from high/positive (MHCC97H, C3A, and JHH5) to MET low/negative (SNU398) were used to determine MET-specific CAR T cells specificity and effector function using MTS assay. We also collected media from the tumor-T cell co-cultures and determined IL-2 and IFNγ secretion using ELISA. Finally, real-time confocal imaging (24 h) was performed to record the progress of MET-CAR T cell mediated killing activity against MHCC97H/mCherry cells.

Results We show that both MET-CAR.CD28.ζ and MET-CAR.4-1BB.ζ -T cells significantly killed MHCC97H, C3A, and JHH5 cells in antigen dependent manner. MET-CAR T cell killing is MET dependent as we observed no killing of MET-negative SNU398 cells. In addition, MET-CAR.4-1BB.ζ and MET-CAR.CD28.ζ- T cells secreted IL-2 and IFNγ when co-cultured with MHCC97H, C3A, JHH5 cells, but not SNU398. Confocal imaging studies showed that both MET-specific CAR T cells migrated toward MHCC97H/mCherry cells, formed aggregations, and induced tumor cell death, while MET-CARΔ T cells failed to do so.

Conclusions Here we demonstrate that MET-CAR.4-1BB.ζ and MET-CAR.CD28.ζ- T cells specifically recognize and kill MET-positive HCC cells in vitro. While animal studies are required to validate the efficacy in vivo, our study has produced a novel therapeutic CAR T cell target for treating malignant HCC and other type of cancers with MET overexpression.

Acknowledgements This independent research was supported by the Gilead Sciences Research Scholars Program in Liver Disease- The Americas, and Department of Defense (DoD) Ideal Award (to QX)

Ethics Approval The study was approved by East Tennessee State University’s Ethics Board, approval number #0619.3s.