Abstract: Objective To investigate the effect of targeting alpha fetoprotein-major histocompatibility complex (AFP-MHC) with CAR T cell therapy for liver cancer.Methods The human liver cancer cell line HepG2 was purchased from Guangzhou Zhenniao Biotechnology Co.Ltd. Healthy human donor peripheral blood leukocytes were obtained from Pacific Blood Center, and EasySep Human T Cell Isolation Kit was used to separate T cells. The bispecific antibody ET1402L1 was designed as a second-generation CAR containing the CD28/CD3 costimulatory domain. The CAR construct was cloned into a lentiviral vector for the transduction of primary human T cells. Forty male BALB/c nude mice (weight 21-25 g; 4-6 weeks) were raised under the condition without specific pathogens. HepG2 cells were used to construct the xenogeneic liver cancer model. The cells were divided into HepG2 group and AFP-CAR T cell group. The mice were divided into tumor model group and AFP-CAR T targeted therapy group. The cell proliferation was detected by CCK-8 kit. T cell toxicity was evaluated by the cellular LDH release assay. The mRNA expression levels of cytokines tumor necrosis factor-α (TNFα), interferon gamma(IFNγ), interleukin-2 (IL2) and IL10 were detected by PCR. The size of xenograft tumor was measured by calipers. The AFP level of tumor mice was detected by ELISA. The contrast of mouse viscera was measured by clinical iodohyalcohol (GE, Omnipaque 350 mg I/ mL).Results There was no significant difference in cell proliferation between the 2 groups at the first day (P>0.05). The cell proliferation in AFP-CAR T cell group at day 3, 5 and 7 was significant lower than that in the HepG2 group (P<0.05). The cell lysis rate of HepG2 group and AFP-CAR T cell group were 8.24±2.33% and 72.19±8.32%, the difference was statistically significant (P<0.05). TNFα, IFNγ, IL2 and IL10 mRNA expression levels in AFP-CAR T cell group were higher than HepG2 group (TNFα: 1.95 ± 0.17 vs 1.04 ± 0.02; IFNγ: 2.01 ± 0.19 vs 1.13 ± 0.06; IL2: 1.96 ± 0.16 vs 1.07 ± 0.05; IL10: 1.99 ± 0.18 vs 1.03 ± 0.04) (P<0.05). There was no difference in tumor size between the 2 groups on day 0 (P>0.05). On day 10d, 20d, 30d and 40d, the tumor size of THE AFP-CAR T-targeted therapy group was decreased compared with that of the tumor model group (P<0.05). AFP levels of tumor model group and AFP-CAR T-targeted therapy group were 2564.18±265.19 μg/ mL and 837.49±63.22 μg/ml, respectively. Serum AFP level in the AFP-CAR T-targeted therapy group was lower than that in the tumor model group (P<0.05). The tumor size of the AFP-CAR T-targeted therapy group was lower than that of the tumor model group (P<0.05).Conclusion The clinical effect of targeting alpha-fetoprotein (AFP)-MHC complex with CAR T cell therapy for liver cancer is satisfactory.

Key words: Alpha-fetoprotein-major histocompatibility complex, Cellular immunotherapy, CAR-T, Liver cancer, Xenotransplantation