摘要Objective:The high heterogeneity of hepatocellular carcinoma(HCC)renders traditional therapies unable to effectively activate the patient's immune system to combat tumors.Patients with advanced HCC and T cell functional deficiencies may benefit more from cellular immunotherapy,especially tumor neoepitope-targeted T cell receptor(TCR)-T cells.Neoepitopes with strong immunogenicity provide precise targets for HCC,further enhancing the efficacy of cellular immunotherapy.Methods:A scalable workflow for identifying neoepitopes from 7 HLA-A*02:01-restricted patients with HCC was established based on whole exome sequencing and bioinformatics analyses,followed by identification of neoepitope-specific TCRs through tetramer-based screening and single-cell TCR cloning technology,which were further validated in the JC4 cell model.The cytotoxicity of CD8+TCR-T cells was evaluated in neoepitope-positive tumor cell lines or NCG mice.Results:Ten specific neoepitopes were identified,among which neoepitope B and T lymphocyte attenuatorP267L[BTLAP267L(SLNHSVIGL)]exhibited advantageous properties as a potential tumor target.Three TCRs(85-3,126-5,and 52-3)were confirmed to specifically recognize the neoepitope BTLAP267L,while no cross-recognition of irrelevant or wild-type epitopes was observed.Activated BTLAP267L-specific CD8+TCR-T cells released extensive perforin,granzyme B,IFN-γ,and TNF-α in vitro,thereby inducing strong cytotoxic effects against BTLAP267L-positive T2 or HCC cell lines.BTLAP267L-specific CD8+TCR-T cells mediated robust tumor regression due to long-lasting survival and released perforin without causing significant cytotoxic effects on normal organs in murine experiments.Conclusions:This preclinical study demonstrated the beneficial effects of neoepitope BTLAP267L-specific TCR-T cell immunotherapy,unlocking a novel strategy for personalized precision therapy in HCC.