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.

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.

Ten specific neoepitopes were identified, among which neoepitope B and T lymphocyte attenuator^P267L [BTLA^P267L (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 BTLA^P267L, while no cross-recognition of irrelevant or wild-type epitopes was observed. Activated BTLA^P267L-specific CD8⁺ TCR-T cells released extensive perforin, granzyme B, IFN-γ, and TNF-α in vitro, thereby inducing strong cytotoxic effects against BTLA^P267L-positive T2 or HCC cell lines. BTLA^P267L-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.

This preclinical study demonstrated the beneficial effects of neoepitope BTLA^P267L-specific TCR-T cell immunotherapy, unlocking a novel strategy for personalized precision therapy in HCC.

Neoantigens arising from gene mutations in tumors can induce specific immune responses, and neoantigen-based immunotherapies have been tested in clinical trials. Here, we characterized the efficacy of altered neoepitopes in improving immunogenicity against gastric cancer.

Raw data of whole-exome sequencing derived from a patient with gastric cancer were analyzed using bioinformatics methods to identify neoepitopes. Neoepitopes were modified by P1Y (the first amino acid was replaced by tyrosine) and P2L (the second amino acid was replaced by leucine). T2 binding and stability assays were used to detect the affinities between the neoepitopes and the HLA molecules, as well as the stabilities of complexes. Dendritic cells (DCs) presented with neoepitopes stimulated naïve CD8⁺ T cells to induce specific cytotoxic T lymphocytes. ELISA and carboxyfluorescein succinimidyl ester were used to detect IFN-γ and TNF-α levels, and T cell proliferation. Perforin was detected by flow cytometry. The cytotoxicity of T cells was determined using the lactate dehydrogenase assay.

Bioinformatics analysis, T2 binding, and stability assays indicated that residue substitution increased the affinity between neoepitopes and HLA molecules, as well as the stabilities of complexes. DCs presented with altered neoepitopes stimulated CD8⁺T cells to release more IFN-γ and had a greater effect on promoting proliferation than wild-type neoepitopes. CD8⁺T cells stimulated with altered neoepitopes killed more wild-type neoepitope-pulsed T2 cells than those stimulated with wild-type neoepitopes, by secreting more IFN-γ, TNF-α, and perforin.

Altered neoepitopes exhibited greater immunogenicity than wild-type neoepitopes. Residue substitution could be used as a new strategy for immunotherapy to target neoantigens.