Engineering In Vitro Organ‐Structured Tumor Model for Evaluating Neoantigen‐Specific T Cell Responses in Hepatocellular Carcinoma

Abstract Neoantigens derived from somatic mutations in cancer cells can induce antigen‐specific T‐cell immune response for cancer immunotherapy. However, the 3D models for assessing neoepitope immunogenicity and efficacy of anti‐tumor T‐cell immune response to neoantigens are less than perfect. Here, a 3D tumor model based on recellularized liver matrix is leveraged with HepG2 cells to investigate T cell cytotoxic reactivity toward hepatocellular carcinoma (HCC) neoantigens. The whole exome sequencing (WES) data of 364 HCC patients in The Cancer Genome Atlas database are collected and 25 highly potential immunogenic neoantigens to human leukocyte antigen (HLA)‐A*02:01 molecule in silico are predicted. Six of the HCC neoantigen candidates are functionally validated with high immunogenicity by measuring cellular interferon‐γ secretion and cytotoxicity during neoantigen‐specific T‐cell immune responses in vitro. Then, the minigene of six functionally identified neoantigen peptides is constructed and the minigene‐modified GFP‐HepG2 cells are generated. Neoantigen‐specific immune response is observed with highly secreted Granzyme B, IFN‐γ, and PD‐1 when targeting the minigene‐modified GFP‐HepG2 cells in the 3D RLM HCC tumor model. Overall, the 3D RLM tumor model provides a novel strategy for preclinical assessment of the efficacy of neoantigen‐specific T cell immune response, which helps develop personalized cancer vaccines and immunotherapy treatments for HCC patients.