Next Generation Sequencing‐Based Identification of T‐Cell Receptors for Immunotherapy Against Hepatocellular Carcinoma

Hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC) remains a global health concern, and HBV proteins may be ideal targets for T cell‐based immunotherapy for HCC. There is a need for fast and efficient identification of HBV‐specific T cell receptors (TCRs) for the development of TCR‐transduced T (TCR‐T) cell‐based immunotherapy. Two widely employed TCR identification approaches, T cell clonal expansion and single‐cell sequencing, involve a TCR singularization process for the direct identification of Vα and Vβ pairs of TCR chains. Clonal expansion of T cells is well known to have tedious time and effort requirements due to the use of T cell cultures, whereas single‐cell sequencing is limited by the requirements of cell sorting and the preparation of a single‐cell immune‐transcriptome library as well as the massive cost of the whole procedure. Here, we present a next‐generation sequencing (NGS)‐based HBV‐specific TCR identification that does not require the TCR singularization process. Conclusion: Two pairing strategies, ranking‐based strategy and α–β chain mixture‐based strategy, have proved to be useful for NGS‐based TCR identification, particularly for polyclonal T cells purified by a peptide‐major histocompatibility complex (pMHC) multimer‐based approach. Functional evaluation confirmed the specificity and avidity of two identified HBV‐specific TCRs, which may potentially be used to produce TCR‐T cells to treat patients with HBV‐related HCC.