Fig. 2

Overview of the necessary steps to develop, integrate, and test one TCR-T product. A Workflow for reforming recipient T cells to express TCRs that aim target antigens. First, target antigens are derived from tumor cells of individual patients. A series of protein acquisitions and multi-omics identifications are performed to identify tumor antigens, and new TSAs are computationally predicted. Acquired natural or synthetic antigenic peptides, or TMG transduction, enable autologous APCs to stimulate T cells with antigens to produce reactive TCRs or to detect T cell reactivity. Reactive productive T cells or in situ tumor-infiltrating cells are sorted and expanded, or screened using barcode tetramers or barcode antibodies, followed by single-cell RNA-seq or TCR sequencing, ultimately yielding the target TCR and gene sequence. Recipient T-cells can be obtained from peripheral T cells from the patient or from an HLA-matched healthy donor. A single tumor-responsive TCR-T is obtained by expressing TCR in recipient T cells via viral or non-viral vectors. B The effectiveness of a TCR-T can be validated by T-cell activity assays and MHC-polymer soluble ligands or a constructed libraries test. T cell activity is determined by antigen titration, HLA-matched cell line culture, and two- or three-dimensional (2D or 3D) tissue cultures. In vivo mouse models are established with human HLA for detection of tumor killing activity and toxicity documentation. Off-target activities of TCR-Ts on self-tissues can also be recorded simultaneously, via computer prediction, screening of reactive peptides for alanine or whole-amino-acid substitutions, screening of a full library of human self-peptides, and co-culturing of a variety of self-HLA-matched cell and tissue lines. Finally, TCR-Ts are subjected to clinical trials in various phases and several results have been reported