Fig. 3

Rapid identification of neoantigen-specific TCRs for personalized engineered TCR-T-cell therapy. Tumor (T) and normal (N) DNA are used to conduct WES and RNA-seq to identify cancer-specific nonsynonymous mutations. Candidate neoantigens are used to design tandem minigenes (TMGs) encoding mutant peptides and synthesize mutant peptide libraries (Step 1). TILs and PBMCs are isolated from single-cell suspensions taken from patient samples. TILs and PBMCs are analyzed using single-cell TCR-CITE-Seq, and a combined tag of gene and surface protein expression is used to predict candidate neoantigen-reactive T cells (Step 2). Candidate antigen-reactive T cells are cocultured with autologous antigen-presenting cells (APCs) expressing candidate neoantigens (TMG or peptides), and antigen-specific T cells are amplified (Step 3). Antigen-specific T cells are selected by flow cytometry, and neoantigen-reactive TCRs are identified and screened by scTCR-CITE-seq or deep sequencing. Then, the TCRα/β chain is reverse transcribed by single-cell multiplex nested RT-PCR, and its related plasmid is constructed (Step 4). T cells expressing candidate reactive TCRs are generated by cloning the selected TCR sequence into a retrovirus vector and transducing T cells. The recognition of neoantigens by T cells transduced by the TCRα/β chain is verified by different screening experiments (Step 5). PBMCs are obtained from patients, and the reactive T cells of the neoantigen-specific TCRα/β chain are amplified by the above methods (Step 6). Validated neoantigen reactive TCRα/β chains are selected to design final personalized TCR-engineered T (TCR-T) cell products that will be injected into patients for cell therapy (Step 7)