Fig. 4.

Schematic overview and validation of neoantigen and cognate TCR discovery technology. Tumor and/or peripheral blood mononuclear cell (PBMC) derived DNA/RNA are used to perform WES/RNA-seq to identify non-synonymous variants. Through deep learning-based prediction of neoantigen epitopes, select candidate epitopes to synthesize TMG/long peptides. The monocyte-derived APCs should be engineered to promote antigen presentation and T cell activation. Then, immortalized/engineered APCs were loaded with antigen library. When APCs co-cultured with tumor-infiltrating lymphocytes, neoantigen-reactive T cells will be labeled and selected by flow cytometry. The neoantigen-specific TCR are screened by scTCR-seq, and clone candidate TCRs to PBMC derived T cells. Finally, the recognition of neoantigens by T cells is verified by several screening experiments, such as neoepitope tetramers/4-1BB staining, IFN-γ ELISPOT, cytotoxic activity of tumor killing, degranulation. Meanwhile, neoantigen-specific TCRs could be rapid cloned through T cell characterizing by a panel of CXCL13, ENTPD1(CD19) and CD200 etc.