NeoPAIR-T: Functional Mapping of Neoantigen–TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System

Targeting mutation-derived neoantigens is a promising strategy for personalized immunotherapies. However, identifying true neoantigens and cognate T cell receptors (TCRs) remains challenging because computational prediction of neoantigen peptides is uncertain and most tumor-infiltrating lymphocytes are bystanders rather than tumor-reactive, necessitating functional validation. Here, we developed NeoPAIR-T (Neoantigen–TCR Pairing Assay using reporter T cells), a functional assay based on co-culture of TCR–T reporter cells and autologous antigen-presenting cells (APCs) to screen neoantigen–TCR pairs. Reporter T cells are Jurkat-derived cells engineered to express a luciferase/eGFP dual reporter, providing quantitative readouts of TCR activation, while APCs are immortalized autologous cells transfected with tandem minigenes (TMGs) encoding predicted neoantigens, bypassing peptide synthesis. NeoPAIR-T also includes TCRα-knockout with targeted knock-in of candidate TCRs at the TCRβ locus to prevent mispairing and enables parallel testing of multiple reporter T cell clones co-cultured with the same APCs for efficient identification of functional pairs. Using lung cancer samples, whole-exome and RNA sequencing predicted 63 candidate peptides assembled into three TMGs. Single-cell RNA/TCR sequencing identified eight TCR clonotypes, introduced into reporter T cells and tested in parallel. Co-culture with TMG-expressing APCs revealed two functional neoantigen–TCR pairs validated by peptide assays (EC50: 10−9.2–10−6.7 M). Collectively, NeoPAIR-T streamlines neoantigen–TCR identification for vaccine and TCR-T applications.