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

## TL;DR

A new system called NeoPAIR-T helps identify which T cell receptors react to tumor-specific neoantigens, improving the development of personalized cancer immunotherapies.

## Contribution

NeoPAIR-T is a CRISPR-engineered platform that enables scalable, functional mapping of neoantigen–TCR pairs using a dual reporter system and tandem minigenes.

## Key findings

- NeoPAIR-T enables parallel screening of neoantigen–TCR pairs using a Jurkat reporter system with luciferase/eGFP readouts.
- Two neoantigen–TCR pairs were validated in lung cancer samples with EC50 values ranging from 10−9.2 to 10−6.7 M.
- The platform integrates transcriptome-guided TCR selection and minigene-based antigen presentation for scalable discovery.

## Abstract

What are the main findings?
A CRISPR/Cas9-engineered Jurkat reporter system (NeoPAIR-T) was developed for multiplexed, functional identification of authentic neoantigen–TCR pairs.The NeoPAIR-T platform integrates transcriptome-guided TCR selection with tandem minigene-based antigen presentation, enabling parallel screening and precise mapping of tumor-reactive TCRs.

A CRISPR/Cas9-engineered Jurkat reporter system (NeoPAIR-T) was developed for multiplexed, functional identification of authentic neoantigen–TCR pairs.

The NeoPAIR-T platform integrates transcriptome-guided TCR selection with tandem minigene-based antigen presentation, enabling parallel screening and precise mapping of tumor-reactive TCRs.

What are the implications of the main findings?
NeoPAIR-T bridges the gap between computational prediction and functional validation, providing a workflow designed for scalability in neoantigen–TCR discovery.This platform offers a practical foundation for accelerating the development of personalized cancer immunotherapies such as neoantigen vaccines and TCR–T cell therapy.

NeoPAIR-T bridges the gap between computational prediction and functional validation, providing a workflow designed for scalability in neoantigen–TCR discovery.

This platform offers a practical foundation for accelerating the development of personalized cancer immunotherapies such as neoantigen vaccines and TCR–T cell therapy.

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.