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

Area of Science:

• Immunology
• Oncology
• Biotechnology

Background:

• Personalized immunotherapies targeting neoantigens show promise but face challenges in identifying true neoantigens and cognate T cell receptors (TCRs).
• Computational predictions of neoantigen peptides are often uncertain, and most tumor-infiltrating lymphocytes are bystanders, necessitating functional validation.
• Current methods require extensive peptide synthesis and lack efficiency in screening neoantigen-TCR pairs.

Purpose of the Study:

• To develop a functional assay, NeoPAIR-T (Neoantigen-TCR Pairing Assay using reporter T cells), for efficient screening and identification of functional neoantigen-TCR pairs.
• To overcome limitations in neoantigen prediction and TCR reactivity assessment for personalized cancer immunotherapy.
• To provide a streamlined approach for identifying neoantigen-TCR pairs for vaccine and TCR-T cell therapies.

Main Methods:

• Developed NeoPAIR-T, a co-culture assay using engineered TCR-T reporter cells (Jurkat-derived with luciferase/eGFP dual reporter) and autologous antigen-presenting cells (APCs) transfected with tandem minigenes (TMGs) encoding predicted neoantigens.
• Incorporated TCRα-knockout with targeted TCRβ knock-in to prevent TCR mispairing and enable parallel testing of multiple reporter T cell clones.
• Applied whole-exome and RNA sequencing to lung cancer samples to predict neoantigens, assembled into TMGs, and used single-cell RNA/TCR sequencing to identify TCR clonotypes for reporter T cell engineering.

Main Results:

• NeoPAIR-T successfully identified two functional neoantigen-TCR pairs from lung cancer samples.
• The assay demonstrated high efficiency by testing eight TCR clonotypes in parallel against TMG-expressing APCs.
• Functional pairs were validated by peptide assays, showing high affinity (EC50: 10^-9.2 to 10^-6.7 M).

Conclusions:

• NeoPAIR-T is an effective and streamlined functional assay for identifying neoantigen-TCR pairs.
• This assay overcomes key challenges in neoantigen and TCR identification, facilitating personalized cancer immunotherapies.
• NeoPAIR-T has significant implications for the development of novel cancer vaccines and TCR-T cell therapies.