Comprehensive self-antigen screening to assess cross-reactivity in promiscuous T-cell receptors

TL;DR

This paper introduces a high-throughput method to screen for potential off-target effects of T-cell receptors used in therapies, helping to improve their safety and effectiveness.

Contribution

The study presents a novel functional screening approach using Tope-seq and large peptide libraries to detect cross-reactive epitopes in therapeutic TCRs.

Findings

The method detected known cross-reactive epitopes from large peptide libraries with high significance.

Iterative biopanning and bioinformatic refinement improved screening sensitivity and accuracy.

The approach was applied to a library of over 20 million peptide-coding DNA fragments, demonstrating its scalability.

Abstract

T cell receptor (TCR) therapeutics are an emerging modality of biologic and cell-based medicines with the unique ability to target intracellular antigens and finely discriminate between healthy and infected or mutated cells. An obstacle to the development of new TCR therapeutics is the difficulty in engineering these proteins for enhanced therapeutic efficacy while avoiding introduction of unexpected off-target autoreactivity. In this study, we conduct functional high-throughput screening to profile all possible genome-encoded peptides for ability to trigger response in an engineered candidate therapeutic TCR and assess risk of off-target toxicity. We used multiple approaches for constructing comprehensive self-antigen cell libraries and highlight key considerations for designing toxicity screening campaigns using high-throughput TCR profiling in a live cell context. We then use Tope-seq to screen epitope libraries against a model therapeutic candidate TCR and show that this strategy can be used to detect known cross-reactive epitopes from libraries of >5 × 105 unique peptide-coding sequences at a significance threshold of p < 0.01 in first-pass bulk screening. We also incorporate strategies for iterative biopanning and bioinformatic refinement to improve sensitivity and accuracy and demonstrate here the first proof-of-principle for functional TCR screening on a library of >2 × 107 peptide-coding DNA fragments, further advancing the potential of in vitro approaches to perform unbiased TCR epitope discovery.

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