Computational design of orthogonal TCR α/β interfaces for dual-TCR therapeutics

Tomoaki Kinjo; Shawn Yu; Nathan Nicely; Andrew Leaver-Fay; William Y Kim; Brian Kuhlman

doi:10.64898/2026.01.02.697397

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Computational design of orthogonal TCR α/β interfaces for dual-TCR therapeutics

Preprint

Open access

Computational design of orthogonal TCR α/β interfaces for dual-TCR therapeutics

Tomoaki Kinjo, Shawn Yu, Nathan Nicely, Andrew Leaver-Fay, William Y Kim and Brian Kuhlman

bioRxiv

Cold Spring Harbor Laboratory

01/02/2026

DOI: 10.64898/2026.01.02.697397

PMID: 41509221

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https://doi.org/10.64898/2026.01.02.697397View

Preprint (Author's original) This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

T-cell receptors (TCRs) recognize peptides presented by MHC, enabling access to intracellular targets that are largely inaccessible to antibodies and difficult to target with small molecules. Despite this potential, their inherent cross-reactivity limits tumor specificity, while single-antigen targeting provides limited coverage of intratumoral heterogeneity. Dual-TCR therapeutics comprising two distinct TCRs could enhance tumor specificity via combinatorial recognition while broadening coverage across heterogeneous antigens. However, practical development of dual-TCR therapeutics has been limited by α/β subunit mispairing that prevents efficient production and creates undesired binding properties. Here, we develop orthogonal TCR α/β interfaces that prevent subunit mispairing. Using computational multistate design and second-site suppressor strategies implemented in Rosetta, we identified over 250 TCR variants for experimental screening to assess protein stability and pairing fidelity. The top-performing designs achieved approximately 95% correct pairing, as validated by mass spectrometry and X-ray crystallography. Focusing mutations on constant domains and conserved framework regions of variable domains enabled broad applicability across diverse TCRs while preserving antigen recognition. Using these orthogonal interfaces, we developed trispecific T-cell engagers (TriTEs) that target two cancer-testis antigens and CD3 on T cells, demonstrating enhanced potency under dual-antigen engagement (EC50 of 380 fM) while maintaining high activity when targeting cells displaying a single antigen (EC50s of 48 pM and 20 pM). This orthogonal TCR interface technology establishes a generalizable platform for engineering multi-specific immune therapeutics targeting diverse cancer antigens.

Details

Title: Subtitle: Computational design of orthogonal TCR α/β interfaces for dual-TCR therapeutics
Creators: Tomoaki Kinjo - University of North Carolina at Chapel Hill
Shawn Yu - University of North Carolina at Chapel Hill
Nathan Nicely - University of North Carolina at Chapel Hill
Andrew Leaver-Fay - University of North Carolina at Chapel Hill
William Y Kim - University of North Carolina at Chapel Hill
Brian Kuhlman - University of North Carolina at Chapel Hill
Resource Type: Preprint
Publication Details: bioRxiv
DOI: 10.64898/2026.01.02.697397
PMID: 41509221
ISSN: 2692-8205
eISSN: 2692-8205
Publisher: Cold Spring Harbor Laboratory; United States
Grant note: P30 CA016086 / NCI NIH HHS R35 GM131923 / NIGMS NIH HHS T32 GM086330 / NIGMS NIH HHS
Language: English
Date posted: 01/02/2026
Academic Unit: Internal Medicine
Record Identifier: 9985177936302771

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