A universal platform for simultaneous TCRα/β removal enables safer and more potent TCR therapies and autoimmune modeling

Sewell, A ORCID: https://orcid.org/0000-0003-3194-3135 2026. A universal platform for simultaneous TCRα/β removal enables safer and more potent TCR therapies and autoimmune modeling. 10.64898/2026.02.19.706929

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Abstract

Adoptive T-cell therapies using tumour-specific T-cell receptors (TCRs) are limited by competition with endogenous receptors, which impairs efficacy and poses risks of off-target autoreactivity. Here we present a CRISPR-based platform that completely and selectively eliminates both endogenous TCR-α and -β chains without affecting introduced transgenic TCRs, irrespective of codon optimization. This approach achieves >90% deletion efficiency in Jurkat and primary human T cells, markedly enhancing the expression, pairing fidelity, and functional potency of transgenic receptors. Using a clinically relevant HLA-A*02:01-restricted DMF5 TCR, we show that dual TCR ablation boosts antigen-specific activation and cytotoxicity in vitro and significantly enhances tumor clearance in vivo in human immune system (HIS) mice, while preventing graft-versus-host disease (GVHD). Targeted locus amplification revealed that CRISPR-induced double-strand breaks did not alter lentiviral integration profiles, confirming genomic safety. Extending this approach to four insulin-reactive TCRs demonstrated that removal of endogenous receptors increased transduction efficiency and functional activity, with one (1E6) showing selective activation and infiltration of stem cell–derived islet grafts (SC-islets) in vivo. This study establishes a universal, safe, and scalable genome-editing platform for generating functionally precise human T cells. By integrating cancer immunotherapy and autoimmune disease modelling within a single framework, it provides a strong preclinical rationale for dual endogenous TCR removal as a route to improved specificity, safety, and therapeutic efficacy in TCR-based cell therapies.

Item Type:	Working paper
Date Type:	Published Online
Schools:	Schools > Medicine
Additional Information:	For full list of authors see: https://doi.org/10.64898/2026.02.19.706929
Last Modified:	03 Mar 2026 13:01
URI:	https://orca.cardiff.ac.uk/id/eprint/185431

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