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Introduction T cell receptor-engineered T cell (TCR-T) therapy is a promising approach for cancer treatment. Expanding the repertoire of functional TCRs across diverse human leucocyte antigen (HLA) alleles and tumour antigens is essential to broaden patient access while minimising off-target toxicities. However, naturally occurring tumour-specific TCRs, which represent an important source of therapeutic candidates, are typically derived from extremely rare peripheral T-cell populations. Therefore, their detection and characterisation remain challenging with conventional methods.Research design and methods Agnostic mass cytometry screening identified a novel HLA-A*11:01-restricted TCR targeting an MSLN epitope. Preclinical efficacy screening of the discovered TCR included cognate peptide and various tumour cell lines expressing MSLN. Safety profiling included alloreactivity testing and an integrated screening approach of predicted off-target epitopes from both (A) conventional alanine-scan (A-scan) and (B) structurally informed TCR-pMHC interaction modelling approaches.Results We used a comprehensive, high-throughput TCR discovery and safety profiling platform to identify an MSLN-targeting TCR and validated a novel HLA-A*11:01-restricted mesothelin epitope as a therapeutic target. The TCR exhibited robust activity against MSLN-expressing tumour cells from ovarian, prostate, lung and pancreatic origins but showed cross-reactivity against an epitope from DHRS11.Conclusions We demonstrate the utility of our described TCR discovery and characterisation platform by applying it to identify a TCR targeting a novel MSLN HLA-A*11:01-restricted epitope. The discovered TCR showed promising anti-tumour activity although DHRS11 cross-reactivity limits its clinical eligibility.