Overcoming Barriers in Cancer Immunotherapy: Technical and Experimental Approaches for Preclinical Discovery, Characterization, and Optimization of Antigen-Specific TCR-T Cells

Background: The T cell receptor (TCR) is central to adaptive immunity, enabling cytotoxic T lymphocytes to recognize and eliminate disease. This specificity underpins TCR-engineered adoptive cell therapy (TCR-T), which redirects T cells against malignancy. However, major translational barriers limit broader clinical application of TCR-T therapy. First, current methodologies for identifying antigen-specific TCR candidates that are both safe and therapeutically potent remain limited. Second, even when suitable candidates are advanced into development, transferred TCR-T cells frequently demonstrate insufficient persistence and suboptimal functionality within the immunosuppressive tumor microenvironment. Methods: To address these challenges, we implemented a three-part technical and experimental strategy encompassing: (i) high-throughput TCR discovery, (ii) streamlined preclinical evaluation, and (iii) functional optimization of TCR-T cell therapies. First, an agnostic high-throughput screening and validation pipeline combining multiplexed mass cytometry and TCR-transduced Jurkat reporter assays was developed to identify HLA class I-restricted TCRs targeting viral and tumor antigens. Next, a prioritized candidate—an HLA A*11:01–restricted TCR recognizing a novel mesothelin (MSLN) epitope—was advanced for detailed preclinical evaluation. Functional efficacy assays assessed antigen-specific activation and tumor cell cytolysis. Integrated safety testing included alloreactivity screening, alanine scanning mutagenesis, and structure-based peptide–MHC–TCR modeling to evaluate epitope specificity and off-target risk. Finally, to enhance therapeutic durability, functional augmentation strategies were explored, including co-stimulatory receptor engineering and characterization of a putatively immunosuppressive CD8⁺ regulatory T cell subset. Results: High-throughput screening identified more than 100 unique physiologically derived HLA class I-restricted TCRs targeting viral and tumor antigens, establishing a broad repertoire of therapeutically relevant candidates. The prioritized HLA A*11:01–restricted TCR recognizing a novel MSLN epitope demonstrated antigen-specific activation and tumor cell cytolysis. MSLN, a tumor-associated antigen overexpressed across several solid tumors, has not previously been targeted by an HLA A*11:01–restricted therapeutic TCR. Integrated predicted in silico and in vitro and safety assessment of the same TCR identified a cross-reactive antigen not readily identified in traditional screening methods, mitigating off-target risk. Among functional optimization strategies, co-expression of the co-stimulatory molecule CD27 most consistently enhanced cytokine release and sustained cytolytic activity, suggesting improved persistence and long-term efficacy potential. Conclusions: These studies address key translational challenges in TCR-T therapy by advancing high-throughput discovery, improving preclinical safety evaluation, and enhancing functional durability. Collectively, this work informs the rational design of next-generation antigen-specific cellular immunotherapies with improved safety, specificity, and therapeutic persistence.