KRASG12V- and CTAG1B-targeted TCR-T cells from phage TCR library suppress colorectal and esophageal tumors

Background: Colorectal cancer (CRC) remains a leading cause of cancer-associated mortality and is difficult to treat due to therapy resistance and limited durable responses. Esophageal cancer (EC) is similarly challenging, exhibiting highly aggressive and metastatic behavior that contribute to its poor prognosis. Adoptive TCR-engineered T cell (TCR-T) therapies show promise in these settings, but their efficacy is limited by suboptimal antigen specificity and an immunosuppressive tumor microenvironment (TME). To address these barriers, we isolated high-affinity TCRs from a phage-displayed TCR library targeting KRASG12V neoantigen in CRC and CTAG1B cancer/testis antigen (CTA) in EC. Methods: KRASG12V (8-16 )/HLA-A11:01-restricted and CTAG1B (157-165) /HLA-A02:01-restricted TCRs were isolated from an optimized phage-displayed TCR library generated from healthy donor CD8⁺ T cells through high-throughput biopanning. Binders were screened against antigen-family peptides and irrelevant controls by phage ELISA to eliminate cross-reactivity. Binding affinities were measured by SPR. Antigen-specific TCRs (asTCRs) were cloned into lentiviral vectors and transduced into primary human T cells. TCR-T function was assessed using peptide-pulsed T2 cells and KRASG12V⁺ or CTAG1B⁺ tumor cells through LDH-release cytotoxicity and cytokine secretion assays. Normal human cells were used to evaluate off-target toxicity. In vivo antitumor activity was tested in NOD/SCID-IL2rgamma⁻/⁻ xenografts with histological confirmation of TCR-T infiltration. Results: Next-generation sequencing confirmed that the 10 9 -scale library contained >90% functional genes and >80% sequence diversity. Three high-affinity TCRs were identified for each antigen (K D as low as 1.07 μM). These TCRs exhibited strict antigen specificity with no detectable cross-reactivity. TCR-T cells expressing asTCRs showed potent antigen-dependent cytotoxicity against KRASG12V⁺/HLA-A11:01⁺ colorectal and CTAG1B⁺/HLA-A02:01⁺ esophageal tumor cells (up to 80% lysis, p<0.01), accompanied by robust IFN-gamma and TNF-alpha secretion (>1000 pg/mL, p<0.01). No killing was observed against any of seven healthy cell types. TCR-T cells remained effective at low effector-to-target ratios and maintained stable proliferation and effector-memory phenotypes. In xenograft models, KRAS- and CTAG1B-targeted TCR-T cells induced significant tumor regression compared to unmodified T cell controls (p<0.05), with increased effector TCR-T infiltration, and reduced antigen escape. Conclusions: This work establishes a rapid and robust platform for isolating potent, safe, antigen-specific TCRs targeting both oncogenic driver mutation (KRASG12V) and an immunogenic CTA (CTAG1B). TCR-T cells demonstrated strong antitumor activity in vitro and in vivo , supporting the translational potential of this approach for solid tumor treatment.