T cell receptors (TCRs), a promising therapeutic strategy for targeting intracellular antigens, can specifically recognize antigen peptide presented by human lymphocyte antigen (HLA) and mediate T cell responses to eliminate tumor cells. However, the low affinity of TCRs presents a great challenge to their broader clinical application. TCR-mimic (TCRm) antibodies, with similar antigen recognition capabilities as TCRs but higher affinity, offer a solution to these limitations, enabling the targeting of previously undruggable cancer antigens. Neverthelss, specificity remains a major challenge to ensure therapeutic accuracy and patient safety, as TCRm antibodies need to precisely target a single tumor-specific peptide antigen presented by HLA rather than other similar peptides.

Using RenTCR-mimic transgenic mice, we describe the generation and preclinical evaluation of fully human TCRm antibodies by fusing them with a humanized anti-CD3 scFv to form T cell engager or assembled on T cell surface to build TCRm-T cells, further to specifically redirect T cells to lysis tumors by our proprietary RenTCR-mimic platform. We determine cross-reactive peptide sequences predicted by in silico or known off-target sequences. We further confirm the specificity and safety of TCRm antibodies. Despite low density antigen copies, TCRm antibodies elicited antibody-mediated T-cell recognition and cytotoxicity against antigen peptide-HLA complexes-restricted cell lines. Based on these methods, we perform screens to validate TCRm antibodies specificity. These experiments led us to discriminate between the target peptide and the potential off-target peptides presented by HLA and highlight the therapeutics potential of TCRm antibodies in tumor immunotherapy, offering a novel approach to expanding the repertoire of druggable cancer targets.