Systematic De-Risking of TCR-Mimic Therapeutics Through Proteome-Wide Off-Target Landscaping and a Generalizable Design Rule Framework

Schuster, S.; Hartl, F. A.; Stehle, J.; Beier, F.; Al-Hasani, H.; Reinhart, C.; Weng, T.-H.; Kraemer, S.; Hamde, P.; Herz, T.; Oesterlin, S.; Schmidt, M.; Gross, T.; Stehl, L.; Kilb, N.; Juenemann, G.; Heiss, K.; Sinclair, A.; Schoettgen, J.; Meyer, P.; Atay, B.; Zaghla, B. K. Q.; Link, S.; Goll, J.; Leoni, B.; Steinmann, B.; Selinger, O.; Welsch, S.; Roth, G.; Michel, H.; Klatt, M.; Birkenfeld, J.

doi:10.64898/2026.07.30.741432

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bioRxiv · 10.64898/2026.07.30.741432

Systematic De-Risking of TCR-Mimic Therapeutics Through Proteome-Wide Off-Target Landscaping and a Generalizable Design Rule Framework

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Abstract

TCR-mimic (TCRm) antibodies targeting peptide-human leukocyte antigen (pHLA) complexes enable precision immunotherapy against intracellular antigens, including cancer-testis antigens (CTAs). Achieving high specificity, however, remains challenging because of the vast diversity of the human immunopeptidome and the associated risk of off-target recognition. Here, we introduce ValidaTe, a unified framework for the proteome-scale prediction, validation, and mitigation of off-target liabilities in pHLA-directed therapeutics. ValidaTe integrates rational target prioritization, peptide-centric binder selection, proteome-wide off-target prediction, and therapeutic engineering into a hierarchical de-risking workflow. Using the CTA MAGE-A4 as a proof-of-concept, we identify the TCRm antibodies VR-4 and VR-6 with superior specificity and demonstrate how this workflow enables the discovery of safer pHLA-targeted binders. Furthermore, ValidaTe establishes the basis for the WiFi (Widened Fingerprint) engineering principle, which rationally combines TCRms with complementary off-target fingerprints in trivalent T-cell engagers to minimize unintended interactions while preserving potent target-specific activity. Together, these findings establish a generalizable framework for the rational development of safer and more selective pHLA-targeted therapeutics. We further discuss how orthogonal proteomic characterization may complement this workflow as a final layer of translational safety assessment prior to clinical development. TeaserValidaTe accelerates safe pHLA-targeted immunotherapy through proteome-wide off-target mapping and WiFi design

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Schuster, S., Hartl, F. A., Stehle, J., Beier, F., Al-Hasani, H., Reinhart, C., Weng, T.-H., Kraemer, S., Hamde, P., Herz, T., Oesterlin, S., Schmidt, M., Gross, T., Stehl, L., Kilb, N., Juenemann, G., Heiss, K., Sinclair, A., Schoettgen, J., Meyer, P., Atay, B., Zaghla, B. K. Q., Link, S., Goll, J., Leoni, B., Steinmann, B., Selinger, O., Welsch, S., Roth, G., Michel, H., Klatt, M., Birkenfeld, J.. 2026-08-04. Systematic De-Risking of TCR-Mimic Therapeutics Through Proteome-Wide Off-Target Landscaping and a Generalizable Design Rule Framework. https://doi.org/10.64898/2026.07.30.741432

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