Goll, J. | Search bioRxiv

bioRxiv2026

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

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

immunology↗

bioRxiv2025

Molecular determinants of selective and high-affinity binding of the scaffold protein PDZK1 to the transporter URAT1

The renal solute carrier URAT1 (SLC22A12) is essential for urate homeostasis, with loss-of-function linked to renal hypouricemia, nephrolithiasis and lower gout risk. URAT1 function depends on binding the multi-PDZ domain scaffold protein PDZK1 (NHERF3), with a similar role suggested for the related NHERF1. The molecular basis of these interactions remains poorly understood. Using fluorescence anisotropy, we show that full-length human PDZK1 binds the C-terminal peptide of URAT1 with high affinity (KD 170 nM), unlike NHERF1 (KD >70 {micro}M). The PDZ1 domain of PDZK1 alone is sufficient for high-affinity binding (KD 160 nM), while PDZ4 provides a secondary site (KD 1.35 {micro}M), with both interactions characterized by rapid kinetics. Gel filtration shows that PDZK1 can bind two URAT1 peptides. X-ray structures of individual PDZ domains from PDZK1 and NHERF1 complexed with the URAT1 peptide reveal the molecular determinants for PDZK1s higher affinity and selectivity. Murine Pdzk1 and Nherf1 bind Urat1 with high affinity indicating species-specific interactions. These data provide insights into URAT1 regulation by PDZ scaffold proteins with relevance for understanding urate homeostasis regulation and related disorders.

biochemistry↗