Zhao, Y.; Yilmaz, M.; Lee, E.; Teh, C.; Guo, L.; Sonmez, K.; Giancardo, L.; Trang, G.; Xu, F.; Espinosa-Cotton, M.; Cheung, N.-K.; Kim, J.; Cheng, X.

Therapeutic antibody discovery remains slow and resource-intensive, with traditional methods providing limited control over epitope selection. We present a workflow for de novo nanobody design applied to a novel Desmoplastic Small Round Cell Tumor target encompassing four stages: (1) epitope identification guided by our hotspot recommendation agent using physical chemistry-based structure and sequence analysis tools with two curated databases (IEDB, PFAM), (2) de novo nanobody generation using three independent methods (RFantibody, IgGM, mBER) across multiple predicted antigen structures and nanobody frameworks, (3) multi-metric scoring including structural metrics from folding models, and in silico binding affinity from our sequence- based predictor, (4) high-throughput yeast surface display (YSD) screening followed by surface plasmon resonance (SPR) characterization of the specific binders. We generated 288,000 nanobody designs spanning eight target epitope regions and three variable domains of heavy chain-only antibody (VHH) frameworks. Multi-objective Pareto filtering with our candidate selection agent yielded 100,000 candidates for YSD screening with fluorescence-activated cell sorting (FACS). Of 116 enriched candidates advanced to SPR characterization, 46/116 (39.7%) produced reliable kinetic fits with Rmax [≥] 30 RU, yielding KD values from 0.66 nM to 305 nM (median 31.7 nM). These results show that an agent-guided computational workflow can design nanomolar to sub-nanomolar nanobody binders against a novel target without experimental structure or prior antibody information.