Agent-Guided De Novo Design of Nanobody Binders Against a Novel Cancer Target

Therapeutic antibody discovery remains slow and resource-intensive, with traditional methods providing limited control over epitope selection. We present an agent-guided workflow for de novo nanobody design against a novel Desmoplastic Small Round Cell Tumor target, encompassing epitope identification via our hotspot recommendation agent, de novo generation using three independent methods (RFantibody, IgGM, mBER) across multiple predicted antigen structures, multi-metric scoring including structural and sequence-based binding affinity metrics, and high-throughput yeast surface display screening followed by surface plasmon resonance (SPR) characterization. From 288,000 designs spanning eight epitope regions and three VHH frameworks, Pareto-based filtering selected 100,000 candidates; of 116 enriched candidates advanced to SPR, 46 (39.7\%) produced reliable kinetic fits ($R_{\max}$ $\geq$ 30 RU) with $K_D$ values from 0.66 nM to 305 nM (median 31.7 nM). These results demonstrate 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.