De novo designed single-domain antibodies protect against lethal cobra venom neurotoxicity in vivo

Generative protein design can now rapidly produce de novo binders with high affinity and functional activity against a wide range of targets, including lethal snake venom toxins. However, so far most reported successes rely on new-to-nature scaffolds with limited therapeutic precedent. Single-domain antibodies (V _H Hs) offer a clinically validated alternative scaffold that can bind and neutralize long-chain α -neurotoxins, which are some of the most lethal components in snake venoms. Here we compare three recently established de novo design models with V _H H-design capabilities (Germinal, RFantibody, and BoltzGen) for their ability to generate V _H Hs against the neurotoxin α -cobratoxin from the monocled cobra ( Naja kaouthia ). Using standardized model inputs and evaluation criteria based on AlphaFold3 interface confidence (ipTM) and RMSD self-consistency, we find that Germinal was the only method to generate designs passing stringent in silico criteria for experimental testing. We therefore performed a larger Germinal design campaign employing three different V _H H frameworks and experimentally validated 46 designs in vitro . Of these, 42 expressed as soluble proteins and we identified four binding hits derived from two of the three tested frameworks. Of the four binders, two lead candidates were further characterized and demonstrated high affinity (K _D s of 4.1 nM and 10.8 nM), monomeric behavior and low polyreactivity, indicating favorable biophysical and developability properties, as well as functional toxin neutralization in vitro . To assess their therapeutic potential we investigated their ability to protect against α -cobratoxin toxicity in vivo . Both candidates fully protected mice after α -cobratoxin challenge, with 100% survival compared to a lethal control. One candidate also retained notable neutralization capacity against whole venom of Naja kaouthia with a survival of 56%, while the other protected 22% when tested in a rescue setting. Together, we demonstrate that de novo V _H H design can generate high affinity single-domain antibodies with in vivo protection against lethal cobra venom neurotoxicity, and provide practical insights into method- and framework-dependent performance.