De novo designed single-domain antibodies protect against lethal cobra venom neurotoxicity in vivo
- 1Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark
Abstract
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 (VHHs) offer a clinically validated alternative scaffold that can bind and neutralize long-chain -neurotoxins, which are some of the most lethal components in snake venoms. Here we compare three recently established de novo design models with VHH-design capabilities (Germinal, RFantibody, and BoltzGen) for their ability to generate VHHs against the neurotoxin -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 VHH 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 (KDs 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 -cobratoxin toxicity in vivo. Both candidates fully protected mice after -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 VHH 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.
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BibTeX
@misc{overath2026novo,
title = {{De novo designed single-domain antibodies protect against lethal cobra venom neurotoxicity in vivo}},
author = {M. D. Overath and E. V. S. Lundquist and K. H. Björnsson and S. Kerwin and C. P. Jacobsen and M. v. B. Moiniche and D. R. Espi and K. Boddum and M. Benard-Valle and M. L. Fernandez-Quintero and S. P. Mackessy and T. P. Jenkins},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.01.748349},
url = {https://www.biorxiv.org/content/10.64898/2026.09.01.748349v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - De novo designed single-domain antibodies protect against lethal cobra venom neurotoxicity in vivo
AU - M. D. Overath
AU - E. V. S. Lundquist
AU - K. H. Björnsson
AU - S. Kerwin
AU - C. P. Jacobsen
AU - M. v. B. Moiniche
AU - D. R. Espi
AU - K. Boddum
AU - M. Benard-Valle
AU - M. L. Fernandez-Quintero
AU - S. P. Mackessy
AU - T. P. Jenkins
PY - 2026
DA - 2026/09/01
PB - bioRxiv
DO - 10.64898/2026.09.01.748349
UR - https://www.biorxiv.org/content/10.64898/2026.09.01.748349v1
AB - 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 (VHHs) offer a clinically validated alternative scaffold that can bind and neutralize long-chain -neurotoxins, which are some of the most lethal components in snake venoms. Here we compare three recently established de novo design models with VHH-design capabilities (Germinal, RFantibody, and BoltzGen) for their ability to generate VHHs against the neurotoxin -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 VHH 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 (KDs 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 -cobratoxin toxicity in vivo. Both candidates fully protected mice after -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 VHH 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.
ER -