Discovery of toxin-‐‑targeting IgGs against elapid neurotoxins Andreas H. Laustsen1,2, Aneesh Karatt-‐‑Vellatt3; Edward W. Masters2, Saioa Oscoz3, Peter Slavny2, Alice M. Luther2, Rachael A. Leah2, Majken L. Olesen2, Bruno Lomonte4, José María Gutiérrez4, John McCafferty3 1Department of Biotechnology and Biomedicine, Technical University of Denmark
2Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen 3IONTAS Ltd., Babraham
Research Institute, Cambridge CB22 3AT, United Kingdom 4Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
Biotechnological snakebite antivenom
Snakebite envenoming represents a serious public health challenge in tropical parts of the world, causing death and morbidity to hundreds of thousands of victims each year. Current treatment against snakebite consists of animal-‐‑derived antiserum, which is associated with a high degree of immunogenicity, high cost, and batch-‐‑to-‐‑batch variation1,2. Here, we report the discovery of the first fully human IgGs targeting key toxins3 from the notorious black mamba (Dendroaspis polylepis)4 and the monocled cobra (Naja kaouthia)5.
Conversion of unique scFv binders to IgG
Following expression of 57 scFvs with unique VH CDR3 regions in E. coli and evaluation of binding strength, the 24 most promising scFvs were selected and converted to IgG format. Of these, 20 displayed good binding after successful expression in HEK cells.
scFv +
+
IgG scaffold
Human IgG
In vivo neutralization of toxins by IgGs Dendrotoxin 1
⍺-‐‑cobratoxin
Discovery of human scFvs by phage display
Based on a combined toxicovenomics and phage display selection approach6, 125 human scFv binders were isolated from the IONTAS phage display library against the medically relevant Dendrotoxin 1 from D. polylepis and ⍺-‐‑cobratoxin from N. kaouthia.
The in vivo neutralization potential of the top 15 toxin-‐‑targeting IgGs was investigated in CD-‐‑1 mice. Of these, several showed the ability to prolong survival significantly, but particularly one IgG against Dendrotoxin 1 and one IgG against ⍺-‐‑cobratoxin provided full protection in mice at toxin:IgG ratios of 1:2 and 1:4, respectively. This first discovery of human IgGs capable of neutralizing snake toxins will hopefully help pave the way for cost-‐‑effective recombinant antivenoms based on oligoclonal mixture of IgGs7,8.
Venom toxins
1. Panning
2. Binding
Human IgGs
Na+
Extracellular Ca2+
Intracellular
3. Washing
Repeat
Analyze 6. ELISA
5. Amplification
4. Elution
References
[1] Gutiérrez JM, León G, Lomonte B, Angulo Y. In-‐‑flammation & Allergy-‐‑Drug Targets 2011, 10; 369-‐‑80. [2] Laustsen AH et al. Current Pharmaceutical Design 2016, 22; 5270-‐‑5293. [3] Laustsen AH, Lohse B, Lomonte B, Engmark M, Gutiérrez JM. Toxicon 2015, 104; 43-‐‑45. [4] Laustsen AH, Lomonte B, Lohse B, Fernández J, Gutiérrez JM. Journal of Proteomics 2015, 119; 126-‐‑142. [5] Laustsen AH, Gutiérrez JM, Lohse B, Rasmussen AR, Fernández J, Milbo C, Lomonte B. Toxicon 2015, 99; 23-‐‑35. [6] Laustsen AH. Recombinant Antivenoms. University of Copenhagen 2016. [7] Laustsen AH, Johansen KH, Engmark M, Andersen MR. Nature 2016, 538; 41. [8] Laustsen AH, Johansen KH, Engmark M, Andersen MR. PLOS Neglected Tropical Diseases 2017, 11; e0005361.
Contact information
Technical University of Denmark Department of Biotechnology and Biomedicine Søltofts Plads 223 DK-‐‑2800 Kgs. Lyngby, Denmark
[email protected]/ (+45) 2988 1134
Acknowledgements
We thank the Novo Nordisk Foundation (NNF16OC0019248), EliteForsk Rejsestipendie, Oticon Fonden, Lundbeckfonden, Fonden for Lægevidenskabens Fremme, Knud Højgaards Fond, Christian og Ottilia Brorsons rejselegat for yngre videnskabsmænd og kvinder,, IONTAS Ltd., and Instituto Clodomiro Picado for financial support.
