lundi 6 mars 2023 11:00
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Richard Stenner1, Johara Stringari1, Sophie Hall1, Imre Berger1,2, Christiane Berger-Schaffitzel1
1Bristol Research Centre for Synthetic Biology BrisSynBio, School of Biochemistry, University of Bristol, 1 Tankard’s Close, Bristol BS8 1TD, UK. 2 Max Planck-Bristol Centre for Minimal Biology, Cantock’s Close, Bristol BS8 1TS, UK.
Aim – Snakebite envenoming is a Neglected Tropical Disease annually responsible for 138,000 deaths and 400,000 disabilities. Current anti-venoms, manufactured from hyperimmunised animals, are weakly effective as only 10-15% of the pooled anti-venom effectively neutralizes toxins. The ADDovenom project aims to use state-of-the-art protein engineering and display technology to generate novel, high-affinity, and safe anti-venoms from naïve libraries.
Methods – Ribosome Display is an in vitro selection and evolution method for generating high-affinity binders from naïve libraries. Using our nanobody and ADDobody libraries, ribosome display will be used to generate high-affinity binders against a diverse range of snake venom antigens. The high-affinity binders will be isolated, biophysically characterized, and their efficacy evaluated in cell-based assays at the Liverpool School of Tropical Medicine (in collaboration with Prof. Nicholas Casewell). The antigens used in the selections include recombinant venom proteins expressed from either bacterial expression systems (in collaboration with Dr. Renaud Vincentelli) or MultiBac insect expression systems, and the EpiString constructs developed by Prof. Robert Harrison.
Results – To-date, multiple Echis phospholipase A2s (PLA2s) and a hyaluronidase have been recombinantly expressed from insect cells, and multiple Echis disintegrins and metalloprotease EpiStrings have been recombinantly produced from bacteria. The activities of all the antigens have been confirm. All the antigens have been biotinylated prior to ribosome display, with this modification having no deleterious effect on activity. An ADDobody library has been generated in a format suitable for ribosome display and is actively being employed in selections to generate high-affinity binders against purified PLA2s and hyaluronidase. Initial rounds of ribosome display, using the ADDobody library, have yielded potential binders against the aforementioned targets, especially for Echis PLA2s and hyaluronidase. In addition, a naïve nanobody library has also been generated (by Dr Huan Sun and Dr Johara Stringari) and is currently being utilized in selections against disintegrins.
Main Conclusions – The primary goal of the ADDovenom project is to combat snakebite envenomation by producing safe, effective, and affordable antivenoms. The preliminary data indicate that ribosome display is an effective methodology for accomplishing this objective by generating high-affinity binders against an assortment of pertinent snake venom targets.
Publié le février 15, 2023