In this project, a plethora of results were achieved, which are summarized below:
Synthetic biology toolbox
To support the development of broadly-neutralizing and pH-sensitive antibodies, we developed an array of synthetic biology tools and protocols, including expression vectors for E. coli and P. pastoris with various solubilization and biotinylation tags, several dozens recombinant toxins (native and consensus), associated vectors, and expression and purification protocols.
Designed toxins and broadly-neutralizing antibodies
We developed a tool for designing antigens to discover broadly-neutralizing antibodies, focusing on snake, scorpion, and spider toxins. Designed immunogens were expressed in bacteria and yeast cells, enabling phage display campaigns that identified antibodies capable of cross-recognizing natural toxins from different orders (e.g. Scorpiones and Aranae) and different elapid snake species, respectively.
Discovery and design of pH-sensing antibodies
We developed biosensor assays (BLI and SPR) to identify cross-reactive antibodies displaying pH-sensitive kinetics against long-neurotoxins. Crystallography pipelines were established to explore the molecular basis of pH-sensitive binding. This involved microbial and mammalian production of antibodies and antibody variable domains, gel filtration purification, and optimization screens at different pH levels.
Structural biology
We established crystallographic screening protocols to study antibody binding properties, particularly broadly-neutralizing and pH-sensitive interactions. Protocols included buffer screening conditions for co-crystallization, analysis of nucleation events, and refinement of crystallographic models. Over 50 crystals were analyzed, leading to structures determined at various resolutions and providing insights into molecular mechanisms underlying pH-sensitive interactions. Further, we developed and patented a universal pH-switch for antibodies, which has resulted in a spinout (Y-king Biologics) that has already secured funding of EUR 3 million.
Nanobodies and in silico designed minibinders
In addition to the above, this project also significantly contributed to the development of a pipeline for nanobody discovery and development as well as the design of minibinders against snake toxins. The results of this were published in Nature Communications and Nature.
Allergy immunotherapy
To address the challenges of Pollen-Food Allergy Syndrome (PFAS), we used the principles from above and developed a novel immunotherapy approach utilizing mRNA-lipid nanoparticles (mRNA-LNPs) to deliver consensus allergens. This strategy elicited broad-spectrum neutralizing IgG responses against diverse food and pollen allergens in murine models, demonstrating potential for more effective and patient-friendly treatments for PFAS and other cross-reactive allergies.
