Periodic Reporting for period 4 - MABSTER (Monoclonal Antibodies with Binding Sensitive To Environmental Regulation)
Reporting period: 2024-07-01 to 2024-12-31
Snakebite envenoming is a Neglected Tropical Disease (NTD) that each year affects 2.5 million victims and kills >100,000. Conventional antivenoms, derived from immunized animals, can inflict serum sickness and anaphylaxis in patients, and are costly to manufacture. Recombinantly produced monoclonal human antibodies with special toxin-binding properties that are sensitive towards regulation by their microenvironment, which may be discovered using phage display selections, may solve this issue, providing significant societal impact by enabling the development of cost-effective antivenoms to victims in low and middle-income countries.
In this project, phage display selections, synthetic biology, and antibody/antigen engineering techniques were harnessed in three scientific objectives in the pursuit of developing novel methodologies for the discovery of therapeutic human monoclonal antibodies that are recyclable (can neutralize more than one snake toxin per antibody), broadly neutralizing (can neutralize similar snake toxins from multiple species), and that are both broadly cross-reactive and recyclable at the same time. This enables entirely new ways of designing biotherapeutics against complex indications, such as snakebite envenoming, but also cancer, infectious, and parasitic diseases, where the targets can be elusive due to hyper-mutability. Additionally, significant contributions were made to the discovery of nanobodies and design of protein minibinders that can effectively neutralize snake toxin in vivo, and to the development of novel allergy immunotherapies based on the principles developed in this project.
In this project, phage display selections, synthetic biology, and antibody/antigen engineering techniques were harnessed in three scientific objectives in the pursuit of developing novel methodologies for the discovery of therapeutic human monoclonal antibodies that are recyclable (can neutralize more than one snake toxin per antibody), broadly neutralizing (can neutralize similar snake toxins from multiple species), and that are both broadly cross-reactive and recyclable at the same time. This enables entirely new ways of designing biotherapeutics against complex indications, such as snakebite envenoming, but also cancer, infectious, and parasitic diseases, where the targets can be elusive due to hyper-mutability. Additionally, significant contributions were made to the discovery of nanobodies and design of protein minibinders that can effectively neutralize snake toxin in vivo, and to the development of novel allergy immunotherapies based on the principles developed in this project.
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.
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.
In this project, we have made progress beyond state of the art in several different thematic areas, presented below together with the expected results going forward:
Designed toxins, allergens, and immunotherapy
We have, for the first time, utilized designed antigens (e.g. consensus toxins) in phage display-based antibody discovery campaigns, demonstrating the ability to discover broadly-neutralizing antibodies targeting structural epitopes, which opens new avenues for antibody discovery and engineering. Building on this innovation, we have also developed a novel allergy immunotherapy platform leveraging mRNA-LNPs to deliver consensus allergens. This approach elicits broad-spectrum neutralizing IgG responses against diverse food and pollen allergens, showcasing its potential for addressing PFAS and other cross-reactive allergies.
Discovery and design of pH-sensing antibodies
We obtained improved cross-reactive binders by using toxins expressed in different bacterial systems for phage display selections, with yeast and mammalian expression systems also being explored. Established protocols enabled the discovery of effective pH-sensitive and cross-reactive antibodies, revealing mechanisms of pH-sensitive antigen binding and broadly-neutralizing properties. We characterized Fc and FcRn binding of selected IgGs at pH 7.4 and 5.4 using ELISA and employed a human endothelial recycling assay (HERA) to assess recycling properties. Our results led to a robust pipeline for designing antibodies with unique binding features.
Structural biology
Structural understanding of antibody-mediated toxin neutralization is largely absent in toxinology. We established a crystallography pipeline that provides atomic-level insights into epitope-paratope interactions, revealing a conformational epitope of conserved toxin residues interacting with acetylcholine receptors (AChR). Some antibodies mimic nAChR residues, demonstrating neutralization through structural mimicry. Notable differences in pH-sensitive binding among neurotoxins suggest an allosteric mechanism mediated by the antibody light chain, potentially universal for creating pH-sensing antibody scaffolds. These insights led to a spinout (Y-king Biologics) that has already secured funding of EUR 3 million.
Nanobodies and in silico designed minibinders
This project contributed to the development of a pipeline for discovery of nanobodies and design of minibinders, computationally designed proteins, targeting snake toxins. The results for the latter, published in Nature, represent a major scientific breakthrough, as this was the first demonstration of in vivo functionality of minibinders, which are currently a highly regarded topic in science, with their foundational concepts recognized via David Baker’s Nobel Prize in 2024.
Designed toxins, allergens, and immunotherapy
We have, for the first time, utilized designed antigens (e.g. consensus toxins) in phage display-based antibody discovery campaigns, demonstrating the ability to discover broadly-neutralizing antibodies targeting structural epitopes, which opens new avenues for antibody discovery and engineering. Building on this innovation, we have also developed a novel allergy immunotherapy platform leveraging mRNA-LNPs to deliver consensus allergens. This approach elicits broad-spectrum neutralizing IgG responses against diverse food and pollen allergens, showcasing its potential for addressing PFAS and other cross-reactive allergies.
Discovery and design of pH-sensing antibodies
We obtained improved cross-reactive binders by using toxins expressed in different bacterial systems for phage display selections, with yeast and mammalian expression systems also being explored. Established protocols enabled the discovery of effective pH-sensitive and cross-reactive antibodies, revealing mechanisms of pH-sensitive antigen binding and broadly-neutralizing properties. We characterized Fc and FcRn binding of selected IgGs at pH 7.4 and 5.4 using ELISA and employed a human endothelial recycling assay (HERA) to assess recycling properties. Our results led to a robust pipeline for designing antibodies with unique binding features.
Structural biology
Structural understanding of antibody-mediated toxin neutralization is largely absent in toxinology. We established a crystallography pipeline that provides atomic-level insights into epitope-paratope interactions, revealing a conformational epitope of conserved toxin residues interacting with acetylcholine receptors (AChR). Some antibodies mimic nAChR residues, demonstrating neutralization through structural mimicry. Notable differences in pH-sensitive binding among neurotoxins suggest an allosteric mechanism mediated by the antibody light chain, potentially universal for creating pH-sensing antibody scaffolds. These insights led to a spinout (Y-king Biologics) that has already secured funding of EUR 3 million.
Nanobodies and in silico designed minibinders
This project contributed to the development of a pipeline for discovery of nanobodies and design of minibinders, computationally designed proteins, targeting snake toxins. The results for the latter, published in Nature, represent a major scientific breakthrough, as this was the first demonstration of in vivo functionality of minibinders, which are currently a highly regarded topic in science, with their foundational concepts recognized via David Baker’s Nobel Prize in 2024.