Engineering of pH-dependent antigen binding properties for toxin-targeting IgG1 antibodies using light-chain shuffling.

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples.

Antibody-dependent enhancement of toxicity of myotoxin II from Bothrops asper.

Improved therapies are needed against snakebite envenoming, which kills and permanently disables thousands of people each year. Recently developed neutralizing monoclonal antibodies against several snake toxins have shown promise in preclinical rodent models. Here, we use phage display technology to discover a human monoclonal antibody and show that this antibody causes antibody-dependent enhancement of toxicity (ADET) of myotoxin II from the venomous pit viper, Bothrops asper, in a mouse model of envenoming that mimics a snakebite.

Phage display assisted discovery of a pH-dependent anti-α-cobratoxin antibody from a natural variable domain library.

Recycling IgG antibodies bind to their target antigen at physiological pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the IgG antibodies to be recycled into circulation via FcRn-mediated cellular pathways, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non-recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing in vitro antibody selection campaigns utilizing histidine doped libraries.

An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules.

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel.

Cross-reactivity trends when selecting scFv antibodies against snake toxins using a phage display-based cross-panning strategy.

Antibodies with cross-reactive binding and broad toxin-neutralizing capabilities are advantageous for treating indications such as infectious diseases and animal envenomings. Such antibodies have been successfully selected against closely related antigens using phage display technology. However, the mechanisms driving antibody cross-reactivity typically remain to be elucidated.

Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes.

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins . Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobra.

discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom.

The monocled cobra () is among the most feared snakes in Southeast Asia due to its toxicity, which is predominantly derived from long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines, major limitations in safety, supply consistency, and efficacy create a need for improved treatments.

Advances in antibody phage display technology.

Phage display technology can be used for the discovery of antibodies for research, diagnostic, and therapeutic purposes. In this review, we present and discuss key parameters that can be optimized when performing phage display selection campaigns, including the use of different antibody formats and advanced strategies for antigen presentation, such as immobilization, liposomes, nanodiscs, virus-like particles, and whole cells. Furthermore, we provide insights into selection strategies that can be used for the discovery of antibodies with complex binding requirements, such as targeting a specific epitope, cross-reactivity, or pH-dependent binding.

Synthetic antibodies block receptor binding and current-inhibiting effects of α-cobratoxin from Naja kaouthia.

Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin.

Orally active bivalent VH construct prevents proliferation of F4 enterotoxigenic in weaned piglets.

A major challenge in industrial pig production is the prevalence of post-weaning diarrhea (PWD) in piglets, often caused by enterotoxigenic (ETEC). The increased use of antibiotics and zinc oxide to treat PWD has raised global concerns regarding antimicrobial resistance development and environmental pollution. Still, alternative treatments targeting ETEC and counteracting PWD are largely lacking.

Cross-Reactive SARS-CoV-2 Neutralizing Antibodies From Deep Mining of Early Patient Responses.

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response.

An in vitro methodology for discovering broadly-neutralizing monoclonal antibodies.

Broadly-neutralizing monoclonal antibodies are of high therapeutic utility against infectious diseases caused by bacteria and viruses, as well as different types of intoxications. Snakebite envenoming is one such debilitating pathology, which is currently treated with polyclonal antibodies derived from immunized animals. For the development of novel envenoming therapies based on monoclonal antibodies with improved therapeutic benefits, new discovery approaches for broadly-neutralizing antibodies are needed.

Unity Makes Strength: Exploring Intraspecies and Interspecies Toxin Synergism between Phospholipases A and Cytotoxins.

Toxin synergism is a complex biochemical phenomenon, where different animal venom proteins interact either directly or indirectly to potentiate toxicity to a level that is above the sum of the toxicities of the individual toxins. This provides the animals possessing venoms with synergistically enhanced toxicity with a metabolic advantage, since less venom is needed to inflict potent toxic effects in prey and predators. Among the toxins that are known for interacting synergistically are cytotoxins from snake venoms, phospholipases A from snake and bee venoms, and melittin from bee venom.

History of Envenoming Therapy and Current Perspectives.

Each year, millions of humans fall victim to animal envenomings, which may either be deadly or cause permanent disability to the effected individuals. The Nobel Prize-winning discovery of serum therapy for the treatment of bacterial infections (tetanus and diphtheria) paved the way for the introduction of antivenom therapies for envenomings caused by venomous animals. These antivenoms are based on polyclonal antibodies derived from the plasma of hyperimmunized animals and remain the only specific treatment against animal envenomings.

Engineering and design considerations for next-generation snakebite antivenoms.

Snakebite envenoming is a devastating Neglected Tropical Disease, the treatment of which has seen relatively little innovation since the invention of antivenom serotherapy in 1894. Current antivenoms have been and continue to be invaluable in saving thousands of lives. However, these medicines are associated with a number of drawbacks pertaining to availability, safety, and efficacy.

Antibody Cross-Reactivity in Antivenom Research.

Antivenom cross-reactivity has been investigated for decades to determine which antivenoms can be used to treat snakebite envenomings from different snake species. Traditionally, the methods used for analyzing cross-reactivity have been immunodiffusion, immunoblotting, enzyme-linked immunosorbent assay (ELISA), enzymatic assays, and in vivo neutralization studies. In recent years, new methods for determination of cross-reactivity have emerged, including surface plasmon resonance, antivenomics, and high-density peptide microarray technology.

Basics of Antibody Phage Display Technology.

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments.