pH-Engineered Venom-Fighting Antibodies

Snakebite envenoming is a neglected tropical disease, with an estimated 1.8 to 2.7 million cases occurring each year. Snake venoms, complex protein cocktails with diverse toxicity profiles, can cause severe and irreversible harm and even death. Antivenoms are the most effective therapeutic option, but they are made from animal-derived antibodies, which can trigger serious allergic reactions. Additionally, only a few of these antibodies specifically target the venom from the snake that caused the bite, requiring higher doses.

To develop more effective human antibodies for antivenoms, Andreas Laustsen-Kiel, a bioengineer at the Technical University of Denmark, homed in on monoclonal antibody technology. In a paper published in Structure, Laustsen-Kiel’s team engineered pH-responsive human antibodies, known as acid-switched antibodies, that targeted snake venom toxins.¹ By exploiting pH-dependent antigen binding properties, the researchers rescued their toxin-targeting monoclonal antibodies from degradation. These findings aid in the development of antibody-based therapies that achieve enhanced therapeutic efficacy at lower doses.

Normally, cells take up antigens bound to immunoglobulin G (IgG) from the bloodstream and shuttle them to endosomes. The organelle’s acidic environment triggers the release of the antigens, which continue onto lysosomal degradation. The now free IgGs bind to the neonatal Fc receptor (FcRn), which protects them from the cell’s “garbage disposal system” and transports them to the cell surface. There, exposure to the neutral pH of the extracellular space triggers the release of IgGs from the receptor. This phenomenon prolongs IgG circulation time and antibody response.

They have effectively implemented a new way to identify antibodies that rely on changes in pH to be recycled, which, in theory, should allow them to have longer half-lives if they were administered to people.
– Nicholas Casewell, Liverpool School of Tropical Medicine

To leverage this process and extend the half-lives of monoclonal antibodies, researchers have synthetically engineered acid-switched antibodies that adopt a similar pH-dependent binding pattern. The most common approach is to introduce histidine residues into the variable regions of the human immunoglobulin G1 (IgG1) antibody. However, Tulika Laprade, then a graduate student in Laustsen-Kiel’s group and coauthor of the study, wanted to circumvent the use of these histidine regions given the risk of immunogenicity from these artificial sequences. Instead, she used light-chain shuffling, an alternative method used to improve affinity by iteratively shuffling the light chain subunit of the IgG1. Then, she screened the library of antibody candidates for versions that exhibited pH-dependent binding to myotoxin II (M-II) and alpha-cobratoxin (α-cbtx), two clinically relevant snake toxins from the Fer-de-Lance pit viper and monocled cobra, respectively. For this, they used phage display selection.²

“Phage display, which is one of the most robust display techniques, is particularly useful, because there you can change the environment, and the phages are fine at low pH, at neutral, [and] at high pH,” said Laustsen-Kiel.

Using this technique, he and his team screened and selected phages that bound to snake toxin antigens at a pH of 7.4 but unbound at a pH of 5.5. “It's a very powerful way of selecting the properties of antibodies that have the properties you want,” said Laustsen-Kiel. But now they needed to test their antibodies inside living cells.

To test the performance of these engineered antibodies, the researchers used an endothelial cell-based model. They treated cells with anti-M-II or anti-α-cbtx IgG1 variants, either bound or unbound to their respective snake antigen. They detected more acid-switched antibodies both during uptake and after recycling, with more of these engineered antibodies being released into the cell medium than wild type IgG1. Even without their snake venom antigen, acid-switched antibodies were recycled at higher levels than their wild type counterparts.

Nicholas Casewell, a molecular biologist from Liverpool School of Tropical Medicine who was not involved in the study, believes that acid-switched monoclonal antibodies show considerable promise for improving snakebite therapy. “They have effectively implemented a new way to identify antibodies that rely on changes in pH to be recycled, which, in theory, should allow them to have longer half-lives if they were administered to people.”

The researchers observed variability in the cellular recycling process depending on the IgG1 clone and the targeted antigen. “[This] was perhaps not super surprising, but often people tend to have a simplistic view of antibodies,” said Laustsen-Kiel. “Cellular uptake of antibodies is complex, and binding of an antigen affects it.” The team observed that IgG1s bound to M-II exhibited increased levels of cellular uptake, recycling, and accumulation. However, these parameters were reduced or unchanged in IgGs bound to α-cbtx. This indicates that acid-switched monoclonal antibodies with different binding kinetics are processed differently by cells and there is no one-size-fits-all combination.

“It is unique in our field in that [this study] explores the potential utility of monoclonal antibodies that are engineered to have particularly desirable properties,” said Casewell.

These findings have sparked new perspectives and ideas for engineering antibodies. Although there are additional parameters to investigate, such as modulating pH sensitivity and its underlying mechanisms, Laustsen-Kiel believes this platform has significant potential for developing more effective snakebite therapeutics.