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Shark Antibodies Make a Splash

Ribbon structure of a shark-derived single-domain antibody

Ribbon structure of a shark-derived single-domain antibody showing the binding loops (red and blue) as well as the hyper variable region 2 (green). Positions mutated are displayed as ball and stick format.

Defense Threat Reduction Agency’s
Chemical and Biological Technologies Department

The shark is one of the oldest species on earth dating back more than 450 million years, but now their antibodies are being used in new research funded by the Defense Threat Reduction Agency’s Joint Science and Technology Office. Shark antibodies provide an alternative, cost-effective approach in developing diagnostic and therapeutic tools for increased warfighter protection from chemical and biological threats.

Current applications use antibody-based recognition elements typically derived from mammals. However, shark antibodies, like camelids, contain only a paired heavy chain, making them smaller and more stable. Termed ‘single-domain’ antibodies, they are approximately 10 times smaller than traditional antibodies, containing the smallest naturally occurring antigen binding domains, while retaining excellent binding ability and exquisite specificity. These features are the basis of recent work managed by JSTO’s Dr. Ilya Elashvili and performed by researchers at the U.S. Naval Research Laboratory (NRL) under the supervision of Dr. George Anderson and Dr. Ellen Goldman.

Because the antibodies are comprised of only one domain, most recover a large percentage of their 3-D structure and binding ability after being heat denatured. Other advantages include the ability of single-domain antibodies to be rationally-selected, engineered to improve their properties and tailored to specific applications, and are easier to mass-produce by standard recombinant technology. The binding region of the shark’s unusual antibodies can be expressed recombinantly in yeast, plants or bacteria while preserving their advantageous properties.

In a previously published report by the United Kingdom’s Defence Science and Technology Laboratory, researchers identified a shark-derived single-domain antibody specific for the nucleoprotein of Ebola virus. NRL researchers recently confirmed the shark antibody’s high affinity for the nucleoprotein, but found its melting temperature (53 °C) and structural recovery (68 percent) following a single denaturing heat cycle to be low compared to most antibodies. Using protein engineering approaches, the melting temperature of the shark-derived single-domain antibody was increased by approximately 10 ?C while maintaining its high affinity and specificity. Increasing melting temperature, thereby preventing the denaturation of the single-domain antibody, facilitates long-term storage at elevated temperatures without loss of function.

The process utilized to stabilize the shark antibody paralleled the road map detailed by the NRL group to raise the melting temperature of camelid single-domain antibodies. Researchers grafted the binding loops of the single-domain antibodies onto a more stable shark-derived framework. Subsequently, the team subjected the graft to mutational studies and found that a single amino acid change in this graft (in the hyper variable region 2) would result in better temperature stability (melting temperature of 63 °C compared to 53 °C for the original.) In addition, the amino acid adjustment allowed better structure recovery (increased to 78 percent compared to 68 percent for the original) following a single denaturing heat cycle while maintaining superb affinity for the Ebola virus protein.

Offering the first demonstration of molecular engineering to increase the thermal stability of a shark-derived single-domain antibody, researchers reported the success of the process in a recent PLOS ONE article “Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.”

As a cost-effective alternative to both conventional and camelid single-domain antibodies for therapeutic, detection and biotechnology applications, recombinant production enables a more uniform and consistent product. Stabilizing high melting temperatures can greatly reduce the logistical cost of shipping and storing for detection, diagnostics and therapeutic reagents as they would no longer need to be maintained at refrigerated temperatures. This would lower development costs for new technologies focused on protecting the warfighter and civilian populations.

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