Researchers at the U.S. Department
of Energy's Argonne National Laboratory have developed a systematic method
to improve the stability of antibodies. The technique could lead to better biosensors,
disease therapeutics and diagnostic reagents and non-laboratory applications,
including environmental remediation.
Protein stability arises from networks of inter-atomic interaction. In this protein, a network is formed when Q37, a surface amino acid residue, forms a hydrogen bond with amino acid residue Y86 and interacts with amino acid residue D82 through a bridging water molecule. Image courtesy Raj Pokkuluri.
Antibodies are proteins produced by humans and animals to defend against infections;
they are also used to diagnose and treat some diseases and detect toxins and
pathogens. "The primary issue with antibodies is that they are fragile
and short-lived outside of cooler temperature-controlled environments, making
their usefulness usually limited to laboratory applications," said Argonne
senior biophysicist Fred Stevens, the project's principle investigator.
Specifically, "stabilized antibodies, with full functionality, could be
used in diagnostic and detection kits that can survive in less than optimal
environments and be stockpiled for years at a time," Stevens said. "They
could be used to combat diseases like cancer. They can also be used as the basis
for biosensors that can continuously detect for pathogens like botulinum, ricin
and anthrax in places such as airports and subway stations—locations where
it is not currently possible to provide ongoing detection of pathogens because
antibodies cannot tolerate the environmental conditions."
Argonne has provided funding toward Stevens’ research. Earlier research
funded by the National Institutes of Health showed that it was possible to stabilize
antibodies after a team led by Stevens unexpectedly discovered that natural
antibodies contain stabilizing amino acid replacements.
Antibodies are made up of four polypeptides—two light chains and two
heavy chains. These chains are made up of modules known as constant and variable
domains. The light and heavy chain each have a variable domain, which come together
to form the antigen binding site. Because of the great diversity of amino acids
in the variable domains, different antibodies are capable of interacting with
an effectively unlimited number of targets.
Sometimes this variability comes at a price; the amyloid-forming light chains
were less stable than their normal counterparts. However, even amyloid-forming
light chains have amino acid substitutions that improve stability. When seven
of these amino acid changes were introduced into an amyloid-forming variable
domain, a billion-fold improvement in thermodynamic stability was obtained reflecting
a much higher ratio of native protein folds to unfolded proteins—a major
determinate of antibody shelf life.
"Our work at this detailed level had taught us that antibody stabilization
is possible, but we needed to find out if antibodies could be stabilized without
compromising their function and do so with moderate experimental investment,"
Stevens said. Recent work suggests these goals are potentially achievable. To
proactively improve the stability of a different antibody variable domain, Argonne
researchers drew up a short list of 11 candidate amino acid changes. Four of
the amino acid changes improved antibody stability and when combined together
in the original domain, they provided a 2,000-fold improvement in stability.
A follow up experiment using a functional antibody fragment was able to improve
antibody stability comparably, with no loss of antibody functionality. Both
experiments required approximately one month to accomplish instead of the potentially
open-ended time required for most protein stabilization projects.
There is a correlation between thermodynamic stability and thermal stability;
the billion-fold improvement in thermodynamic stability increased the thermal
resistance of the protein to heating, resulting in a “melting temperature”
of about 160 degrees Fahrenheit. "However, still unanswered is whether
it is possible to be confident about improving the stability of any antibody
generated against a particular target," Stevens said. "Our research
indicates that stabilization of antibodies is possible. We project that it could
be possible to generate the data to guide stabilization of every future antibody
in the near future."
Argonne’s Office of Technology Transfer is actively seeking participation
from industry for licensing as well as funding for further development of this
technology.