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.
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 issues 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 has 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 has taught us that
antibody stabilization was 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 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.
Image of protein
stability
SOURCE: Argonne National Laboratory
