More than 30 distinct monoclonal antibodies are now FDA-approved for treatment of cancers, autoimmune disorders, and infectious diseases. In addition, monoclonal antibodies are used in numerous diagnostic tests and sensors. The global market for monoclonal antibody therapeutics was$40 billion, $45 billion, and $47.78 billion in 2011, 2012, and 2013, respectively. Many therapeutic monoclonal antibodies are coming off patent between 2015 and2020, which will serve to attract many new entrants to this field. This market is estimated to be $73.8 billion in 2018.
However, significant challenges still exist in application development: monoclonal antibodies are relatively fragile and short lived outside of cooler, temperature controlled environments. The majority of commercially available monoclonal antibodies are relatively unstable. Antibody therapies face dosing challenges, some of which could be attributed to their serum half-life.
Argonne researchers Rosemarie Wilton and the late Fred Stevens have developed a proprietary monoclonal antibody engineering strategy to improve the antibody thermal stability. Fred Stevens’s earlier research had unexpectedly discovered that unstable, amyloid-forming antibody fragments (which occur in certain disease states) contain stabilizing as well as destabilizing amino acid variations. This finding launched the team into research on antibody stabilization. For example, when seven stabilizing 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 factor in antibody shelf-life. The billion-fold improvement in thermodynamic stability increased the protein’s thermal resistance to heating, resulting in a “melting temperature” of about 70°C. Higher antibody melting temperatures have also been achieved in our recent studies.
In focusing on a different antibody variable domain, 11 candidate amino acid changes were identified: four of these changes improved stability and, when combined together in the original domain, a 2,000-fold improvement in stability resulted. Results were comparable in a follow-up experiment using a functional antibody fragment. Both experiments were performed in a month’s time, whereas most protein stabilization projects often require open-ended timeframes.
Argonne researchers have developed a systematic method for improving the thermal stability of monoclonal antibodies and antibody fragments by incorporation of limited amino acid changes into the variable domain framework. Our approach has also been tested with a known therapeutic monoclonal antibody as an example. Antibody clearance studies using a transgenic mouse model system that expresses human FcRn, the receptor responsible for maintaining antibodies in the serum, were also conducted. Our results confirm that these specific amino acid substitutions in the key regions have a positive stabilizing effect on the monoclonal antibody, which is observed as a significant increase in the serum half-life of the antibody. Extending these rum half-life of an antibody could lead to lower therapeutic dose and better pharmacokinetic properties. Furthermore, stabilized antibodies may also prove to be less immunogenic by reducing aggregates that can accumulate during storage. Stabilized antibodies could also offer better diagnostic properties. Results show that serum half-life can be manipulated by altering antibody stability.
This new method is systematic, fast, economical, and highly predictable. Because stabilized antibodies show a longer serum half-life, we expect them to have better pharmacokinetic and therapeutic profiles — translating tithe benefit of the patients. Stabilized antibodies are also expected to perform better as diagnostic and detection tools that must operate in less-than-optimal environments. The method can also be used to generate multiple products from the same antibody by fine-tuning stability levels. For instance, antibodies with low stability are well suited for imaging and radiotherapy applications. Antibodies with medium stability are preferred for therapeutic applications, and antibodies with high stability are desirable for diagnostic and biosensor applications.
A stabilized version of commercially available monoclonal antibodies has been produced to demonstrate the concept.
Ready for development under a research partnership and licensing model. Argonne would like to partner with companies to stabilize their proprietary antibodies under a research partnership.