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Engineering therapeutic antibodies for enhanced stability

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Tags: Bioprocess

Antibodies (Abs) are widely used in therapeutic, diagnostic, imaging, biosensor, and research applications. Antibody-based therapeutics have been highly successful clinically and represent one of the fastest growing sectors in the pharmaceutical industry. Paul Ehrlich laid the early foundation for advancing antibody therapy by postulating the concept of “magic bullets” (Strebhardt and Ulrich, 2008). Monoclonal antibodies (mAbs), through their attractive specificity, have revolutionized the field of biotechnology research by being able to pick the proverbial molecular needle from the surrounding cellular haystack. They are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. Researchers require mAbs to bind numerous important biological targets, such as lipids, metabolites of small molecule drugs, and other protein-based markers. Researchers are searching for ways to further enhance mAbs' sensitivity, selectivity, and multiplexing ability. Of the new 45 new molecular entities approved by the FDA in 2015, nine were mAbs. To be practically useful, Abs must be highly stable and bind their target antigens with high affinity. Despite their potential, few antibodies possess ideal biophysical properties. It is, therefore, pertinent that natural antibodies be tailored by a variety of methods to suit a particular therapeutic use.

Antibodies are the ultimate example of combinatorial biochemistry since each human is capable of producing on the order of one billion different antibodies, generating a library that exceeds the diversity of any product synthesized by combinatorial efforts. Some of the favorable pharmaceutical properties desired in therapeutic antibodies include high thermal stability, low aggregation propensity in order to facilitate manufacturing and storage, as well as long serum half-life. When an Ab is designed as a drug, features such as size, tissue penetration and distribution, half-life, affinity, stability, and immunogenicity should be taken into consideration and optimized accordingly. Furthermore, Abs used for diagnostic purposes and antibody-based biosensors benefit from minimal need for cold-chain storage. In view of a highly organized and differentiated molecular structure, Abs are amenable to be engineered for desired functionality and physico-chemical properties to broaden their application. A general approach consists of utilizing as a starting point, a stable IgG structural scaffold that can be engineered to improve desired features while maintaining antigen binding affinity.

The development of Ab therapeutics is an iterative process. Antibody stability engineering strategies begin by improving the residues linked to stability or using directed evolutionary strategies to identify aggregation-resistant frameworks (Dudgeon et al., 2012). By far, the most popular recombinant Ab region is the single-chain variable fragment (scFv), which represents the antigen-binding site of an antibody. Compared to other Ab fragments with more domains or interfaces, scFv can be conveniently expressed in a variety of hosts and have broad applications in medicine. Strategies to engineer scFv fragments with increased stability can be divided into rational and evolutionary approaches. For rational approaches, structure- or sequence-based knowledge is used to predict stabilizing mutations into a given scFv fragment, which can then be confirmed by site-directed mutagenesis. Grafting complementarity determining regions (CDRs) of defined specificity onto known stable frameworks is a simple and straightforward rational approach to antibody stabilization (Jung et al., 1999). Evolutionary strategies involve fine tuning consensus sequences associated with protein stability. For example, introduction of consensus residues in the candidate antibody has been demonstrated to improve stability of immunoglobulin (IgG). A disulfide bond linking N- and C-terminal ® strands of an isolated CH2 domain has been shown to significantly increase its stability. These approaches, however, have not yet been applied to the more complex, full length immunoglobulin.

McConnell and colleagues have reported antibody stabilization by combining rational and computational design to generate a human IgG with a melting temperature (Tm) of 90°C. They have provided elegant data obtained with a Thermofluor assay on the utility of CDR grafting loops to generate high-affinity antibody with enhanced biophysical characteristics. A therapeutic or diagnostic tumor-binding scFv fragment is required to retain its activity for several hours or days after injection without precipitating or being degraded. Improving protein thermostability is an important goal of protein engineering that makes Abs easier to handle. Increased thermostability offers storage options and facilitates the commercial development of therapeutic products. Systematically altering the makeup of proteins is an exciting frontier of computational biology. Applying these techniques to the development of the next generation of Ab therapies would result in many effective new treatments for cancer and an array of diseases and disorders over the next decade. Engineering human Ab technologies represents a technical tour de force that has been built through extensive knowledge of the human immunoglobulin heavy and light chains. Recent success with immunotherapy is a testimony for Ehrlich's vision and offers promise for a more efficacious “magic bullets” against cancer.

Enzo Life Sciences is a strong contributor to the Bioprocess market with tools to monitor product integrity, purity and efficiency in a high-throughput manner such as our PROTEOSTAT® Protein aggregation assay and PROTEOSTAT® Thermal shift stability assay.

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  1. K. Strebhardt and A.Ullrich. Paul Ehrlich's magic bullet concept: 100 years of progress. Nat. Rev. Cancer. (2008) 8: 473.
  2. K. Dudgeon et al. General strategy for the generation of human antibody variable domains with increased aggregation resistance. Proc. Natl. Acad. Sci. U S A. (2012) 109: 10879.
  3. S. Jung, et al. Selection for improved protein stability by phage display. J. Mol. Biol. (1999) 294: 163.
  4. A.D. McConnell et al. An integrated approach to extreme thermostabilization and affinity maturation of an antibody. Protein Eng. Des. Sel. (2013) 26: 151.

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