Genetic Engineering & Biotechnology News

AUG 2013

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TUTORIAL OMICS Membrane Protein Monoclonal Antibodies DNA Immunization Elicits Diverse High-Performance Abs James W. Stave, Ph.D., Ross S. Chambers, and Michael C. Brown Therapeutic antibodies are a key driving force in the biotechnology industry thanks to their ability to safely and effectively target a broad range of diseases, including cancer, autoimmune disorders, and infectious diseases. Global sales of therapeutic antibodies reached $38 billion in 2009, and the market continues to grow. Transmembrane proteins, including multipass membrane proteins (MMP) like G-protein coupled receptors (GPCRs) and ion channels, are essential for cellular function and important targets for therapeutic monoclonal antibody (mAb) discovery and companion diagnostic assays. Greater than 40% of all drug targets are MMP but only a few have been successfully targeted by antibodies. While antibodies to a relatively large number of MMP are offered for commercial sale, the performance of the majority of these antibodies is poor, and for those that work, their utility is generally restricted to research applications like Western blot. MMP mAbs useful in fow cytometry and functional assays with living cells are rare and diffcult and expensive to make. Membrane protein function is dictated by three-dimensional structure, and for an antibody to exert a physiologic effect on a cell it must bind to the structure of a protein as it exists in the intact membrane. In living cells, only the regions of membrane proteins that are extracellular are available for antibody binding. For MMP, the extracellular structure is composed of discontinuous regions, or loops, of the protein sequence that associate with each other on the surface of the cell. The complex 3D structure is membrane dependent. Without the membrane the native structure does not form. X-ray crystallography studies have shown that the "footprint" of an antibody binding site (CDR) encompasses an area of approximately 20x30 Å. Figure 1 illustrates the relative sizes of a mouse mAb CDR and the extracellular region of the GPCR ADORA2A. The size of the antibody footprint is similar to the size of the entire extracellular region of ADORA2A (56 extracellular amino acids), and large enough to make contact with multiple extracellular loops. The total number of GPCR extracellular amino acids is frequently between 50 and 125. Thus, antibodies could interact with signifcant portions of the entire extracellular region of most GPCRs. High-Performance Antibodies To develop high-performance antibod- Figure 1. X-ray crystal structures of a mouse monoclonal antibody and multipass membrane protein ADORA2A illustrating the size of the antibody binding site (CDR) relative to the extracellular domain of the GPCR. ies to the extracellular regions of MMP it is necessary to immunize animals and select antibodies that recognize membrane-dependent protein antigen structures that are large Journal of Computational Biology A Journal of Computational Molecular Cell Biology atics meesdearch ioinfge,rpmr-reviewed r tho s ns inhbutting-ed o ee to Innovaroviiding you wit c atics and bioinform P S 28 | onal biology in computati b.com /cmb ww.liebertpu C Aler ts w ign up for TO August 2013 | GENengnews.com | Genetic Engineering & Biotechnology News enough to result in numerous contacts with antibody and high-affnity binding. Short linear peptide antigens do not fold into native 3D structures and do not represent signifcant portions of MMP extracellular structures. Purifed, full-length native and recombinant MMP may contain all of the extracellular regions, but they are devoid of the membrane and are not folded into native structures useful for functional antibody development. Virus-like particles (VLPs) and transfected cells expressing the target of interest are frequently used for MMP antibody development; however, a limitation of these approaches is the existence of other, nontarget proteins that create a needle in a haystack problem, and success using these approaches has been disappointingly low. Due to the value of functional mAbs, the dearth of available antibodies, and the diffculties and high cost associated with making them, SDIX has established methods for development of high-performance antibodies to the extracellular regions of MMP. At the heart of this process is a DNA immunization protocol that elicits specific antibody responses significantly higher than those elicited using VLP and James W. Stave, Ph.D. (stavej@gmail.com), is former senior research fellow, Ross S. Chambers, Ph.D., is director, technology development, and Michael C. Brown is research fellow at SDIX. Web: www.sdix.com.

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