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 flow cytometry and functional assays with living cells are rare and difficult 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 significant portions of the entire extracellular region of most GPCRs.