G-protein-coupled receptors (GPCR) are the largest class of signal-transducing receptors on human cells. As a result, GPCRs play essential roles in many human diseases, and today constitute more than 30% of all clinically approved therapeutic targets. It is widely recognized that many GPCRs would make excellent therapeutic targets for antibodies. However, for a variety of reasons, most GPCRs are poor antigens. GPCRs span the plasma membrane 7 times, and thus, most GPCRs cannot be obtained in a usable form for antibody selection in vitro or in vivo. They are also difficult to express at high levels in heterologous hosts, and do not elicit strong responses from animals immunized with GPCR-expressing cells. As a result of these obstacles, no anti-GPCR antibodies have yet been approved for therapeutic use in humans. Peptides corresponding to the extra-cellular structures of GPCRs have been used to raise or select antibodies, but since they lack native conformations, the resulting antibodies have not had adequate affinities or specificities for therapeutic use. We have developed a method for stabilizing extra-cellular structures of receptors in native-like conformations by scaffolding them onto carrier proteins and selecting peptide """"""""chaperones"""""""" from random peptide libraries inserted between the extra-cellular domains and the carrier protein, using natural ligands or conformation-sensitive antibodies as the selectors. The carrier of choice is beta-lactamase because it allows for efficient selection for ligand-stabilized receptor conformations from very large chaperone libraries in E. coli by antibiotic resistance. We will use this strategy to stabilize the extra-cellular domains of GPCRs in soluble form, so that high-affinity antibodies can be obtained and developed for therapeutic use. We will focus on chemokine receptors because they play essential roles in many inflammatory diseases, and high-affinity antibodies could have therapeutically useful anti-inflammatory activities with greater specificity and less toxicity than currently available small molecule anti-inflammatorites. In Phase I, we will optimize the extra-cellular domains of 2 key human inflammatory chemokine receptors, CXCR1 and CXCR2, using the neutrophil chemokine IL-8 to select native conformations of these GPCRs on the surface of beta-lactamase. A panel of stable IL-8 binders will then be used to obtain anti-CXCR1 and anti-CXCR2 antibodies. In Phase II, the best antibodies obtained in Phase I will be studied in animal models of inflammatory lung disease. Additional GPCRs will be engineered for antibody selection, and corporate partners will be sought for the development of anti-GPCR antibody products for therapeutic, diagnostic, and research markets. ? ?
