Abstract
Receptors and their ligands are important therapeutic targets for about one third of marketed drugs. Here, we describe an epitope-guided approach for selection of antibodies that modulate cellular signaling of targeted receptors. We chose CXC chemokine receptor 2 (CXCR2) in the G-protein coupled receptor superfamily as receptor and a CXCR2 N-terminal peptide for antibody selection. We obtain a highly selective, tight-binding antibody from a 1011-member antibody library using combinatorial enrichment. Structural and Hydrogen-Deuterium-Exchange mass spectrometry analyses demonstrate antibody interaction with an N-terminal region of CXCR2 that is part of the IL-8 epitope. The antibody strongly inhibits IL-8-induced and CXCR2-mediated neutrophil chemotaxis in vitro and alleviates hCXCR2-dependent experimental autoimmune encephalomyelitis symptoms in mice. As inappropriate neutrophil migration accompanies many diseases including inflammatory bowel disease, glomerulonephritis, allergic asthma, chronic obstructive pulmonary disease, and cancer, this antibody has potential for development as a therapeutic agent, akin to anti-TNF antibodies. However, an important difference here is that the antibody targets the chemokine receptor and competes with natural ligand, rather than targeting the ligand itself.
Highlights
Receptors and their ligands are important therapeutic targets for about one third of marketed drugs
Each of the three extracellular loops of human CXCR2 (hCXCR2) was implicated in binding of IL-8 in different studies[39,40,41]
Nine scFv sequences showing high enrichment in the panning were sub-cloned into a pFuse expression vector and their expression and affinity confirmed by ELISA screening of the cellular supernatants with pepN48 (Supplementary Fig. 1a)
Summary
Receptors and their ligands are important therapeutic targets for about one third of marketed drugs. Multiplex signaling endows GPCRs with parallel functions in a cell, making it difficult to design relevant assays linking any single target receptor to specific downstream cellular activities It remains a challenge for the pharmaceutical industry to develop highly effective therapeutics against these important targets. While antibodies have emerged as an ever increasing source of new therapies, as yet only two therapeutic antibodies to GPCRs have been developed (to CCR4 and CGRPR)[5,6], others have been generated, including to CXC chemokine receptor 1 (CXCR1) and CXCR2, but not advanced to therapy[7,8,9,10] This paucity is largely due to the difficulty in generating antibodies that bind to functional conformations of these membrane proteins. CXCR2 is involved in neutrophil chemotaxis, which normally follows inflammatory stimuli[19,20,21,22,23]
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