Selection of a picomolar antibody that targets CXCR2-mediated neutrophil activation and alleviates EAE symptoms

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.

In Silico Maturation of a Nanomolar Antibody against the Human CXCR2

“…To provide a proof of concept that in silico affinity maturation is possible in a practical setup, we considered the pool of antibodies obtained in [ 4 ] by phage panning and in vitro affinity maturation using a peptide corresponding to the first 48 residues of the N-terminus of CXCR2 (pepN48) as bait.…”

“…To force the sampling of sequences toward a binding energy minimum, we relied on the Monte Carlo multi-dimensional search method with the Metropolis algorithm [ 33 ]. Mutants were designed with the same protocol of the in vitro maturation performed in [ 4 ]: mutations were allowed only in the CDR3 of the heavy chain of the antibody excluding Gly100, Cys102, and Cys107. Furthermore, no amino acid could be mutated into Gly, Leu, Met, Trp, Tyr, Cys, or His.…”

“…The large diversity in antibody sequences assures that it is theoretically possible to find at least one antibody that recognizes and tightly binds to any given protein, including those produced by the organism itself. Antibodies can regulate proteins on the cell surface [ 3 , 4 , 5 , 6 ] or inside the cell [ 7 ], and thanks to these properties, they have been engineered to become not only powerful biotechnological tools but also, more importantly, the largest and fastest-growing class of therapeutic proteins [ 8 , 9 ].…”

“…Monoclonal antibodies have several benefits, such as fewer off-target adverse effects, fewer drug–drug interactions, higher specificity, and potentially increased efficacy through targeted therapy. The selection of antibodies from a phage-display library can be performed on cells overexpressing the target protein [ 14 ] or using the purified protein or part of it, such as extracellular domains or peptides in the hypothetical or desired binding regions [ 4 , 5 ]. The library is screened for phage binding to the antigen through its expressed surface mAb by a technique called (bio-)panning.…”

“…As a proof of concept, we tested our method to improve an antibody that binds to the extracellular N-terminus of the interleukin 8 receptor beta (CXCR2) in the G-protein-coupled receptor family. This antibody was obtained by panning a combinatorial antibody library of phages expressing the single-chain variable fragment (scFv) [ 35 ] and using part of the extracellular N-terminus of the CXCR2 as a bait peptide [ 4 ]. Our results showed that affinity maturation in silico can be achieved and that the newly designed antibodies could improve the original one, even when the starting antibody had a nanomolar affinity.…”