Abstract 4742: Comprehensive epitope mapping of antibodies against CXCR4 and Claudin-4 by Shotgun Mutagenesis

Abstract

Identifying antibody epitopes on membrane proteins can help differentiate antibody binding mechanisms, identify immunodominant structures, distinguish and classify antibodies, and identify cancer state-specific antigens, providing valuable information for therapeutic and diagnostic applications. However, membrane proteins represent challenging targets for epitope mapping studies using conventional methodologies such as X-ray crystallography or peptide scanning. We have developed a technology, ‘Shotgun Mutagenesis Epitope Mapping’ for rapidly mapping antibody epitopes on structurally-intact proteins expressed in mammalian cells. Here, we applied Shotgun Mutagenesis to create a comprehensive mutation library for CXCR4, a GPCR associated with tumor metastasis. The CXCR4 mutation library, which comprised >700 clones with 2.7x coverage of each of the 352 residues, was screened in triplicate with 5 different anti-CXCR4 MAbs to identify their epitopes. Our analysis revealed that all CXCR4 MAb epitopes were conformational and showed a requirement for charged residues located on the second extracellular loop. CXCR4 MAb epitopes were mapped onto the crystal structure to visualize and identify common structural features. Using epitope information, we generated point mutations in CXCR4 at positions of critical contact residues, incorporated them into virus-like particles (‘Lipoparticles’) and measured their binding kinetics. These approaches helped define the electrostatic contribution of individual amino acid residues on CXCR4 to the association rate, dissociation rate, and overall binding interaction with CXCR4 MAbs. Taking the same approach, we generated a comprehensive mutation library for the biomarker Claudin-4, a tight junction protein upregulated in epithelial cancers. The Claudin-4 mutation library, which comprised >400 clones with complete coverage of each of the 209 residues, was screened with a panel of 7 different anti-Claudin-4 antibodies to map their epitopes. We identified several distinct conformational epitopes that all map exclusively to the large first extracellular loop, identifying this domain as an immunodominant region of claudin-4. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4742. doi:1538-7445.AM2012-4742