Engineering And Mapping Difficult Proteins Using Comprehensive Mutagenesis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAAlanine (Ala) scanning is a widely used mutagenesis approach for systematic evaluation of protein function, which for large targets can be laborious and time-consuming. We have developed a platform technology, Shotgun Mutagenesis, for rapid and comprehensive alanine scanning mutagenesis for protein mapping and engineering applications. Using this technology, every residue in a target protein is individually mutated, expressed in human cells, and assayed for desired functions. Critical mutations are rapidly identified using high-throughput 384-well assays including flow cytometry. Here we used Shotgun Mutagenesis to create comprehensive mutation libraries for different classes of targets including the GPCR CXCR4, bitter taste receptor TAS2R16 and the therapeutic antibody Palivizumab (Synagis). For the cancer target CXCR4, we identified critical residues for antibody binding (epitope mapping) and ligand-dependent activation (functional domain mapping). We also discovered TAS2R16 residues that can be altered for increased expression (protein optimization) and Palivizumab residues which tolerate germline substitutions permitting engineering of a maximally humanized antibody variant (antibody humanization). This platform allows comprehensive protein mapping and engineering of challenging targets previously considered prohibitively time-consuming or expensive, enabling discovery pathways for many untapped therapeutic targets.Citation Format: Cheryl Paes, Jason Goodman, Melanie Wescott, Yana Thaker, Anu Thomas, Joseph Couto, Joseph Rucker, Benjamin J. Doranz. Engineering and mapping difficult proteins using comprehensive mutagenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2015-2441