title: Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: a FACS-based, high-throughput selection and analytical tool.
abstract: Low expression, poor solubility, and polyspecificity are significant obstacles that have impeded the development of antibodies discovered from in vitro display libraries. Current biophysical characterization tools that identify these 'developability' problems are typically only applied after the discovery process, and thus limited to perhaps a few hundred candidates. We report a flow cytometric assay using a polyspecificity reagent (PSR) that allows for the identification and counter selection of polyspecific antibodies both during and after the selection process. The reported assay correlates well with cross-interaction chromatography, a surrogate for antibody solubility, as well as a baculovirus particle enzyme-linked immunosorbent assay, a surrogate for in vivo clearance. However, unlike these assays, PSR labeling is compatible both with screening of individual antibodies as well as selections of large antibody libraries. To this end, we demonstrate the ability to counter-select against polyspecificity while enriching for antigen affinity from a diverse antibody library, which enables simultaneous evolution of both antigen binding and superior non-target-related properties during the discovery process.
authors.affiliation: Adimab LLC, 7 Lucent Drive, Lebanon, NH 03766.
authors.forename: Yingda
authors.initials: Y
authors.lastname: Xu
authors.forename: William
authors.initials: W
authors.lastname: Roach
authors.forename: Tingwan
authors.initials: T
authors.lastname: Sun
authors.forename: Tushar
authors.initials: T
authors.lastname: Jain
authors.forename: Bianka
authors.initials: B
authors.lastname: Prinz
authors.forename: Ta-Yi
authors.initials: TY
authors.lastname: Yu
authors.forename: Joshua
authors.initials: J
authors.lastname: Torrey
authors.forename: Jerry
authors.initials: J
authors.lastname: Thomas
authors.forename: Piotr
authors.initials: P
authors.lastname: Bobrowicz
authors.forename: Maximiliano
authors.initials: M
authors.lastname: Vásquez
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
authors.forename: Eric
authors.initials: E
authors.lastname: Krauland
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D061505:Cell Surface Display Techniques / Q000379:methods*; D003429:Cross Reactions; D019020:Directed Molecular Evolution; D005434:Flow Cytometry; D006801:Humans; D055550:Protein Stability; D012995:Solubility; D013696:Temperature; D015003:Yeasts / Q000166:cytology*
chemical_list: D000906:Antibodies
keywords: antibody; cross interaction chromatography; directed evolution; flow cytometry; polyspecificity