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.

Target-independent variable region mediated effects on antibody clearance can be FcRn independent

“…Additionally, briakinumab displays a higher tendency for selfinteractions, with a greater wave length shift in the AC-SINS assay (31.1 vs. One.6 nm). Compared to the antibody sets used in previous studies, 24,25,31,32 ustekinumab scores close to baseline in all 4 assays, while briakinumab scores above the predictive threshold for poor developability in all assays.…”

“…TMDD effects may be ameliorated by engineering pH-dependent binding, 44 and nonspecific binding 32 and FcRn affinity [9][10][11][12][13][14] can be modulated by antibody engineering. The somewhat limited success of engineering efforts to improve antibody clearance PK by modulating FcRn affinity 5 may be in many instances attributable to dominant nonspecific tissue clearance mechanisms that operate independently of FcRn recycling, as demonstrated for the antibodies in this report.…”

“…To further understand the relative contributions of FcRn affinity and other biophysical properties to PK properties, ustekinumab and briakinumab were subjected to a panel of early stage screening assays. These included 3 cross-interaction related assays (cross interaction chromatography (CIC), 31 baculovirus ELISA, 24 and polyspecificity reagent (PSR) binding assay), 32 as well as one self-interaction related assay (affinity capture self-interaction nanoparticle spectroscopy (AC-SINS)). 33,34 The CIC plots for the 2 antibodies are shown in Fig.…”

Biophysical properties of the clinical-stage antibody landscape

“…For monoclonal antibodies, these properties include high-level expression, high solubility, covalent integrity, conformational and colloidal stability, low polyspecificity, and low immunogenicity. The high cost of failing any of these criteria at a late stage in drug development has led to considerable efforts at predicting developability on the basis of sequence motifs and experimentally determined biophysical properties (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

Assessing the function of pneumococcal neuraminidases NanA, NanB and NanC inin vitroandin vivolung infection models using monoclonal antibodies

“…NanA-specific mAbs were generated by in vitro selection using full length human IgG1 libraries expressed by yeast as previously published [61–64] and biotinylated NanA-variants as baits. Antibody clones were characterized as described below and selected for affinity-maturation (light chain diversification and subsequent heavy chain mutagenesis).…”