Chaperone proteins as single component reagents to assess antibody nonspecificity

Abstract

Early stage assays that evaluate monoclonal antibody drug-like properties serve as valuable tools for selection of lead candidates. One liability for clinical development, off-target reactivity, is often assessed by binding to a mixture or panel of noncognate proteins. While robust, these mixes are often ill-defined, and can suffer from issues such as lot-to-lot variability. In this study, we discovered in immunoprecipitation experiments that certain chaperones are present in one of these mixtures;we then explored the use of recombinant chaperone proteins as well-characterized agents to predict antibody nonspecificity. Antibody binding to the heat shock proteins HSP70, HSP90, or trigger factor all served as predictors of cross-interaction propensity, with HSP90 providing the greatest ability to predict antibody clearance rates in mouse. Individual chaperone binding correlates surprisingly closely with binding to complex cell extracts, with the exception of a few “false negatives” (assuming a complex cell extract as the “true” value). As defined reagents, these chaperone reagents present advantages for high throughput assays of nonspecificity.

Affinity Maturation Enhances Antibody Specificity but Compromises Conformational Stability

“…We used mAbs targeting infectious disease agents to ensure that the reported serum half-lives were not affected by target-mediated clearance. The 16 anti-pathogen mAbs were cloned into an IgG 1 expression vector, produced in HEK293 cells, and tested for non-specific binding to (1) a panel of non-cognate antigens (Tiller et al, 2007;Wardemann et al, 2003); (2) baculovirus particles (BVPs) (Hö tzel et al, 2012); (3) a mixture of membrane and cytosolic proteins (polyspecificity reagent [PSR]) ; and (4) the HSP90 chaperone protein (Kelly et al, 2017) ( Figure S2A). The correlation between the degree of polyreactivity and human serum half-life was the strongest for the PSR assay (R 2 = 0.492) and BVP assay (R 2 = 0.512) ( Figure S2A).…”

“…Complementary assays have also been developed to measure antibody cross-interaction with either a panel or heterogeneous mixture of non-cognate antigens. For example, ELISA-and flow cytometry-based assays have been developed that measure antibody polyreactivity to defined sets of structurally diverse antigens (e.g., single-stranded DNA [ssDNA], double-stranded DNA [dsDNA], flagellin, insulin, and lipopolysaccharide), baculovirus particles, mixtures of membrane and cytosolic proteins, or individual chaperone proteins (Hö tzel et al, 2012;Kelly et al, 2017;Wardemann et al, 2003;Xu et al, 2013). Finally, analytical hydrophobic interaction chromatography (HIC) is routinely used for measuring antibody hydrophobicity.…”

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

“…The first and most common one is to perform positive selections for antigen binding and negative selections for non-specific binding to eliminate variants with low specificity [34–37]. Several types of polyspecificity reagents have been developed for eliminating non-specific variants and enabling the isolation of antibodies with specificities that rival those of natural antibodies [37, 38]. For example, one particularly effective polyspecificity reagent is composed of soluble membrane proteins generated from mammalian cell lysates [34, 37, 39, 40].…”

“…Negative selections performed with this polyspecificity reagent and positive selections performed with the target antigen led to the isolation of antibodies with greatly reduced levels of non-specific binding relative to antibodies obtained without negative selections [37]. Moreover, chaperone proteins (e.g., Hsp90) have also been shown to be effective and well-defined polyspecificity reagents [38]. These examples demonstrate that various types of polyspecificity reagents are invaluable tools for performing negative selections to remove non-specific antibody variants during library sorting.…”

“…It will be important to further develop effective and well-defined polyspecificity reagents for negative selections to eliminate antibodies with low specificity and solubility. It is not clear to what extent this is possible using a single polyspecificity reagent [37, 38] or whether multiple reagents are needed to eliminate antibodies with defects in different properties. It will also be important to identify new conformational ligands that recognize diverse classes of human variable regions to achieve robust selection of antibody variants with high stability.…”