Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG

Abstract

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This "panel-specific" feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.

Application of a Plug-and-Play Immunogenicity Assay in Cynomolgus Monkey Serum for ADCs at Early Stages of Drug Development

“…Although the UNISA is a simple ELISA, it required the production of a chimeric mouse-cynomolgus monkey IgG assay control. Our laboratory has a key reagent, a mouse monoclonal antibody that is reactive to many Genentech humanized mAb biotherapeutics [ 20 ] that could be used to produce similar assay control chimeras. However, a broader binding specificity was desirable as this antibody showed limited binding to human derived antibodies.…”

Development and Characterization of a Neutralizing Anti-idiotype Antibody Against Mirvetuximab for Analysis of Clinical Samples

“…We drove selection to favor paratope binders through a series of counter selection steps, including random human IgG, and screening against numerous humanized monoclonal antibodies carrying a framework similar to mirvetuximab. Several alternative strategies have been reported to generate high-specificity anti-idiotypic reagents, including phage display in combination with either in vitro panning (14) or affinity maturation through ribosome display (15), as well as other techniques (16)(17)(18). Each approach has its advantages and can be successfully applied to raise useful tools for bioanalytical assays.…”

Preexisting Antibodies to an F(ab′)2Antibody Therapeutic and Novel Method for Immunogenicity Assessment

“…Cynomolgus monkey serum samples from untreated animals were purchased from BioreclamationIVT (Hicksville, NY) and Covance (Westbury, NY). Detection conjugate for the PK assay was prepared using 10C4, a mouse monoclonal antibody that recognizes anti-DLL4 in the presence of human IgG [ 24 ], coupled to horseradish peroxidase (HRP) as described by the manufacturer (Pierce Plus Activated Peroxidase, ThermoFisher Scientific, Waltham, MA).…”

“…The plate was washed, and captured anti-DLL4 F(ab′) 2 was detected by adding 10C4-HRP conjugate, a mouse monoclonal antibody that recognizes anti-DLL4 in the presence of human IgG [ 24 ] coupled to HRP. After incubation and washing, signal was generated by adding TMB (KPL), stopping the reaction with phosphoric acid.…”