An Affinity Capture Elution (ACE) assay for detection of anti-drug antibody to monoclonal antibody therapeutics in the presence of high levels of drug

Abstract

Monoclonal antibody therapeutics typically have relatively long half-lives and can be dosed at high levels. Although formation of anti-drug antibodies (ADA) is relatively rare, detection of these antibodies can be very difficult in the presence of high circulating levels of drug. Typically these ADA are detected by bridging ELISAs which can be very sensitive to even low levels of drug. We describe an ELISA method based on affinity capture of ADA on solid-phase drug followed by removal of excess free drug, release and transfer of bound ADA and subsequent detection using biotinylated drug. The assay is both sensitive and highly tolerant to free drug with detection of 500 ng/ml of ADA readily achieved in the presence of 500 μg/ml of drug.

Introduction

Monoclonal antibody therapeutics are finding increased usage for treatment of a wide variety of clinical indications with over 18 approved products. The incidence of anti-drug antibodies (ADA) varies widely from almost 100% for some murine monoclonals to virtually undetectable for some humanized antibodies (Bourdage et al., 2005). These antibodies can have significant clinical consequence. Bendtzen et al. reported detection of anti-infliximab antibodies that apparently caused loss of bioavailability, infusion reactions and treatment failure (Bendtzen et al., 2006). Anti-rituximab antibodies caused reduced B-cell depletion and reduced blood levels of rituximab (Looney et al., 2004). There have been cases where neutralizing antibodies to endogenous proteins have caused life-threatening situations (Casadevall et al., 2002, Basser et al., 2002).

Humanized monoclonal antibodies typically have half-lives of 10–20 days and can often be administered at relatively large doses leading to blood levels of more than 500 μg/ml (Maloney et al., 1997). Preclinical studies typically use even higher doses to establish safety margins with necessary short washouts prior to evaluation. Bridging or double antigen format assays are often used to evaluate ADA for monoclonals due to their ability to detect multiple classes of antibody (Feldman et al., 2003: Pendley et al., 2003) . However, there is concern for the ability to detect low affinity antibody due to the requirement for monovalent binding to the solid phase and labeled drug during bridge formation. This monovalent binding also causes the assay to be very sensitive to free drug since blocking of only a single arm of the ADA molecule will prevent bridge formation. A double antigen format assay described for infliximab has drug interference at drug levels above 1.4 μg/ml (Baert et al., 2003). This presents a dilemma when developing preclinical and clinical protocol sampling points since antibodies may be undetectable while drug levels are high thus causing a requirement for washouts of several weeks to several months. These potentially long washout periods could in turn cause decreasing ADA titers, thus making detection of ADA difficult or impossible.

We have therefore developed a method that first dissociates ADA-free drug complexes with acid treatment followed by neutralization in the presence of solid-phase drug giving the ADA an opportunity to be affinity captured. After washing away excess free drug, ADA are eluted off with acid and subsequently bound to a fresh solid surface. Bound ADA are subsequently detected by addition of biotinylated drug followed by streptavidin-HRP and substrate. Results presented here indicate that this affinity capture elution (ACE) assay format is capable of detecting low ng/ml levels of ADA in the presence of a 1000-fold excess of free drug.