Drug Removal Strategies in Competitive Ligand Binding Neutralizing Antibody (NAb) Assays: Highly Drug-Tolerant Methods a
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Research Article Drug Removal Strategies in Competitive Ligand Binding Neutralizing Antibody (NAb) Assays: Highly Drug-Tolerant Methods and Interpreting Immunogenicity Data Michael A. Partridge,1,2 Elif Kabuloglu Karayusuf,1 Gary Shyu,1 Camille Georgaros,1 Albert Torri,1 and Giane Sumner1
Received 7 June 2020; accepted 4 August 2020 Abstract.
Neutralizing anti-drug antibody (NAb) assays often have lower drug tolerance (DT) than trough drug concentrations, potentially under-estimating NAb incidence. To improve DT, drug-specific proteins were coupled to magnetic beads to deplete drug in the sample. To avoid interference from carryover, drug-specific proteins that did not interfere in the NAb assay, such as target or non-blocking anti-drug antibodies, were selected. With the drug depletion step, DT improved by > 10-fold in two competitive ligand binding NAb assays. Analysis of anti-drug antibody positive clinical samples with elevated drug levels demonstrated that NAb incidence was under-estimated without the drug depletion step. However, these NAb-positive samples had low titer and no impact on drug concentrations.
KEY WORDS: competitive ligand binding assays; drug depletion; drug tolerance; immunogenicity; neutralizing antibody.
INTRODUCTION Administration of biological therapeutics can induce an undesirable immunogenic response in patients that can lead to the development of anti-drug antibodies (ADA) (1). Neutralizing antibodies (NAbs) are a subset of ADAs that inhibit binding of the drug to its target, rendering it biologically inactive. Neutralizing anti-drug antibodies (NAbs) have potentially important consequences for both the efficacy and safety of biological therapeutics. By definition, they neutralize the effect of the drug, potentially reducing clinical activity. In addition, where the drug is a biological mimic of an endogenous protein, NAbs may cross react with the drug’s endogenous analogue which can have critical consequences for drug safety (2,3). Detection of an immunogenic response usually involves a tiered approach where a sample is first tested for the presence of ADA, often using a ligand binding immunoassay (1). Further characterization of the ADA response may include a titer assay to determine the relative amount of ADA, and an assay to determine whether the antibody response is neutralizing (4–6). NAb assays are usually very sensitive to the presence of drug in the sample (7–10). The tolerance for drug in NAb assays is generally lower than the ADA assays that initially 1
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591, USA. 2 To whom correspondence should be addressed. (e–mail: [email protected])
detect the immunogenic response, and often lower than the steady-state concentrations of circulating drug in patients. Therefore, some neutralizing antibody responses may not be detected in the NAb assay due to interference from drug in the sample. A number of approaches for improving drug tolerance (DT) in ADA assays have been reported, including
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