Quantification of neutralizing anti-drug antibodies and their neutralizing capacity using competitive displacement and tandem mass spectrometry: Infliximab as proof of principle

Mohsin El Amrani; Camiel Göbel; Annelies C Egas; Stefan Nierkens; C Erik Hack; Alwin D R Huitema; Erik M van Maarseveen

doi:10.1016/j.jtauto.2019.100004

Quantification of neutralizing anti-drug antibodies and their neutralizing capacity using competitive displacement and tandem mass spectrometry: Infliximab as proof of principle

J Transl Autoimmun. 2019 May 28:1:100004. doi: 10.1016/j.jtauto.2019.100004. eCollection 2019 Apr.

Authors

Mohsin El Amrani¹, Camiel Göbel¹, Annelies C Egas¹, Stefan Nierkens², C Erik Hack², Alwin D R Huitema^{1

3}, Erik M van Maarseveen¹

Affiliations

¹ Department of Clinical Pharmacy, Division of Laboratory Medicine and Pharmacy, University Medical Center Utrecht, Utrecht University, the Netherlands.
² Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, the Netherlands.
³ Department of Pharmacy & Pharmacology, Netherlands Cancer Institute, Amsterdam, the Netherlands.

Abstract

Background: The development of anti-drug antibodies (ADA) in patients treated with therapeutic proteins can result in treatment failure. The clinically most relevant fraction of these antibodies are the neutralizing anti-drug antibodies (NAb) that block the pharmacological function of the drug. Consequently, the detection of NAb in plasma is a better predictor of loss of therapeutic response than increased levels of total anti-drug antibodies (ADA) test. Traditional assays to detect ADA and NAb have limited specificity, sensitivity and linear dynamic range.

Method: Here, we demonstrate for the first time the potential of a LC-MS/MS method to measure the concentration of NAb against therapeutic proteins in plasma as exemplified with infliximab (IFX). We designed a competitive screening assay in which the presence of NAb in patients plasma prevents the binding of stable isotopically labeled (SIL) mAb infliximab to TNF-α ligand fixed on a 96-well plate.

Results: After washing, eluting and digesting, the signal intensity of SIL IFX-derived signature peptides was inversely and strongly correlated with NAb concentration in the sample: R² = 0.999. Evaluation data showed that the assay has a high specificity (100%) and a high sensitivity (94%) to predict NAb presence. Cross-validation against total ADA measured by a reference laboratory using radio immunoassay assay (RIA) for ADA provided a good correlation (r² = 0.79).

Conclusion: We developed for the first time a robust and fast screening method on the basis of LC-MS/MS to determine the presence of NAb and its neutralizing capacity in plasma. The analyses of NAb can be combined with therapeutic mAb quantification. Furthermore, the quantification of the neutralizing capacity expressed as mAb mass equivalents opens the door to new personalized dosing strategies in patients with NAb.

Keywords: Anti-drug antibodies; Cell-based assay; Competitive immunoaffinity purification; Liquid chromatography tandem-mass spectrometry; Neutralizing antibodies.