Generation and characterization of a unique panel of anti-adalimumab specific antibodies and their application in therapeutic drug monitoring assays

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Highlights

  • Ten highly specific monoclonal antibodies towards adalimumab were generated.

  • Two adalimumab ELISAs were developed, showed equal characteristics and had excellent agreement with two available ELISAs.

  • Comparative studies using different adalimumab assays will facilitate implementation of treatment algorithms.

Abstract

A number of assays are currently available to support therapeutic drug monitoring of adalimumab. A complete characterization of the assays and comparison of different assays has not been performed. The aim of this study, therefore, is to generate and characterize of a panel of monoclonal antibodies towards adalimumab (MA-ADM); to use this panel to develop novel assays to determine adalimumab concentrations; to assess the impact of tumor necrosis factor (TNF) and (non-)neutralizing antibodies on adalimumab detection and to compare the performance of assays.

In total, ten specific MA-ADM were generated of which four revealed a neutralizing potency of >78%. At least six different clusters were identified using principal component analysis. MA-ADM40D8 was selected as detecting antibody to determine adalimumab in the TNF-coated ELISA (A) and the MA-ADM28B8/MA-ADM40D8 antibody pair was chosen for use in the MA-coated ELISA (B). The impact of TNF and (non-) neutralizing antibodies was similar in both ELISAs. Finally, serum samples of adalimumab-treated Crohn’s disease patients were collected and used for an external validation using the assay of Sanquin (C) and the apDia kit (D). All adalimumab assays showed excellent Pearson correlation: r = 0.96 for A versus B, 0.96 for A versus C, 0.94 for A versus D, 0.97 for B versus C, 0.95 for B versus D and 0.94 for C and D. The excellent agreement with the two commercially available ELISAs allows harmonization of treatment algorithms in and between different hospitals/infusion centers.

Graphical abstract

Diagram 1, flowchart with an overview of the key trials. The abbreviations used are as follows: ADM, adalimumab; MA, monoclonal antibody; MA-ADM, monoclonal antibodies towards adalimumab; TNF, tumor necrosis factor.

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Introduction

Clinical trials show that adalimumab (Humira®), a fully human IgG1 monoclonal antibody targeting tumor necrosis factor alpha (TNFα), is highly effective in inducing and maintaining clinical remission in patients with moderate-to-severe ulcerative colitis and Crohn’s disease, rheumatoid arthritis, psoriasis and ankylosing spondylitis [1], [2], [3], [4], [5]. Despite its therapeutic efficacy, up to 60% of patients with an initial benefit lose response to adalimumab [6], [7], [8]. The production of antibodies, especially the neutralizing antibodies, contributes to the secondary loss of response through drug neutralization and accelerated drug clearance [9].

Therapeutic drug monitoring (TDM) is an emerging strategy to optimize biologic treatment of chronic inflammatory disease. Adalimumab serum concentrations correlate positively with clinical remission and mucosal healing [10], [11], [12], [13], [14]. Currently, healthcare costs are mainly driven by medication cost, in particular anti-TNF biologicals [15]. TDM can determine patients with supra-optimal concentrations of adalimumab. In this way TDM can support reduction of costs by having a clear idea how much the dose can be decreased based on measurements. A Dutch study employing a cohort of 272 adalimumab-treated rheumatoid arthritis patients for 3 years supports this. The study shows that a total amount of € 2,561,648 was saved when treatment dosing schedule, after 6 months of therapy, was adjusted based on clinical response combined with serum trough drug concentrations when compared to the cost of usual care which consists of standard dosing [16].

A number of assays are currently available for adalimumab (enzyme-linked immunosorbent assay, homogeneous mobility shift assay, reporter gene assays) and have been used to study dose-response relationships [13], [17], [18]. Most assays use non-specific, polyclonal antibodies to detect adalimumab concentrations. This implies less specific detection of adalimumab and batch-to-batch variation due to their production process. Here, we developed ELISAs for the determination of adalimumab serum concentrations using specific, monoclonal antibodies. In addition, we investigated the impact of neutralizing and non-neutralizing anti-drug antibodies on the detection of adalimumab concentrations. Finally, the performance of the assays was compared, both internally and externally, using serum samples of Crohn’s disease patients treated with adalimumab.

Section snippets

Materials

Bovine serum albumin (BSA), horseradish peroxidase (HRP), Tween 20, Tween 80, sulphuric acid (H2SO4), mannitol, sucrose, fluorodinitrobenzene and citric acid were purchased from Sigma-Aldrich (Steinheim, Germany). VWR International (Haasrode, Belgium) delivered disodium hydrogen phosphate dihydrate (Na2HPO4∙2H2O) and sodium chloride (NaCl). Potassium dihydrogen phosphate (KH2PO4) was purchased from Fisher Scientific (Loughborough, UK). H2O2 and sodium metaperiodate were purchased from Merck

Cross-reactivity of monoclonal antibodies

Upon screening of hybridomas for production of antibodies towards adalimumab and counter-screening of the conditioned medium for binding to infliximab, a panel of 19 MA-ADM producing hybridomas was selected. Monoclonal antibody production typically yielded between 1.0 and 4.5 mg/mL conditioned medium. Based on the reactivity of the purified monoclonal antibodies towards golimumab and Multigam®, the panel was divided into three different groups. Group 1 (10/19) reacts exclusively with adalimumab,

Discussion

In this project, we created a unique panel of ten highly specific, monoclonal antibodies towards adalimumab. These antibodies were generated in mice and characterized for cross-reactivity with other anti-TNF drugs, neutralizing potency and were grouped in clusters through epitope binning. This allowed us to develop highly specific and accurate assays to measure adalimumab and thereby supporting TDM of adalimumab, which can optimize adalimumab therapy and further reduce costs.

In this panel, we

Conclusions

In this study, ten highly specific monoclonal antibodies towards adalimumab were generated. Two solid-phase adalimumab ELISAs were developed, showed equal characteristics and could be utilized for TDM of adalimumab. External validation revealed that extrapolation of results obtained with assays from different academic groups is possible and this will help harmonization of treatment algorithms. Future studies will benefit from this as adalimumab concentrations can be interchanged in and between

Conflict of interest

A Gils has received financial support for research from Pfizer, speaker fees for Pfizer, Abbvie, Janssen Biologicals and MSD, consultant fees from UCB and has licensed infliximab ELISA to apDia. F Baert is an advisory board member for MSD, Abbvie, UCB, Vifor, Mundipharma and Hospira, has received consultancy fee from Falk, grants/grants pending from MSD, Abbvie, Roche and Mundipharma, lecture fees from MSD, Abbvie, Ferring, Tramedico and Takeda and meeting expenses from Abbvie, Ferring, Falk,

Acknowledgements

This research was supported by grant G.0617.12 from the Fund for Scientific Research Flanders (FWO, Flanders) and grant IOFKP/12/002 from the KU Leuven. SM Bian acknowledges the China Scholarship Council (CSC) for the scholarship. T Van Stappen is a PhD fellow of the Agency for the Promotion and Innovation by Science and Technology in Flanders (IWT, Flanders).

SM Bian designed the experiments, performed analyses and drafted the manuscript. T Van Stappen designed the experiments and drafted the

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