Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

doi:10.1016/j.ab.2011.01.001

Analytical Biochemistry

Volume 411, Issue 2, 15 April 2011, Pages 271-276

https://doi.org/10.1016/j.ab.2011.01.001 Get rights and content

Abstract

Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. An example is natalizumab, a recombinant IgG4 antibody directed against α4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus, IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions.

Introduction

Immunoglobulin G4 (IgG4)¹ is the least abundant of the human IgG subclasses, comprising approximately 4% of the total IgG in serum. However, antigen-specific antibody levels may be dominated by IgG4, usually in situations of chronic or repeated antigenic challenge [1]. The correlation of allergen-specific IgG4 with beneficial effects of immunotherapy suggests that IgG4 is tolerogenic in nature [2]. Indeed, IgG4 is the least effective IgG antibody in terms of effector function, with diminished affinities to most Fc gamma receptors and lacking the ability to activate complement [3], [4]. Furthermore, in vivo, IgG4 typically is effectively monovalent due to Fab arm exchange, resulting in antibodies that can block formation of (large) immune complexes [5], [6], [7], [8]. Therefore, in a therapeutic setting, IgG4 may be selected to serve as blocking antibody, largely avoiding triggering of effector mechanisms.

An example is natalizumab, a recombinant antibody directed against α4-integrin used for treatment of multiple sclerosis (MS). Natalizumab selectively inhibits α4-integrin-mediated adhesion of lymphocytes to endothelial receptors (vascular cell adhesion molecule 1 and mucosal addressin cell adhesion molecule 1), thereby preventing migration across the blood–brain barrier into the central nervous system (CNS). Natalizumab is derived from a murine monoclonal antibody humanized by complementarity-determining region (CDR) grafting onto a human IgG4 framework [9].

Efficient treatment using therapeutic monoclonal antibodies such as natalizumab requires that serum drug levels can be accurately determined, for example, to optimize dosing regimens. Furthermore, patients treated with natalizumab may develop progressive multifocal leukoencephalopathy (PML) [10], [11]. The outcome of PML depends mainly on fast and complete removal of the drug using, for example, plasma exchange. Obviously, decisions on whether or not plasma exchange is needed to wash out the drug may also depend on timely measurements of natalizumab concentrations, and detailed pharmacokinetic studies are needed.

In the case of natalizumab, measurement of drug levels requires special precautions due to structural peculiarities of human IgG4. As noted, IgG4 will undergo Fab arm exchange in vivo, and it was shown that natalizumab will also participate in the exchange process [12]. Furthermore, IgG4 is able to bind to other human and nonhuman IgG [13], [14], [15], complicating the setup of a reliable specific assay. Here we describe the development of an assay to determine serum levels of a therapeutic monoclonal IgG4 antibody. We demonstrate that the above-mentioned factors do indeed complicate development of a quantitative assay. An assay format that takes into account the special features of IgG4 and can be used to accurately measure natalizumab in serum is presented.

Section snippets

Materials and reagents

Natalizumab (Tysabri, Biogen Idec and Elan Pharmaceuticals) was supplied as a 20-mg/ml liquid. This formulation also contains sodium chloride, sodium phosphate, and polysorbate 80. Other humanized monoclonal antibodies used were omalizumab (Xolair, Novartis) and efalizumab (Raptiva, Genentech, Merck Serono). Anti-IgG (MH16-1–horseradish peroxidase [HRP] conjugate) and anti-IgG4 (MH164.1– and MH164.4–HRP conjugate) mouse monoclonal antibodies were obtained from Sanquin Reagents (Amsterdam).

Immunization of rabbits with natalizumab

Specific antibodies to natalizumab were raised by immunizing two rabbits with F(ab)2 fragments of natalizumab. IgG antibodies were isolated using protein A Sepharose, and the resulting antibodies were repeatedly depleted for anti-human Fab antibodies using an IVIG–Sepharose column. The resulting antibodies did not show reactivity (<10,000-fold) to human IgG or other monoclonal therapeutic antibodies, such as omalizumab and efalizumab, or to an IgG4 myeloma protein (not shown).

Exchange reaction of natalizumab

In Fig. 1, the

Discussion

Measurement of drug levels of therapeutic monoclonal antibodies can be challenging. Therapeutic antibodies are designed to resemble human antibodies as much as possible to minimize their immunogenicity. This sometimes makes it difficult to design an assay that is truly specific for the drug. Although most therapeutic antibodies are based on human IgG1, several therapeutic IgG4 antibodies are on the market or in development. Detection of IgG4 antibodies offers several specific challenges that

Acknowledgments

The authors thank Steven Stapel and Chris H. Polman for useful discussions and critical reading of the manuscript.

References (23)

P. Bruhns et al.
Specificity and affinity of human Fc gamma receptors and their polymorphic variants for human IgG subclasses
Blood
(2009)
D.B. Clifford et al.
Natalizumab-associated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: lessons from 28 cases
Lancet Neurol.
(2010)
C.W. Damen et al.
Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma
Anal. Biochem.
(2009)
R.C. Aalberse et al.
Serologic aspects of IgG4 antibodies: I. Prolonged immunization results in an IgG4-restricted response
J. Immunol.
(1983)
R.C. Aalberse et al.
Immunoglobulin G4: an odd antibody
Clin. Exp. Allergy
(2009)
J.G. Salfeld
Isotype selection in antibody engineering
Nat. Biotechnol.
(2007)
R.C. Aalberse et al.
The apparent monovalency of human IgG4 is due to bispecificity
Int. Arch. Allergy Immunol.
(1999)
K.M. van der Neut et al.
Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange
Science
(2007)
J.S. Van der Zee et al.
Inhibition of complement activation by IgG4 antibodies
Clin. Exp. Immunol.
(1986)
J.S. Van der Zee et al.
Serologic aspects of IgG4 antibodies: II. IgG4 antibodies form small nonprecipitating immune complexes due to functional monovalency
J. Immunol.
(1986)

M. Subramanyam

Case study: Immunogenicity of natalizumab

Cited by (62)

Liquid chromatography - tandem mass spectrometry method for determination of natalizumab in serum and cerebrospinal fluid of patients with multiple sclerosis
2023, Journal of Pharmaceutical and Biomedical Analysis
Natalizumab is a humanized recombinant monoclonal IgG4 antibody used in the treatment of multiple sclerosis. Commonly used methods for natalizumab and anti-natalizumab antibodies quantification are enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Measurement of therapeutic monoclonal antibodies can be challenging due to the resemblance to human plasma immunoglobulins. Recent developments in mass spectrometry enables to analyze vast variety of large protein molecules. The aim of this study was to develop a LC-MS/MS method for determining natalizumab in human serum and cerebrospinal fluid (CSF) and apply it to clinical settings. For successful quantification, it was necessary to find specific sequences of peptides in natalizumab. This immunoglobulin was treated with dithiothreitol and iodoacetamide, cleaved with trypsin into short specific peptides and determined on a UPLC-MS/MS system. An Acquity UPLC BEH C18 column at 55 °C and gradient elution was used for analysis. Intra- and interassay accuracies and precisions were tested at four concentration levels. Precision was determined by coefficients of variation and was in the range of 0.8–10.2 %, with accuracy in the range of 89.8–106.4 %. The concentration of natalizumab in patient samples ranged from 1.8 to 193.3 μg/mL. The method was validated according to the European Medicines Agency (EMA) guideline, met all acceptance criteria for accuracy and precision, and is suitable for clinical applications. In comparison to immunoassay, which can be elevated by cross-reaction with endogenous immunoglobulins, the results of developed LC-MS/MS method are more accurate and specific.
Clinical Impact of Antibodies against Ustekinumab in Psoriasis: An Observational, Cross-Sectional, Multicenter Study
2020, Journal of Investigative Dermatology
Ustekinumab is an effective treatment for psoriasis, but response varies between patients. The formation of anti-drug antibodies (ADAs) may explain part of this variation by reducing the free ustekinumab level. Currently, published analyses of the clinical impact of ADAs are incomplete. In this observational cross-sectional multicenter study of 340 patients, we evaluated the impact of ADAs on ustekinumab level and clinical response as assessed by the PASI. Circulating ADA levels were measured using two assays: a drug-sensitive radioimmunoassay and a drug-tolerant ELISA. Circulating ustekinumab levels were measured using an ELISA. ADAs were detected in 3.8% (95% confidence interval [CI] = 3.2–4.2) and in 10.6% (95% CI = 7.9–13.9) of patients using the radioimmunoassay and drug-tolerant ELISA, respectively. At least 85% of the ADAs were neutralizing. Compared with patients negative for ADAs, ADA positivity in the radioimmunoassay and drug-tolerant ELISA were associated with lower median ustekinumab levels (−0.62 μg/ml [95% CI = −1.190 to −0.30] and −0.74 μg/ml [95% CI = −1.09 to −0.47], respectively) and higher absolute PASI (6.6 [95% CI = 3.0–9.9] and 1.9 [95% CI = 0.4–4.0], respectively). Absence of detectable ustekinumab regardless of ADA status correlated with poor clinical outcome (median sample PASI 10.1, 6.5 [95% CI = 3.9–8.8] compared with patients positive for ustekinumab). In conclusion, substantially reduced drug exposure resulting from ADAs formation is associated with impaired clinical response.
Abnormalities and erythroblasts in peripheral blood of multiple sclerosis patients treated with natalizumab
2019, Multiple Sclerosis and Related Disorders
In natalizumab treated patients several hematopoietic abnormalities including erythroblasts, myeloblasts and neutrophilic precursors in peripheral blood have been described. So far, long term effects of the hematopoietic changes have not been reported.
To describe hematopoietic abnormalities in longitudinally monitored MS patients treated with natalizumab. Patients treated with dimethyl fumarate, teriflunomide and fingolimod served as controls. Secondly, the relation between natalizumab serum levels and the occurrence of hematopoietic abnormalities was explored.
212 natalizumab treated patients were included, 91 patients with available baseline samples (998 follow-up samples) were compared with patients with dimethyl fumarate (n = 166, 1154 samples), teriflunomide (n = 38, 228 samples) and fingolimod (n = 114, 853 samples). One hundred twenty one patients without baseline samples (1952 follow-up samples) were included in the follow-up group.
More than half of all natalizumab treated patients developed hematopoietic abnormalities, almost a quarter had erythroblasts. Natalizumab use was associated with an increased risk of developing abnormalities in comparison to oral treatment, with a corrected hazard ratio of 2.3, 10.0 and 8.1 in comparison to fingolimod, dimethyl fumarate and teriflunomide respectively. No difference in developing abnormalities was observed in relation to natalizumab serum concentrations. None of the patients developed a hematologic malignancy during follow up.
Hematopoietic abnormalities are common during natalizumab treatment. Given the lack of consequences of this finding during long-term follow-up, it is generally justifiable to refrain from further diagnostic procedures when observing hematopoietic abnormalities in patients using natalizumab.
Vedolizumab Induces Endoscopic and Histologic Remission in Patients With Crohn's Disease
2019, Gastroenterology
We evaluated the ability of vedolizumab to induce endoscopic and histologic remission in patients with Crohn's disease (CD).
We performed a prospective study of 110 patients with active CD, based on CD activity index (CDAI) scores >220 and mucosal ulcerations, who received open-label vedolizumab (300 mg) infusions at weeks 0, 2, and 6, and every 8 weeks thereafter through week 52 at tertiary centers in Europe. Patients received an additional infusion at week 10 if their CDAI score had not decreased by 70 points. Patients underwent ileocolonoscopy with collection of biopsies at baseline and weeks 26 and 52; a local and central reader determined simple endoscopic index for CD (SES-CD) scores. Histologic features were assessed by a blinded pathologist at week 26. Serum concentrations of vedolizumab were measured at serial time points. The primary outcome was endoscopic and histologic remission in patients with active CD treated with vedolizumab for 52 weeks.
At weeks 26 and 52, 36 patients (29%) and 34 patients (31%), respectively, were in corticosteroid-free clinical remission (CDAI score <150), respectively. Based on intent-to-treat analysis, endoscopic remission (SES-CD score <4) was achieved by 36 patients (33%) and 40 patients (36%) at weeks 26 and 52. Endoscopic responses (decrease in SES-CD score ≥50%) occurred in 44 patients (40%) at week 26 and 5 patients (45%) at week 52. Serum concentrations of vedolizumab were higher at weeks 2, 10, and 22 in patients with lower SES-CD scores. Histologic remission at week 26 was observed in 43 (64%) of 67 patients based on Geboes Score and 37 (66%) of 56 patients based on Robarts Histopathology Index scores in analyses of paired biopsies with inflammation at baseline. Serum concentrations of vedolizumab above 10 mg/L at week 22 were associated with endoscopic remission at week 26.
In a prospective trial, we found that approximately one-third of patients with CD achieve endoscopic remission after 52 weeks of treatment with vedolizumab and two-thirds achieve histologic remission at week 26. Higher serum concentrations of vedolizumab were associated with better outcomes. EUDRACT no: 2014–005376–29.
Pharmacodynamic assessment of cell-bound natalizumab on PBMC samples stored in liquid nitrogen
2019, Journal of Immunological Methods
Citation Excerpt :
To obtain a completely saturated reference sample, PBMCs were post saturated with saturating concentrations natalizumab (2 μg/ml) for 15 min at room temperature, followed by extensive washing with PBS to remove excess natalizumab. Natalizumab serum concentrations were measured using a cross-linking assay, in which specific polyclonal rabbit anti-natalizumab F(ab)2 fragments are used as capture reagent and a mouse antihuman IgG4 (anti-hIgG4) monoclonal antibody is used for detection as described elsewhere (Rispens et al., 2011a). The expression of alpha-4 integrin subunit and natalizumab binding to alpha-4 integrin on PBMCs was analyzed either together or in separate samples by flow cytometry using CD49d-PE (clone 9F10 (BD Biosciences) and in-house APC fluorescent labelled anti-hIgG4 clone MH164.4 in a concentration of 2 μg/ml (Sanquin, The Netherlands).
Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.
Cross-Reactivity of Antibodies to Rituximab with Other Therapeutic Anti-CD20 Antibodies
2024, Journal of Immunology

View all citing articles on Scopus

View full text

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Abstract

Introduction

Section snippets

Materials and reagents

Immunization of rabbits with natalizumab

Exchange reaction of natalizumab

Discussion

Acknowledgments

Blood

Lancet Neurol.

Anal. Biochem.

Serologic aspects of IgG4 antibodies: I. Prolonged immunization results in an IgG4-restricted response

J. Immunol.

Immunoglobulin G4: an odd antibody

Clin. Exp. Allergy

Isotype selection in antibody engineering

Nat. Biotechnol.

The apparent monovalency of human IgG4 is due to bispecificity

Int. Arch. Allergy Immunol.

Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange

Science

Inhibition of complement activation by IgG4 antibodies

Clin. Exp. Immunol.

Serologic aspects of IgG4 antibodies: II. IgG4 antibodies form small nonprecipitating immune complexes due to functional monovalency

J. Immunol.

Case study: Immunogenicity of natalizumab