A novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression

doi:10.1016/j.jim.2006.06.016

Journal of Immunological Methods

Volume 316, Issues 1–2, 30 October 2006, Pages 8-17

https://doi.org/10.1016/j.jim.2006.06.016 Get rights and content

Abstract

The presence of neutralizing antibodies against protein therapeutics is a concern in the biomedical field. Such antibodies not only reduce the efficacy of protein therapeutics, but also impose potential dangers to the patients receiving them. To date, a small number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. Most of the existing assays, however, either involve the use of radioactive materials or have limited sensitivities and/or poor specificities. With advances in mRNA profiling and detection techniques, we have established a novel and non-radioactive bioassay system using branched DNA (bDNA) technology for detecting protein-therapeutic neutralizing antibodies in patient serum. Our assay measures the variations of target gene expression that reflect the biologic effect of the therapeutic agent and the capability of the antibodies, if present, to neutralize the therapeutics. Compared with most existing assays, the new assay is more sensitive and specific, and completely eliminates the use of radioactive materials. Application of the new assay system can be widely expanded if new target genes and responding cell lines for other therapeutics are identified or engineered.

Introduction

The need to detect antibodies capable of neutralizing therapeutic proteins has increased. The publication by Casadevall and colleagues (2002) underscores this importance as it describes the effects of anti-erythropoietin antibodies on development of pure red blood cell aplasia in patients who have received epoetin as treatment for anemia. In pure red cell aplasia, antibodies generated against one type of recombinant human erythropoietin (rEPO) neutralize not only the drug but also endogenous erythropoietin. Affected patients may require lifetime red cell transfusions. A key success factor for therapeutic proteins is the ability to be administered without generating a neutralizing immune response in the patient. Manufacturers of these therapeutic proteins must develop assays capable of identifying antibodies that not only bind to the drug but also can neutralize the biologic effect of the drug. In most cases, such development is done with traditional methodology that assesses the ability of an antibody to block drug-induced cellular responses, including cell proliferation, differentiation, and apoptosis, expression of cellular markers or enzymes, release of cytokines, etc. (reviewed by Mire-Sluis, 2001, Wei et al., 2004). A limitation of these traditional methods is their specificities and sensitivities. The specificity of cell-based bioassays is influenced by various contents of the assay matrix, such as plasma or serum, since cells can also respond to many of these contents. On the other hand, the sensitivity of these assays is often limited by the amount of host serum the assay can tolerate. In addition, most of these assays require a long time, usually multiple days to allow the cells to respond to the therapeutic agent.

A new technology has been developed with the potential to improve the ability to detect neutralizing antibodies in serum samples. Using branched DNA (bDNA) technology, a very early but reliable signal can be measured to identify the cellular response to a therapeutic protein. bDNA technology is a sandwich nucleic acid hybridization system that detects and quantifies target RNA molecules by amplifying the reporter signal using branched DNA probes (Urdea and Wuestehube, 2000). In this report we describe the use of bDNA technology to detect the presence of antibodies capable of neutralizing therapeutic proteins. This technique is applicable as long as the cell responds to the presence of the therapeutic protein by altering the expression of a specific mRNA that can be used as a target for probes. This technique is able to tolerate human serum concentrations from 10% to 20%. This promising new technology has the potential to improve the ability to detect neutralizing anti-therapeutic protein antibodies.

Section snippets

Cells and proteins

UT-7, a human acute megakaryocytic leukemia cell line, originally established by Komatsu et al. (1991), was obtained from Amgen Cell Bank and maintained in growth media [RPMI/1640 (Gibco, NY) containing 10% fetal calf serum (Hyclone, Logon, UT)] supplemented with 10 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF). Recombinant human erythropoietin (rEPO), stem cell factor (rSCF), granulocyte colony-stimulating factor (rG-CSF), rGM-CSF, mouse interleukin-3 (rIL-3), and rabbit

EPO induces PIM-1 expression

mRNA microarray experiments determined which genes in UT-7 cells had altered expression after rEPO treatment. UT-7 cells quieted in rGM-CSF-free media were treated with 20 ng/ml rEPO at 37 °C for 2, 4, 6, or 24 h. Messenger RNAs extracted from the rEPO-treated or untreated control cells were used to generate probes for the subsequent microarray experiments. The mRNA expression level of a number of genes has been changed in rEPO-treated cells compared with that in the untreated control cells (

Discussion

Cytokines or growth factors exert their biologic effects by binding to their receptors and activating various intracellular signal transduction processes (Schlessinger and Ullrich, 1992, Kishimoto et al., 1994, Ihle, 1995, Wells, 1996, Dhanasekaran, 1998). The synergistic action of the activated intracellular signaling pathways causes alterations in gene expression and further leads to changes in cell survival, proliferation or apoptosis (Kishimoto et al., 1994, Ihle, 1995, Appleby et al., 1996

Acknowledgements

The authors thank Wei Liu for help in biostatistical data analysis; Shalini Gupta and Xin Wei for anti-human EPO antibody 29123; Iwona Wala and Jenny Hu for technical help and discussions; and Drs. MaryAnn Foote, Shalini Gupta, and Francesca Civoli, and Nicola Bond for critical reading.

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Research paperA novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression

Abstract

Introduction

Section snippets

Cells and proteins

EPO induces PIM-1 expression

Discussion

Acknowledgements

Cell

Methods Enzymol.

Cell

Exp. Hematol.

Neuron

Biochem. Biophys. Res. Comm.

J. Biol. Chem.

J. Immunol. Methods

Biochim. Biophys. Acta

Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin

N. Engl. J. Med.

Cell signaling: an overview

Oncogene

Type I procollagen production and cell proliferation is mediated by transforming growth factor-beta in a model of hepatic fibrosis

Endocrinology

Research paper
A novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression