JOURNAL OF INTERFERON & CYTOKINE RESEARCH 24:455–469 (2004) © Mary Ann Liebert, Inc. Regulation of Gene Expression by Pegylated IFN-2b and IFN-2b in Human Peripheral Blood Mononuclear Cells DIANA L. BRASSARD, 1,2,6 MARC M. DELORENZO, 1,6 STUART COX, 1,6 DOUGLAS W. LEAMAN, 3 YAPING SUN, 3 WEI DING, 4,6 SEAN GAVOR, 1,6 JEFFREY SPOND, 1,6 FEDERICO GOODSAID, 5,6 RONALD BORDENS, 1,6 and MICHAEL J. GRACE 1,6 ABSTRACT The pleiotropic biologic effects of interferon (IFN) are mediated through regulation of the expression of nu- merous IFN-sensitive genes. Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were analyzed to study the immunoregulatory and antiviral messenger RNAs (mRNAs) and proteins regulated by pegylated IFN-2b (PEG-IFN-2b) and IFN-2b. A dose-dependent and time-dependent response for multi- ple IFN-regulated genes was observed. IFN-dependent protein production and secretion were correlated with IFN-regulated mRNA induction. Overall regulation of gene expression patterns for PEG-IFN-2b and IFN- 2b was comparable, even though the antiviral activity of PEG-IFN-2b demonstrated a longer biologic half- life in vitro compared with IFN-2b. To study the heterogeneity of responses, PBMCs obtained from over 25 healthy donors were analyzed. Within a particular donor dataset, gene-specific and dose-dependent responses to PEG-IFN-2b treatment, demonstrated in both the amplitude of transcriptional upregulation and the du- ration of sustained mRNA upregulation, were observed. However because of donor heterogeneity, the ampli- tude of a given transcriptional response could not be predicted for a specific dose of PEG-IFN-2b. Notably, mRNA levels of oligoadenylate synthetase (OAS), double-stranded RNA (dsRNA)-activated protein kinase (PKR), IP-10, IFN-stimulated gene 54 (ISG54), and ISG15 were upregulated after 120 h of continuous PEG- IFN-2b treatment. These results suggest that the use of antiviral and immunoregulatory protein mRNA lev- els as markers to assess the therapeutic efficacy of IFN-2b and PEG-IFN-2b against viral and neoplastic diseases in clinical trials is promising but will require further analysis using clinical patient samples. 455 INTRODUCTION T HE ANTIVIRAL, ANTIPROLIFERATIVE, and immunomodulatory activities exhibited by interferon- (IFN-) have made it useful in the treatment of both viral diseases and cancer. (1,2) Binding of IFN- as well as IFN- with the type I IFN recep- tor (IFNAR-1/2) on the target cell is required for expression of all its biologic activities; both high-affinity and low-affinity li- gand-receptor binding sites have been implicated in this inter- action. (3–6) Biochemical and mutational analyses have dem- onstrated good correlation between ligand-receptor binding affinity and antiviral and antiproliferative activities. (7) The signal transduction cascade initiated following IFN- li- gand binding to IFNAR-1/2 has been well characterized. IFNAR-1/2 activates Jak family members (Jak1 and Tyk2), which in turn phosphorylate the receptor subunits and the sig- nal transducer and activator of transcription type 1 and 2 (Stat1 and Stat2) proteins. The Stat1/2 heterodimer translocates to the cell nucleus and interacts with IFN-stimulated gene factor 3 (ISGF3) (p48) to induce a transcriptional response by recog- nizing the IFN-stimulated regulatory element (ISRE) se- quence. (8,9) A variety of individual genes regulated by IFN- mediates its many biologic activities. (10–12) For example, the in- duction of various endogenous cytokines and chemokines am- plifies the initial IFN- response and orchestrates the immune system’s defense against microbial, viral, or neoplastic dis- ease. (2,13–15) Various proteins and enzyme activities have been shown to play a key role in classic antiviral responses to Departments of 1 Biotechnology, 4 Bioinformatics, and 5 Drug Safety/Metabolism, Schering-Plough Research Institute, Union, NJ 07083. 2 Present address: Medical Affairs-Oncology, Aventis Pharmaceuticals, Bridgewater, NJ 08807. 3 Department of Biological Sciences, University of Toledo, Toledo, OH 43606. 6 These authors are or were employed by Schering-Plough, whose product was studied in the present work.