Therapeutic effect of PEGylated TNFR1-selective antagonistic mutant TNF in experimental autoimmune encephalomyelitis mice Tetsuya Nomura a,c , Yasuhiro Abe a , Haruhiko Kamada a,d , Hiroko Shibata a , Hiroyuki Kayamuro a,c , Masaki Inoue a , Tomoyuki Kawara a,c , Shuhei Arita a,c , Takeshi Furuya a,c , Takuya Yamashita a,c , Kazuya Nagano a , Tomoaki Yoshikawa a,c , Yasuo Yoshioka a,c,d , Yohei Mukai c , Shinsaku Nakagawa c,d , Madoka Taniai e , Tsunetaka Ohta e , Satoshi Serada b , Tetsuji Naka b , Shin-ichi Tsunoda a,c,d, ⁎, Yasuo Tsutsumi a,c,d a Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan b Laboratory for Immune Signal, National Institute of Biomedical Innovation (NiBio), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan c Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan d The Center for Advanced Medical Engineering and Informatics, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan e Hayashibara Biochemical Laboratories, Inc., 675-1 Fujisaki, Okayama 702-8006, Japan abstract article info Article history: Received 21 October 2009 Accepted 16 December 2009 Available online 24 December 2009 Keywords: Multiple sclerosis Tumor necrosis factor-α TNFR1-selective antagonist PEG-R1antTNF Experimental autoimmune encephalomyelitis Multiple sclerosis (MS) is an inflammatory demyelinating disease, the pathogenesis of which is related to elevated serum levels of tumor necrosis factor-α (TNF). Although anti-TNF therapy has been tested as a potential treatment for MS, no remission of symptoms was observed. Recent reports indicated that the TNFR1 signal was responsible for the pathogenesis of murine experimental autoimmune encephalomyelitis (EAE), while the TNFR2 signal was responsible for recovery of the pathogenesis of EAE. Therefore, selective blocking of TNFR1 appears to be a promising strategy for the treatment of MS. In this regard, we previously succeeded in developing a novel TNFR1-selective antagonistic TNF mutant (R1antTNF) by using phage display technology. Here, we have examined the therapeutic potential of R1antTNF using EAE mice. Treatment with PEGylated R1antTNF (PEG-R1antTNF) significantly improved the clinical score and cerebral demyelination at the onset of EAE. Considerable suppression of Th1 and Th17-type response was also observed in spleen and lymph node cells of mice given PEG-R1antTNF. Moreover, the administration of PEG- R1antTNF suppressed the infiltration of inflammatory cells containing Th1 and Th17 cells into the spinal cord. These results suggest that selective blocking of TNFR1 by PEG-R1antTNF could be an effective therapeutic strategy against MS. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Multiple sclerosis (MS) is an inflammatory demyelinating disease, with a prevalence rate ranging between 2 and 150 per 100,000. While the pathogenesis of MS is not fully understood, previous studies reported that elevated serum proinflammatory cytokines, such as TNF, results in the pathogenesis of MS. Thus, it was anticipated that anti-TNF therapy might improve the symptoms for MS patients [1,2]. However, unexpected results were observed because anti-TNF therapy brought about an exacerbation of MS and enhanced the risk of demyelinating disorder [3–5]. Therefore, there is an urgent need to fully understand the biology of the TNF receptor mediated signaling pathway leading to MS. Recently, functional analysis using TNF receptor knockout mice indicated that TNFR1 signal was responsible for the pathogenesis of EAE, while TNFR2 signal was for the recovery of the pathogenesis. These studies showed that EAE symptoms were milder in TNFR1 knockout mice or TNFR1/TNFR2 double knockout mice than in wild- type mice. However, in TNFR2 knockout mice an exacerbation of clinical score and severe inflammation and demyelination were observed [6,7]. Moreover, other papers reported that TNFR2 mediates protective action, including oligodendrocyte regeneration and sup- pression of activated lymphocytes [8]. Therefore, it is anticipated that blocking of TNFR1 mediated signaling will lead to the development of a therapeutic drug for MS [9]. In this regard, we have succeeded in developing a novel TNFR1-selective antagonistic TNF mutant (R1antTNF) by using phage display technology [10]. R1antTNF suppressed arthritis in a collagen-induced arthritis model. In addition, due to its selective TNFR1 binding, R1antTNF might avoid the risk of Journal of Controlled Release 149 (2011) 8–14 ⁎ Corresponding author. Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Tel.: +81 72 641 9811; fax: +81 72 641 9817. E-mail address: tsunoda@nibio.go.jp (S. Tsunoda). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.12.015 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel