Development of a novel site-specific pegylated interferon beta for antiviral therapy for chronic

29 Pages·2017·3 MB·English

AAC Accepted Manuscript Posted Online 3 April 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00183-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
Tsuge M et al. Page 1
1 Development of a novel site-specific pegylated interferon beta for antiviral therapy for
2 chronic hepatitis B
3 Running title: Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
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5 Masataka Tsuge1,2,3, Takuro Uchida1,3, Nobuhiko Hiraga1,3, Hiromi Kan1,3, Grace Naswa
6 Makokha1,3, Hiromi Abe-Chayama1,3, Daiki Miki1,3,4, Michio Imamura1,3, Hidenori Ochi1,3,4, C.
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7 Nelson Hayes1,3, Rieko Shimozono5, Tomokatsu Iwamura5, Hideki Narumi5, Tomohiko Suzuki5, o
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8 Mie Kainoh5, Tadatsugu Taniguchi6, Kazuaki Chayama1,3,4 nlo
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11 1 Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical m

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12 & Health Sciences, Hiroshima University, Hiroshima, Japan. t
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13 2Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, ://
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14 Japan. c
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15 3Liver Research Project Center, Hiroshima University, Hiroshima, Japan. sm
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16 4Laboratory for Liver Diseases, SNP Research Center, Institute of Physical and Chemical or
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17 Research (RIKEN), Hiroshima, Japan. o
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18 5Pharmaceutical Research Laboratories, Toray Industries Inc., Kanagawa, Japan A
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19 6Department of Molecular Immunology, Institute of Industrial Science, The University of Tokyo, ril
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20 Tokyo, Japan 2
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21 19

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22 Key words: HBV, pegylated interferon beta, antiviral effect, human hepatocyte chimeric mouse, y

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23 gene expression u
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Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
Tsuge M et al. Page 2
27 Corresponding author:
28 Prof. Kazuaki Chayama, MD, PhD
29 Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical
30 and Health Sciences
31 Hiroshima University
32 1-2-3 Kasumi, Minami-ku
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33 Hiroshima 734-8551, Japan o
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34 Tel: [phone]. Fax: [phone]. E-mail: [email protected] nlo
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35 d
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36 Abbreviations:
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37 HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; m

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38 cccDNA, covalently closed circular DNA; chimeric mouse, human hepatocyte chimeric mouse; t
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39 HCC, hepatocellular carcinoma. //
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40 c
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41 Financial support: sm
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42 Ministry of Education, Sports, Culture and Technology and Ministry of Health, Labor and Welfare or
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43 (Grants-in-Aid for scientific research and development). This research is partially supported by o
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44 research funding from the Research Program on Hepatitis from the Japan Agency for Medical A
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45 Research and Development, AMED (grant number: 15fk0210001h0002). il
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46 2
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47 Potential conflicts of interest: 19

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48 Masataka Tsuge - Grant/Research Support: Bristol-Meyers Squibb y

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49 Michio Imamura - Grant/Research Support: Bristol-Meyers Squibb u
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50 Kazuaki Chayama - Speaking and Teaching: Sumitomo Dainippon Pharma, AbbVie GK, MSD t
51 KK, Bristol-Myer Squibb, Gilead Inc; Grant/Research Support: Sumitomo Dainippon Pharma,
52 AbbVie, MSD K.K., Bristol-Myer Squibb, Gilead Inc., Eisai, Toray
53 Rieko Shimozono, Tomokatsu Iwamura, Hideki Narumi, Tomohiko Suzuki and Mie Kainoh -
54 Employee of Toray industries, Inc.
55
Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
Tsuge M et al. Page 3
56 Word count:
57 Abstract: 245 words
58 Main text (Abstract, Introduction, Material and Methods, Results, Discussion, Acknowledgement):
59 3,921 words
60 Figures: 4, Tables: 0
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Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
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64 Abstract
65 Although nucleot(s)ide analogues and pegylated-interferon-α2a (PEG-IFNα2a) can suppress
66 hepatitis B virus (HBV) replication, it is difficult to achieve complete HBV elimination from
67 hepatocytes. A novel site-specific pegylated recombinant human IFNβ (TRK-560) was recently
68 developed. In the present study, we evaluated the antiviral effects of TRK-560 on HBV replication
69 in vitro and in vivo. In vitro and in vivo HBV replication models were treated with antivirals
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70 including TRK-560, and changes in HBV markers were evaluated. To analyze antiviral o
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71 mechanisms, cDNA microarray analysis and an enzyme-linked immunoassay (ELISA) were nlo
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72 performed. TRK-560 significantly suppressed the production of intracellular HBV replication d
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73 intermediates and extracellular HBsAg (P<0.001, P<0.001, respectively), and the antiviral effects
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74 of TRK-560 were enhanced in combination with nucleot(s)ide analogues, such as entecavir and m

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75 tenofovir disoproxil fumarate. The reduction in HBV DNA levels by TRK-560 treatment was t
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76 significantly higher than that by PEG-IFNα2a treatment both in vitro and in vivo (P=0.004, //
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77 P=0.046, respectively), and intracellular HBV cccDNA reduction by TRK-560 treatment was also c
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78 significantly higher than by PEG-IFNα2a treatment in vivo (P=0.0495). cDNA microarrays and sm
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79 ELISA for CXCL10 production revealed significant differences between TRK-560 and PEG- or
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80 IFNα2a in the induction potency of interferon stimulated genes. TRK-560 shows a stronger o
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81 antiviral potency via higher induction of interferon stimulated genes and stronger stimulation of A
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82 immune cell chemotaxis compared to PEG-IFNα2a. As HBsAg loss and HBV cccDNA il
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83 eradication are important clinical goals, these results suggest a potential role for TRK-560 in the 2
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84 development of more effective treatment for chronic hepatitis B infection. 19

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85 (245 words ≤ 250words) y

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Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
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87 Introduction
88 Hepatitis B virus (HBV) infection is a serious global health problem. More than 500,000
89 people per year die due to HBV-related liver diseases, including chronic hepatitis, liver cirrhosis,
90 and hepatocellular carcinoma (1). To prevent the progression of liver diseases, antiviral therapies
91 based on interferon (IFN) and/or nucleos(t)ide analogues (NAs) have been used in the treatment
92 of chronic HBV infection (2-4). Although these antiviral therapies can strongly suppress viral
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93 replication, it is difficult to achieve complete HBV elimination from hepatocytes. The main o
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94 impediment to viral clearance is the presence of HBV covalently closed circular DNA (cccDNA) in nlo
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95 the nucleus. HBV cccDNA is a minichromosome composed of viral DNA supplemented with d
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96 histone and non-histone host proteins (5-10). In general, cccDNA is refractory to IFN and NA
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97 therapy. m

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98 Currently, loss of the HBV surface antigen (HBsAg) is considered a crucial goal of antiviral t
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99 therapy for preventing the progression to advanced liver diseases and for decreasing the //
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100 mortality related to chronic hepatitis B infection (11). To achieve HBsAg loss, several clinical c
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101 trials using pegylated-interferon alpha 2a (PEG-IFNα2a) in combination with NAs have been sm
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102 performed, and some positive results have been reported (12-16). In one clinical trial using or
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103 tenofovir disoproxil fumarate (TDF) and PEG-IFNα2a, the cumulative HBs seroclearance rate in o
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104 patients with TDF and PEG-IFNα2a combination therapy was significantly higher than in patients A
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105 treated with either TDF or PEG-IFNα2a monotherapy (13). However, the HBs seroclearance rate il
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106 was inadequate (~9.1%), and it is necessary to improve the success rate for treatment of chronic 2
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107 hepatitis B. 19

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108 Interferon beta (IFNβ) is a type I interferon that has been used in antiviral therapy for HBe y

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109 antigen-positive chronic hepatitis B patients (17). IFNβ binds to IFN receptors (IFNAR1 and u
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110 IFNAR2), which are the same receptors used by IFNα, and induces activation of interferon t
111 stimulated genes (ISGs) via JAK/STAT signaling (18). Because IFNβ is administrated
112 intravenously, the blood concentration of IFNβ increases immediately; however, the
113 concentration rapidly reduces to levels insufficient for antiviral effects. Therefore, only 20~50% of
114 chronic hepatitis B patients were able to achieve HBV DNA reduction and ALT normalization
115 during IFNβ monotherapy, and ALT re-elevation or HBV DNA rebound sometimes occurred after
Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
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116 completion of the therapy in some responders(19). However, prolonged stimulation with IFNα
117 has been shown to induce a state of refractoriness, desensitizing cells to further interferon
118 stimulation; on the other hand, Makowska et al. reported that repeated stimulation of hepatocytes
119 with IFNβ or IFNλ, unlike IFNα, does not lead to refractoriness, at least in the case of hepatitis C
120 virus infection (20). Therefore, we considered that the improved potency of IFNβ might suppress
121 HBV replication or reduce HBV cccDNA more effectively than IFNα. To improve the potency of
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122 IFNβ, the blood concentration of IFNβ should be maintained at a sufficient level for a longer o
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123 period of time, and ISGs should be induced continuously without desensitizing the interferon nlo
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124 signaling pathway. We have recently developed a novel site-specific pegylated recombinant d
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125 human IFNβ (PEG-IFNβ; TRK-560) that contains a single PEG molecule (43 kDa) covalently
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126 linked to the amino group of lysine (Lys) amino acid 134 of recombinant human IFN-β produced m

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127 in Escherichia coli and has a longer blood half-life without loss of activity compared to t
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128 conventional IFNβ (Cancer Science, 2017, in press). To avoid interfering with binding to IFN- //
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129 receptors due to pegylation and to avoid inducing neutralizing antibodies, in TRK-560, c
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130 polyethylene glycol was site-specifically introduced into a single optimal amino acid within the sm
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131 epitope region of IFNβ. In the present study, we evaluated the antiviral effects of TRK-560 using or
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132 in vitro and in vivo HBV replication models. o
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134 il
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Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
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135 Results
136 Induction of interferon stimulated genes by novel pegylated interferon β TRK-560
137 To verify the effects on interferon signaling by the novel pegylated interferon β, TRK-560,
138 Huh7 cells were treated with either PEG-IFNα2a (100 IU/ml, 7.14 ng/ml) or TRK-560 (100 IU/ml,
139 0.887 ng/ml) for 4 hours, and then cDNA microarray analysis was performed using the cells. The
140 expression profiles of interferon stimulated genes (ISGs) were compared between cells treated
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141 with TRK-560 and cells treated with PEG-IFNα2a. As shown in Figure 1A, most ISGs were o
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142 regulated similarly by both TRK-560 and PEG-IFNα2a treatment. However, although the cells nlo
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143 were treated with the same unit dose (100 IU/ml) of PEG-IFNα2a or TRK-560, the induction d
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144 levels of several ISGs differed between treatment with PEG-IFNα2a and TRK-560. To verify
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145 these differences, we also analyzed the induction levels of specific ISG products (MxA and m

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146 CXCL10) by western blotting and ELISA. As shown in Figure 1B, the concentration-response t
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147 curve of TRK-560-induced CXCL10 production was shifted to the left compared with PEG- //
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148 IFNα2a. A difference in potency was also observed for MxA production (Figure 1C). These c
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149 results indicate that TRK-560 could activate interferon signaling and may have a higher potency sm
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150 for ISG induction. or
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151 o
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152 Anti-HBV effects of TRK-560 in vitro A
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153 As the potency of ISG induction by TRK-560 was confirmed, the anti-HBV effects of TRK- il
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154 560 were evaluated using HBV expression cells, HepG2 cells into which HBV expression 2
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155 plasmids were stably transfected. HBV expression cells were treated with several concentrations 19

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156 of PEG-IFNα2a or TRK-560 for 5 days, and then the change in intracellular core-associated HBV y

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157 replication intermediates in the harvested cells and the change in HBV markers in the collected u
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158 culture media were evaluated. As shown in Figure 2A, intracellular replication intermediates were t
159 significantly reduced by both PEG-IFNα2a and TRK-560 treatments. However, the potency of
160 HBV reduction by TRK-560 was approximately 100-fold higher than that by PEG-IFNα2a. A
161 similar result was also observed by the analysis of HBV DNA reduction in culture media (Figure
162 2B). Furthermore, although PEG-IFNα2a could not reduce HBeAg and HBsAg titers in culture
163 media, HBsAg level could be reduced with a high concentration of TRK-560 (Figure 2C and D).
Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
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164 These results indicate that the anti-HBV activity of TRK-560 might be stronger than that of PEG-
165 IFNα2a and suggest that HBsAg reduction could be achieved using TRK-560.
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167 Anti-HBV effects of TRK-560 in vivo
168 To confirm the high potency of anti-HBV effects by TRK-560 treatment, HBV-infected
169 chimeric mice were generated from PXB mice and were administrated 30μg/kg of PEG-IFNα2a
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170 or TRK-560 twice a week for 6 weeks. During PEG-IFN treatment, serum HBV DNA titers were o
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171 evaluated every 2 weeks. As shown in Figure 3A, serum HBV DNA titers were reduced by both nlo
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172 PEG-IFNα2a and TRK-560 treatment. However, the reduction level by TRK-560 treatment was d
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173 significantly greater than that by PEG-IFNα2a treatment. After 4 weeks of TRK-560 treatment, we
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174 also evaluated intracellular HBV cccDNA levels in the mouse livers and HBsAg and HBeAg in the m

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175 mouse sera (Figure 3B). Each of these HBV markers were significantly reduced following TRK- t
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176 560 treatment, and these results indicated that TRK-560 treatment could reduce not only HBV //
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177 DNA or HBsAg in serum but also HBV cccDNA in HBV infected hepatocytes. c
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178 sm
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179 Synergistic anti-HBV effects by PEG-IFN and nucleos(t)ide analogue combination therapy or
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180 These results suggest that TRK-560 can potently suppress HBV. To investigate the o
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181 synergistic effects of PEG-IFNs in combination with NAs, HBV expression cells were treated with A
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182 both PEG-IFN and NA for 5 days, and changes in intracellular core-associated HBV replication il
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183 intermediate levels were evaluated. As shown in Figure 4A, the potency of the antiviral effect of 2
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184 10nM of ETV is similar to that of 1μM of TDF, which appears to be in line with previous reports 19

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185 indicating an EC50 of 8~9nM for ETV and 0.25~2.4μM for TDF (21-23). Therefore, we analyzed y

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186 the synergistic anti-HBV effect of PEG-IFN used concomitantly with 10nM of ETV or 1μM of TDF. u
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187 By in vitro analysis, similar synergistic effects with NAs were observed between PEG-IFNα2a t
188 and TRK-560 treatments (Figure 4B). The reduction levels of intracellular core-associated HBV
189 replication intermediates by TDF plus PEG-IFN treatment was higher than that by ETV plus
190 PEG-IFN treatment. To confirm the in vitro results, we also performed an in vivo study using
191 HBV-infected chimeric mice. As the level of HBV DNA reduction by TRK-560 was significantly
192 higher than that of PEG-IFNα2a (Figure 3A), synergistic effects were also analyzed by
Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
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193 combination treatment with NA plus PEG-IFNα2a or TRK-560. Although no difference in HBV
194 DNA reduction was observed between TRK-560 plus ETV and PEG-IFNα2a plus ETV or TRK-
195 560 plus TDF and PEG-IFNα2a plus TDF combination treatment (Figure 4C and 4D), HBsAg and
196 HBeAg reduction rates by TRK-560 treatment were significantly higher than those by PEG-
197 IFNα2a treatment (P=0.0495, P=0.0495, respectively) (Figure 4E and 4F), and HBsAg and
198 HBeAg reduction rates were enhanced by NA plus PEG-IFN combination treatments. To
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199 compare the amounts of HBV cccDNA remaining in the hepatocytes, HBV cccDNA levels were o
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200 also measured using human liver tissue extracted from chimeric mouse livers. As shown in nlo
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201 Figure 4G, HBV cccDNA levels in mice treated with TRK-560 or TRK-560 plus NAs were d
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202 significantly reduced compared with those in mice treated with PEG-IFNα2a.
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203 m

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Antiviral effects of a novel site-specific pegylated interferon beta to hepatitis B virus
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204 Discussion
205 In the present study, we compared antiviral effects of PEG-IFNα2a and TRK-560
206 treatment. Although IFN dose is usually reported using international units, we could not use
207 international units to represent PEG-IFN because of differences in the units used among
208 manufacturers. Therefore, we decided to report treatment doses based on the mass of the PEG-
209 IFNs. Both PEG-IFNs were conjugated with around 40kDa of polyethylene glycol and the
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210 molecular weight of each was around 60kDa. Therefore, 1ng/ml of TRK-560 corresponds to o
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211 50nM and 100ng/ml of PEG-IFNα2a corresponds to 5.1μM. In the in vivo study, we injected nlo
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212 30μg/kg of PEG-IFNs into the chimeric mice. In clinical practice, we normally use 180μg/body of d
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213 PEG-IFNα2a (3μg/kg body) for treatment of chronic hepatitis B. Based on FDA (Food and Drug
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214 Administration) recommendations for dosage calculation for animal testing, the optimal dose of m

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215 PEG-IFNα2a for our mouse model was determined to be 30μg/kg/day. t
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216 As shown in Figure 1B and 1C, induction levels of MxA and CXCL10 following treatment //
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217 with 1ng/ml of TRK-560 were similar to that of 100ng/ml of PEG-IFNα2a. Reduction levels of c
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218 intracellular core-associated HBV replication intermediates were also similar between TRK-560 sm
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219 1ng/ml treatment and PEG-IFNα2a 100ng/ml treatment (Figure 2A). Therefore, these results or
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220 suggest that TRK-560 might have a higher potency for ISG induction and HBV suppression than o
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221 PEG-IFNα2a. Furthermore, significant reductions of intracellular HBV cccDNA, serum HBsAg A
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222 levels and serum HBV DNA levels were observed in vivo using TRK-560 (Figure 3B) indicating il
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223 that TRK-560 has higher potencies not only for HBV DNA but also for HBsAg and cccDNA. 2
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224 As IFN is known to have pleiotropic effects on the HBV life cycle, including epigenetic 19

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225 modification or degradation of HBV cccDNA, inhibition of transcription of viral RNA, and y

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226 prevention of encapsidation, treatment with PEG-IFN treatment could be expected to reduce not u
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227 only levels of HBV DNA but also those of other HBV-related markers (HBsAg, HBeAg) (5, 24, 25). t
228 Although intracellular HBV replication intermediates were reduced by PEG-IFN treatments in a
229 dose-dependent manner (Figure 2A), reductions of HBsAg and HBeAg in culture media following
230 PEG-IFN treatment was not observed (Figure 2C and 2D). Considering the diverse effects of IFN,
231 these results seemed to be discrepant. However, the HBV expression cell line used in the
232 experiment was generated from stable transfection of an HBV expression plasmid into HepG2