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Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model

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Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model bioRxiv preprint doi: https://doi.org/10.1101/2020.04.22.056754; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a 2 Human Liver Chimeric Mouse Model 3 4 Matthew Denta, Krystal Hamorskyb,c,d, Thibaut Vausseline, Jean Dubuissone, Yoshinari Miyataf, 5 Yoshio Morikawaf, Nobuyuki Matobaa,c,d,* 6 7 aDepartment of Pharmacology and Toxicology, University of Louisville School of Medicine, 8 Louisville, KY, USA 9 bDepartment of Medicine, University of Louisville School of Medicine, Louisville, KY, USA 10 cJames Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, 11 KY, USA 12 dCenter for Predictive Medicine, University of Louisville School of Medicine, Louisville, KY, 13 USA 14 eUniversity of Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 15 – CIIL – Center for Infection & Immunity of Lille, Lille, France 16 fPhoenixBio USA Corporation, New York, NY, USA 17 18 Running Head: HCV Inhibition of a Lectin-Fc Fusion Protein 19 20 *Address correspondence to Dr. Nobuyuki Matoba, [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.22.056754; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 21 ABSTRACT 22 Infection with hepatitis C virus (HCV) remains to be a major cause of morbidity and mortality 23 worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope 24 glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans 25 (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with 26 cell-entry receptors. However, currently there is no approved therapeutic targeting this 27 potentially druggable biomarker. Here, we investigated the therapeutic potential of the lectibody 28 Avaren-Fc (AvFc), a HMG-binding lectin-Fc fusion protein. In vitro assays showed AvFc’s 29 capacity to neutralize cell culture-derived HCV in a genotype independent manner with IC50 30 values in the low nanomolar range. A histidine buffer-based AvFc formulation was developed 31 for in vivo studies using the PXB human liver chimeric mouse model. Systemic administration 32 of AvFc was well tolerated; after 11 consecutive doses every other day at 25 mg/kg, there were 33 no significant changes in body or liver weights, nor any impact noted in blood human albumin 34 levels or serum alanine aminotransferase activity. Gross necropsy and liver pathology further 35 confirmed the lack of discernible toxicity. This treatment regimen successfully prevented 36 genotype 1a HCV infection in all animals, while an AvFc mutant lacking HMG binding activity 37 failed to block the infection. These results suggest that targeting envelope HMGs is a promising 38 therapeutic approach against HCV infection. In particular, AvFc may provide a safe and 39 efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end- 40 stage liver disease patients. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.22.056754; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 41 INTRODUCTION 42 Hepatitis C virus (HCV) is an enveloped monopartite positive sense ssRNA virus in the 43 family Flaviviridae and the causative agent of hepatitis C disease. Its genome encodes three 44 structural (core, E1, E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, 45 NS5B) (1). HCV is highly heterogenous and globally distributed, consisting of seven genotypes 46 each further subdivided into multiple subtypes. Genotype 1 is the predominant genotype 47 worldwide and particularly concentrated in high-income and upper-middle income countries, 48 whereas genotype 3 and 4 are more common in lower-middle and low-income countries (2). In 49 the United States, injection drug use represents the primary risk factor for contracting HCV 50 infection (3, 4). Around 15-25% of people acutely infected with HCV will clear the virus, while 51 the remainder will develop chronic infection that can persist largely unnoticed for decades. 52 Indeed, many HCV carriers discover their chronic infection after they have developed cirrhosis 53 (5). Chronic HCV infection is also associated with the development of hepatocellular carcinoma, 54 and those with the disease are more likely to develop cryoglobulinemia and non-Hodgkin’s 55 lymphoma (6). 56 There is no vaccine currently available for HCV. Prior to 2011, the standard chronic 57 HCV treatment was a non-specific antiviral medication using ribavirin combined with a 58 pegylated interferon-α, which was associated with significant toxicity and limited treatment 59 efficacy (7). In 2011, the U.S. Food and Drug Administration approved the first generation of 60 direct-acting antivirals (DAAs) for HCV: boceprevir and telaprevir, both of which inhibit the 61 viral protease (NS3/4A) but required cotreatment with ribavirin and peginterferon (8, 9). Further 62 approval of more potent DAAs, such as NS3/4A, NS5B and NS5A inhibitors led to the 63 development of oral ribavirin/peginterferon-free regimens (5). Multi-DAA regimens achieve 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.22.056754; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 64 sustained virologic response (defined as a period of time with no viral RNA detection) rates as 65 high as 100%, and are less toxic and more tolerable than their predecessors (10-13). While the 66 cure rates are remarkable, there exist populations of patients who may not benefit from DAA 67 therapy (14), especially patients with decompensated cirrhosis due to chronic HCV infection, for 68 whom liver transplantation may be a last resort (15). Moreover, recurrent infection occurs 69 universally and rapidly post liver transplantation (16, 17), which increases the risk of accelerated 70 cirrhosis, graft failure and death (18). DAAs, by their nature, cannot prevent recurrent infection. 71 Therefore, alternative or complementary therapies to DAAs that can block viral entry to target 72 cells, such as antibodies or other molecules acting alike, may need to be considered in these 73 circumstances (18, 19). However, there is currently no entry inhibitor approved for HCV 74 treatment. 75 The HCV envelope proteins E1 and E2 are heavily glycosylated and, like glycoproteins 76 of other enveloped viruses (HIV and the coronaviruses, for instance), have a high proportion of 77 high-mannose-type N-glycans (HMGs) on their surface (20-22). These glycans are typically 78 processed to hybrid and complex forms on glycoproteins secreted by healthy cells (23). Thus, the 79 HMGs on the surface of HCV may be considered a druggable target. We have previously 80 described the development of an HMG-targeting lectin-Fc fusion protein, or “lectibody”, called 81 Avaren-Fc (AvFc), which was shown to bind with high affinity to clusters of HMGs on the HIV 82 envelope protein gp120 and effectively neutralize multiple HIV clades and groups including 83 HIV-2 and simian immunodeficiency virus (24). Further analysis indicated that AvFc can bind to 84 HCV E2 protein (24). Therefore, in this study, we aim to investigate the anti-HCV therapeutic 85 potential of AvFc in in vitro neutralization assays and an in vivo HCV challenge study using 86 PXB-mice®, a chimeric uPA/SCID mouse model transplanted with human hepatocytes (25). 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.22.056754; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 87 88 MATERIALS AND METHODS 89 Animal Care 90 The use of animals was approved by the University of Louisville’s Institutional Animal 91 Care and Use Committee and the Animal Ethics Committee of PhoenixBio Company, Ltd. 92 (Resolution No.: 2281). All animals were given a standard diet and water ad libitum and housed 93 in a temperature and humidity-controlled facility with a 12-hour day/night cycle. 94 95 Production of AvFc and non-HMG-binding AvFc variant. 96 AvFc and a non-HMG-binding variant (AvFclec-) were produced by agroinfiltration with 97 magnICON® vectors in Nicotiana benthamiana plants as previously described (24). AvFc was 98 purified from plants after a 7-day incubation period using protein A and ceramic hydroxyapatite 99 (CHT) chromatography. 100 101 HCV neutralization assays 102 Huh-7 cells (26) and HEK-293T cells (ATCC) were cultured in Dulbecco's modified 103 Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and 1% 104 penicillin/streptomycin. To produce cell cultured HCV (HCVcc), we used a modified version of 105 the plasmid encoding JFH1 genome (genotype 2a), provided by T. Wakita (National Institute of 106 Infectious Diseases, Tokyo, Japan) (27, 28). The H77/JFH1 chimera, which expresses the core- 107 NS2 segment of the genotype 1a polyprotein within a genotype 2a background, has been 108 described previously (29). The genotype 4a ED43/JFH1 (30), genotype 5a SA13/JFH1 (31), and 109 genotype 6a HK6a/JFH1 (32) infectious HCV recombinants were provided by J Bukh 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.22.056754; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
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