Combinations of Interferon Lambda with Direct-Acting Antiviral Agents

25 Clinical efficacy of a pegylated form of human interferon (IFN) lambda 1 (referred herein 26 as Lambda) has been demonstrated in patients chronically infected with HCV 27 representing genotypes 1 through 4. In these proof-of-concept studies, Lambda showed 28 an improved safety profile compared to the pegylated form of interferon alfa (referred 29 herein as alfa). In this report, an assessment of the in vitro antiviral activity of type III 30 IFNs toward different HCV replicons revealed that the recombinant (r) unpegylated form 31 of IFN lambda 1 (rIFNλ1) exerted the most robust effect while rIFNλ3 exhibited greater 32 activity than rIFNλ2. More importantly, cross-resistance to rIFNλ1 was not observed in 33 replicon cell lines known to have reduced susceptibility to investigational direct-acting 34 antiviral (DAA) agents targeting the essential HCV non-structural proteins NS3, NS5A or 35 NS5B. When combined with either rIFNα, the NS3 protease inhibitor (NS3 PI) 36 asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir 37 (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFNλ1 38 displayed a mixture of additive and synergistic effects. In three-drug combination 39 studies, inclusion of Lambda with ASV and DCV also yielded additive to synergistic 40 effects. In line with these observations, it was demonstrated that combination of rIFNλ1 41 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in 42 genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant 43 sequences. Overall, these data support further clinical development of Lambda as part of 44 alternative combination treatments with DAAs for patients chronically infected with 45 HCV. 46 Abstract word count: 248/250 47 on M ay 2, 2021 by gest httpaac.asm .rg/ D ow nladed fomword count: 248/250 47 on M ay 2, 2021 by gest httpaac.asm .rg/ D ow nladed fom Introduction 48 Hepatitis C virus (HCV), a positive-strand RNA virus that belongs to the Flaviviridae 49 family, is a major causative agent of chronic liver disease, affecting an estimated 170 50 million individuals worldwide.(1) Until recently, treatment options for chronic HCV 51 infection comprised of the combination of alfa with ribavirin (RBV). This regimen is 52 associated with significant side effects resulting in high rates of noncompliance and 53 demonstrates variable efficacy against numerous HCV genotypes. Although various host 54 and viral factors are believed to influence the outcome of infection, different genotypes 55 are also associated with variable responses to alfa-based treatment.(2-4) More 56 specifically, increasing risk of treatment failure is observed against the most predominant 57 HCV genotype (GT) 1 (subtypes 1a and 1b), which account for approximately 60% of 58 global infections, and where an extended duration of therapy (48 to 72 weeks) is required 59 to enhance response. Successful treatment referred to sustained virological response 60 (SVR) is achieved in only 40 to 50% of patients with HCV GT1, whereas higher rates (78 61 to 86%) have been reported with those infected with HCV GT2 and GT3.(5) In addition, 62 completion of treatment often suffers from poor adherence by patients due to drug-related 63 adverse events, including psychiatric disorders, flu-like symptoms and/or hematological 64 abnormalities such as hemolytic anemia and neutropenia.(6) Recently, the addition of a 65 direct-acting antiviral (DAA) targeting the HCV NS3 protease activity (telaprevir and 66 boceprevir) to the alfa / RBV regimen was approved as the new standard-of-care for the 67 treatment of chronic GT1 infection, a consequence of enhanced SVR rates to around 7068 75% in patients.(7, 8) Unfortunately, the side effects associated with alfa-containing 69 on M ay 2, 2021 by gest httpaac.asm .rg/ D ow nladed fom treatments remain. This highlights a medical need for new HCV therapeutic agents that 70 are more effective and tolerable. 71 72 Human interferon lambda 1 (IFNλ1), also known as interleukin-29 (IL-29), is a recently 73 described human type III interferon (IFN) which has a close evolutionary relationship to 74 the IL-10 cytokines family, and is distantly related to the type I IFNs.(9) Two other IFNλ 75 cytokines simultaneously identified, IFNλ2 (IL-28A) and IFNλ3 (IL-28B), share 76 approximately 81% sequence identity with IFNλ1. The biological characteristics of these 77 cytokines are comparable to those of a type I IFN, such as IFN-alfa (IFNα) and IFN-beta 78 (IFNβ) although sequence homology is low. These various classes of IFN exert their 79 antiviral activities by inducing the expression of IFN-stimulated genes (ISGs) through 80 activation of the janus kinases, Jak1 and Tyk2, and subsequent phosphorylation of the 81 signal transducer and activator of transcription (STAT) factors, STAT1 and STAT2. The 82 complete spectrum of ISGs that mediate an antiviral effect on HCV replication has not 83 yet been defined.(10) Additionally, similar to IFNα/β, expression of IFNλ is induced 84 upon viral infection or stimulation with double-stranded RNA, and has demonstrated 85 broad antiviral activity in vitro including inhibition of viral RNA replication in the HCV 86 replicon model.(11, 12) However, the receptor complex for type III IFNs is structurally 87 unique with a more restricted cell and tissue distribution than that of the type I IFN 88 receptor, being minimally expressed on cells of hematopoietic lineage.(13) These 89 observations suggest that systemic administration of IFNλ1 may be associated with fewer 90 side effects, in comparison with alfa-based treatments. Indeed, proof-of-concept clinical 91 studies with Lambda indicated minimal adverse events and hematologic effects while 92 on M ay 2, 2021 by gest httpaac.asm .rg/ D ow nladed fom demonstrating promising antiviral activity in patients with chronic HCV GT2 or 93 GT3.(14) Moreover, a series of genome-wide association studies (GWAS) independently 94 reported a strong association between common host genetic polymorphisms in the region 95 of the IL28B gene locus (which encodes for IFNλ3), spontaneous viral clearance, and a 96 favorable treatment outcome to alfa-based therapies in chronic HCV subjects.(15, 16) 97 Although a causal immunological mechanism involved in the IL28B genotype remains 98 elusive, a link between HCV clearance and type III IFN regulation underlie the potential 99 importance of Lambda in future therapeutic indications for patients refractory to current 100 treatment options. 101 102 IFNλ1 has been shown to exert comparable antiviral response to alfa in patients infected 103 with HCV GT1 through 4.(17) As with alfa, it will be important to determine that 104 Lambda can be combined with DAAs and also result in improved SVR rates. We 105 recently described three classes of investigational DAAs in clinical development that 106 target the activity of different HCV proteins; the NS3 protease inhibitor (NS3 PI) 107 asunaprevir (ASV; BMS-650032), the first-in-class NS5A replication complex inhibitor 108 (RCI) daclatasvir (DCV; BMS-790052), and the NS5B RNA-dependent RNA 109 polymerase non-nucleoside site I inhibitor (NS5B I) BMS-791325.(18-20) Combination 110 of each of these inhibitors, respectively, with alfa / RBV were shown to be generally 111 well-tolerated and demonstrated greater antiviral activity than a placebo regimen of alfa / 112 RBV in treatment-naive patients infected with HCV GT1.(21-23) In this report, we 113 document the antiviral activity of the various recombinant (r) forms of IFNλ cytokines in 114 a number of HCV cell culture systems representing GT1a, GT1b, and GT2a. In addition, 115 on M ay 2, 2021 by gest httpaac.asm .rg/ D ow nladed fom we show that a rIFNλ1-based regimen compares favorably with a rIFNα-based regimen 116 in reducing the emergence of HCV resistance in replicon cells when combined with 117 various classes of DAAs currently in clinical development. Overall, these data support 118 the inclusion of Lambda as part of new treatment options to HCV therapy. 119 120 on M ay 2, 2021 by gest httpaac.asm .rg/ D ow nladed fom Materials and Methods 121 Compounds. 122 Recombinant (r) IFNα-2b (Intron A; rIFNα) and the pegylated form of IFNα-2a 123 (PEGASYS; alfa) were purchased from Myoderm Medical Supply (Norristown, PA) 124 and Hoffman-La Roche, Inc. (Nutley, NJ), respectively. Human recombinant forms of 125 IL-29 (rIFNλ1), IL-28A (rIFNλ2), IL-28B (rIFNλ3) and IL-28B K70R (rIFNλ3-K70R) 126 were synthesized at ZymoGenetics, Inc (Seattle, WA). The pegylated form of IFNλ1 127 (Lambda) is a covalent conjugate of rIFNλ1 (molecular weight of 19.6 kDa) and a 20 128 kDa linear polyethylene glycol chain. All forms of IFNλ have been extensively 129 characterized by a number of methods, including amino acid analysis, N-terminal 130 sequencing, size exclusion / chromatography-multi-angle light scattering (SEC-MALS), 131 peptide map analysis, and showed equivalent whole mass values (ranging from 19.6 to132 20kDa). ASV, DCV and BMS-791325, synthesized by Bristol-Myers Squibb Co. (BMS), 133 have been previously described.(19, 18, 20) 134 135 Cell lines and viral constructs. 136 Human hepatoma cells, Huh-7 and Huh-7.5, were maintained in Dulbecco’s modified 137 Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum 138 (FBS) and L-glutamine (2 mM). HCV sub-genomic replicon cell lines representing 139 GT1b (Con1 strain), GT1a (H77c strain) and GT2a (JFH1 strain) wild-type (WT) 140 sequences were generated at BMS as previously described.(24) Stable Huh-7 cells 141 containing HCV replicon variants with specific NS3, NS5A, or NS5B amino acid 142 substitutions were established following selection with G418 as previously described.(24) 143 on M ay 2, 2021 by gest httpaac.asm .rg/ D ow nladed fom