DR.

KOSH AGARWAL (Orcid ID : 0000-0002-4754-828X)

PROF. PIETRO ANDREONE (Orcid ID : 0000-0002-4794-9809)

Accepted Article
DR. MINDIE H. NGUYEN (Orcid ID : 0000-0002-6275-4989)

Article type : Original Paper

Safety and Efficacy of Vesatolimod (GS-9620) in Patients with Chronic

Hepatitis B Who Are Not Currently on Antiviral Treatment

Short Title: Vesatolimod for Chronic Hepatitis B

Kosh Agarwal1, Sang Hoon Ahn2, Magdy Elkhashab3, Audrey H. Lau4, Anuj

Gaggar4, Anuradha Bulusu4, Xinyu Tian4, Andrea L. Cathcart4, Jacky Woo4, G.

Mani Subramanian4, Pietro Andreone5, Hyung Joon Kim6, Wan Long Chuang7,

Mindie H. Nguyen8

1
Institute of Liver Studies, Kings College Hospital, London, United Kingdom;
2
Yonsei University College of Medicine, Brain Korea 21 Project of Medical

Science, Seoul, Republic of Korea;

3
Toronto Liver Centre, Toronto, Ontario, Canada;
4
Gilead Sciences, Inc., Foster City, CA;
5
Center for the Study and Research on Hepatitis, Department of Medical and

Surgical Sciences, University of Bologna, Bologna, Italy;

6
Chung-Ang University Hospital, Seoul, South Korea;

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/jvh.12942
This article is protected by copyright. All rights reserved.
 7
Kaohsiung Medical University Hospital, Kaohsiung Medical University,

Kaohsiung, Taiwan;
Accepted Article
8
Stanford University Medical Center, Palo Alto, California

Corresponding Author:

Kosh Agarwal

Institute of Liver Studies

King's College Hospital

Denmark Hill

London SE5 9RS

Phone: +44 (0) 203 299 4015

Fax: +44 (0) 203 299 3167

Email: kosh.agarwal@nhs.net

Funding: This study was supported by Gilead Sciences, Inc., Foster City, California,

USA.

Conflicts of interest: K. Agarwal: Gilead, AbbVie, BMS, MSD; S.H. Ahn: BMS,

Gilead, Roche, MSD, AbbVie, and Green Cross; H.J. Kim: Roche, Abbie, MSD,

BMS, Dong A ST, Sam Jin, Chong Geun Dang; M. Elkhashab: Gilead, AbbVie,

Celgene, Eisai, Genfit, Intercept, Janssen, MSD, Roche, Shire, Spring Bank; A.H.

Lau, A. Gaggar, A.L. Cathcart and G.M. Subramanian: Gilead; P. Andreone: Gilead,

AbbVie, BMS, Intercept, MSD; W-L. Chuang: Gilead, AbbVie, BMS, MSD,

PharmaEssentia, Roche; Mindie H. Nguyen: Gilead, Alnylam, BMS, Dynavax,

Intercept, Janssen, Pfizer, Novartis. No other disclosures were reported.

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 Clinical Trial Number: GS-US-283-1062; NCT02579382
Accepted Article
Abstract

Vesatolimod is an oral agonist of toll-like receptor 7 designed to minimize systemic

exposure and side effects. We assessed the safety and efficacy of vesatolimod in

viremic chronic hepatitis B (CHB) patients not currently on oral antiviral treatment

(OAV) in a Phase 2, multicenter, double-blind, randomized, placebo-controlled study.

Methods: 192 patients stratified by HBeAg status and alanine aminotransferase level

were randomized 2:2:2:1 to receive oral vesatolimod (1-, 2-, or 4-mg) or placebo

once weekly for 12 weeks; tenofovir disoproxil fumarate (300-mg daily) was

administered daily for 48 weeks. Efficacy was assessed by quantitative serum

HBsAg decline at Week 24 from baseline. In addition to safety assessments,

changes in whole blood interferon-stimulated gene (ISG) transcripts and serum

cytokines were explored.

Results: Most patients were male (64.1%) and HBeAg-negative (60.9%) at baseline.

Among vesatolimod-treated patients, most (60.4-69.1%) experienced ≥1 treatment

emergent adverse event; the majority were mild or moderate in severity. No clinically

meaningful differences in HBsAg changes from baseline were observed between

treatment groups. No patients experienced HBsAg loss, while 3 patients experienced

HBeAg loss and hepatitis B e-antibody seroconversion. HBV DNA suppression rates

were similar across all treatment arms at Week 24. ISG15 induction was dose-

dependent and did not correlate with HBsAg changes. A small proportion of patients

exhibited dose-dependent interferon-induction that correlated with grade of

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 influenza-like adverse events. Overall, vesatolimod is safe and well-tolerated in CHB

patients. Though consistent dose-dependent pharmacodynamic induction of ISGs

Accepted Article
was demonstrated, it did not result in clinically significant HBsAg decline.

Key Words: vesatolimod; hepatitis B virus; HBsAg; HBeAg; TLR7; immune response

Introduction

The World Health Organization estimates that nearly 260 million people worldwide

are chronically infected with the hepatitis B virus (HBV) [1]. As one of the leading

causes of chronic liver disease, cirrhosis and HCC development [2-4], chronic

hepatitis B (CHB) infection results in the death of 686,000 individuals annually [5].

Clearance of HBsAg is considered functional cure and the ultimate goal of CHB

therapy, though HBsAg-loss rates are low with currently available treatments and

viral rebound upon discontinuation is near universal [6-9]. CHB is characterized by

compromised innate and adaptive immune responses, including suboptimal antigen

presentation, antigen-specific T cell exhaustion, and insufficient antibody production

[10, 11]. Patients treated with 48 weeks of pegylated-interferon (IFN) concurrently

with tenofovir disoproxil fumarate (TDF) experience greater HBsAg loss than those

receiving either treatment alone [12].

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 Vesatolimod (GS-9620), an oral small molecule toll-like receptor 7 (TLR7) agonist,

was developed to induce a protective, virus-specific immune response to enhance

Accepted Article
HBsAg loss with hepatitis B surface antibody (HBsAb) seroconversion. TLR7 is a

pattern-recognition receptor located in the endolysosomal compartment of

plasmacytoid dendritic cells (pDC) and B cells [13]. Activation of TLR7 via its natural

ligand, single-stranded RNA [14], results in innate and adaptive immune stimulation

through secretion of type I interferon by pDC [15], increased expression of molecules

associated with antigen presentation and T cell co-stimulation (e.g., human

leukocyte antigen-D related, cluster of differentiation 86) on virally-activated pDC

[16], and the induction of B cell differentiation into immunoglobulin-producing plasma

cells [17].

Vesatolimod targets liver and/or gut-associated TLR7-expressing plasmacytoid

dendritic cells (DC) for on-target, pre-systemic induction of local IFN expression

with minimal systemic exposure [18] and induces activation of the TLR7 pathway,

enhancing HBV-specific cytotoxic T cell activation [19]. In a previous Phase 2 study

in virally-suppressed chronic HBV patients administered 1-, 2- or 4-mg vesatolimod

doses for different treatment durations up to 12 weeks, no change in HBsAg decline

was observed [20]. In this companion Phase 2, double-blind, randomized, placebo

(PBO)-controlled study, the safety and efficacy of vesatolimod was assessed in

viremic CHB patients not on oral antiviral therapy.

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 Patients and Methods
Accepted Article
Patients and Study Design

This study is a Phase 2, multicenter, randomized, double-blind, PBO-controlled

sequential-dosing study conducted in 30 centers in the United States, Korea, Italy,

United Kingdom, Canada, Taiwan, Hong Kong-China, and New Zealand from

November 2015 to August 2017 (GS-US-283-1062; clinicaltrials.gov NCT02579382).

Study participants were adults (21-65 years) with CHB infection (documented

evidence of HBsAg positivity for >6 months) not currently on oral antiviral (OAV)

treatment for ≥3 months. At screening, patients were required to have HBV DNA

≥2000 IU/ml. Major exclusion criteria included the presence of cirrhosis (i.e. the

presence of extensive bridging fibrosis [Metavir ≥3 or Ishak fibrosis score ≥4] on liver

biopsy within 5 years of screening or by non-invasive alternatives to liver biopsy [e.g.

screening Fibrotest plus aspartate aminotransferase/platelet ratio index [APRI] or

Fibroscan [21] within 6 months of screening]), and coinfection with HCV, Delta virus

or HIV. Full eligibility criteria are provided in the supplementary information.

All patients provided written informed consent. The study was approved by the

institutional review board at participating sites and conducted in compliance with the

Declaration of Helsinki, Good Clinical Practice guidelines, and local regulatory

requirements.

Eligible patients were randomized in a 1:2:2:2 ratio to receive PBO or 1 of 3 (1-, 2-,

or 4-mg) weekly oral vesatolimod doses for 12 weeks (Gilead Sciences, Inc., Foster

City, CA), with approximately 50 patients per treatment cohort stratified by HBeAg

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 status (positive vs. negative) and ALT (>19 vs. ≤19 IU/L for females; >30 vs. ≤30

IU/L for males) according to American Association for the Study of Liver Diseases
Accepted Article
(AASLD) guidelines [9]. All patients were co-administered TDF 300-mg once daily at

start of vesatolimod treatment until at least end of study (Week 48). At Week 48,

duration of follow-up and/or treatment was dependent upon the principal

investigator’s discretion (i.e. 24-144 weeks follow-up  TDF or until initiation of

alternative CHB therapy). An external data monitoring committee will provide

external review of all safety data through the end of the vesatolimod dosing period

(study is currently ongoing).

Study assessments

Adverse events (AEs) with an onset date on or after the first dose date through

discontinuation of treatment were recorded. AEs were investigator-assessed and

graded according to the Gilead Grading Scale for Severity of AEs and Laboratory

Abnormalities (supplementary information). Dose-limiting and study drug

discontinuation guidelines are specified in the protocol (supplementary information).

All assessments were conducted according the schedule provided in the

supplementary information. Screening assessments included standard laboratory

and clinical tests, measurement of serum HBV DNA and HBsAg levels, and TLR7

genotyping. Quantitative HBV DNA was determined by COBAS Taqman assay

(Roche, Indianapolis, IN) (linear range: 20-170,000,000 IU/mL). Quantitative HBsAg

levels were determined by Quantitative Abbott ARCHITECT i2000SR (Abbott Ireland

Diagnostic Division, Ireland) (linear range: 0.05–125,000.00 IU/mL).

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 The magnitude and kinetics of the pharmacodynamic response were determined by

comparing the expression levels of mRNA from IFN-stimulated gene (ISG)

Accepted Article
transcripts (ISG15, MX1, and OAS1) and control genes (TLR7 and TLR9) to the

expression of a housekeeping gene mRNA (glyceraldehyde 3-phosphate

dehydrogenase [GAPDH]) using quantitative reverse transcriptase PCR (Covance

Genomics, Redmond, WA). Fold change in ISG and control gene mRNA expression

relative to baseline were calculated according to the ddCt method [22] and described

as mean fold change.

Whole blood and serum samples were collected for gene expression analysis and

cytokine and chemokine quantification after the first dose or the last dose (week 11).

All samples except the 7-hour post-dose time point were collected pre-dose. Serum

concentrations of IFN, IFN-induced protein (IP-10), IFN-inducible T cell alpha

chemoattractant (ITAC), macrophage inflammatory protein-1 (MIP-1), and MIP-1,

were quantified by chemiluminescent Ciraplex assay (Aushon BioSystems, Billerica,

MA) at weeks 12, 24, and 48 according to the schedule in the supplementary

information.

Endpoints

The primary endpoints were safety and tolerability; primary efficacy was assessed at

Week 24 as change from baseline in HBsAg expression (log10 IU/mL). Secondary

efficacy endpoints included the proportions of patients with HBsAg and HBeAg loss

and hepatitis B surface antibody (HBsAb) and hepatitis B e antibody (HBeAb)

seroconversion at Weeks 24 and 48; mean changes in HBsAg (log10 IU/mL) from

baseline at Weeks 12 and 48; the proportion of patients with ≥0.5 log10 IU/mL decline

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 in serum HBsAg titers from baseline to Weeks 12, 24, and 48; the proportions of

patients with HBV DNA < lower limit of quantitation (LLOQ) (<20 IU/mL) at Weeks 24
Accepted Article
and 48; the proportion of patients experiencing HBV viral breakthrough (i.e. 2

consecutive occurrences of HBV DNA ≥69 IU/mL after a HBV DNA <69 IU/mL or an

increase in HBV DNA ≥1.0 log10 IU/mL from nadir), and the proportion of patients

with drug resistance mutations through Week 48.

HBsAg and HBeAg loss and HBsAb and HBeAb seroconversion were defined

according to standard convention (supplementary information) and patients with

HBeAg loss and HBeAb seroconversion were included only if a positive HBeAg

baseline result had been reported for those patients. Exploratory objectives included

evaluation of the effects of vesatolimod on changes in serum cytokines or

chemokines and peripheral blood gene expression levels.

Virologic Resistance Analysis

Resistance surveillance included all patients with at least 24 weeks of TDF treatment

as previously described [23]. Population sequencing of the polymerase/reverse

transcriptase (pol/RT) region was performed for all patients with HBV DNA at or

above the viral load cut off of the sequencing assay (69 IU/mL) at Week 48 or last

visit on TDF.

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 Statistical analysis

All randomized patients who received at least one dose of study medication were
Accepted Article
included in the safety and efficacy analyses. Patients who completed the dosing

period and had specific laboratory data available for calculating estimates of

pharmacodynamics parameters were included in pharmacodynamic analyses.

No formal statistical analysis was performed to determine sample size or to assess

safety outcomes. The change from baseline to Week 24 or other secondary time

points (Weeks 12 and 48) in HBsAg (log10 IU/mL) was analyzed using mixed effect

model repeat measurement (MMRM) (full details in the supplement). An unstructured

within-patient covariance matrix was employed. Estimated least-square means of

treatment effects comparing the TDF + vesatolimod groups with the TDF + PBO

group at Week 24 were presented along with the 95% CIs and unadjusted p-values

(supplementary information).

Descriptive statistics were used to summarize the absolute values and change from

baseline values in HBsAg (log10 IU/mL) by analysis visit and treatment group, both

overall and by baseline ALT level (ALT > ULN vs ALT ≤ ULN) and baseline HBeAg

status (positive vs negative).

The impact of dose on biomarker fold changes relative to baseline changes was

assessed using non-parametric Jonckheere’s trend test and confirmed using linear

regression models adjusting for baseline biomarker levels; the latter analyses p-

values are not reported herein. No multiple testing adjustments were applied to the

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 nominal p-values from different biomarkers, time points, and analysis models. Full

details are provided in the supplement.

Accepted Article
Multivariate analyses (forward selection followed by backward elimination) were

performed to assess the impact of baseline demographic and disease characteristic

variables (full details are provided in the supplement).

Results

Baseline characteristics

Of the 260 patients initially screened across the 4 treatment groups, 192 were

randomized and received at least one oral dose of vesatolimod (n=164) or PBO to

match (n=28).

The mean age (range) of the patients was 42 (20-65) years across all treatment

groups and the majority male (64%), HBeAg-negative (61%) and Asian (81%). The

mean HBsAg baseline titer range among groups was between 3.5 and 3.8 log10

IU/mL and the proportions of patients with normal ALT at baseline were 39%, 26%,

30%, and 29% for PBO, 1-, 2- and 4-mg dose, respectively. Twenty patients (10%)

had prior IFN experience (Table 1). Fifty patients (26%) had previously received oral

antiviral treatment, and the mean time (range) from end of oral antiviral treatment to

study screening was 3.5 years (3 months – 15 years).

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 Efficacy

Mean changes in serum HBsAg (log10 IU/mL) from baseline through Week 48 are
Accepted Article
shown in Figure 1A. The mean declines at the primary endpoint (24 weeks) for the 1-

, 2, and 4-mg groups were -0.056, -0.146, and -0.036 log10 IU/mL, respectively, and

statistically no different to the change from baseline in the PBO group (-0.163 log10

IU/mL). Only 10 (7%) patients experienced ≥0.5-log10 declines (two 1-mg dose; six 2-

mg dose; two 4-mg dose) in HBsAg at Week 24 in any vesatolimod-treated arms

compared to 3 patients (11%) receiving PBO at Week 24. At Week 48, 3 (5.7%), 9

(16.1%), and 8 (14.5%) of patients in the vesatolimod 1-, 2-, and 4-mg groups,

respectively, achieved median ≥0.5 log10 IU/mL declines in HBsAg levels compared

to PBO (5 [17.9%]). The greatest median declines in HBsAg from baseline were

observed in HBeAg-positive patients with elevated ALT (Figure 1B).

No patient in any treatment group experienced HBsAg loss or HBsAb

seroconversion through Week 48. No patient experienced HBeAg loss or HBeAb

seroconversion at Week 24; 3 patients (1 each in the vesatolimod 1-, 2-, and 4-mg

groups) experienced HBeAg loss and HBeAb seroconversion at Week 48. A total of

33 (62.3%), 41 (75.9%), and 40 (75.5%) patients in the vesatolimod 1-, 2-, and 4-mg

groups, respectively, had HBV DNA <LLOQ at Week 48 compared to PBO (n=18

[64.3%]). No clinically significant differences were observed between treatment

groups in the kinetics of HBV DNA decline (Figure 1C).

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 Virologic Resistance Analysis

Thirty patients met the criteria for sequence analysis at Week 48, with 26 patients
Accepted Article
remaining viremic in the absence of confirmed virologic breakthrough, 3 patients (two

1-mg dose; one 2-mg dose) experiencing virologic breakthrough at Week 48, and 1

patient (4-mg dose) experiencing virologic breakthrough at early discontinuation.

Sequence analysis of the post baseline samples for these 30 patients detected no

changes in the HBV pol/RT sequence from baseline in 18 patients, polymorphic site

substitutions in 8 patients, or conserved site changes in 2 patients. Two patients

were unable to be sequenced, likely due to low viral loads (<2 log10IU/mL). Among

the patients who experienced virologic breakthrough at Week 48, no patient had

sequence changes compared to baseline. Overall, no HBV pol/RT amino acid

substitutions associated with increases in HBV DNA were detected.

Safety

Treatment with vesatolimod was generally well tolerated in all groups. Nine (4.7%)

patients did not complete vesatolimod treatment and 5 (2.6%) patients did not

complete TDF treatment through week 48. Of the 183 patients dosed with

vesatolimod, 15 (8%) patients discontinued vesatolimod early due to AEs (n=6),

withdrawal of consent (n=3), loss to follow-up (3), study drug non-compliance (n=2)

and lack of efficacy (n=1). Of the 6 patients who discontinued vesatolimod early due

to AEs, 5 met the protocol defined dose limiting toxicity discontinuation criteria. Two

patients who discontinued vesatolimod due to AE were in the 2-mg group (Grade 2

ALT related to both vesatolimod and TDF [n=1]; Grade 3 liver function not related to

vesatolimod but related to TDF [n=1]) and 4 were in the 4-mg group (Grade 3 ALT

related to both vesatolimod and TDF [n=1]; Grade 2 neutropenia related to

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 vesatolimod [n=1]; Grade 1 pyrexia and nausea and Grade 2 chills and myalgia all

related to vesatolimod [n=1]; Grade 2 headache, vomiting, chills, and arthralgia and
Accepted Article
Grade 3 pyrexia all related to vesatolimod [n=1]). Of the 187 patients who completed

TDF through week 48, 5 (2.7%) patients discontinued TDF due to withdrawal of

consent (n= 2), lack of efficacy (n=1), AE (n=1), and loss of follow-up (n=2) (Table 2).

No patients experienced an AE leading to modification or interruption of vesatolimod;

4 (2.1%) patients experienced an AE leading to modification or interruption of TDF

(1-mg group [n=1]; 2-mg group [n=1]; 4-mg group [n=2]) as detailed in the

supplementary information.

Four patients had treatment-emergent serious AEs (SAEs; n=4, two 1-mg dose

patients, one 2-mg patient, and one 4-mg dose patient). No SAEs occurred in more

than 1 patient and all SAEs were considered not related to either study drug (i.e.

vesatolimod or TDF). One posttreatment-emergent SAE (Grade 2 pelvic congestion)

was reported in the vesatolimod 2-mg group on Day 344, was considered not related

to any study drugs, and resolved on Day 387.

Overall, patients from both vesatolimod-treated and PBO groups experienced a

similar number of TEAE (66% vesatolimod vs. 61% PBO). Of vesatolimod-treatment

related AEs, 30% of patients treated with vesatolimod reported at least 1 AE

compared to 25% of PBO patients, all of which were mild to moderate in severity

(data not shown). A total of 6 (2 [3.6%] 2-mg group; 4 [7.3%] 4-mg group) patients

experienced a Grade 3 (severe) AE of which 3 were considered related to

vesatolimod (1 [1.8%] in the 2-mg group; 2 [3.6%] in the 4-mg group); no patients in

this study had a Grade 4 TEAE (Table 2). No notable changes in median vital signs

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 (systolic blood pressure, diastolic blood pressure, and pulse) were observed during

the study. The majority (%) of TE laboratory abnormalities in all of the groups were
Accepted Article
Grade 1 or 2 in severity. A small percentage of 2-mg (n=3 [5%]) and 4-mg (n=6

[11%]) vesatolimod-treated patients demonstrated dose-dependent treatment-related

grade ≥2 influenza-like (i.e. chills, fatigue, headache, influenza, joint pain, myalgia,

and/or pyrexia) AEs (Table 3). Overall, no dose-dependency for laboratory

abnormalities was observed across groups.

Overall, 7 patients had an ALT flare during the study (ALT >2 × baseline and

>5 × ULN): 2, 2, 2, and 1 patient in the vesatolimod 1-, 2-, and 4-mg or PBO,

respectively. The flares occurred early during treatment and ALT levels generally

normalized thereafter.

Vesatolimod-Induced Changes in Inflammatory Protein Expression

Similar to our previous report [20], medium serum levels of IP-10 and ITAC

demonstrated dose-dependent, transient increases with vesatolimod treatment

(trend test p<0.05 7 hours and p<0.0001 24 hours post-dose for both chemokines)

while changes in MIP1 and MIP1 were negligible (Table 4 and supplementary

information). Most patients had IFN levels BLQ at baseline that remained BLQ at

post-baseline time points. A small proportion of patients with BLQ IFN levels at

baseline, however, had IFN levels above LLOQ after vesatolimod treatment that

was dose-dependent (Figure 2A). A statistically significant association between this

proportion and grade of influenza-like AE was also observed at 7-hours post-dose

[p=0.001] but was not determined to be statistically significant at 24-hours post dose

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 [p=0.007]. Assessments of the association of grade of influenza-like AE with ITAC or

IP-10 levels were not statistically significant at either time point (analyses were
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performed in all patients, including those with baseline IFN above LLOQ).

Vesatolimod-Induced ISG15 Responses

As informed from previous studies [20, 24], whole blood ISG mRNA responses were

measured 24 hours post-vesatolimod dose. ISG15 response was dose-dependent,

with patients receiving the highest vesatolimod dose (4-mg) demonstrating the

highest levels of ISG15 induction (i.e. 13.2-fold median increase, compared to 4.98-

fold increase for 2-mg and 3.7-fold increase for 1-mg) (Figure 2B). No association of

24 hour-ISG15 change with HBsAg-level change at Week 24 was observed (Figure

2C).

Multivariate Analyses of ISG15 mRNA Response Associations

To determine factors that may affect pharmacodynamic responses to vesatolimod,

univariate and multivariate analyses were performed to assess the relationships

between a 2-fold or greater baseline ISG15 mRNA induction and specific baseline

demographic and disease characteristics of interest. In multivariate analyses, female

gender (OR 3.41, 95% CI 1.27-10.3, p=0.0136), Asian race (OR 3.83, 95% CI 1.07-

13.9, p=0.0396) and lower baseline ISG15: GAPDH mRNA (OR 2.02, 95% CI 1.2-

3.55, p=0.0079) expression were associated with ≥2 fold change in ISG15

(supplementary information). When comparing dose-dependency of ISG15-induced

responses, the 4-mg vesatolimod dose was associated with ISG15 upregulation

compared to the 1-mg treatment dose (OR 5.87, 95% CI 1.86-21.2, p=0.0021) while

the 2-mg dose was not (supplementary information).

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 Discussion

This report summarizes the second of two Phase 2 clinical trials to examine the
Accepted Article
efficacy and safety of vesatolimod in patients with chronic HBV infection. Overall, in

viremic CHB patients not on OAV, vesatolimod was safe and well-tolerated at all

doses compared to PBO. However, similar to the companion Phase 2 trial in virally

suppressed CHB patients, no statistically significant differences in HBsAg serum

concentration (log10 IU/mL) from baseline were observed between PBO and

vesatolimod treatment groups at the primary efficacy endpoint (Week 24).

As vesatolimod is a selective and potent agonist of TLR7 that induces type I IFN

production by pDC upon stimulation, we expected IFN production by TLR7-

expressing gut-associated and/or liver pDC induced by oral vesatolimod [24, 25] to

enhance HBV-specific cytotoxic T cell activation in the absence of detectable plasma

interferon. Through this mechanism, vesatolimod-treated patients were anticipated to

experience improved outcomes similar to or better than those associated with

current nucleos(t)ide analogue treatment regimens.

We have previously reported significant reductions in HBV/woodchuck hepatitis virus

(WHV) DNA and HBsAg/woodchuck hepatitis surface antigen (WHsAg) in

chronically-infected chimpanzees [19] and woodchucks [26] in response to

vesatolimod treatment. However, in both studies, the animals received higher

vesatolimod doses (in concentration and/or frequency) than the dose levels deemed

safe to administer in CHB patients [20, 24-27]. As described in our other Phase 2

vesatolimod study in virally suppressed patients, inadvertent overdoses of 10 and 20

mg were not safe or tolerated by 2 patients. Thus, the lack of efficacy, as assessed

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 by HBsAg decline in the present study may be due to differences in vesatolimod

dose administration frequency and/or concentration, both of which were chosen for
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safety and tolerability assessment.

Another dose-related limitation of this study is the 12-week duration of therapy; given

the lower doses administered to patients compared to those in the aforementioned

animal studies, a longer vesatolimod treatment duration may be required to

precipitate the appropriate immune response (e.g. extended local exposure to TLR7-

dependent mediators, including IFN) within the liver. However, the lack of more

robust HBsAg declines in vesatolimod treatment groups at Week 12 (Figure 1A)

compared to PBO argues against a greater efficacy effect with longer treatment

course.

As suggested by our previous Phase 1b and 2 clinical trial studies, treatment with

vesatolimod was safe and well-tolerated at all doses evaluated (1-, 2- and 4-mg) in

patients with chronic HBV infection [20, 24]. With up to 12 consecutive, oral, once-

weekly doses, few clinically significant AEs or laboratory abnormalities were

observed in any of the vesatolimod groups, including no clinically significant

increases in gamma-glutamyl transferase (GGT), bilirubin, anemia or

thrombocytopenia as previously observed in preclinical large animal studies [26]. No

SAEs occurred in more than 1 patient and all SAEs were considered not related to

either study drug (i.e. vesatolimod or TDF). One post-treatment-emergent SAE

(Grade 2 pelvic congestion), reported in the 2-mg dose TDF + vesatolimod group on

Day 344, was considered not related to study drugs and resolved on Day 387.

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 In virally suppressed CHB patients, when overall TEAEs were compared, there was

no dose dependency observed across any cohorts [20]; a similar lack of dose-
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dependent effects of vesatolimod treatment on TEAEs compared to PBO was

observed in this study. However, in viremic patients, Grade 3 and 4 TEAEs were

more frequently observed in vesatolimod treated patients receiving the highest doses

of vesatolimod (2- or 4-mg) evaluated. These distinct AE profiles in viremic vs. virally

suppressed patients could be related to inherent differences in the patient

populations. For example, the differential median fold-change in ISG15 induction

after 12 doses of vesatolimod was observed to be higher in viremic patients in this

study (supplementary information) than the induction measured in virally suppressed

patients under the same dosing conditions in a companion study [20]. Further, serum

inflammatory protein analyses have demonstrated significant, dose-dependent,

transient increases with vesatolimod doses in IP-10, ITAC and IFN concentrations

at 7- and 24-hour time points, consistent with the previous Phase 2 study [20]. While

on-target induction of IP-10 and ITAC was demonstrated herein, these levels may

not be sufficient to mount a meaningful antiviral immune response. Overall, as only a

small patient sample size was available from both studies to make these

assessments, whether these observed variations in patient AE profile are related to

different baseline levels of patient immune mediators (e.g. chemokines [IP-10, ITAC],

cytokines [IFN, IFN]) or specific immune cell subsets (pDC, B cells) is yet to be

determined.

Overall, once-weekly, oral, vesatolimod was safe and well-tolerated in viremic

patients with chronic HBV infection [20]. In both Phase 2 studies, dose-dependent,

on-target, pharmacodynamic induction of cytokines and ISGs was demonstrated,

although this did not result in significant HBsAg declines.

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 Acknowledgements

We extend our thanks to the patients and their families and to all of the participating
Accepted Article
investigators. We thank Bridget Colvin for medical writing and editing assistance in

preparation of the manuscript.

Authors’ contributions: Kosh Agarwal, Anuj Gaggar, G. Mani Subramanian

contributed to the conception and design of the study. Audrey H. Lau, Anuj Gaggar,

Anuradha Bulusu, Xinyu Tian, Andrea L. Cathcart, Jacky Woo and G. Mani

Subramanian contributed to the generation, collection, assembly, analysis and/or

interpretation of data. All authors contributed to drafting or revision of the manuscript

and approved the final version.

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 Table 1. Patient Demographics and Baseline Characteristics.
TDF + 1-mg TDF + 2-mg TDF + 4-mg
TDF + PBO
Vesatolimod Vesatolimod Vesatolimod
N=28
Accepted Article
N=53 N=56 N=55

Mean age, y (range) 41 (23-61) 41 (21-62) 44 (20-63) 44 (21-65)

Male, n (%) 16 (57) 32 (60) 40 (71) 35 (64)

Race, n (%)

Asian 22 (79) 41 (77) 48 (86) 44 (80)

White 4 (14) 6 (11) 5 (9) 10 (18)

Other 2 (7) 6 (11) 3 (5) 1 (2)

HBeAg +, n (%) 11 (39) 20 (38) 23 (41) 21 (38)

Mean HBV DNA,
5.9 (2) 5.9 (2) 5.6 (2) 5.9 (2)
log10 IU/mL (SD)
HBsAg ≤1000 IU/mL, n (%) 3 (11) 7 (13) 14 (25) 10 (18)
Mean baseline HBsAg,
3.8 (0.8) 3.6 (0.8) 3.5 (0.9) 3.6 (0.7)
log10 IU/mL (SD)
HBV genotype, n (%)

A 0 0 1 (2) 3 (6)

B 6 (21) 15 (28) 15 (27) 12 (22)

C 15 (54) 24 (45) 30 (54) 31 (56)

D 6 (21) 9 (17) 7 (13) 8 (15)

Other 1 (4) 5 (9) 3 (5) 1 (2)

ALT ≤ULN, n (%) 11 (39) 14 (26) 17 (30) 16 (29)

Prior IFN experience, n (%) 4 (14) 4 (8) 6 (11) 6 (11)

Prior OAV experience, n (%) 10 (36) 13 (25) 17 (30) 10 (18)

ALT, alanine aminotransferase; IFN, interferon; SD, standard deviation; ULN, upper
limit of normal.

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 Table 2. Treatment Discontinuation, Treatment-Emergent Adverse Events, and
Treatment Emergent Laboratory Abnormalities
TDF + 1-mg TDF + 2 mg TDF + 4 mg
TDF + PBO
Patients, n (%) Vesatolimod Vesatolimod Vesatolimod
Accepted Article
N=28
N=53 N=56 N=55
AEs 17 (61) 32 (60) 38 (68) 38 (69)

Grade 3 AEs* 0 0 2 (4) 4 (7)

Serious AEs 0 2 (4) 1 (2) 1 (2)

Overall Safety
Vesatolimod treatment
discontinuation 0 0 2 (4) 4 (7)
due to AEs

Death 0 0 0 0

Grade 3/4 7 (25) 8 (15) 4 (7) 10 (18)

Hemoglobin (Grade 3) 0 0 1 (2) 0

Lymphocytes (Grade 3) 0 0 0 2 (4)

Neutrophils (Grade 4) 0 0 0 1 (2)

White Blood Cells

0 0 0 1 (2)
(Grade 3)

ALT

Grade 3 1 (4) 1 (2) 2 (4) 1 (2)

Grade 4 0 2 (4) 1 (2) 1 (2)

Lab AST (Grade 3) 0 2 (4) 1 (2) 1 (2)
Abnormalities†
Amylase (Grade 3) 1 (4) 0 0 1 (2)

Creatine kinase

Grade 3 1 (4) 1 (2) 1 (2) 0

Grade 4 2 (7) 0 0 1 (2)

Fasting glucose or
Glucose 0 0 0 1 (2)
(Hyper, Grade 3)
Hyperkalemia
1 (4) 0 0 0
(Grade 4)
Urine RBC or Occult
3 (11) 5 (9) 2 (4) 2 (4)
Blood (Grade 3)
*No Grade 4 AEs. Grade 3 AEs reported: abdominal pain, pyrexia, ALT increased, liver
function test increased, headache, sciatica; †Maximum severity across all laboratory tests.

AE, adverse event; ALT, alanine aminotransferase; AST, aspartate

aminotransferase; PBO, placebo; RBC, red blood cell; TDF, tenofovir disoproxil
fumarate.

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 Table 3. Influenza-Like AEs
TDF + TDF + 1-mg TDF + 2-mg TDF + 4-mg
PBO Vesatolimod Vesatolimod Vesatolimod
Accepted Article
Patients, n (%) N=28 N=53 N=56 N=55

Grade 1 10 (36) 14 (26) 11 (20) 21 (38)

Grade 2 0 5 (9) 4 (7) 6 (11)

Grade 3/4 0 0 1 (2) 1 (2)

Treatment-related Grade
≥2 0 0 3 (5) 6 (11)
influenza-like* AE
*Influenza, influenza-like illness, fatigue, pyrexia, chills, myalgia, joint pain, or headache
AE, adverse event; PBO, placebo; TDF, tenofovir disoproxil fumarate.

Table 4. Median (Q1, Q3) Differences in Fold Changes of Cytokines in Each

Treatment Arm at Wk 11 + 24H.
Trend
TDF + TDF + 1-mg TDF + 2-mg TDF + 4-mg
Test p-
PBO Vesatolimod Vesatolimod Vesatolimod
value*
0.843 0.957 1.055 2.044 <0.000
IP-10
(0.686,1.077) (0.716,1.362) (0.743,1.458) (1.207,3.160) 1
1.131 1.215 1.521 2.832 <0.000
ITAC
(0.878,2.146) (0.960,2.406) (0.932,2.067) (1.637,5.108) 1

MIP-1 1 (0.488,1) 1 (0.498,1) 1 (0.516,1.254) 0.913 (0.463,1) 0.22

0.925 0.896 0.880 0.940

MIP-1 0.48
(0.765,1.076) (0.727,1.202) (0.708,1.085) (0.832,1.187)
IP-10, IFN-induced protein; ITAC, IFN-inducible T cell alpha chemoattractant; MIP,
Macrophage inflammatory protein; PBO, placebo; TDF, tenofovir disoproxil fumarate.
*Jonckheere Tepestra trend test

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 Figure Legends
Accepted Article
Figure 1. Changes in HBsAg level over time and by disease characteristic.

Mean changes in HBsAg (log10 IU/mL) were measured through Week 48 (A). Median

HBsAg (log10 IU/mL) decline at Week 24 by HBeAg and ALT status were compared

across treatment arms; ULN, upper limit of normal (B). Mean changes in HBV DNA

(IU/mL) were measured through Week 48 (C). ALT, alanine aminotransferase; BL,

baseline; EOT, end of treatment; EOS, end of study; SD, standard deviation; TDF,

tenofovir disoproxil fumarate; ULN, upper limit of normal.

Figure 2. Pharmacodynamic responses with Vesatolimod Treatment. The

proportion of patients with below the limit of quantitation IFN at baseline that

demonstrated IFN above LLOQ at Day 1 + 7 hours and Week 11 + 24 hours was

compared; p-values are from Cochran-Armitage trend test comparing all dose

groups (A). ISG15 induction was quantified at specific time points through Week 24

including Week 11 + 24 hours; p-values by trend test. Q, quartile (B). The

relationship of the change in HBsAg level at Week 24 against whole blood ISG

mRNA responses measured 24 hours post-vesatolimod dose at Week 11 + 24 hours

was assessed (C). IFN, interferon; ISG, interferon-stimulated gene; LLOQ, lower limit

of quantification; PBO, placebo; TDF, tenofovir disoproxil fumarate.

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Accepted Article

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Accepted Article

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Accepted Article

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