Bepirovirsen is an antisense oligonucleotide that targeting all hepatitis B virus (HBV) messenger RNAs to reduce the levels of viral proteins.
A phase 2b, randomized, investigator-unblinded trial was conducted involving participants with chronic HBV infection who were receiving or not receiving nucleoside or nucleotide analogue (NA) therapy. Participants were randomly assigned (in a 3:3:3:1 ratio) to one of four groups. Group 1 received weekly subcutaneous injections of bepirovirsen at a dose of 300 mg for 24 weeks; group 2 received bepirovirsen at a dose of 300 mg for 12 weeks then 150 mg for 12 weeks; group 3 received bepirovirsen at a dose of 300 mg for 12 weeks then placebo for 12 weeks; and group 4 received placebo for 12 weeks then bepirovirsen at a dose of 300 mg for 12 weeks. Groups 1, 2, and 3 additionally received loading doses of bepirovirsen. The composite primary outcome was established as the hepatitis B surface antigen (HBsAg) level below the limit of detection and an HBV DNA level below the limit of quantification maintained for 24 weeks after the planned end of bepirovirsen treatment, without newly initiated antiviral medication.
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The intention-to-treat population comprised 457 participants (227 receiving NA therapy and 230 not receiving NA therapy). Among those receiving NA therapy, 6 participants in group 1 (9%; 95% credible interval, 0 to 31), 6 in group 2 (9%; 95% credible interval, 0 to 43), 2 in group 3 (3%; 95% credible interval, 0 to 16), and no participants in group 4 (0%; post hoc credible interval, 0 to 8) experienced primary-outcome events. Among participants not receiving NA therapy, 7 experienced primary-outcome events (10%; 95% credible interval, 0 to 38), 4 in group 2 (6%; 95% credible interval, 0 to 25), 1 in group 3 (1%; post hoc credible interval, 0 to 6), and none in group 4 (0%; post hoc credible interval, 0 to 8). During weeks 1 through 12 of the study period, it was observed that adverse events such as injection-site reactions, pyrexia, fatigue and increased alanine aminotransferase levels were more common among those taking bepirovirsen (groups 1, 2 and 3) compared with those taking placebo (group 4).
Chronic hepatitis B virus (HBV) infection is a major global health concern, with an estimated 1.5 million new infections and 820,000 deaths each year. Predominantly, these cases arise from cirrhosis and hepatocellular carcinoma. The aim of therapy is to achieve functional cure; this is defined as long-term hepatitis B surface antigen (HBsAg) loss with or without seroconversion (positive for antibodies against HBsAg [anti-HBs]), and sustained undetectable HBV DNA after cessation of therapy. Despite prolonged treatment with nucleoside or nucleotide analogue (NA) therapy, which is the first-line treatment for HBV infection, fewer than 5% of patients have achieved HBsAg loss after 12 months of treatment. This highlights the need for therapies that are capable of achieving functional cure.
A phase 2a trial of Bepirovirsen (GSK3228836), a 2′-O-methoxyethyl modified antisense oligonucleotide, demonstrated that 4 weeks’ treatment caused a rapid and dose-dependent decrease in HBsAg levels, and transient HBsAg loss in some participants. The immunostimulatory effect of the drug via TLR8 may have been responsible for these effects. A phase 2b trial (B-Clear) was therefore conducted to investigate the efficacy and safety of 12- and 24-week bepirovirsen treatment in participants with chronic HBV infection who were either receiving or not receiving NA therapy. The primary efficacy outcome measured was the duration of HBsAg and HBV DNA loss 24 weeks after cessation of bepirovirsen treatment, in the absence of new antiviral therapy.
Participants: Participants for this study were required to be 18 years of age or older with a documented chronic hepatitis B virus (HBV) infection for at least six months, an HBsAg level of more than 100 IU per milliliter, and an alanine aminotransferase (ALT) level less than or equal to two times the upper limit of the normal range. Furthermore, participants receiving nucleos(t)ide analog (NA) therapy needed to be on a stable NA regimen, have an HBV DNA level of less than 90 IU per milliliter, and an ALT level less than or equal to two times the upper limit of the normal range. Participants not receiving NA therapy needed to have either never received such therapy or had finished NA therapy at least six months before screening, have an HBV DNA level of more than 2000 IU per milliliter, and an ALT level less than three times the upper limit of the normal range. Exclusion criteria included presence of hepatitis C, human immunodeficiency virus infection or hepatitis D virus infection; cirrhosis; hepatocellular carcinoma; and interferon-containing therapy within 12 months before screening.
Table in the Supplementary Appendix, which is available with the full text of this article at NEJM.org, presents the baseline characteristics of the study population.
Outcomes: The primary composite efficacy outcome was specified as an HBsAg level below the lower limit of detection (0.05 IU per milliliter) and an HBV DNA level below the lower limit of quantification (20 IU per milliliter) maintained for 24 weeks after the planned end of bepirovirsen treatment in groups 1, 2, and 3, in the absence of any medication initiated for the purpose of suppressing HBV replication. Group 4 (placebo-first group) was included to allow evaluation of the effect of a loading dose and evaluation of safety in the first 12 weeks; however, this trial did not have sufficient power to assess the primary outcome in this group. Additionally, a prespecified analysis used a modified version of the primary outcome which allowed “blips” (single-time-point increases in the HBsAg level to greater than or equal to the lower limit of detection or in the HBV DNA level to greater than or equal to the lower limit of quantification) in response.
Secondary efficacy outcomes were compared between groups 1 and 2, groups 1 and 3, and groups 2 and 3 in the form of a proportion of participants having a primary-outcome event; the proportion of participants having an HBsAg level below the lower limit of detection and an HBV DNA level below the lower limit of quantification at the end of treatment; log changes from baseline in HBsAg and HBV DNA levels (according to category of HBsAg or HBV DNA level); actual values and change from baseline in HBsAg, HBV DNA, HBeAg, and anti-HBs levels; as well as ALT normalization (ALT level less than or equal to the upper limit of the normal range) among participants whose ALT level was above the upper limit at baseline but not receiving newly initiated antiviral treatment.
The safety outcomes of this study were assessed through clinical assessments, laboratory measurements, and the observation of adverse events. Furthermore, increases in ALT levels, as well as class effects of antisense oligonucleotides (renal injury, injection-site reactions, thrombocytopenia, and vascular inflammation and complement activation) were evaluated as adverse events of special interest.
Statistical Analysis: Approximately 440 participants were planned to be enrolled, including 66 participants receiving NA therapy and 66 not receiving NA therapy in each of Groups 1, 2, and 3, and 22 participants receiving NA therapy and 22 not receiving NA therapy in Group 4. This sample size was chosen with a Bayesian model that was selected to provide a posterior probability of 75% or higher for the true response rate exceeding a fixed threshold under a range of assumed values for the threshold of interest and true response rate. An estimation approach with no hypothesis testing was used to analyze the primary outcome. A Bayesian hierarchical model (including baseline stratification factors) was employed to calculate the point estimate of the primary outcome and 95% credible interval. In case the Bayesian hierarchical model did not converge, a post hoc unstratified Bayesian analysis was performed (as detailed in the Methods section of the Supplementary Appendix).
The efficacy objectives were assessed through the use of estimands, which were precise descriptions of the treatment effect and reflected the clinical question posed by the clinical trial objective. The primary estimand was the proportion of participants in groups 1, 2 and 3 who had a primary outcome event, regardless of whether they had completed their treatment or whether any interruptions or adherence issues had arisen due to disruptive events such as the coronavirus disease 2019 pandemic. A receiver-operating-characteristic analysis was conducted in order to explore a range of baseline HBsAg cutoff points as potential predictors of response.
Safety analyses included a descriptive summary of adverse-event incidence, comparison of the first 12 weeks of treatment in groups 1, 2, and 3 with placebo in group 4 for vital signs and laboratory data as well as an efficacy analysis conducted in the intention-to-treat population, comprising all randomly assigned participants based on their trial-group assignment. Subsequently, safety analyses were performed in all participants who had undergone randomization and received at least one dose of bepirovirsen or placebo and were based on the trial agent received. As previously specified, outcomes were separately analyzed for participants receiving NA therapy and those not receiving it. Additional details can be found in the Supplementary Appendix.
Trial Design: B-Clear was a Phase 2b, randomized, parallel-cohort trial conducted from July 27, 2020 through March 18, 2022 at 123 sites in 22 countries (see the Supplementary Appendix). The trial sponsor and participants were blinded to the assignments of the trial groups, which were known to the investigators. Randomization was performed using an interactive Web-response system, with stratification based on hepatitis B e antigen (HBeAg) status (positive or negative) and baseline HBsAg level (less than or equal to 3 or greater than 3 log10 IU per milliliter).
Participants were randomly assigned in a 3:3:3:1 ratio to one of four groups. Group 1 received weekly subcutaneous injections of bepirovirsen at a dose of 300 mg for 24 weeks; Group 2 received bepirovirsen at a dose of 300 mg for 12 weeks then 150 mg for 12 weeks; Group 3 received bepirovirsen at a dose of 300 mg for 12 weeks then placebo for 12 weeks; and Group 4 received placebo for 12 weeks then bepirovirsen at a dose of 300 mg for 12 weeks (Figure 1). Loading doses of bepirovirsen (300 mg, in groups 1, 2, and 3) or placebo (in group 4) were administered on days 4 and 11. The participants were followed up to 55 weeks with a 24-week treatment period and 24-week follow-up period. Those who were receiving NA therapy continued such therapy during the trial.
Trial Oversight: GSK designed and oversaw the trial conduct, data collection and analysis, with professional writers paid by the sponsor composing the first draft of the manuscript under the authors’ direction. The manuscript was subsequently reviewed and edited by all authors before submission for publication. All authors concurred in making this decision, and vouched for the accuracy and completeness of the data, as well as for the fidelity of the trial to the protocol. An independent Data Monitoring Committee reviewed unblinded data throughout. The trial was conducted in accordance with the Declaration of Helsinki, Council for International Organizations of Medical Sciences’ International Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical Practice Guidelines of International Council for Harmonisation and all applicable laws and regulations in participating countries.
Local institutional review boards or independent ethics committees reviewed and approved the protocol and amendments. All participants gave written informed consent.
Participants: The intention-to-treat population for the trial included 457 participants, of whom 227 were receiving NA therapy and 230 were not receiving it. 6% (13) of those receiving NA therapy and 10% (23) of those not receiving it prematurely discontinued their usage of either bepirovirsen or placebo. Out of these early discontinuations, 2% (5) and 3% (8) respectively owed to adverse events. Demographic and clinical characteristics between the two trial groups at baseline were generally similar, with the participants being representative of the population with chronic HBV infection.
Primary Outcome: In Group 1, a primary-outcome event occurred in 6 participants (9% CI 0 to 31) who received NA therapy and 7 participants (10% CI 0 to 38) who did not receive such therapy. For Group 2, a primary-outcome event was observed in 6 participants (9% CI 0 to 43) who received NA therapy and 4 participants (6% CI 0 to 25) who did not. Lastly, in Group 3, a primary-outcome event occurred in 2 participants (3% CI 0 to 16) who received NA therapy and 1 participant (post hoc CI 0 to 6) who did not.
When employing the modified primary-outcome definition that allowed for «blips,» a total of seven participants in Group 1 receiving nucleos(t)ide analog (NA) therapy and ten not receiving NA therapy had a response, equating to 10% (95% credible interval, 0 to 36) and 14% (95% credible interval, 0 to 64), respectively.
A total of 16% of participants receiving NA therapy and 25% not receiving NA therapy with a low HBsAg level (≤3 log10 IU per milliliter) at baseline had a primary-outcome event, whereas 6% and 7%, respectively, with a high HBsAg level (>3 log10 IU per milliliter) experienced the same event. A receiver-operating-characteristic analysis in group 1 determined that an HBsAg level of approximately 3000 IU per milliliter (3.5 log10 IU per milliliter) is an appropriate cutoff point for predicting response. At that cutoff point in group 1, 5 of 43 participants (12%) receiving NA therapy and 6 of 24 (25%) not receiving NA therapy had a primary-outcome event.
Among HBeAg-negative participants, there was a primary-outcome event in 10% of those receiving NA therapy and 14% of those not receiving NA therapy. Conversely, among HBeAg-positive participants, the primary-outcome event occurred only among those receiving NA therapy (6% of participants). None of the participants not receiving NA therapy experienced such an event.
Secondary Outcomes: Changes in HBsAg and HBV DNA levels were dependent on the duration of bepirovirsen treatment. An increase in both HBsAg and HBV DNA levels was observed in certain participants after treatment discontinuation.
Of the participants in Group 1, 63% receiving NA therapy and 59% not receiving NA therapy had an HBsAg level of less than 100 IU per milliliter by the end of treatment. The corresponding values at 24 weeks after the end of treatment were 38% and 29% respectively. Additionally, 26% and 29%, respectively, achieved a level below the lower limit of detection at the end of treatment. At 24 weeks after the end of treatment, 8% and 14%, respectively, achieved a level below this lower limit. Furthermore, 50% in each group had a decrease of at least 3 log10 IU per milliliter in their HBsAg level by the end of treatment. Relapse was lowest in Group 1 at the end of the trial.
Among participants not receiving NA therapy, the number in Group 1 who had an HBV DNA level below the lower limit of quantification was 26 (37%) at the end of treatment, and 19 (27%) at 24 weeks after the end of treatment. This was compared to 20 (29%) and 15 (22%), respectively, in Group 2; 18 (26%) and 9 (13%), respectively, in Group 3; and 4 (17%) at both time points in Group 4. Data excluding participants who received newly initiated antiviral medication are shown in Figure S8. Furthermore, among participants in Group 1 not receiving NA therapy, a decrease of at least 3 log10 IU per milliliter in the HBV DNA level was observed in 27 participants (39%) at the end of treatment and 18 (26%) at 24 weeks after the end of treatment.
During Weeks 1-12, when comparison between bepirovirsen and placebo was possible, adverse events were reported more frequently in groups 1, 2, and 3 (78%, 85%, and 76% respectively for those receiving NA therapy; 90%, 82%, and 87% respectively for those not receiving NA therapy) than in Group 4 (43% of those receiving NA therapy; 54% of those not receiving NA therapy). For example, injection-site reactions, pyrexia, fatigue, and increased ALT levels were reported more frequently with bepirovirsen than with placebo. Notably, the majority of adverse events reported fell under the category of adverse events of special interest.
During Weeks 1-12, no participants in Group 4 (Placebo) had serious adverse events; however, five participants receiving NA therapy (one in Group 1, one in Group 2 and three in Group 3) and three participants not receiving NA therapy (Group 1) experienced serious adverse events. Grade 3 or 4 adverse events occurred in 7-13% of those receiving NA therapy and 10-14% of those not receiving NA therapy among Groups 1, 2, and 3; the frequency was 0 for Group 4. After the treatment period (Weeks 25-48), the frequency of reported adverse events was generally similar among the trial groups and of low frequency for individual reported events.
Between Weeks 1 and 48, a higher proportion of participants who reported adverse events were not receiving NA therapy compared to those who were receiving NA therapy. Furthermore, there was a higher incidence of increased ALT level, increased aspartate aminotransferase level, decreased platelet count, decreased complement factor C3 level and decreased complement factor C4 level in this group. For both groups receiving and not receiving NA therapy, the most common adverse events were injection-site reactions like erythema, pain or pruritus. In total, seventeen participants experienced adverse events that caused discontinuation of either bepirovirsen or placebo; the frequency ranged from 0 to 4% among those receiving NA therapy and from 0 to 7% among those not receiving it.
Overall, 74 participants experienced Grade 3 or 4 adverse events, with frequency ranging from 0 to 16% among those receiving NA therapy and 17 to 23% among those not receiving NA therapy. Additionally, 6 participants who received NA therapy and 11 who did not receive NA therapy had serious adverse events; of these, one in the former group and three in the latter were deemed to be related to bepirovirsen treatment. No deaths occurred. Furthermore, injection-site reactions were the most common adverse events of special interest, comprising 48 to 74% of all participants across trial groups and those receiving or not receiving NA therapy.
From the University of Hong Kong, Hong Kong (M.-F.Y.), the Faculty of Health Sciences in Bytom, Medical University of Silesia, Katowice (E.J.), Erasmus Medical Center, Rotterdam, the Netherlands (H.L.A.J.), GSK, Stevenage (R.E., G.Q., L.M., S.K., F.C.), and Luigi Sacco Hospital, Milan (G.R.); the State Key Laboratory of Liver Research, University of Hong Kong, Hong Kong, Nanfang Hospital, Southern Medical University, Guangzhou (J. Hou), and Regional Institute of Gastroenterology and Hepatology and Babeş-Bolyai University, Department of Clinical Psychology and Psychotherapy, International Institute for Advanced Study of Psychotherapy and Applied Mental Health, Cluj-Napoca (C.P.); the Department of Medicine, Queen Mary Hospital, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, and National University Health System, Singapore (S.-G.L.); the University of Medicine and Hospital for Active Treatment Sofiamed, Sofia, Bulgaria (N.I.), and Hospital Italiano de Buenos Aires, Buenos Aires (A.G.); the University of Rzeszow, College of Medical Sciences, Centrum Medyczne w Lancucie, Lancut (R.P.), Dr. Victor Babes Clinical Hospital of Infectious and Tropical Diseases, Carol Davila University of Medicine and Pharmacy, Bucharest (C.P.P.), and Korea University Ansan Hospital, Ansan (H.J.Y.); Toronto General Hospital, Toronto (H.L.A.J.), and Spitalul Clinic de Boli Infectioase si Pneumoftiziologie, Craiova (G.D.); the Department of Gastroenterology, Hiroshima Red Cross Hospital and Atomic-Bomb Survivors Hospital (K. Tsuji), Sfanta Cuvioasa Parascheva Infectious Diseases Clinical Hospital, Galati (M.A.), and Modern Medicine Clinic, Moscow (T.S.); Kagawa Prefectural Central Hospital, Takamatsu (K. Takaguchi), Université de Paris-Cité and INSERM Unité Mixte de Recherche 1149, Department of Hepatology, Assistance Publique–Hôpitaux de Paris Hôpital Beaujon, Clichy, France (T.A.), and the Department of Medicine, University of Saskatchewan, Regina (A.W.); «Grigore T. Popa» University of Medicine and Pharmacy, Iasi (A.V.), and Azienda Ospedaliero–Universitaria di Modena, Baggiovara Hospital, Modena (P.A.); GSK, Durham, NC (J.C., D.T.), GSK, Brentford (H.P.), and College of Medicine, Pusan National University and Biomedical Research Institute, Pusan National University Hospital (J. Heo), and Inje University Busan Paik Hospital (S.-J.P.), Busan — all in South Korea; and GSK, Dubai, United Arab Emirates (T.L.), and GSK, Collegeville, PA (M.P.).