INTRODUCTION
Chronic hepatitis B (CHB) is a global health threat. Half of the global population lives in regions of high hepatitis B virus (HBV) endemicity, and over 820,000 patients die from HBV-related complications annually [1]. In the context of clinical trial design, functional cure is defined as sustained seroclearance of hepatitis B surface antigen (HBsAg) with unquantifiable HBV DNA at 24 weeks off treatment [2]. Functional cure is a desired endpoint in CHB, as it is associated with reduced liver fibrosis [3,4] and hepatocellular carcinoma (HCC) risks [5].
Currently approved drugs for HBV infection include nucleos(t)ide analogues (NUCs) and pegylated interferon alpha (Peg-IFNα), with NUCs being the predominant treatment option globally. NUCs can effectively suppress HBV replication with high barriers to resistance [6]. However, HBsAg seroclearance rarely occurs with NUC treatment. Yeo et al. [7] performed a meta-analysis on HBsAg seroclearance, demonstrating annual HBsAg seroclearance rates of 0.8% and 1.3% in NUC-treated and untreated patients, respectively. It is estimated that HBsAg reduces by 0.1 log IU/mL per year in NUC-treated patients, equating to HBsAg seroclearance after 36–52 years of treatment [8,9]. Most NUC-treated patients will not achieve functional cure and will require indefinite therapy.
The other approved HBV drug is Peg-IFNα—an immunomodulator that stimulates both innate and adaptive immune responses against HBV. Peg-IFNα is administered subcutaneously, usually for a finite course of 48 weeks [10]. The effects of Peg-IFNα on HBsAg seroclearance are more easily observed in patients with HBV genotype A, hence its role is limited in Asian patients with predominant non-genotype A HBV [10,11]. Furthermore, Peg-IFNα is contraindicated in decompensated liver disease, and is associated with autoimmune, hematological, and psychiatric side effects [12].
Given the limitations of current drugs, novel HBV treatment strategies aiming for functional cure are now under investigation. The first group of novel drugs are virus-targeting agents—which inhibit specific steps of the virus lifecycle, whereas the second group comprises immunomodulators—which augment host immune responses against HBV [12]. This article will review the novel treatment strategies for HBV functional cure, with a focus on agents that are currently under clinical development.
VIRUS-TARGETING AGENTS
HBV naturally exists as an enveloped virus with a partially double-stranded relaxed circular DNA (rcDNA) genome of 3,200 base pairs [13]. The HBV lifecycle is depicted in Figure 1. HBV first enters hepatocytes through interaction with sodium taurocholate cotransporting polypeptide (NTCP) receptors [14]. After cellular entry, the virus uncoats, and the rcDNA is transported into the host nucleus. Through host cell DNA repair machinery, rcDNA is ligated to form covalently closed circular DNA (cccDNA), which can subsequently transcribe messenger RNAs (mRNA) and pregenomic RNA (pgRNA) [15]. mRNAs are translated to viral proteins including hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), HBV polymerase and the HBV X protein. HBcAg is a key component for the formation of viral capsids, whereas HBsAg is required for the formation of the viral envelope. HBsAg may also be released into the circulation as subviral particles [16].
Novel drugs have been developed to target the key steps of the viral lifecycle, and the main virus-targeting agents in clinical trials are summarized in Table 1.
Entry inhibitors
Entry inhibitors are competitive inhibitors of the HBsAg binding site on NTCP receptors. Through interfering with HBsAg binding, entry inhibitors block HBV entry into hepatocytes. Entry inhibition has primarily been studied in HBV and hepatitis D virus (HDV) coinfected patients, as HDV has limited therapeutic targets otherwise [12].
Bulevirtide is the first-in-class entry inhibitor that was approved by the European Medicines Agency for chronic HDV infection. In the MYR301 trial, patients were randomized to receive bulevirtide 2 mg daily for 144 weeks, bulevirtide 10 mg daily for 144 weeks, or to receive no treatment for 48 weeks followed by bulevirtide 10 mg daily for 96 weeks (control group). The primary endpoint was normalization of alanine aminotransferase (ALT) plus undetectable or 2 log reduction in HDV RNA at week 48. 45% and 48% of patients in the bulevirtide 2 mg and bulevirtide 10 mg groups achieved the primary endpoint, respectively, whereas only 2% of control patients reached this endpoint. Despite its potent effects on HDV, HBsAg seroclearance or HBsAg reduction by ≥1 log IU/mL did not occur in any patients after 48 weeks of bulevirtide treatment [17]. Given the availability of other novel agents, bulevirtide would likely have a limited role for patients with HBV mono-infection. Nonetheless, bulevirtide would remain as an important drug for patients with HBV and HDV coinfection.
Transcription inhibitors
Farnesoid X receptor (FXR) are nuclear receptors that regulate bile acid homeostasis [18]. FXR also interacts with the HBV enhancer II core promoter sequences and with the HBV X protein to regulate HBV transcription. FXR agonists can inhibit the transcription of cccDNA, inhibiting downstream translation and viral replication [19].
Vonafexor is a selective FXR agonist that has been studied in CHB patients. After administration of vonafexor 400 mg daily for 29 days, a statistically significant yet modest reduction of HBsAg by 0.1 log IU/mL was achieved. Vonafexor had no significant effects on other HBV markers after 29 days of treatment [20].
RNA interference agents
RNA interference (RNAi) agents can be subdivided into antisense oligonucleotides (ASOs) and small-interfering RNAs (siRNAs). Both types of drugs are designed with complementary nucleotide sequences to conserved regions of the HBV genome, enabling potent post-transcriptional gene silencing of all HBV transcripts [21]. As HBsAg can be produced from both HBV cccDNA and integrated HBV DNA [22], RNAi agents are designed to target sequences upstream from integration hotspots. This in turn enables knockdown of RNA transcribed from both cccDNA and integrated HBV DNA [21]. Aside from their direct effect in suppressing viral protein production and viral replication, RNAi agents also indirectly enhance host immune reconstitution through reducing host exposure to viral proteins.
Despite their similar action, ASOs and siRNAs have distinct structures. ASOs are single-stranded DNA that can enter hepatocytes in the unconjugated form. ASOs accumulate in cytoplasm, and operate through RNA splicing by RNAse-H. siRNAs are double-stranded RNA which enter hepatocytes through conjugated carriers. siRNAs accumulate in endosomes, and operate through RNA splicing by the RNA-induced silencing complex [21].
Bepirovirsen is an unconjugated ASO that is the most advanced novel agent in the developmental pipeline now. In the phase IIb B-Clear trial on bepirovirsen, the primary outcome was undetectable HBsAg with unquantifiable HBV DNA at 24 weeks post end-of-treatment (EOT). In patients who received subcutaneously injected bepirovirsen 300 mg weekly for 24 weeks with a loading dose, 9% of NUCtreated patients and 10% of non-NUC patients achieved the primary outcome, respectively. Among the patients who received 24 weeks of 300 mg weekly bepirovirsen, baseline HBsAg ≤3 log IU/mL was associated with higher response rates to bepirovirsen (NUC-treated patients: primary outcome in 16% of patients with baseline HBsAg ≤3 log IU/mL vs. 6% of patients with baseline HBsAg >3 log IU/mL; non-NUC patients: primary outcome in 25% of patients with baseline HBsAg ≤3 log IU/mL vs. 7% of patients with baseline HBsAg >3 log IU/mL) [23]. It is noteworthy that the intrinsic Toll-like receptor (TLR) 8 activity of bepirovirsen may provide additional control on HBV. Long-term monitoring of bepirovirsen responders is underway, with interim results suggesting sustainable drug effects for more than 9 months [24]. Bepirovirsen has now entered phase III trials.
GSK3389404 is an alternate form of bepirovirsen that is conjugated with N-acetylgalactosamine (GalNAc). GSK3389404 was studied in a phase II trial, yielding HBsAg suppression by 0.13–0.75 log IU/mL after 85 days of treatment. No incidences of HBsAg seroclearance were observable [25]. GSK3389404 has weaker HBsAg suppression effects than bepirovirsen, and its development has hence been halted. It is hypothesized that GalNAc conjugation enhanced hepatocyte uptake and limited GSK3389404 uptake in liver sinusoidal cells and Kupffer cells, which in turn reduced immune signaling and overall anti-HBV effects [26].
A range of siRNAs including daplusiran/tomligisiran, elebsiran, imdusiran, RBD-1016 and xalnesiran have entered phase II trials. In general, the siRNAs have 1 or more triggers targeting conserved sequences in the HBV genome. Daplusiran/ tomligisiran has 2 triggers targeting the S and X open reading frames (ORFs), xalnesiran has 1 trigger targeting the S ORF, while elebsiran, imdusiran and RBD-1016 are all designed with 1 trigger targeting the X ORF. As a class, siRNAs can potently suppress HBsAg, with over 90% of patients achieving HBsAg reduction by ≥1 log IU/mL and over 50% of patients achieving HBsAg suppression to <100 IU/mL [27-31]. Importantly, siRNA effects are sustainable after EOT. Mak et al. [32] followed up siRNA treated patients (15 patients with 4 doses of ARC-520, and 38 patients with 3 doses of daplusiran/ tomligisiran) for a mean time of 52.5 months, demonstrating sustainable HBsAg suppression for up to 6 years in some patients.
Capsid assembly modulators
The HBV nucleocapsid is essential for the HBV lifecycle, as it encapsidates HBV pgRNA and HBV polymerase, acting as a site for reverse transcription. Nucleocapsids containing mature nucleic acids can be enveloped by HBsAg to form new virions or may be recycled to replenish the cccDNA pool inside the hepatocyte nucleus [33].
Capsid assembly modulators (CAMs) are oral drugs that target HBcAg to interfere with nucleocapsids. CAMs are further divided into CAM-As—which induce aberrant nucleocapsid formation, and CAM-Es—which induce empty nucleocapsid formation [34]. Through its direct action on nucleocapsids, CAMs enable potent suppression of viral replication. As a class, CAMs can suppress both HBV DNA and HBV RNA to different degrees depending on individual drug potency. However, the early generation CAMs had minimal effects on viral proteins such as HBsAg [35-37].
Aside from its direct effect on nucleocapsids, CAMs also have secondary effects in inhibiting nucleocapsid recycling, in turn blocking cccDNA replenishment. Newer generation CAMs such as ALG-000184 have stronger secondary effects and are being actively studied [33]. In the ongoing ALG-000184-201 phase I trial, ALG-000184 (+/- entecavir) is administered for 96 weeks in HBeAg positive patients. In the week 72 interim analysis, a maximum mean HBV DNA reduction of 7 log IU/mL was achieved. 90% of patients on ALG-000184 300 mg monotherapy and 50% of patients on ALG-000184 300 mg+entecavir achieved unquantifiable HBV DNA at week 72. More importantly, significant reductions in HBV antigens were observed at week 72, with maximum mean reduction of HBsAg, HBeAg and HBrAg by 1.0 log IU/mL, 1.9 PEI log U/mL, and 2.1 log U/mL, respectively [38]. Based on the positive phase I data, ALG-000184 will be further studied in phase II trials.
Protein export inhibitors
Nucleic acid polymers (NAPs) are phosphorothioate oligonucleotides that inhibit HBV subviral particle transport from hepatocytes into the circulation. REP2139 (and its analogue REP2165) are NAPs administered by weekly intravenous infusions. In a phase II trial, REP2139/ REP2165 for 48 weeks led to HBsAg suppression by more than 1 log IU/mL in 87.5% of patients (35 out of 40), with 60% of patients (24 out of 40) having HBsAg suppressed to <0.05 IU/mL. At 48 weeks after EOT, 35% of patients (14 out of 40) had sustained response with HBsAg <0.05 IU/mL [39]. S antigen Transport-inhibiting Oligonucleotide Polymers (STOPS) are structurally altered forms of NAPs [40]. However, the development of STOPS has been discontinued after a negative phase I trial.
IMMUNOMODULATORS
Host immune tolerance is a critical pathogenetic factor contributing to HBV persistence. With long-term exposure to viral proteins (such as HBsAg), the host immune system develops tolerance towards HBV. HBV has evolved evasion mechanisms against the innate immune system [41], and can also induce T-cell dysfunction [42]. In contrast to virus-targeting agents, immunomodulators aim to reverse immune tolerance to generate host anti-HBV responses. The currently approved Peg-IFNα is an immunomodulator targeting the innate immune system, which can induce downstream cytokine release to trigger lymphocyte responses. Figure 2 depicts the main modes of action of novel HBV immunomodulators, and the immunomodulators in clinical trials are summarized in Table 2.
Toll-like receptor agonists
TLRs are important pattern recognition receptors in the innate immune system. TLRs detect pathogen molecular patterns and trigger downstream interferon and cytokine pathways to initiate host antiviral responses [43].
Selgantolimod is an oral TLR8 agonist that has entered phase II clinical trials. In 39 NUC-treated CHB patients who received selgantolimod for 24 weeks, 18% and 26% of patients had >0.1 log IU/mL HBsAg decline at EOT and at 24 weeks after EOT, respectively. 5% of patients (2 out of 39) had HBsAg seroclearance at 24 weeks after EOT [44]. Another phase II trial studied the use of selgantolimod+tenofovir alafenamide (TAF) in viremic CHB patients. Selgantolimod induced significant changes in serum cytokines, chemokines and immune cell subsets, yet no significant HBsAg suppression was noted. None of the viremic patients treated with selgantolimod achieved HBsAg decline by 1 log IU/mL or more at EOT. The mean HBsAg reduction at week 48 was 0.12, 0.16 and 0.12 log IU/mL in the selgantolimod 3 mg+TAF arm, the selgantolimod 1.5 mg+TAF arm, and the placebo+TAF arm, respectively [45].
TLR7 agonists have also been studied in CHB. While TLR7 agonists have shown target engagement with downstream immune signaling, the HBsAg reduction by TLR7 agonist monotherapy is modest. Ruzotolimod (RO7020531) treatment for 6 weeks led to mean HBsAg decline ranging from 0.07–0.15 log IU/mL at 6 weeks post-EOT [46]. Vesatolimod, another TLR7 agonist, showed no significant effects on HBsAg after 12 weeks of treatment [47,48].
T-cell modulation
T-cell dysfunction and deficit are important contributing factors to adaptive immunity dysfunction in CHB [42]. The programmed cell death protein 1 (PD1)/programmed cell death ligand 1 (PDL1) pathway is inhibitory towards T-cells and is overexpressed in CHB. Anti-PD1 or anti-PDL1 agents have hence drawn interests as novel immunomodulators for CHB.
Envafolimab is the only subcutaneously administered anti-PDL1 checkpoint inhibitor, while others are administered intravenously. In a phase II trial, lower baseline HBsAg level was significantly associated with higher magnitude of HBsAg reduction. After 24 weeks of envafolimab, the EOT HBsAg reduction was 1.38, 0.19, and 0.09 log IU/mL in patients with baseline HBsAg ≤100, 100–1,000, and >1,000 IU/mL, respectively [49]. In an extension trial studying 19 patients with baseline HBsAg <100 IU/mL, 21.1% of patients (4 out of 19) achieved HBsAg seroclearance after 24 weeks of envafolimab [50]. Cemiplimab and nivolumab are other anti-PD1/anti-PDL1 checkpoint inhibitors that have entered phase II trials.
Inhibitors of apoptosis (IAP) antagonist and immune mobilizing monoclonal T-cell receptors against virus (ImmTAV) are other novel T-cell modulating techniques that have shown anti-HBV effects in preclinical studies [51,52]. Both IAP antagonists and ImmTAV are being studied in ongoing clinical trials.
Therapeutic vaccination
Therapeutic vaccination utilizes host active immunity to generate anti-HBV responses. HBsAg-based therapeutic vaccines have entered development for almost two decades, yet older HBsAg-based therapeutic vaccines had limited antiviral effects [53]. Emerging data showed that the incorporation of non-HBsAg viral proteins may enhance vaccinal effects, and this has since reignited interest in HBV therapeutic vaccines [54].
VTP-300 is a prime-boost therapeutic vaccine targeting the polymerase, core and S regions of HBV. In a phase II trial, VTP-300 led to HBsAg decline by >0.5 log IU/mL in 16.7% of patients (3 out of 18 patients), with sustained HBsAg suppression at 8 months post-EOT. Of note, the three patients with HBsAg decline >0.5 log IU/mL all had baseline HBsAg levels <50 IU/mL [55].
BRII-179 is a therapeutic vaccine that targets all three HBV surface proteins (HBsAg, pre-S1 and pre-S2). In a phase II trial on BRII-179, >30% of patients developed anti-HBs responses with BRII-179 treatment, yet no significant HBsAg reductions were observed [56].
Monoclonal antibodies
Passive immunity through administration of anti-HBV monoclonal antibodies has been studied as a novel immunomodulation strategy for CHB. In the VIR-3434-1002 trial, a single dose of the monoclonal antibody tobevibart was administered. In an interim analysis up to week 8, patients who received tobevibart 300 mg achieved mean HBsAg and HBV DNA decline from baseline to nadir by 1.83 log IU/mL and 2.03 log IU/mL, respectively [57].
Lenvervimab is another monoclonal antibody that has entered clinical trials. Lenvervimab was administered at weekly doses for 4 doses in a phase I trial on patients with baseline HBsAg <1,000 IU/mL. 10.3% of patients (3 out of 29) who received multiple lenvervimab doses achieved undetectable HBsAg during treatment, although the HBsAg levels subsequently rebounded after EOT [58].
COMINBATION STRATEGIES
As reviewed in the prior sections, the novel HBV therapeutics are unable to consistently induce HBsAg seroclearance at high rates. Some virus-targeting agents, such as RNAi agents, have demonstrated potent effects in HBsAg suppression. Nonetheless, baseline HBsAg level appears to heavily influence treatment responses [23]. In contrast, novel immunomodulators all have modest effects on HBsAg, and have limited roles in monotherapy. It is hypothesized that combination strategies with novel HBV therapeutics may yield synergistic antiviral effects by targeting multiple pathways simultaneously [59]. Table 3 depicts the novel combination strategies that have entered clinical trials.
Combination of virus-targeting agents
The combination of daplusiran/tomligisiran (siRNA)+ bersacapavir (CAM) has been studied in the REEF-1 and REEF-2 trials. In the REEF-1 trial, the primary outcome was the proportion of patients reaching a predetermined criteria for stopping NUCs (ALT below 3x upper limit normal, unquantifiable HBV DNA, negative HBeAg, and HBsAg <10 IU/mL). After 48 weeks of treatment, the primary outcome occurred in 19% of patients on daplusiran/tomligisiran 200 mg monotherapy, 16% of patients on daplusiran/ tomligisiran 100 mg monotherapy, 9% of patients on daplusiran/ tomligisiran 100 mg+bersacapavir 250 mg, and 0% of patients on bersacapavir 250 mg monotherapy. Patients receiving daplusiran/tomligisiran 100 mg+bersacapavir 250 mg achieved HBsAg decline by 1.8 log IU/mL at EOT. In comparison, the treatment arms on daplusiran/tomligisiran 200 mg and daplusiran/tomligisiran 100 mg achieved EOT HBsAg decline by 2.6 log IU/mL and 2.1 log IU/mL, respectively. No patients in the combination arm achieved HBsAg seroclearance, whereas two patients on daplusiran/ tomligisiran 200 mg and one patient on daplusiran/ tomligisiran 100 mg achieved HBsAg seroclearance at 24 weeks post-EOT [60].
In the REEF-2 trial, the combination of daplusiran/tomligisiran 200 mg+bersacapavir 250 mg was compared against placebo in NUC-treated patients. 46.9% of patients on combination therapy achieved HBsAg <100 IU/mL at 48 weeks post-EOT, yet no patients achieved HBsAg seroclearance. All treatment including NUCs were withdrawn after 48 weeks of therapy. Patients in the treatment arm, when compared with controls, were less likely to have post-EOT biochemical flares (3.6% in active arm vs. 28.6% in control arm), and were less likely to require NUC resumption (9.1% in active arm vs. 26.8% in control arm) [61].
Imdusiran (siRNA)+vebicorvir (CAM) is another virus-targeting agent combination that has been studied. A phase II trial compared NUC-treated patients on imdusiran+ vebicorvir, imdusiran monotherapy, and vebicorvir monotherapy, respectively. An outcome of interest was the proportion of patients reaching a predetermined criteria to stop NUCs at week 48 (ALT below 2x upper limit normal, unquantifiable HBV DNA, and HBsAg <100 IU/mL). This stop NUC criteria was achieved by 80.0% of patients on imdusiran monotherapy, 61.5% of patients on combination therapy, and 0% of patients on vebicorvir monotherapy [62].
These studies suggest that the combination of siRNA+ CAM does not have synergistic effects on HBsAg suppression. The HBsAg reduction in this combination regimen appears to be primarily driven by siRNAs.
Combination of immunomodulators
The combination of therapeutic vaccination with anti-PD1 checkpoint inhibitors can simultaneously augment the function and abundance of anti-HBV T-cells. This strategy was studied in a phase II trial on VTP-300 (therapeutic vaccine)+nivolumab (anti-PD1). VTP-300 is a prime-boost vaccine with the prime and boost doses given at baseline and day 28, respectively. The trial was hence designed with treatment arms of 1) nivolumab administered with the day 28 boost dose only, or 2) nivolumab administered at baseline with the prime dose and at day 28 with the boost dose. In patients who received VTP-300+day 28 nivolumab, mean HBsAg decline of0.76 log IU/mL and 0.80 log IU/mL were achieved at 2 months post-EOT and 7 months post-EOT, respectively. 11.1% of patients (2 out of 18 patients) in the VTP-300+day 28 nivolumab arm achieved HBsAg seroclearance, and this response was sustained at 7 months post-EOT. In contrast, patients who received VTP-300+nivolumab at both baseline and day 28 did not achieve significant HBsAg decline or HBsAg seroclearance [55]. A potential explanation is that nivolumab with the prime dose vaccine may interfere with initial T-cell activation, which in turn negates vaccinal effects.
An ongoing open label trial is studying different regimens of VTP-300+nivolumab in patients with baseline HBsAg <200 IU/mL. The trial includes arm 1—vaccine boost with nivolumab on day 29, arm 2—vaccine boost with nivolumab on day 29 and day 85, and arm 3—vaccine boost on day 29 and day 85 with nivolumab on day 36. 43% of patients in arm 2 (3 out of 7) and 14% in arm 3 (1 out of 7) achieved undetectable HBsAg, and two of these patients from arm 2 had sustained undetectable HBsAg at day 169 [63].
Another immunotherapy combination that has been studied is BRII-179 (therapeutic vaccine) with or without IFNα. This regimen was able to mount antibody responses to HBsAg, although there was no significant HBsAg reduction observed [56].
Combination of virus-targeting agents and immunomodulators
The combination of virus-targeting agents with immunomodulators targets both the host and the virus, which should yield maximal synergistic effects. Given the promising results seen in monotherapy with RNAi agents, they have become the backbone in most combination regimens with immunomodulators. The RNAi agents can also induce immune reconstitution through reducing viral antigens, which should theoretically augment immunomodulator effects.
The phase II B-Together trial studied the combination of bepirovirsen with Peg-IFNα in NUC-treated patients. The trial enrolled 55 patients in treatment arm 1—bepirovirsen 300 mg weekly with loading dose for 24 weeks followed by 24 weeks of Peg-IFNα, and 53 patients in treatment arm 2—bepirovirsen 300 mg weekly with loading dose for 12 weeks followed by 24 weeks of Peg-IFNα. Overall, 9% of patients in treatment arm 1 and 15% of patients in treatment arm 2 achieved HBsAg seroclearance at 24 weeks post-EOT. While Peg-IFNα did not enhance the magnitude of HBsAg suppression on top of bepirovirsen, it was effective in reducing post-EOT relapse [64].
Peg-IFNα has also been studied in combination with multiple siRNAs including imdusiran, elebsiran, and daplusiran/tomligisiran, respectively. Patients generally had excellent HBsAg suppression with siRNA+Peg-IFNα. In the combination of imdusiran+Peg-IFNα, 28% of patients who received 24 weeks of Peg-IFNα achieved HBsAg seroclearance with anti-HBs development at EOT [65]. Whereas with the elebsiran (6–13 doses)+Peg-IFNα (44–48 weeks) regimen, 25.8% of patients achieved HBsAg seroclearance at EOT, with 16.1% of patients having sustained HBsAg seroclearance at 24 weeks post-EOT [66]. Daplusiran/tomligisiran+Peg-IFNα has been studied in immunotolerant patients with baseline HBV DNA >4 log IU/mL, and the combination treatment led to sustained HBsAg seroclearance in 14.8% of patients [67].
Another commonly studied combination strategy involves siRNA+therapeutic vaccines. In the trial on imdusiran+ VTP-300, 94.7% on combination therapy achieved HBsAg <100 IU/mL at EOT, while 84.2% in the imdusiran monotherapy arm reached this endpoint [68]. Daplusiran/tomligisiran+JNJ-0535 (therapeutic vaccine) and elebsiran+BRII-179 (therapeutic vaccine) are other combinations that have entered phase II trials, demonstrating mean EOT HBsAg reduction of 2.18 log IU/mL and 1.75 log IU/mL, respectively [69,70].
The phase II PIRANGA trial studied the combination of xalnesiran+ruzotolimod (TLR7 agonist) or Peg-IFNα in NUC-treated patients. Among patients with baseline HBsAg <3 log IU/mL, patients on xalnesiran+Peg-IFNα appeared to have the best response, with 23% achieving HBsAg seroclearance at 24 weeks post-EOT. In contrast, this endpoint was achieved by 12% of patients on xalnesiran+ ruzotolimod, and in 3% of patients on xalnesiran monotherapy [71].
In the phase II trial on daplusiran/tomligisiran+nivolumab (anti-PD1), EOT HBsAg reduction of 2.0–2.1 log IU/mL was achieved, with 35.3–50.0% of patients achieved HBsAg <10 IU/mL at EOT, depending on different nivolumab administration regimens [72].
Finally, the phase II MARCH trial studied the combination of elebsiran+tobevibart (anti-HBV monoclonal antibody) +/- Peg-IFNα. The combination of elebsiran+tobevibart+PegIFNα appeared to enhance the sustainability of HBsAg suppression, as 14.3% of patients on this triple regimen had HBsAg seroclearance at EOT with 9.5% sustaining HBsAg negativity at 12 weeks post-EOT. In contrast, 15.0% on elebsiran+tobevibart had HBsAg seroclearance at EOT, with no patients sustaining this response at 12 weeks post-EOT [73].
SAFETY OF NOVEL TREATMENT STRATEGIES
As the novel agents in development have all been meticulously evaluated in animal studies and phase I trials, they are generally well-tolerated with favorable safety profiles. Nonetheless, several agents have indeed shown unexpected adverse effects, requiring termination of development. Terminated agents include inarigivir (pattern recognition receptor agonist) – with drug-induced mortality in patients, ARC-520 (siRNA)—with mortality in non-human primates due to the drug excipient, and ABI-H2158 (CAM)—with severe drug-induced hepatotoxicity [12]. It is hence critical to collect long-term safety data from novel agents before they are routinely implemented in clinical practice.
As reviewed above, Peg-IFNα is commonly utilized as an adjunct in combination regimens. Peg-IFNα is contraindicated in patients with decompensated cirrhosis. Furthermore, Peg-IFNα is associated with a diverse range of adverse effects. Clinicians should be vigilant with Peg-IFNα use, and some patients will inevitably require regimens without Peg-IFNα.
Ongoing trials frequently include NUC discontinuation phases to assess for sustained HBsAg seroclearance after NUC withdrawal. This is necessary for achieving functional cure, yet the evidence on NUC discontinuation after novel therapies is limited. Furthermore, NUC discontinuation is not without risks. In the REEF-2 trial (daplusiran/tomligisiran+bersacapavir), a patient enrolled in the control arm (receiving NUC alone) developed severe HBV flare with liver failure after NUC withdrawal [74]. Future research should aim to study viral marker kinetics after NUC discontinuation, and universal criteria for safe NUC withdrawal after novel therapies should be established [75].
DISCUSSION
The field of novel HBV therapeutics is rapidly growing. Due to varying patient populations and trial designs, it is difficult to directly compare the efficacy of different novel therapeutics. Nonetheless, RNAi agents appear to be the most potent in HBsAg suppression and in inducing HBsAg seroclearance. Among patients who received 300 mg weekly bepirovirsen for 24 weeks, the best performance was achieved in patients with HBsAg ≤3 log IU/mL (16% of NUC-treated patients and 25% of non-NUC patients achieving sustained undetectable HBsAg at 24 weeks post-EOT) [23]. Whereas for combination regimens of ASOs or siRNAs+Peg-IFNα, sustained HBsAg seroclearance at 24 weeks post-EOT was achievable in ≥15% of patients in specific treatment arms [64,66,67,71]. Long-term data has also been accumulated in siRNAs, demonstrating sustainable HBsAg responses for up to 6 years [32]. The maintenance of treatment responses after EOT is critical for novel therapeutics, as sustained HBsAg seroclearance is required for the definition of functional cure. Viral rebound after EOT is suboptimal as it represents a state of inadequate viral control, and treatment withdrawal may not be safe.
Aside from RNAi agents, newer-generation CAMs can also effectively suppress HBsAg. The efficacy in HBsAg suppression likely corresponds to the targeted pathways by the virus-targeting agents. RNAi agents target cccDNA transcriptional activity, whereas newer-generation CAMs prevent cccDNA replenishment, highlighting the importance of targeting cccDNA-related pathways in HBsAg suppression.
Among immunomodulators, incidences of HBsAg seroclearance have been reported in trials for specific agents, including selgantolimod, envafolimab, and lenvervimab. Nonetheless, immunomodulators generally have modest effects on HBsAg, and are unlikely to be used as monotherapy.
Various combination strategies including combination of virus-targeting agents, combination of immunomodulators, and combination of virus-targeting agents with immunomodulators have been tested. The combinations involving RNAi agents+immunomodulators appear to have the most promising results, as immunomodulators ride on the RNAi-induced HBsAg reduction and increase durability. It is important to study the influence of dosing sequence and timing in combination regimens, as these factors can induce unforeseen negative effects on treatment responses (such as in the combination of VTP-300+nivolumab) [55]. Safety, costs, and patient acceptability are also important considerations before these novel combination strategies can be widely utilized.
Current clinical trials on novel therapeutics focused on highly selected HBV patient cohorts, and our understanding on treatment response predictors remains limited. Lower baseline HBsAg is associated with HBsAg seroclearance with different novel agents [23,55,63], and will likely be a key factor in patient selection. Age is also negatively correlated with HBsAg reduction in siRNA therapy [32]. Ongoing studies are assessing immune-based markers in both peripheral blood and in the liver as predictors of treatment response [76,77]. Ultimately, a better understanding of response predictors will guide personalized application of novel therapeutics. Patients with low likelihood of HBsAg seroclearance may require combination regimens with specific tailoring of treatment duration and dosing. In contrast, patients with high likelihood of HBsAg seroclearance may only require a single course of RNAi therapy to achieve functional cure.
With progressive advancements in novel HBV therapeutics, functional cure is now an attainable treatment goal. However, functional cure should not be the final target in HBV therapy. The persistence of integrated HBV DNA and cccDNA after functional cure remains as risk factors for liver related complications [2]. In particular, potentially hepatocarcinogenic HBV integrations are present in all HBV disease phases, even after HBsAg seroclearance [78]. Complete HBV cure—the sterilization of cccDNA and integration HBV DNA from the host, should hence be the ultimate treatment target. Complete cure should theoretically eliminate risks of HBV-related complications. Preclinical studies have utilized gene editing and epigenetic techniques in targeting integrated HBV DNA and cccDNA to achieve complete cure [79-81]. The results from these novel treatment approaches are keenly anticipated, and the development of these novel techniques may enable complete HBV cure in the future.
While the current clinical trials have shown exciting results on virological responses and HBsAg seroclearance, it remains uncertain whether novel agents can reduce liver-related complications. Future studies must collect long-term data to determine whether virological responses to novel therapies can be translated to clinically relevant outcomes.
From a population health perspective, development of effective novel therapeutics would dramatically boost our progress in hepatitis elimination. However, practical issues such as linkage to care and drug provision are equally important in hepatitis care. It is estimated that only 10.3% of CHB patients worldwide know of their infection status [82], while only 8.2% of treatment-eligible patients actually receive therapy [83]. Public health interventions to optimize HBV care pathways will be necessary to maximize the beneficial effects from novel therapeutics.
To conclude, multiple novel virus-targeting agents and immunomodulators are under active development. Combination strategies have also emerged, demonstrating potent and sustainable effects in inducing HBsAg seroclearance. Functional cure is now an attainable treatment goal in CHB. Multiple clinical trials are ongoing, and the emerging novel agents will revolutionize CHB management.
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