Prospect of emerging treatments for hepatitis B virus functional cure

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.

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].

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].