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Reply to: “Bepirovirsen/GSK3389404: Antisense or TLR9 agonists?”

Published:November 28, 2022DOI:https://doi.org/10.1016/j.jhep.2022.11.023

      Linked Article

      To the Editor:
      Dr Andrew Vaillant has hypothesized that the principal mechanism of action of GSK3389404 (N-acetylgalactosamine [GalNAc]-conjugated antisense oligonucleotide [ASO]) and bepirovirsen (non-GalNAc-conjugated ASO with the same target sequence as GSK3389404) is through immune stimulation rather than mRNA degradation.
      • Vaillant A.
      Bepirovirsen/GSK3389404: antisense or TLR9 agonists?.
      Although the relative contribution of mRNA/pregenomic RNA degradation and immune stimulation is still under investigation, we would like to make the following comments.
      Dr Vaillant asserts that the mechanism of action of ASOs and small-interfering RNAs (siRNAs) is inconsistent with mRNA degradation due to the rapid selection of individual point mutations.
      • Vaillant A.
      Bepirovirsen/GSK3389404: antisense or TLR9 agonists?.
      While it is true that HBV reverse transcriptase (RT) has no proofreading activity, its substitution rate is lower than that of other RT-dependent viruses, and is variable along the genome, with lower substitution rates in overlapping vs. non-overlapping regions.
      • McNaughton A.L.
      • D'Arienzo V.
      • Ansari M.A.
      • Lumley S.F.
      • Littlejohn M.
      • Revill P.
      • et al.
      Insights from deep sequencing of the HBV genome-unique, tiny, and misunderstood.
      The binding site of bepirovirsen and GSK3389404 is highly conserved across different HBV genotypes, where single point mutations potentially affect both the polymerase and X protein.
      • Yu Y.
      • Koenig A.
      • Livingston C.
      • van Gijzel H.
      • Elston R.
      • Theodore D.
      • et al.
      RNA launch system enables in vitro evaluation of bepirovirsen efficacy against HBV variants.
      Replication of HBV can be effectively inhibited by nucleos(t)ide analog (NA) therapy without selection of NA resistance despite long-term clinical use.
      • Roade L.
      • Riveiro-Barciela M.
      • Esteban R.
      • Buti M.
      Long-term efficacy and safety of nucleos(t)ides analogues in patients with chronic hepatitis B.
      Consequently, the addition of an siRNA or ASO to a patient already receiving stable NA therapy with suppressed HBV replication is unlikely to lead to the rapid development of resistant infectious virus particles. Participants in the GSK3389404 study were on stable NA therapy prior to enrollment and had suppressed viral replication.
      • Yuen M.F.
      • Heo J.
      • Kumada H.
      • Suzuki F.
      • Suzuki Y.
      • Xie Q.
      • et al.
      Phase IIa, randomised, double-blind study of GSK3389404 in patients with chronic hepatitis B on stable nucleos(t)ide therapy.
      We note that the “saw tooth” HBsAg response observed with dosing of ARB-1467 mentioned by Dr Vaillant may be accounted for by other explanations such as drug exposure. No resistance information was provided in the reference cited.
      Dr Vaillant also affirms that single mutations within the binding site will abolish activity. In the article referenced in support of this statement, double nucleotide mismatches within the central region of the binding site resulted in complete loss of activity for both siRNAs and RNase H-dependent oligonucleotides, whereas a double mutant (single nucleotide substitution in the 5’ and 3’-terminal domains) reduced, but did not abolish, activity. This is consistent with mutations having differential effects depending on their location but does not support that single mutations will abolish activity. Work to characterize nucleotide substitutions within the bepirovirsen/GSK3389404 binding site is ongoing using next-generation sequencing. An in vitro-selected single mutation within the center of the bepirovirsen/GSK3389404 binding site conferred a 4–5-fold reduction in susceptibility;
      • Yu Y.
      • Koenig A.
      • Livingston C.
      • van Gijzel H.
      • Elston R.
      • Theodore D.
      • et al.
      RNA launch system enables in vitro evaluation of bepirovirsen efficacy against HBV variants.
      the clinical significance of these results is not yet established.
      We and Dr Vaillant share a mutual understanding of the secondary pharmacology of bepirovirsen by activating innate immune sensors such as toll-like receptors (TLRs). Dr Vaillant hypothesized that bepirovirsen acts as a TLR9 agonist via one CpG motif present in the DNA part of the gapmer. Typically, two CpG motifs are required for TLR9 activation in humans, while only one motif may be sufficient in rodents.
      • Pohar J.
      • Yamamoto C.
      • Fukui R.
      • Cajnko M.M.
      • Miyake K.
      • Jerala R.
      • et al.
      Selectivity of human TLR9 for double CpG motifs and implications for the recognition of genomic DNA.
      However, the location of the motif and composition of RNA wings may impact TLR9 activation.
      • Pohar J.
      • Yamamoto C.
      • Fukui R.
      • Cajnko M.M.
      • Miyake K.
      • Jerala R.
      • et al.
      Selectivity of human TLR9 for double CpG motifs and implications for the recognition of genomic DNA.
      It is therefore unclear if bepirovirsen is a bona fide TLR9 agonist, and in vitro studies indicate this is not the case.
      • You S.
      • Delahaye J.
      • Ermler M.
      • Singh J.
      • Jordan W.
      • Ray A.
      • et al.
      Bepirovirsen, antisense oligonucleotide (ASO) against hepatitis B virus (HBV), harbors intrinsic immunostimulatory activity via Toll-like receptor 8 (TLR8) preclinically, correlating with clinical efficacy from the Phase 2a study.
      If an ASO can trigger TLR9 activation, it should show elevation of key serum markers typically induced by TLR9 activation in wild-type mice or cell lines.
      • Ushach I.
      • Zhu R.
      • Rosler E.
      • Pandey R.K.
      • De Costa N.T.S.
      • Pourshahian S.
      • et al.
      Targeting TLR9 agonists to secondary lymphoid organs induces potent immune responses against HBV infection.
      However, cytokines that are typically part of a type I immune response upon TLR9 activation, such as IL-12p40 and interferon alpha (IFN-α),
      • Ushach I.
      • Zhu R.
      • Rosler E.
      • Pandey R.K.
      • De Costa N.T.S.
      • Pourshahian S.
      • et al.
      Targeting TLR9 agonists to secondary lymphoid organs induces potent immune responses against HBV infection.
      were not observed with bepirovirsen in wild-type C57BL/6 mice.
      • You S.
      • Delahaye J.
      • Ermler M.
      • Singh J.
      • Jordan W.
      • Ray A.
      • et al.
      Bepirovirsen, antisense oligonucleotide (ASO) against hepatitis B virus (HBV), harbors intrinsic immunostimulatory activity via Toll-like receptor 8 (TLR8) preclinically, correlating with clinical efficacy from the Phase 2a study.
      Furthermore, IL-12p40, but not IFN-α, was induced by bepirovirsen and selgantolimod, a well-characterized TLR8 agonist, in the plasma of human TLR8 knock-in mice, supporting that bepirovirsen mimics the responses of a TLR8 agonist.
      • You S.
      • Delahaye J.
      • Ermler M.
      • Singh J.
      • Jordan W.
      • Ray A.
      • et al.
      Bepirovirsen, antisense oligonucleotide (ASO) against hepatitis B virus (HBV), harbors intrinsic immunostimulatory activity via Toll-like receptor 8 (TLR8) preclinically, correlating with clinical efficacy from the Phase 2a study.
      Dr Vaillant strongly disputed that bepirovirsen can activate TLR8 because 2’-O-methylation in the RNA part of the ASO would abrogate TLR8 activation. While methylation may decrease TLR8 responsiveness, there have been instances where methylated RNA can still activate TLR8 or even enhance TLR8 recognition over TLR7.
      • Nicolai M.
      • Steinberg J.
      • Obermann H.L.
      • Solis F.V.
      • Bartok E.
      • Bauer S.
      • et al.
      Identification of an optimal TLR8 ligand by alternating the position of 2'-O-ribose methylation.
      In the B-Clear Phase IIb study, bepirovirsen produced robust reductions in HBsAg in participants on stable NA therapy and those not on NA.
      • Yuen M.-F.
      • Lim S.-G.
      • Plesniak R.
      • Tsuji K.
      • Janssen H.L.A.
      • Pojoga C.
      • et al.
      Efficacy and safety of bepirovirsen in chronic hepatitis B infection.
      In contrast, only very modest reductions have been seen with TLR agonists.
      • Kayesh M.E.H.
      • Kohara M.
      • Tsukiyama-Kohara K.
      Toll-like receptor response to hepatitis B virus infection and potential of TLR agonists as immunomodulators for treating chronic hepatitis B: an overview.
      At the end of 24-week treatment with bepirovirsen 300 mg, approximately 30% of participants achieved HBsAg below the lower limit of detection,
      • Yuen M.-F.
      • Lim S.-G.
      • Plesniak R.
      • Tsuji K.
      • Janssen H.L.A.
      • Pojoga C.
      • et al.
      Efficacy and safety of bepirovirsen in chronic hepatitis B infection.
      something rarely observed with siRNAs.
      • Yuen M.F.
      • Locarnini S.
      • Lim T.H.
      • Strasser S.I.
      • Sievert W.
      • Cheng W.
      • et al.
      Combination treatments including the small-interfering RNA JNJ-3989 induce rapid and sometimes prolonged viral responses in patients with CHB.
      This suggests an important contribution of the immunostimulatory properties of bepirovirsen towards the observed clinical data. Our results therefore support a primary mode of action of bepirovirsen through RNA degradation, and the immunostimulatory activity of bepirovirsen via immune sensors, such as TLR8, in the liver may be synergized with HBsAg reduction, leading to the clinical responses observed. Efforts to better understand the molecular mechanisms behind the virological and immunological responses to bepirovirsen are ongoing.

      Financial support

      The study was funded by GSK (study 205670).

      Conflict of interest

      M-F Yuen reports grant/research support from AbbVie, Assembly Biosciences, Arrowhead Pharmaceuticals, Bristol Myers Squibb, Fujirebio Incorporation, Gilead Sciences, Immuncore, Merck Sharp and Dohme, Springbank Pharmaceuticals, Sysmex Corporation and Roche, consultancy for AbbVie, Aligos Therapeutics, AiCuris, Antios Therapeutics, Arbutus Biopharma, Arrowhead Pharmaceuticals, Assembly Biosciences, Bristol Myers Squibb, Clear B Therapeutics, Dicerna Pharmaceuticals, Finch Therapeutics, Fujirebio Incorporation, GSK, Gilead Sciences, Immunocore, Janssen, Merck Sharp and Dohme, Roche, Springbank Pharmaceuticals, Silverback Therapeutics, Sysmex Corporation and Vir Biotechnology, and lecture fees from AbbVie, Dicerna Pharmaceuticals, Fujirebio Incorporation, Gilead Sciences, Merck Sharp and Dohme, Roche and Sysmex Corporation. S You and R Elston are employees of GSK and hold stock/shares in GSK.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      M-F Yuen, was involved in the conception or design of the study and data acquisition. M-F Yuen, R Elston and S You were involved in data analysis or interpretation. The manuscript was reviewed and edited by all authors. All authors approved the manuscript for submission and vouch for the accuracy/completeness of the data.

      Acknowledgments

      The authors would like to thank Jerome Bouquet, Megan Ermler, Jenn Singh, Jennifer Cremer, Melanie Paff and Dickens Theodore for their contributions to this letter. Editorial support (in the form of writing assistance, including development of the initial draft based on author direction, collating authors’ comments, grammatical editing, and referencing) was provided by Chrystelle Rasamison, of Fishawack Indicia Ltd, UK, part of Fishawack Health, and was funded by GSK.

      Supplementary data

      The following are the supplementary data to this article:

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