Background & Aims: Immune targeting is likely required for functional cure of chronic hepatitis B (CHB). Tobevibart, a human monoclonal antibody against hepatitis B virus (HBV) surface antigen (HBsAg), neutralizes HBV and hepatitis delta virus (HDV). This study aimed to characterize effects of the engineered GAALIE Fc of tobevibart on HBV immune responses. Methods: We studied tobevibart and its equivalent HBC34*-GAALIE in vitro using electron microscopy, FcgR reporter cells, and primary human or mouse immune cells to assess HBsAg binding, dendritic cell (DC) activation, and T cell stimulation. Tobevibart-mediated binding of HBsAg to immune cells was evaluated also in a phase 1 clinical trial in patients with CHB. Results: The GAALIE Fc of tobevibart mediated gain of function in FcgR signaling in immune complexes (ICs) with HBsAg compared to wild-type (WT) Fc and increased binding of HBsAg to neutrophils and monocytes in vitro. Similarly, dosing of 300 mg tobevibart in patients with CHB mediated binding of HBsAg to these cells in vivo, concomitant with reducing HBsAg in circulation. In vitro, ICs of HBC34*-GAALIE and HBsAg activated human DCs significantly more than HBC34*-WT. These DCs presented antigen and stimulated HBsAg-specific human T cells. Similarly, ICs of HBC34*-GAALIE and HBsAg activated DCs from mice transgenic for human FcgRs and stimulated CD4+ T cells from immunized animals more than ICs with HBC34*- WT. Conclusions: We demonstrate that tobevibart combines the advantages of potent neutralization of HBV and HDV, FcgR-mediated reduction of HBsAg, and Fc-dependent enhancement of T cell responses. Tobevibart is currently under clinical investigation alone or in combination with other agents to treat patients with chronic hepatitis delta and to induce functional cure of patients with CHB. ### Competing Interest Statement LV, RW, RM, BG, ES, SVG, LER, YPC, JdI, AM, KET, SD, JME, LW, NC, JD, NS, AP, LS, DCloutier, GS, CHT, FAL, CHD, FB, AL, AA, DCorti, MAS are or were employees of Vir Biotechnology and may hold shares in Vir Biotechnology. LER, NC, GS, FAL, DC and MAS are listed as inventors on patent applications, which disclose the subject matter described in this manuscript. KA, MFY, HW, and EG served as advisors or received grant support from various industry partners. The remaining authors declare no conflict of interest. ### Clinical Trial NCT04423393 ### Funding Statement This clinical trial was designed, conducted and funded by Vir Biotechnology, Inc. as sponsor. David Belnap received funding from Vir Biotechnology through an agreement with University of Utah, related to the work described in this paper. The tobevibart phase 1 clinical study received editorial support by Lumanity Scientific Inc., which was funded by Vir Biotechnology. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: (A) For the phase 1 clinical trial (VIR-3434-1002, ClinicalTrials.gov, [NCT04423393][1]) Approval from the local Institutional Review Board or Independent Ethics Committee was obtained and informed consent was obtained from all participants prior to their participation in the study. These local review boards or committees were of the Medical Faculty of the University of Duisburg-Essen, Germany; London City & East Research Ethics Committee, Bristol, United Kingdom; University Hospital Birmingham, United Kingdom; Queen Mary Hospital, Hong Kong; University of Hong Kong, Hong Kong; Ethics Committee at Medical Center of Arensia Exploratory Medicine Limited Liability Company, Kyiv, Ukraine; Asan Medical Center, Seoul, South Korea; Gangnam Severance Hospital Yonsei University, South Korea; Korea University Anam Hospital, South Korea; Pusan National University Hospital, South Korea; Seoul National University Hospital, South Korea; Health and Disabilities Ethics Committee, Ministry of Health, Wellington, New Zealand; Romania Academy of Sciences, Bucharest, Romania; Singapore Health Services, Singapore. (B) For human samples obtained outside the above study for research in vitro, Whole blood, PBMCs, sera or plasma samples were obtained from human subjects under study protocols approved by the local Institutional Review Boards (Ethics Committees of the Canton Ticino, Switzerland or IRB of the University of California San Francisco or Advarra IRB for Quest Diagnostics, USA). All donors provided written informed consent for the use of blood and blood components. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT04423393&atom=%2Fmedrxiv%2Fearly%2F2025%2F01%2F14%2F2025.01.13.25320453.atom

BACKGROUND & AIMS:Effective immune targeting is likely essential for achieving functional cure of chronic hepatitis B (CHB). Tobevibart, a human monoclonal antibody against hepatitis B surface antigen (HBsAg), neutralizes HBV and HDV. This study aimed to characterize the effects of the engineered GAALIE Fc of tobevibart on HBV immune responses. METHODS:We studied tobevibart and its equivalent, HBC34∗-GAALIE, in vitro using electron microscopy, reporter cells, and primary human or mouse immune cells to assess dynamics of HBsAg reduction, dendritic cell (DC) activation, and T-cell stimulation. Tobevibart-mediated binding of HBsAg to immune cells was also evaluated in a phase I clinical trial in patients with CHB. RESULTS:The GAALIE Fc of tobevibart increased binding to activating FcγRs, decreased binding to inhibitory FcγRIIb, mediated gain of function in FcγR signaling in immune complexes with HBsAg, and increased binding of HBsAg to neutrophils and monocytes in vitro compared to wild-type Fc. Similarly, administration of 300 mg tobevibart in patients with CHB resulted in binding of HBsAg to these cells in vivo, concurrent with a reduction in circulating HBsAg. In vitro, immune complexes of HBC34∗-GAALIE and HBsAg activated human DCs significantly more than HBC34∗-wild-type. These DCs presented antigen and stimulated HBsAg-specific human T cells. Similarly, HBC34∗-GAALIE augmented HBsAg-specific CD4+ T-cell responses from mice transgenic for human FcγRs. CONCLUSIONS:We demonstrate that tobevibart combines the advantages of potent neutralization of HBV and HDV with FcγR-mediated reduction of HBsAg and enhancement of T-cell responses. It is currently under clinical investigation, alone and in combination with other agents, for the treatment of chronic hepatitis delta and to induce functional cure of CHB. CLINICAL TRIAL:VIR-3434-1002, phase 1, ClinicalTrials.gov identifier NCT04423393. IMPACT AND IMPLICATIONS:Chronic infection with HBV or co-infection with HDV can cause severe liver disease and cancer. We have previously shown that the monoclonal antibody tobevibart potently neutralizes HBV and HDV. Here we demonstrate that the engineered Fc region of tobevibart effectively interacted with several immune cell types, potentially facilitating the rapid removal of damaging viral proteins from the circulation and activating T-cell responses that may control HBV infection long term.

Background Chronic hepatitis B virus (HBV) remains a global concern, with current treatments achieving low rates of HBsAg seroclearance. VIR-2218 (elebsiran), a small interfering RNA agent against HBV transcripts, reduces HBsAg concentrations. We aimed to evaluate the safety and antiviral activity of VIR-2218 with and without pegylated interferon-alpha-2a treatment in participants with chronic HBV. Methods This open-label, phase 2 study was conducted at 23 sites in six countries (New Zealand, Australia, Hong Kong, Thailand, South Korea, and Malaysia). Adults (aged 18-65 years) with chronic HBV infection without cirrhosis and with HBsAg more than 50 IU/mL and HBV DNA less than 90 IU/mL who were on continued nucleoside or nucleotide reverse transcriptase inhibitor (NRTI) therapy for 2 months or longer were eligible. Participants were enrolled into one of six cohorts to receive VIR-2218 200 mg subcutaneously every 4 weeks, with or without 180 mu g subcutaneous pegylated interferon-alfa-2a once per week. Cohort 1 received six doses of VIR-2218 (total 20 weeks); cohort 2 received six doses of VIR-2218 starting at day 1, plus 12 doses of pegylated interferon-alfa-2a starting at week 12 (total 24 weeks); cohort 3 received six doses of VIR-2218 and 24 doses of pegylated interferon-alfa-2a (total 24 weeks); cohort 4 received six doses of VIR-2218 and up to 48 doses of pegylated interferon-alfa-2a (total 48 weeks); cohort 5 received up to 13 doses of VIR-2218 and up to 44 doses of pegylated interferon-alfa-2a (total 48 weeks); and cohort 6 received three doses of VIR-2218 and 12 doses of pegylated interferon-alfa-2a (total 12 weeks). The primary endpoints were the incidence of adverse events and clinical assessments (including results of laboratory tests). Secondary endpoints were the mean maximum reduction ofserum HBsAg at any timepoint; the proportion ofparticipants with serum HBsAg seroclearance at any timepoint and for more than 6 months after the end of treatment; and the proportion of participants with antiHBs seroconversion at any timepoint. For patients who were HBeAg-positive, we also assessed the proportion with HBeAg seroclearance or anti-HBe seroconversion at any timepoint. This study is registered with ClinicalTrials.gov, NCT03672188, and is ongoing. Findings Between July 2, 2020, and Nov 2, 2021, 124 individuals were screened for eligibility, 84 of whom were enrolled (15 in cohort 1, 15 in cohort 2, 18 in cohort 3, 18 in cohort 4, 13 in cohort 5, and five in cohort 6). Participants were predominantly HBeAg-negative, Asian, and male (66 [79%] participants were male and 18 [21%] were female). Most treatment emergent adverse events were grades 1-2. Three (20%) participants in cohort 1, four (27%) in cohort 2, eight (44%) in cohort 3, seven (39%) in cohort 4, six (46%) in cohort 5, and two (40%) in cohort 6 reported treatment- emergent adverse events related to VIR-2218. 12 (80%) participants in cohort 2, 12 (67%) in cohort 3, 14 (78%) in cohort 4, 13 (100%) in cohort 5, and three (60%) in cohort 6 reported treatment-emergent adverse events related to pegylated interferon-alfa-2a. Two (13%) participants in cohort 1 had elevations in alanine aminotransferase, compared with 13 (87%) participants in cohort 2, 15 (83%) in cohort 3, 17 (94%) in cohort 4, 11 (85%) in cohort 5, and three (60%) in cohort 6. The mean maximum change from baseline at any timepoint in HBsAg concentration was -20 log10 IU/mL (95% CI -21 to -18) in cohort 1, -22 log10 IU/mL (-25 to -18) in cohort 2, -25 log10 IU/mL (-28 to -21) in cohort 3, -24 log10 IU/mL (-31 to -18) in cohort 4, -30 log10 IU/mL (-37 to -23) in cohort 5, and -17 log10 IU/mL (-21 to -14) in cohort 6. 11 participants (one in cohort 2, one in cohort 3, five in cohort 4, and four in cohort 5) receiving VIR-2218 plus pegylated interferon-alfa-2a had HBsAg seroclearance at any timepoint. Of these, ten (91%; one in cohort 2, five in cohort 4, and four in cohort 5) had anti-HBs seropositivity. Six participants (one in cohort 2, three in cohort 4, and two in cohort 5) had sustained HBsAg seroclearance through to 24 weeks after the end of treatment. No participants receiving VIR-2218 monotherapy (cohort 1) or VIR-2218 plus pegylated interferon-alfa-2a 12-week regimen (cohort 6) had HBsAg seroclearance. 12 (42%) of 26 participants (one of four in cohort 1, two of six in cohort 2, four of seven in cohort 3, four of six in cohort 4, and one of three in cohort 5) who were HBeAg positive at baseline had HBeAg seroclearance or anti-HBe seroconversion. Interpretation The results of this phase 2 study support further development of VIR-2218 as a potential therapy for patients with chronic HBV infection. Additional clinical trials of VIR-2218 with and without pegylated interferon-alfa-2a in combination with an HBsAg-targeting monoclonal antibody are ongoing.

for HBcrAg, Roche Cobas TaqMan system with an inhouse assay for HDV RNA).Results: At baseline, mean HBV RNA levels were 1.71 ± 1.18 log cp/ml and HBV RNA levels were undetectable in 68.8% of patients.During treatment, mean HBV RNA levels did not differ between patients receiving either tenofovir or placebo.When separating between treatment responders (HDV RNA negative at FU24) and nonresponders, mean levels of HBV RNA were significantly lower in responders at treatment week 24, 48 and end of treatment.Accordingly, the proportion of patients with undetectable HBV RNA was significantly higher in treatment responders at week 24 and 48 (79% vs. 50%, p = 0.0031; 75% vs. 50%, p = 0.0276) (Figure).However, HBV RNA levels of patients with viral relapse did not differ from those with maintained response at the end of treatment.No correlation between HBV RNA levels and HBV DNA, qHBsAg, HBcrAg or HDV RNA levels was detected at any time-point during the study.Interestingly, only 36% of patients with undetectable HBV RNA showed undetectable HBcrAg levels at baseline, whereas all but two patients with undetectable HBcrAg were HBV RNA negative (n = 24/26, 92%). Conclusion:On-treatment HBV RNA levels differ significantly between responders and non-responders.Along with other virological parameters, HBV RNA could be a supportive marker to identify patients benefiting from treatment with pegIFNa.However, overall HBV RNA levels were low throughout the study population.

ALT) levels throughout the study; 2 participants in each regimen had grade 1 ALT elevations.No treatment-related serious AEs were reported. Conclusion:These preliminary data support that a longer duration of treatment with VIR-2218 results in deeper and more sustained reductions in HBsAg.In both regimens of VIR-2218, no differences in safety or tolerability were observed.

VIR-2218 is an investigational N-acetylgalactosamine–conjugated RNA interference therapeutic in development for chronic hepatitis B virus (HBV) infection. VIR-2218 was designed to silence HBV transcripts across all genotypes and uses Enhanced Stabilization Chemistry Plus (ESC+) technology. This study was designed to evaluate the single-dose pharmacokinetics of VIR-2218 in preclinical species and healthy volunteers. Preclinically, a single subcutaneous dose of VIR-2218 (10 mg/kg) was administered to rats and nonhuman primates (NHPs), and the pharmacokinetics were assessed in plasma, urine, and liver using standard noncompartmental analysis (NCA) methods. Clinically, healthy volunteers were randomized (6:2 active:placebo) to receive a single subcutaneous dose of VIR-2218 (50–900 mg) or placebo. Pharmacokinetics were similarly assessed within human plasma and urine using NCA methods. In rats and NHPs, VIR-2218 was stable in plasma and was converted to AS(N-1)3’VIR-2218, the most prominent circulating metabolite, at < 10% plasma exposure compared with parent. VIR-2218 rapidly distributed to the liver, reaching peak liver concentrations within 7 and 24 h in rats and NHPs, respectively. In humans, VIR-2218 was rapidly absorbed, with a median time to peak plasma concentration (tmax) of 4–7 h, and had a short median plasma half-life of 2–5 h. Plasma exposures for area under the plasma concentration–time curve up to 12 h (AUC0–12) and mean maximum concentrations (Cmax) increased in a slightly greater-than-dose-proportional manner across the dose range studied. Interindividual pharmacokinetic variability was low to moderate, with a percent coefficient of variation of < 32% for AUC and < 43% for Cmax. A portion of VIR-2218 was converted to an active metabolite, AS(N-1)3’VIR-2218, with a median tmax of 6–10 h, both of which declined below the lower limit of quantification in plasma within 48 h. The pharmacokinetic profile of AS(N-1)3’VIR-2218 was similar to that of VIR-2218, with plasma AUC0–12 and Cmax values ≤ 12% of VIR-2218. VIR-2218 and AS(N-1)3’VIR-2218 were detectable in urine through the last measured time point, with approximately 17–48% of the administered dose recovered in urine as unchanged VIR-2218 over 0–24 h postdose. Based on pharmacokinetics in preclinical species, VIR-2218 localizes to the liver and likely exhibits prolonged hepatic exposure. Overall, no severe or serious adverse events or discontinuations due to adverse events were observed within the dose range evaluated for VIR-2218 in healthy volunteers (Vir Biotechnology, Inc., unpublished data). VIR-2218 showed favorable pharmacokinetics in healthy volunteers supportive of subcutaneous dosing and continued development in patients with chronic HBV infection. NCT03672188, September 14, 2018.