Challenges, Considerations, and Principles to Guide Trials of Combination Therapies for Chronic HBV

Transcription

1 Accepted Manuscript Challenges, Considerations, and Principles to Guide Trials of Combination Therapies for Chronic HBV Ryan Taylor Anderson, MS, Seng Gee Lim, MBBS, FRACP, FRCP, MD, Poonam Mishra, MD, MPH, Filip Josephson, MD, PhD, Eric Donaldson, PhD, Bruce Given, MD, Veronica Miller, PhD PII: S (18) DOI: Reference: YGAST To appear in: Gastroenterology Please cite this article as: Anderson RT, Lim SG, Mishra P, Josephson F, Donaldson E, Given B, Miller V, Challenges, Considerations, and Principles to Guide Trials of Combination Therapies for Chronic HBV, Gastroenterology (2019), doi: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 1 Title: Challenges, Considerations, and Principles to Guide Trials of Combination Therapies for Chronic HBV Short title: HBV treatment combination guiding principles Authors: 1. Ryan Taylor Anderson, MS, Graduate Student Assistant, Forum for Collaborative Research, University of California, Berkeley, School of Public Health, Washington, District of Columbia 2. Seng Gee Lim, MBBS, FRACP, FRCP, MD, Professor, National University of Singapore, Singapore, Republic of Singapore 3. Poonam Mishra*, MD, MPH, Deputy Director for Safety, US Food and Drug Administration, Silver Spring, Maryland 4. Filip Josephson*, MD, PhD, Clinical Assessor, Swedish Medical Products Agency, Uppsala, Sweden 5. Eric Donaldson*, PhD, Clinical Virology Reviewer, US Food and Drug Administration, Silver Spring, Maryland 6. Bruce Given, MD, Chief Operating Officer, Arrowhead Pharmaceuticals, Inc., Pasadena, California 7. Veronica Miller, PhD, Executive Director, Forum for Collaborative Research, University of California, Berkeley, School of Public Health, Washington, District of Columbia * Refer to disclaimer Grant Support: The Hepatitis B Forum receives support from Abbott Molecular, Aicuris, Altimmune, Arbutus Biopharma, Arrowhead Pharmaceuticals, Assembly

3 2 Biosciences, ContraVir, DDL Diagnostics, Gilead Sciences, GlaxoSmithKline, Hepatitis B Foundation, Janssen, MEDIAN Technologies, MedImmune (a member of AstraZeneca), Novartis, PPD, Quest Diagnostics, Roche Molecular Systems, and Springbank Pharma. Abbreviations: ALT: alanine aminotransferase, cccdna: covalently closed circular DNA, CHB: Chronic Hepatitis B, CHMP: Committee for Medicinal Products for Human Use, DILI: drug-induced liver injury, DDI: drug-drug interaction, DNA: deoxyribonucleic acid, EMA: European Medicines Agency, FDA: US Food and Drug Administration, HBsAg: hepatitis B surface antigen, HBeAg: hepatitis B e-antigen, HBV: Hepatitis B Virus, HCC: hepatocellular carcinoma, HCV: Hepatitis C Virus, ICH: International Conference on Harmonisation, IFN: interferon, INR: International Normalized Ratio, IU: international units, ml: millilitre, MPA: Medical Products Agency NAFLD: non-alcoholic fatty liver disease, NASH: non-alcoholic steatohepatitis, NrtI: nucleos(t)ide reverse transcriptase inhibitors, PD: pharmacodynamic, PK: pharmacokinetic, RNA: ribonucleic acid, SVR24: sustained virologic response at 24-weeks Correspondence: Veronica Miller, Ph.D., Executive Director, Forum for Collaborative Research, 1608 Rhode Island Ave, Suite 212, Washington DC 20036, veronicam@berkeley.edu, P: , F: Disclosures: Ryan Anderson: reports nothing to disclose. Seng Gee Lim: reports that he receives research support from Gilead Sciences and Abbott Diagnostics. In addition, he serves on the advisory board for Gilead Sciences.

4 3 Abbott Diagnostics, AbbVie, MSD, Springbank, and Roche, as well as the speaker bureau for Gilead Sciences, Abbott Diagnostics, and AbbVie. Poonam Mishra: reports nothing to disclose. Filip Josephson: reports nothing to disclose. Eric Donaldson: reports nothing to disclose. Bruce Given: reports that he is an employee and shareholder of Arrowhead Pharmaceuticals, Inc. Veronica Miller: reports support from Abbott Molecular, Aicuris, Altimmune, Arbutus Biopharma, Arrowhead Pharmaceuticals, Assembly Biosciences, Astra Zeneca, ContraVir, DDL Diagnostics, Gilead Sciences, GlaxoSmithKline, Hepatitis B Foundation, Janssen, MEDIAN Technologies, Novartis, PPD, Quest Diagnostics, Roche Molecular Systems, and Springbank Pharma. Author Contribution: Ryan Taylor Anderson drafted and provided critical revisions the manuscript. Seng Gee Lim, Poonam Mishra, Filip Josephson, Eric Donaldson, Bruce Given and Veronica Miller developed the manuscript concept, and contributed to the critical revision and final approval of the manuscript.

5 4 Introduction Currently approved clinical interventions for chronic hepatitis B (CHB) are limited to treatments that require long-term therapy for the majority of patients. While observational studies have shown that sustained HBV DNA suppression achieved with current therapies is associated with improvement in clinical outcomes, an excess risk of hepatocellular carcinoma (HCC) remains 1, and HBsAg seroclearance is uncommon 2. Furthermore, evidence exists that HBsAg seroclearance is associated with decreases in long-term disease complications 3. There is also a need for effective and safe finite therapies to avoid life-long medication. Renewed interest in the advancement of novel drugs for the treatment of hepatitis B virus (HBV) infection has led to the ongoing development of a broad diversity of agents targeting both the virus and host. The over-arching goal of such development is achieving high rates of functional cure; defined as sustained undetectable hepatitis B surface antigen (HBsAg) concurrent with suppressed plasma HBV DNA. The Treatment Combinations Working Group (TCWG) of the HBV Forum, a project of the Forum for Collaborative Research, is tasked with developing a conceptual framework for clinical trials that provides adequate safeguards for trial participants while allowing rapid testing and innovation of new treatment combinations aiming at functional cure. In the following article, principles of combination drug development for both trials with two or more investigational drugs as well as novel therapeutics combined with already approved treatment options are considered.

6 5 Discerning Meaningful Clinical Endpoints and the Need for Novel Therapies Our work builds upon the main outcomes of the Hepatitis B Treatment Endpoints Workshop 4, during which the utility of using sustained virologic response at 24- weeks following the completion of a finite treatment course (SVR24); characterized as plasma HBV DNA suppression accompanied by sustained undetectable serum HBsAg, with or without seroconversion to hepatitis B surface antibody, was discussed as a primary efficacy endpoint. As the aim of such therapies would be sustained HBsAg negativity, long-term follow-up studies would be required to ascertain the maintenance of virological suppression and HBsAg loss off-treatment. Data from these studies could also be used to assess the impact of functional cure on the risk of events such as HCC, hepatic decompensation, liver transplantation and mortality. Individual clinical trials and meta-analyses have demonstrated excellent therapeutic responses using nucleos(t)ide reverse transcriptase inhibitors (NrtIs), as measured by sustained suppression of viral replication while on therapy, improvement in liver histology, regression of liver cirrhosis, and reductions in liver decompensation, HCC and death 5. However, NrtI therapy rarely leads to long-term off-treatment viral suppression, thus requiring indefinite treatment to maintain suppression of viral replication 6. Post-treatment persistent virological relapse, defined as having a serum HBV DNA level >2000 IU/ml on 2 consecutive measurements at least 3- months apart following treatment discontinuation 7, occurred in 51-66% of patients

7 6 who met cessation criteria and/or had at least 1-year follow-up after therapy discontinuation, indicating that the cccdna reservoir is maintained during longterm NrtI treatment 6, 8. Antiviral drug resistance remains an issue, especially with early-generation NrtIs, which are still widely used in some low-income settings 9. More importantly, viral suppression decreases, but does not completely abrogate, the risk for HCC 1. Interferon (IFN) historically produced higher rates of functional cure, with responses partly dependent upon genotype, but is rarely used for reasons of convenience, tolerability and lack of efficacy in the majority of patients 10. The expectation that all patients will adhere to long-term or lifelong non-curative treatment is not realistic, and there is a strong patient preference for finite therapy options 11. Effective, finite HBV treatment therefore remains an unmet medical need. New therapeutic strategies and novel drugs are necessary to increase the likelihood of achieving functional cure. Intracellular cccdna is the permanent reservoir of HBV replication, and a main target for new therapeutic strategies 12. New drugs will have to target and clear, silence, or reduce the cccdna reservoir, and/or permanently suppress HBV replication to generate a sustained response off-treatment. While cccdna is the major driver of viral replication, the role of genomically integrated HBV DNA in promoting HBV chronicity, immune tolerance, and triggering HCC will also need to be considered 13.

8 7 As for HIV and HCV, combining therapies may have additive or synergistic antiviral effects and may reduce or delay resistance. Combination therapy, likely required for successful treatment of HBV aiming at functional cure is essentially uncharted territory 5 ; though treatment with NrtIs in combination with IFN has shown only slight improvement, if any, over monotherapy 2. In HBV, co-development of drugs for functional cure is challenging due to the lack of an appropriate animal model, and the fact that new therapeutics will act in the context of and ongoing interaction between virus, hepatocytes and the immune system. Applying Current FDA Combination Guidance to CHB The 2013 FDA guidance addressing the co-development of two or more investigational drugs in combination 14 states that co-development may be an appropriate strategy should the following criteria be met: 1. The combination is intended to treat a serious disease or condition. 2. There is a strong biological rationale for use of the combination. 3. A full nonclinical characterization of the activity of both the combination and the individual new investigational drugs, or a short-term clinical study on an established biomarker, suggests that the combination may provide a significant therapeutic advance over available therapy and is superior to the individual agents. A nonclinical model should demonstrate that the

9 8 combination has substantial activity and provides greater activity, a more durable response, or a better toxicity profile than the individual agents. 4. There is a compelling reason why the new investigational drugs cannot be developed independently. The first criterion is fulfilled because untreated CHB can lead to serious and lifethreatening complications. Even with approved treatments the risk of HCC remains elevated, most patients cannot maintain viral suppression off therapy, and adherence to long-term therapy may be a problem. A combination that targets two or more HBV viral targets with the goal of post-treatment sustained response would qualify as a strong rationale for co-development. For the third point, in vitro evidence is recommended to support the mechanism of action and establish antiviral activity of the new agent to be combined with an approved drug due to the lack of an adequate animal model. Cell culture combination studies, or in vivo data showing that the new drug does not antagonize the approved drug, and vice versa, would be necessary prior to dosing with the combination in humans. As for the fourth point, for the foreseeable future new therapeutics other than novel NrtIs will likely be tested on the background of already approved NrtIs until there is sufficient data to allow for the combination of new agents. It may be that a NrtI would be required to control infection and reduce the chances of resistance by suppressing HBV DNA replication while a new complementary agent targets one or more viral or host proteins involved in the HBV replication cycle 15. Furthermore, an

10 9 investigational therapeutic effective in treatment combination regimens may not produce measurable or impressive antiviral effects as monotherapy 16. This strategy may be appropriate provided there is no concerning drug-drug interaction (DDI) or combination safety issues, and that pharmacodynamic (PD) biomarker(s) can be measured despite virological suppression by the NrtI. Conversely, the developer of a candidate drug that might produce improved rates of SVR24 may have an incentive to show efficacy as a monotherapy. A reasonable course would be to initially demonstrate activity on the background of NrtIs, followed by monotherapy studies or subcombinations of regimen components to demonstrate the contribution of each if warranted and appropriate. Considerations and Challenges for Clinical Trials of HBV Combination Therapies All early phase clinical trials investigating combination therapies should be founded on a solid scientific rationale and explicit justification; further, there should be vigilance to identify new or worsened adverse events, and close monitoring of adverse effects of specific interest. The possibility of overlapping toxicities and potential DDI must be considered 17. Initial trials in patients could include those who are untreated and HBeAg-positive with active disease, or virally suppressed HBeAgnegative or -positive with at least 6-months of treatment with NrtIs 4. While we may hope that future drugs/combinations will be effective regardless of genotype,

11 10 HBeAg status or prior treatments received, this may not be the case. As such, development programs will need to recognize potential differences in these parameters, by study design and stratification, particularly in phase 3 trials. Phase 1a-1b/2a clinical development of new HBV therapies After first in human trials, if the novel agent has the potential to show activity on serum markers of HBV infection, such activity should subsequently be assessed in CHB patients. These first trials in patients may be of short duration and could be conducted either as monotherapy or as an add-on to a background of NrtI therapy. Adding an experimental agent onto a NrtI treatment schedule may be more suitable for the initial safety assessment in patients, as well as for dose-finding, provided there is no anticipated combination toxicity or DDI concerns, and that the main PD marker(s) of activity is measurable despite suppression of plasma HBV DNA. In case there is no relevant PD endpoint except for plasma HBV DNA, early studies can be conducted in treatment-naïve patients, or in previously-treated viremic patients currently not on treatment. Treatment-naïve patients, or previously-treated viremic patients currently not on treatment, provide a rapid readout on early antiviral activity endpoints, especially HBV DNA, and are often preferred for inclusion in early proof-of-concept trials. Challenges in recruiting treatment-naïve patients include: (a) patient willingness to enroll in a commitment-intensive trial of an unproven agent or combination regimen before receiving standard of care

12 11 therapy, (b) widespread availability of affordable NrtIs, and (c) high rates of treatment initiation in large clinical centers, especially those that were high enrollers in previous HBV treatment trials. Logistically, NrtI-suppressed patients may be easier to enroll in phase 1b-2a proof-of-concept trials for novel CHB therapies. However, viremic patients will remain important for early phase studies to determine virologic efficacy if viral replication is the primary therapeutic target. Consideration should be given to the fact that viral genotypes have distinct geographical distributions and could potentially influence the disease severity and response to treatment 18. Relevant DDI studies would be appropriate given a mechanistic rationale for the possibility of clinically relevant pharmacokinetic (PK) drug-drug interactions 14. Phase 2-3 efficacy evaluations of HBV treatment regimen SVR24 as defined in the introduction may be an appropriate primary efficacy endpoint for phase 3 trials for finite treatment of CHB, if complemented by a longterm follow-up to confirm the durability of post-treatment response 4. However, this endpoint may be impractical for decision-making in phase 2 trials exploring novel drugs or combinations, as this considerably extends the duration of the exploratory work.

13 12 An important aspect of drug development for HBV combination therapy aiming at functional cure is evaluating novel immunological and virological markers of drug response that might identify promising activity in phase 2, meriting progression to confirmatory trials, where their potential use in response-guided therapy could be assessed prospectively 19. Such exploratory biomarkers include quantitative assays for HBV RNA, HBeAg, HBsAg, and hepatitis B core-related antigen 20. There is also considerable interest in developing methods and effective markers to evaluate and monitor the host immune response to treatment 21. Alanine Aminotransferase (ALT) Flares ALT elevations are commonly seen as both part of the natural course of CHB and in response to effective therapy 22, 23. Increases in ALT may be a consequence of the desired drug effect and may be necessary for achieving functional cure 24, 25. Treatments that are effective in converting HBsAg status may also be associated with increased risk of ALT elevations (generally referred to as flares in the HBV community). Thus, special attention must be given to distinguishing the cause and severity of abnormalities in liver function tests observed during treatment. A discussion of the clinical characteristics associated with different types of flares is found in the supplementary material. Differentiating between treatment-associated flares related to response and ALT elevations due to drug-induced liver injury (DILI) during treatment may be difficult

14 13 for investigators as well as regulatory authorities. It is important to institute monitoring in clinical trials that is adequate to recognize ALT flares early enough to avoid severe clinical consequences, and stringent stopping criteria should be included in clinical trial protocols. Considering the importance of distinguishing DILI from hepatic flares, natural disease progression, and virologic relapse in CHB clinical trials, a separate HBV Forum working group has been convened to draft consensus recommendations for liver safety assessment and management. Guiding Principles for Working with Novel Combinations The development of finite CHB therapies with the goal of functional cure must be appropriately balanced by concern for the safety of clinical trial participants. To achieve the proper balance, we propose that the principles included in Table 1 be consistently applied. These principles are further elaborated upon in the supplement. Conclusion The Forum for Collaborative Research provides an open, highly adaptive, and iterative platform for experts from industry, academia, patient advocacy, and regulatory authorities to facilitate a thorough discussion among relevant

15 14 stakeholders regarding the challenges of drug development for the treatment of CHB. The recommendations and proposed guiding principles presented here are based on the expertise of working group members and supported by non-clinical and clinical data cited in this manuscript. These considerations represent the consensus view of the stakeholders. It is our intention that this information will inform clinical trial design and help expedite clinical development of novel therapies. Definitive guidance and clarifications pertaining to the design of clinical investigations can be obtained by consulting with regulatory agencies during the development process. Disclaimer This article represents the opinions of the authors and does not represent the official policy or views of the FDA, the EMA/CHMP or the Swedish Medical Products Agency (MPA). Appendix A Working Group Members Ibironke Addy, MBBS, MSc, AiCuris; Nezam Afdal, MD, Spring Bank Pharmaceuticals; Ryan Taylor Anderson, MS, Forum for Collaborative Research; Tanvir Bell, MD, US Food and Drug Administration; Carol Brosgart, MD, University of California, San Francisco; Nathaniel Brown, MD, Hepatitis B Foundation; Eric Donaldson, PhD, US Food and Drug Administration; Jordan Feld, MD, PhD, Toronto Western Hospital

16 15 Liver Center; Anuj Gaggar, MD, PhD, Gilead Sciences, Inc.; Ed Gane, MBChB, MD, FRACP, MNZM, Auckland City Hospital; Bruce Given, MD, Arrowhead Pharmaceuticals; Pedro Goicochea, MSc, MA, Forum for Collaborative Research; Radhakrishnan Iyer, PhD, Spring Bank Pharmaceuticals, Inc.; Chris Kukka, Hepatitis B Foundation; Pietro Lampertico, MD, PhD, University of Milan; Seng Gee Lim, MD, National University of Singapore; Stephen Locarnini, PhD, Victorian Infectious Diseases Reference Laboratory; Uri Lopatin, MD, Assembly Biopharmaceuticals; Mala Maini, PhD, University College of London; Eduardo Bruno Martins, MD, DPhil, Consultant; Patricia Mendez, MD, Arbutus Biopharma, Inc.; Veronica Miller, PhD, Forum for Collaborative Research; Jules O Rear, PhD, US Food and Drug Administration; Sandra Palleja, MD, PPD, Inc.; Daniela Paulsen, AiCuris; Jean-Michel Pawlotsky, MD, PhD, Henri Mondor University Hospital; Kimberly Struble, PharmD, US Food and Drug Administration; David Suhy, PhD, Benitec Biopharma Ltd; John Sullivan-Bolyai, MD, MPH, ContraVir Pharmaceuticals, Inc.; Andrew Vaillant, PhD, Replicor; Cynthia Wat, MFPM, Roche; Kelly Wong, PhD, Novartis Institutes for BioMedical Research; Teresa Wright, MD, AGAF, San Francisco Veterans Affairs Hospital.

17 16 References: 1. Honda K, Seike M, Murakami K. Benefits of nucleos(t)ide analog treatments for hepatitis B virus-related cirrhosis. World J Hepatol 2015;7: Li WC, Wang MR, Kong LB, et al. Peginterferon alpha-based therapy for chronic hepatitis B focusing on HBsAg clearance or seroconversion: a metaanalysis of controlled clinical trials. BMC Infect Dis 2011;11: Kim GA, Lim YS, An J, et al. HBsAg seroclearance after nucleoside analogue therapy in patients with chronic hepatitis B: clinical outcomes and durability. Gut 2014;63: Lok AS, Zoulim F, Dusheiko G, et al. Hepatitis B cure: From discovery to regulatory approval. J Hepatol 2017;67: Lok AS, McMahon BJ, Brown RS, Jr., et al. Antiviral therapy for chronic hepatitis B viral infection in adults: A systematic review and meta-analysis. Hepatology 2016;63: Papatheodoridis G, Vlachogiannakos I, Cholongitas E, et al. Discontinuation of oral antivirals in chronic hepatitis B: A systematic review. Hepatology 2016;63: EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67: Chong CH, Lim SG. When can we stop nucleoside analogues in patients with chronic hepatitis B? Liver Int 2017;37 Suppl 1: Ghany MG, Doo EC. Antiviral resistance and hepatitis B therapy. Hepatology 2009;49:S

18 Su TH, Liu CJ. Combination Therapy for Chronic Hepatitis B: Current Updates and Perspectives. Gut Liver Lim SG, Aung MO, Chung SW, et al. Patient preferences for hepatitis B therapy. Antivir Ther 2013;18: Nassal M. HBV cccdna: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 2015;64: Wooddell CI, Yuen MF, Chan HL, et al. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci Transl Med 2017; FDA. Codevelopment of Two or More New Investigational Drugs for Use in Combination, Dawood A, Abdul Basit S, Jayaraj M, et al. Drugs in Development for Hepatitis B. Drugs Pham EA, Perumpail RB, Fram BJ, et al. Future Therapy for Hepatitis B Virus: Role of Immunomodulators. Curr Hepatol Rep 2016;15: Paller CJ, Bradbury PA, Ivy SP, et al. Design of phase I combination trials: recommendations of the Clinical Trial Design Task Force of the NCI Investigational Drug Steering Committee. Clin Cancer Res 2014;20: Fung SK, Lok AS. Hepatitis B virus genotypes: do they play a role in the outcome of HBV infection? Hepatology 2004;40: Lin D, Yang HI, Nguyen N, et al. Reduction of chronic hepatitis B-related hepatocellular carcinoma with anti-viral therapy, including low risk patients. Aliment Pharmacol Ther 2016;44:

19 Honer Zu Siederdissen C, Maasoumy B, Cornberg M. What is new on HBsAg and other diagnostic markers in HBV infection? Best Pract Res Clin Gastroenterol 2017;31: Gehring AJ. New treatments to reach functional cure: Rationale and challenges for emerging immune-based therapies. Best Pract Res Clin Gastroenterol 2017;31: Chang ML, Liaw YF. Hepatitis B flares in chronic hepatitis B: pathogenesis, natural course, and management. J Hepatol 2014;61: Nair S, Perrillo RP. Serum alanine aminotransferase flares during interferon treatment of chronic hepatitis B: is sustained clearance of HBV DNA dependent on levels of pretreatment viremia? Hepatology 2001;34: Chang TT, Gish RG, de Man R, et al. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B. N Engl J Med 2006;354: Marcellin P, Heathcote EJ, Buti M, et al. Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B. N Engl J Med 2008;359: Author names in bold designate shared co-first authorship.

20 19 Table 1. Guiding Principles for Combination Drug Development in CHB 1. Provide a solid scientific rationale for pursuing the proposed combination based on appropriate non-clinical studies. 2. Perform a careful comparison of the nonclinical toxicology findings with the candidate combination drugs to determine if target organs of toxicity overlap. Where appropriate, conduct combination toxicology evaluations. 3. Review clinical adverse events and/or pharmacologic effects information to evaluate areas of overlapping toxicity. If found, address the potential impact on the clinical trial design to assure patient safety and whether additional non-clinical evaluations are indicated. 4. Review the routes and mechanisms of absorption, metabolism, and elimination of the candidate drugs to assess the potential for PK drug interactions. Likewise, assess the impact of each drug on metabolic pathways and transporters, again with an eye toward identifying any possible PK interactions. This work might identify the need for non-clinical or clinical assessments of such interactions. 5. Confront the issue of anticipated ALT increases and any special concerns, such as known potential for hepatotoxicity. Assure that the protocol includes appropriate measures for the early recognition and evaluation of such increases and appropriate stopping rules to ensure safety of trial participants. 6. Exclude patients with advanced cirrhosis until combinations have demonstrated adequate efficacy and an acceptable safety profile. Furthermore, patients with decompensated cirrhosis (Child-Pugh B/C) should not be an initial clinical target for HBV cure trials until safety in compensated cirrhosis has been established.

21 Supplementary Material 1 Nonclinical Safety Considerations for Experimental Agents and Drug Combinations HBV is a DNA hepadnavirus with a complex replication cycle, including establishment of an episomal structure, often referred to as a mini-chromosome in the nucleus of host hepatocytes 1. A complex HBV virus-host interaction and replication cycle contributes to low rates of functional cure for CHB infection, spontaneously or in response to therapy. Several therapeutic agents targeting multiple steps of the HBV replication cycle, and/or seeking to improve the host response to HBV infection, are in nonclinical and clinical development. These agents can be broadly classified into four categories based on their intended mode of action: 1) direct-acting anti-viral agents, 2) agents aimed at modifying the host innate immunity and/or adaptive anti-hbv T and B cell responses, 3) agents for selective sensitization of HBV infected hepatocytes to immune elimination, and 4) agents directly targeting cccdna 2. There is currently no nonclinical model available that has been shown to predict likely efficacy in achieving functional cure in humans. Chimpanzees remain the only immunocompetent nonhuman model system fully susceptible to HBV infection, but are no longer available for study due to concerns regarding ethical implications 3. Related viruses infect ducks and woodchucks. Such models are often studied with new agents, but their relevance to humans remains uncertain. Nonetheless, a promising drug candidate that has good antiviral activity in

22 nonclinical studies and no disqualifying safety signals in animals can be used in 2 humans to determine efficacy and safety. A discussion of the strengths and limitations of nonclinical models used in HBV, especially with regard to various new drug classes and viral targets, can be found elsewhere and is beyond the scope of this report 4, 5. In most instances, demonstrating the nonclinical equivalent of functional cure will likely not be possible. For the treatment of chronic HBV with combination therapy, the safety of combining antiviral agents and characterization of the toxicology, at present, are the key nonclinical consideration. As such, novel combinations should be scrutinized for any potential PK or PD interactions that could have safety implications 6. Choosing appropriate drug combinations, and deciding the extent of nonclinical evaluations of these combinations, should include 3 major considerations: 1. A comparison of nonclinical toxicology findings for drugs to be combined, 2. A review of clinically-relevant adverse events (if available) and/or pharmacologic effects to identify any potential overlap between drugs, 3. An identification of the routes of metabolism and clearance for the candidate drugs, and the potential for PK drug-drug interactions. The HBV Forum has not been tasked to consider the nonclinical activity and safety requirements for individual new drugs in development prior to their first use in humans. In any event, such guidance is already available to drug developers in ICH guidance documents 7, 8.

23 3 Additional Considerations and Challenges for Clinical Trials of HBV Combination Therapies New drugs coming into clinical development will in the general case be initially explored as single agents in healthy volunteers. The principles of first-in-human trials are well described in ICH guidance 9. The well demonstrated safety and tolerability of NrtIs constitutes a high bar with respect to safety assessments for new agents. The benefit-risk assessment would take into consideration the therapeutic effect of the new agent in the context of current treatment options available. Early stage clinical studies are likely to either focus on specific subpopulations of CHB patients or may include both HBeAg-positive and -negative patients, but those on therapy will be addressed separately. Anti-viral activity should be studied across genotypes for novel therapies. While pan-genomic efficacy would be ideal, there may be scenarios where a drug could be approved for a specific genotype. SVR24 originated as an endpoint for studies investigating chronic HCV infection and is likely also appropriate for CHB 10. It is possible that extending the SVR assessment to a later timepoint, such as 48-weeks off-treatment (SVR48), may be necessary for some drugs. Indeed, there is increasing evidence that low levels of qhbsag may improve potential for HBsAg loss, and this is most evident in patients who lose HBsAg after

24 stopping long-term NrtI therapy 11. In a small randomized control trial, stopping 4 NrtIs was associated with greater reduction in qhbsag and HBsAg loss in four patients (19%) while those who continued NrtIs had no patients with HBsAg loss and less reduction in qhbsag 12. However, it is yet to be demonstrated that there is immunological recovery with low qhbsag and the level where this recovery occurs is yet to be established. Integrated viral genomes producing HBsAg is a challenging subject and it is likely that some patients are not able to achieve HBsAg loss due to viral integration 13. Identifying these patients will no doubt be a subject of future research within the field. It may also not be possible to distinguish between eradication or silencing of viral genomes, hence the goal of removal of these viral sequences is quite aspirational as we do not have the tools to do so currently. Sponsors are encouraged to work with regulatory authorities to develop strategies for addressing these issues in their clinical development programs. Of particular note, normalization of ALT is important to indicate the improvement of necroinflammatory activity that is anticipated as a consequence of successful therapy. However, it is recognized that due to the high incidence of obesity-driven non-alcoholic fatty liver disease (NAFLD or NASH) concurrent with CHB, ALT normalization as an endpoint to detect drug activity may be less sensitive in some populations 14, 15. Non-invasive assessment of the severity of liver disease in patients with NAFLD or NASH is currently a challenge for hepatology even in the absence of additional pathologies such as CHB 16. Again, sponsors will need to work with regulatory authorities to develop strategies for addressing this issue.

25 5 The process of drug discovery and combination therapy development is resource intensive, requiring substantial fiscal and temporal commitment. Central to the success of candidates is the difficult balancing act of developing potentially lifesaving drug combinations in an efficient manner, defining adequate nonclinical safety margins before first-in-human trials, and providing appropriate therapeutic evidence to support the use of these regimens in the clinic 17. It remains unknown which drug combinations might lead to a sustained viral response off-treatment, as well as or whether a sequential approach will prove more effective. However, successful combinations may require multiple viral or host targets. Regulatory input at various phases of clinical development can be helpful in outlining a feasible pathway to approval of new drugs and combinations based on the current science of chronic HBV biology. Characterization of 4 Distinct Types of ALT Flares Observed in CHB Patients HBV flares are likely related to immune responses, and have unique considerations for CHB patients 18, 19. The dynamic state of interactions between hepatocytes, HBV, and immune cells during infection and treatment provides the basis for phenomenological characterization of 4 distinct types of ALT flares: spontaneous ALT flares during the natural history of CHB infection, and 3 types of ALT flares observed during patient treatment. Spontaneous ALT flares during the natural history of CHB infection are precipitated by reactive infection, disease progression, or related to host immune

26 response leading to spontaneous HBeAg/HBsAg seroconversion 20. Flares related 6 to reactive infection are important clinically because they are occasionally severe, may occur in viremic individuals with cirrhosis or patients after a curative resection of HCC, and may account for 2% of deaths due to CHB infection 21. One key differentiator in this context is that HBV DNA is usually rising, often accompanied by rising viral antigenemia. Early on-treatment flares, observed during the first 3 months of treatment, are often associated with rapid multi-log10 HBV DNA reductions. Hepatitis flares induced by short course IFN-α treatment of patients with HBV during the second or third month of treatment were found to be the most powerful predictor of sustained response in a retrospective investigation of 121 IFN-treated patients 22. During several phase 3 trials investigating response to NrtI therapy, approximately 10% of patients experienced early on-treatment flares with an ALT increase >10x ULN that correlated with a reduction >2 log10 of serum HBV DNA, and were not associated with hepatic decompensation In a study of patients who developed spontaneous ALT flares with hyperbilirubinemia, increased mortality was associated with higher baseline HBV DNA levels, coagulopathy, lower platelet count and older age 26. With early on-treatment flares, absolute ALT levels can sometimes be quite high (ALT levels ranging from 1-3,000 IU/L), but these flares are generally not associated with declining hepatic synthetic function (decreasing albumin or INR) or declining excretory function (increasing bilirubin). As it is likely that multi-targeted regimens will produce early on-treatment flares with greater frequency and, possibly, severity, it is important that trials institute close monitoring to recognize patients with early on-treatment ALT flares.

27 7 It is important to note that this perspective for management of early on-treatment ALT flares with NrtI and IFN therapies may not prove optimal for agents with differing mechanisms of action, or with agents for which non-clinical toxicology or early clinical data suggest an appreciable potential for hepatoxicity. The HBV community is intrigued but also concerned regarding what sort of ALT flares may be seen with immunomodulatory agents that enhance cytotoxic immune responses to HBV-infected hepatocytes. There is a perceived risk of hepatic decompensation in circumstances of potent killing of infected hepatocytes, or when hepatic functional reserve is limited by advanced fibrosis. It therefore seems more appropriate to study patients with sufficient hepatic reserve, deferring the study of cirrhotic CHB patient population until the safety profile of new investigational agents is better understood. Note that DILI in general also often presents in the first 3 months of treatment 27. Later on-treatment flares (>3 months from start of treatment) can be related to viral breakthroughs, to DILI in general, to host immune response initiated by effective therapy, or to adventitious causes. Following the emergence of viral drugresistance during long-term NrtI therapy, ALT increases gradually and there is an increased likelihood of flare, hepatic decompensation, or liver failure 18. Later onset ALT flares are also associated with immune restoration related to treatment response and following immunosuppression during pregnancy or following certain treatments for various diseases in patients with coexisting maladies. Patients presenting with ALT increases during this time period on-treatment require close monitoring and diagnostic evaluations. Treatment interruption or discontinuation

28 is recommended for patients with biochemical evidence of hepatic 8 decompensation at any time during clinical trials. Post-treatment flares often occur within 6 months following treatment cessation. ALT flares following therapy that are related to the return of viral replication are preceded by increasing serum HBV DNA levels, are likely to be due to immune responses, and the temporal relationship between rising ALT and HBV DNA levels is similar to what is observed during spontaneous hepatitis B flares 19, 28. Posttreatment flares can occasionally be severe, possibly leading to hepatic decompensation or even death (albeit, rarely) if not retreated in time 29. Additional Considerations for the Table 1 Guiding Principles Principle 1. Non-clinical evidence supporting the proposed combination should include the mechanism of action (when known), establishing antiviral activity, defining cell culture-based resistance and cross-resistance, and showing no antagonism in cell culture-based combination studies. Principle 2. This is especially important if the drugs are to be co-administered and target organ toxicity overlap exists, consider conducting pilot combination toxicologic experiments of sufficient duration to cover the onset of overlapping toxicities with both agents to determine if there is as additive or synergistic toxicological effect.

29 Principle 3. If overlapping toxicity is found, these adverse events may influence the 9 safety monitoring plans to include specific evaluations to be conducted within the clinical trial, and may influence such elements as starting doses, titration rules, and stopping rules. In some instances, prior clinical trial information might also point to the need for conducting combination toxicology work. Principle 5. Effective treatment combinations may produce more frequent, and possibly more exaggerated, flares than encountered typically with currently available drugs. This issue will be particularly complicated when one or more drugs in the combination has a hepatic toxicity signal in nonclinical toxicology studies. As such, the management of flares is an issue that should be predefined in the trial protocol thoroughly. Mechanisms should be included in trial procedures to be certain that flares are identified early, followed carefully as specified in protocol and that experimental therapy is terminated if these flares are associated with emergent signs of symptoms of changes in liver synthetic function, or stopping rules are met.

30 10 References: 1. Nassal M. HBV cccdna: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 2015;64: Levrero M, Testoni B, Zoulim F. HBV cure: why, how, when? Curr Opin Virol 2016;18: Wieland SF. The chimpanzee model for hepatitis B virus infection. Cold Spring Harb Perspect Med 2015;5. 4. Dandri M, Petersen J. Animal models of HBV infection. Best Pract Res Clin Gastroenterol 2017;31: Iannacone M, Guidotti LG. Mouse Models of Hepatitis B Virus Pathogenesis. Cold Spring Harb Perspect Med 2015;5. 6. Lodola A. Developing combination drugs in preclinical studies. Methods Mol Biol 2011;691: International Conference on Harmonisation; Guidance on M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals; availability. Notice. Fed Regist 2010;75: International Conference on Harmonisation; addendum to International Conference on Harmonisation Guidance on S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals; availability. Notice. Fed Regist 2012;77: General Considerations for Clinical Trials. Fed Regist 1997;62:66113.

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32 19. Tan AT, Koh S, Goh W, et al. A longitudinal analysis of innate and adaptive 12 immune profile during hepatic flares in chronic hepatitis B. J Hepatol 2010;52: Perrillo RP. Acute flares in chronic hepatitis B: the natural and unnatural history of an immunologically mediated liver disease. Gastroenterology 2001;120: Liaw YF, Chien RN, Yeh CT, et al. Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy. Hepatology 1999;30: Nair S, Perrillo RP. Serum alanine aminotransferase flares during interferon treatment of chronic hepatitis B: is sustained clearance of HBV DNA dependent on levels of pretreatment viremia? Hepatology 2001;34: Chang TT, Gish RG, de Man R, et al. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B. N Engl J Med 2006;354: Lai CL, Gane E, Liaw YF, et al. Telbivudine versus lamivudine in patients with chronic hepatitis B. N Engl J Med 2007;357: Marcellin P, Heathcote EJ, Buti M, et al. Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B. N Engl J Med 2008;359: Hsu YC, Wu CY, Chang CY, et al. Pretreatment viral DNA stratifies mortality risk in patients receiving antiviral therapy for severe acute exacerbation of chronic hepatitis B. Antivir Ther 2013;18: Iorga A, Dara L, Kaplowitz N. Drug-Induced Liver Injury: Cascade of Events Leading to Cell Death, Apoptosis or Necrosis. Int J Mol Sci 2017;18.

33 28. Honkoop P, de Man RA, Niesters HG, et al. Acute exacerbation of chronic 13 hepatitis B virus infection after withdrawal of lamivudine therapy. Hepatology 2000;32: Zhang NP, Reijnders JG, Perquin M, et al. Frequency and clinical outcomes of flares related to nucleos(t)ide analogue therapy in patients with chronic hepatitis B. J Viral Hepat 2011;18:e Author names in bold designate shared co-first authorship.