Entry inhibition. Stephan Urban, PhD

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1 Medical Faculty Heidelberg Entry inhibition Stephan Urban, PhD Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Germany German Center for Infectious Diseases (DZIF), TTU Hepatitis, Heidelberg, Germany

2 OPINIONS EXPRESSED DO NOT REFLECT ANYONE S POSITION BUT MY OWN I HAVE FINANCIAL RELATIONSHIPS WITHIN THE LAST 12 MONTHS RELEVANT TO MY PRESENTATION WITH: GILEAD, HUMABS, BMS, GALAPAGOS; MSD, MYR-GMBH. I AM CO-APPLICANT AND CO-INVENTOR OF PATENTS PROTECTING MYRCLUDEX B FOR THE USE AS HBV/HDV ENTRY INHIBITORS

3 Entry of HBV and HDV into hepatocytes HBV and HDV share the same envelope proteins and use identical receptors Attachment to Heparan Sulfate Proteoglycans (HSPG) low specificity; S- and pres-dependent; prerequisite for NTCP binding 2. Release of the NTCP binding site within the L-protein at the hepatocyte membrane or after endocytosis High affinity binding to NTCP sensitive against NTCP substrates (Myrcludex B) 4. Membrane fusion and nucleocapsid release requirement of additional host factors Urban, Bartenschlager, Kubitz & Zoulim, Gastroenterology, 2014; Lempp & Urban, Intervirology, 2014 Yan et al., elife 2012

4 Infectivity determinants within the HBV envelope proteins Urban et al., Gastroenterology, 2014; N-terminal Myristoylation of the L-protein The N-terminal 75 pres1 amino acids The antigenic loop of the S-domain a hydrophobic cluster in the TM1 of the S-domain

5 Entry inhibitors and their targets 1. Neutralizing antibodies: Target the antigenic loop of the S-domain or N-terminal epitopes in the pres1-domain no neutralizing antibodies against cellular targets (e.g. NTCP) are available so far highly specific; some recognize all genotypes, neutralization of SVPs required; intravenous/intraperitoneal administration 2. Attachment inhibitors: Negatively or positively charged drugs that bind the virus (e.g. heparin) or cellular HSPGs (e.g. poly-lysin) efficient, since HSPG-interaction is a prerequisite for pres-mediated NTCP binding; not very specific 3. Substrates of NTCP: conjugated bile salts (e.g. TCA) or small molecules (e.g. irbesartan) that are transported by NTCP orally available; very high concentrations required; very short half-life time at the receptor; no clinical use unless dead end substrates available 4. Irreversible NTCP inhibitors: Myrcludex B, Cyclosporin A and derivatives. allosteric inhibitors of NTCP; irreversibly block receptor function at non saturating concentrations; long half life time at the receptor, some have high specificity some not; block transport of bile salts and other NTCP substrates at higher concentrations; no small molecule available so far. Lempp & Urban, Intervirology, 2014

6 General considerations for the use of entry inhibitors for HBV and HDV Prophylactic use Prevent vertical transmission from mothers to newborns Prevent reinfection after liver transplantation/post exposure prophylaxis Prevent flares under immune suppressive therapy Chronic setting Protect naïve and regenerated hepatocytes from de novo cccdna/hdv RNA formation Sustained entry inhibition bears curative potential, either alone or in combination with strategies aiming at eliminating infected cells. (Mailly et al., Nat Biotechnol. 2015) Does entry inhibition contribute to clearance of cccdna or HDV RNA in chronically infected patients?

7 Myrcludex B as a specific inhibitor of NTCP Myrcludex B Myrcludex B, shows strong inhibitory potential for HBV and HDV infection (IC 50 ca 80 pm in PHH). It specifically inhibits NTCP at the basolateral membrane of differentiated hepatocytes (t 1/2 = 16 h). It exclusively targets parenchymal liver cells (ideal pharmacokinetics).

8 Myrcludex B blocks HBV infection of primary human hepatocytes (PHH) with picomolar IC 50 Infection of PHH with HBV in the absence and the presence of 100 nm Peptide; detection of newly synthesized intracellular HBsAg day 15 p.i. no peptide 5% Myrcludex B treated (IC 50 : 80 pm) HBsAg = day 15 p.i. DAPI = nuclei Myrcludex B completely blocks cccdna formation in PHH

9 Myrcludex B specifically targets NTCP in the liver Myrcludex B control peptide WT +/- knock-out WT Slijepcevic et al, Hepatology, 2015

10 Myrcludex B blocks de novo infection and spread in humanized mice HBV HBsAg, HBeAg, HBV, Histology PHH upa-scid Myrcludex B Post infection Myrcludex B Prior to infection control Block of establishment Block of spread No amplification of cccdna during Myrcludex B treatment Petersen et al., Nature Biotechnol., 26: (2008) Volz et al., Journal of Hepatology 2013, vol. 58, => Intrahepatic spread of HBV depends on NTCP-mediated de novo infection of hepatocytes.

11 The HBV replication cycle: How to diminish the cccdna pool incoming nucleocapsid reimported nucleocapsid cccdna can be formed from incoming viral rcdna...or by replenishment via de novo synthetized nucleocapsids cccdna What is the contributions of de novo incoming nucleocapsids and intracellularly reimported nucleocapsids to the maintenance of cccdna in the liver?

12 Key questions related to the clinical efficacy of entry inhibitors 1. Does intrahepatic spread of viral templates require de novo entry of virions? 2. Can HBV cccdna be propagated through mitosis of hepatocytes? 3. What is the contribution of de novo synthesized nucleocapsids on cccdna maintenance? 4. What are the turnover rates of HBV- and HDV/HBV-infected hepatocytes? 5. Are there differences in the half-life times of naïve, HBV- or HDV-infected hepatocytes? Yes No Minor?? Yes Many of these basic questions are still unresolved...but we are beginning to get answers that favor entry inhibition as an important concept for reducing cccdna levels!

13 Clinical effects of Myrcludex B in chronically infected patients HDV HBV Bogolomov et al., J. Hepatol Bogolomov et al., unpublished Myrcludex reduces HDV RNA by 1,67log (w24) and HBV DNA by 0,8log (w12) survival of 2.14% of HDV producing cells (w24) and 15,8 % of HBV producing cells (w12) => turnover rates are higher in infected hepatocytes compared to naïve cells => entry inhibition for prolonged times (e.g. 10-fold t 1/2 ) may result in cure

14 Synergism of entry inhibitors with other drugs Expected synergism with entry inhibitors drugs that restore immune recognition of infected hepatocytes or eliminate them immune modulators, engineered T-cells, HBsAg-secretion inhibitors?, cyclosporins? Synergism with IFNa has been clinically demonstrated (Bogolomov et al. 2016) Drugs that block intracellular replenisment of cccdna (e.g. CaMs) A complete block of de novo cccdna formation and cccdna replenishment may have the most pronounced effect on cccdna. Durantel & Zoulim, J. Hepatol., 2016

15 Ongoing Myrcludex B trials in HDV/HBV infected patients Phase 2b in Combination with Tenofovir (TDF) Trial in HDV Infection: MYR 202 Phase 2 in Combination with Interferon Trial in HDV Infection: MYR 203 Patients with cirrhosis, or interferon nonresponders or non-eligible On background on anti-hbv therapy with TDF Primary endpoint HDV RNA negativation or 2log decline at end of treatment 30 pts per arm, 120 in total 20 centers in Germany and Russia Parallel 5: Talk 37 H. Wedemeyer Patients eligible for interferon treatment Combination with PegIFNa Primary endpoint HDV RNA negativation at EoFU Secondary endpoints include HBsAg levels and negativation 15 patients per arm, 60 in total 10 centers in Russia => Results are very promising and the 202 study will be presented at the AASLD meeting 2017.

16 Summary and conclusions Entry inhibitors addressing the NTCP receptor of HBV and HDV show clinical efficacy Virological responses probably reflect the loss of infected hepatocytes Entry inhibition is an efficient way to block de novo HBV cccdna and HDV RNA formation NTCP-blockade result in bile salt elevations in patients without clinical symptoms Outlook and ongoing activities Myrcludex B received orphan drug status by the FDA and EMA Myrcludex B received prime eligibility status from EMA A Phase I study with tenofovir demonstrated safety and no drug drug interference Phase II efficacy trials in Russia and Germany are ongoing (will be reported at AASLD 2017) Development of an oral formulation is in progress Aiming at fast approval for HBV/HDV co-infected patients A combination of Myrcludex B with immune activators or inhibitors that prevent intracellular nucleocapsid replenishment might be a key for curative therapies

17 Thanks, Acknowledgments and Funding Thanks to all the patients and to all people involved in clinical work in Russia Yi Ni, Florian A. Lempp, Katrin Schöneweis, Andreas Schulze, Alexa Schieck, Anja Meier, Stefan Mehrle, Caroline Gähler, Sarah Engelhard, Christa Kuhn, Christina Filzmeyer, Jessika Sonnabend, Christina Kaufman, Shirin Nkongolo, Oscar Lamas, Martina Spille, Stefanie Held, Claudia Tolliver, Matthias Engelke, Berit Lange; Stefan Seitz, Kerry Mills, Paul Schnitzler, Steffen Geiss, Anja Rippert, Franzi Schlund. Ralf Bartenschlager et al., Molecular Virology, Heidelberg A. Alexandrov, Myr-GmbH; P. Bogomolov et al., Moscow Research Clinical Institute H. Wedemeyer & M. Manns, MH Hannover, Kompetenznetz Hepatitis, HepNet Study House W. Haefeli, Clinical Pharmacology, University Hospital Heidelberg (DZIF Clinical trial unit) Walter Mier et al., Nuclear Medicine, Heidelberg M. Dandri et al., University Medical Center Hamburg-Eppendorf, TTU Hepatitis Hamburg Deutsches Zentrum für Infektionsforschung (DZIF) (clinical and accompanying studies) BMBF Innovative Therapieverfahren (preclinical research) HEPATERA, Moscow; Myr-GmbH Burgwedel, High Tech Gründerfonds (industrial partners) DFG, EU, WHO, Landesstiftung Baden Württemberg, CellNetwork-Heidelberg HBIGS, Graduate School, Heidelberg