Endpoints and New Targets for

Curing Hepatitis BProfessor Stephen Locarnini

WHO Regional Reference Laboratory for Hepatitis BVictorian Infectious Diseases Reference Laboratory,

Doherty InstituteMelbourne, Victoria 3000, AUSTRALIA

Disclosure

Gilead Sciences

Inc

Arrowhead Research Corp

Spring Bank Pharmaceuticals,

Inc.

Roche Molecular

AusBioLtd

Janssen (J&J)

Consulting Fees (eg. Advisory Boards)

yes yes yes yes yesContract Research (grant)

yes yes yes

Types of Chronic HBV Control and Cure

Peters, MG & Locarnini, S. 2016. Gastroenterol and Hepatol;13(6):348-356.

Inactive StateSustained, off drug:• No inflammation: normal ALT and liver biopsy• HBV DNA low or undetectable• HBsAg-positiveFunctional Cure (Clinical Resolution)Sustained, off drug:• No inflammation: normal ALT and liver biopsy• HBsAg loss• Anti-HBs gainComplete Cure (Virologic Cure)• All of the above plus• Loss of cccDNA in the liver

New Viral Targets

1. Entry and Pathways of Intrahepatic Spread (Re-Entry)

2. Cytosolic Transport of Nucleocapsids3. cccDNA Generation & Processing

(HBcAg and HBx)4. HBV Transcription5. HBV Nucleocapsid Assembly (HBcAg)6. HBsAg7. Putting It All Together

Electron Microscopy: HBV in Serum

Wang, J et al 2016. J Hepatol;65:700-710

1. Entry & Re-Entry and HBV

• HBV WT• HBV SPLICE VARIANTS• HBV FL RNA

HBx RNA Was Detected Very Early After HBV Infection

Beran, R et al 2017. EASL

*Mock infection. LHB, large HBV surface protein; d, day; h, hour; MHB, middle HBV surface protein; ORF, open reading frame; SHB, small HBV surface protein.

Li, J et al 2016. Hepatol;63(1):11-13

HBV Entry into Hepatocytes

• Internalization via Clathrin-Mediated Endocytosis• IMPORTANT CONCEPT EMERGING: PROTECT CELLS FROM

MULTIPLE ROUNDS OF VIRAL RE-INFECTION (Urban, S)

2. Cytosolic Transport of NucleocapsidsA. Primary infection

– Mature nucleocapsids transported via importin α/β whilst nucleoporin 153 delivers nucleocapsids to nuclear basket where they uncoat at nuclear pore

– Genomic RC DNA enters nucleoplasm with Pol covalently attached and is then converted to PF-RC DNA

– (?) HBV RNA species eg: HBx mRNA released and translated

B. Persistent infection– Intracellular conversion pathway provides low level recycling

of nucleocapsids- replication complexes from hepatocyte cytoplasm

– Multiple rounds of re-infection replenish key intermediates

TARGET: NUCLEOCAPSID DISASSEMBLY WITH CpAM COMPOUNDS: BLOCK cccDNA GENERATION

Key Step 2: Reverse Transcription

Host Enzymes

Covalently Closed Circular

(ccc) DNAHBV RC Genomic DNA

(-) (+)

HBV DNA Polymerase Host RNA

Polymerase II

HBV Reverse Transcriptase

HBV Minus (-) DNA

(-)

Pregenomic HBV (pg) RNA

AAAA

3. Key Step 1: Conversion of RC DNA into cccDNA

1. Removal of RT2. Removal of r3. Ligation of (-) DNA4. Completion of (+) DNA5. Removal of capped RNA6. Ligation of (+) DNA

Bock, T. et al 1994. Virus Genes;8:215

Bock, T. et al 2001. JMB;307:183

Newbold, J and Locarnini, S 1995. J. Virol;69:3350

activated nucleosomal spacing

High Replication Phenotype

Transcriptionally ActiveHigh Viraemia

repressive nucleosomal spacing

Low Replication Phenotype

Quiescent or activeMedium to Low Viraemia

HBV cccDNA is a Minichromosome

“OPEN”

“CLOSED”

HBcAg and HBx are key Components of the HBV Minichromosomes

Bock, T. et al 2001. JMB;307:183.;Belloni, L et al 2009. PNAS;106:19975-19979.;

Guo, YH et al 2011. Epigentics;6:720.;Levrero, M & Zucman-Rossi, J. 2016. J Hepatol; 64(1 Suppl):S84-S101

HBx blocks methylation complexHBcAg binds HBV DNA (CpG Island II)HBx knock-out result in transcriptional arrest of MC

HBx Contributes to Establish an Active cccDNA Chromatin State

Alarcon, V et al 2015. Sci Rep;6:1-11

HBx Promotes Degradation of the Smc5/6 Complex to Prevent Silencing of HBV cccDNA

cccDNA, covalently closed circular DNA; Cul4, Cullin 4; DDB1, damage-specific DNA-binding protein 1; HBV, hepatitis B virus; HBx, HBV X protein; ND10, nuclear domain 10; Smc5/6, structural maintenance of chromosome 5/6 complex Beran, R et al 2017. EASL.

Livingstone et al

HBx Inhibitors: To Silence Minichromosome

• Crystal structure– CUL4A-DDB1 ubiquitin ligase complex

interacts with HBx

• Small molecule inhibitors– in development

HBcAg and Transcription: Preferential Binding of HBcAg to HBV Minichromosome

Bock, CT et al 2001. J Mol Biol;307:183

A B

• HBcAg binds to CpG Island II (Guo, YH et al 2011. Epigenetics;6:720)

• in the presence of HBcAg peak number of nucleosomes increased from 15 to 16, resulting in a 20bp decrease in nucelosomal spacing

• HBc enhances upstream NK-κβ binding thereby promoting activation of Enh II/pregenomic promoter (Kwon, JA & Rho HM. 2002. Biochem Cell Biol;80:445)

Interaction of APOBEC 3A/3B, HBV Core Protein (HBc) and cccDNA

Lucifora, J et al 2014. Science;343(6176):1221-8

Targeting HBV Nucleocapsids

Antimicrob Agents Chemother. 2002 Sep;46(9):3057-60. Yuen M-F, et al. AASLD 2015, San Francisco. #LB-10

Deres et al, Science 2003 Klumpp et al, PNAS 2015

#2 Heteroaryldihydropyrimidines

Destabilization of nucleocapsids

#1 Novel classes of capsid inhibitors based on the 3D structure of HBcNovira, Assembly Biosciences, Janssen, Roche, and othersPhase 1/2 studies with Novira compound (NVR3-778) completed

Lam A, et al. AASLD 2015, San Francisco. #33

[sulphonamide/sulfamoyl benzimide derivatives]

How Can cccDNA be Cleared? An Immunological Perspective

Fourel, I et al 1994. J Virol;68:8321.; Guo, JT et al 2000. J Virol;74:1495.Thimme, R et al 2003. J Virol;77:68.; Guidotti, L et al 1999. Science;284:825

CytolyticNon-Cytolytic

How Can cccDNA be Cleared? A Virological Perspective

• directly purge all of the pre-existing cccDNA [topoisomers] (Summers, J and Mason, WS. 2004. Proc Natl Acad Sci (USA);101(2):638-640)

OR• permanently silence cccDNA (condensed

chromatin) transcription (Locarnini, S and Newbold, J. 1997. J Antimicrob Chemoth;39:559-562)

• “cccDNA targeting” small molecule antivirals. eg: di-substituted sulfonamides(Cai, D et al 2012. Antimicrob Agents Chemoth;56:4277)

Direct cccDNA Destruction: Molecular Based Therapies

• cleave cccDNA moleculesVIAcccDNA sequence-specific endonuclease using

– zinc-finger nucleases (Hoeksema, KA & Tyrell, DL. 2010. Meth MolBiol;649:97-116)

– transcription activator-like effector nucleases (TALENS) (Chen, J et al 2014. Mol Ther;22:303-311)

– CRISPR/Cas9 technology (Seeger, C & Sohn, JA. 2014. Mol TherNucleic Acids;3:e216)

• clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform for efficient gene knockout

• can inhibit HBV replication up to eight fold• inhibition due to introduction of mutations and

deletions in cccDNA from recruited Cas9 repaired by non-homologous end joining (NHEJ)

• inhibition not affected when IFN-α used in combination

• independently confirmedKennedy, EM et al 2015. Virology;476:196.

Lin, S-R et al 2014. Mol Ther Nuc Acids;3:e186.

Modulating Transcription: Epigenetics• Structure of Chromatin

– altered by post-translational modification of histones– acetylation, phosphorylation, methylation and ubiquitylation

• Relaxed Chromatin: Histone Acetylase (HAT)

– transcription activation complex containing HATs– HATs acetylate lysine residues of the histone tails

• Compacted Chromatin: Histone Deacetylases (HDAC)

– transcription repression complex containing HDAC– HDACs deacetylate histone lysine tails

• Conclusion– acetylation status of HBV minichromosome (cccDNA-bound H3 & H4

histones) regulates HBV transcription/replication and is reflected in viral load

Activation of Gene Expression

Repression of Gene Expression

Pollicino, T. et al 2006. Gastroenterology;130:823

Haematologica. 2009;94(11):1618-22

Antiviral activity of GS-5801, a liver-targeted prodrug of a lysine demethylase-5 inhibitor, in an HBV primary human hepatocyte infection model

Gilmore S, et al. EASL 2017, Amsterdam. #SAT-160

AIM: To characterize the relationship between the PD effect (increase in H3K4 methylation) and antiviral activity of GS-5801 in primary human hepatocytes

HMT, histone methyltransferases; K4, lysine 4; KDM5i, lysine demethylase-5 inhibitor; Me, methyl.

Wang, J et al 2016. J Hepatol;65:700-710

4. HBV Replication: RNA PathwayModel on the Production of Enveloped

pgRNA Virion and its Infectious Potential

HBV SPLICING / PACKAGING

HBV Transcription

The RNA Sensor RIG-I Dually Functions as an Innate Sensor and Direct Antiviral Factor

for Hepatitis B Virus

• RIG-I senses the HBV genotype A, B, and C for the induction of type I and III IFNs

• The 5’-ε region of HBV pgRNA is a key element for the RIG-I mediated recognition

• RIG-I counteracts the interaction of HBV polymerase with pgRNA to suppress viral replication

• Type III IFNs are predominantly induced in human hepatocytes during HBV infection

Sato et al., 2015, Cell Immunity 42, 123–132

Inarigivir (SB9200)• Small molecule nucleic acid

hybrid (SMNH)• Orally bioavailable prodrug• Active metabolite SB9000 • Actively transported into

hepatocytes via OATP1 • 30:1 liver to plasma ratio• Not metabolized, not

phosphorylated. • Biliary excretion of intact

molecule• No direct activity against DNA

polymerase

Antiviral Activity of SB9200 (Inarigivir) HBV RNA and HBcrAg Profile of ACHIEVE Trial

See AASLD Abstract #39 and Late Breaker Poster

In HBeAg-NEG group [3 log rapid decline HBV RNA ~1 log gradual decline HBV DNA]

kU/m

L

Mechanism of RNA Interference (RNAi)

Natural Process of

RNAi

cleaved mRNA

Selective GeneSilencing

mRNAdegradation

dicerdicerdsRNA

siRNAs

cleavage

RISC

strand separation

cleavage

Therapeutic Gene Silencing complementary pairing

mRNA(A)n

SyntheticsiRNAs

29

Yuen MF, et al. EASL 2017, Amsterdam. #PS-045

HBsAg reduction in HBeAg-positive patients

Smaller reductions in HBsAg in HBeAg– patients:Mean max –0.7 Log10; max observed –1.4 Log10

Multiple doses of ARC-520 resulted in additional reductions in all markers

Reduction in HBeAg+ patients greater than in HBeAg– patients

Difference reflects reductions in HBsAg from cccDNA in HBeAg+ patients vs. integrated DNA in HBeAg– patients

Immediate reductions in HBsAg in HBeAg+ patients:Mean max –2.2 Log10; max observed –3.1 Log10

7/8 patients reported at least one mild AE; none serious of that led to D/C

Prolonged RNAi therapy with ARC-520 in treatment-naive, HBeAg-positive and negative patients with chronic HBV results in significant reductions in HBsAg

HBsA

g (IU

/mL)

80

100

–1020 40 600

Week

1000

10000

100000

Single dose cohort 7

Extensioncohort 10

Patient 01-7981Patient 01-7982Patient 01-7985First dose of ext.Last dose of ext.

2.4kb S

HBV Transcripts Differ Between HBeAg+ and HBeAg-Chimps PacBio Single Molecule Real-Time (SMRT) Sequencing

DR1

DR2 HBV Poly(A) signal

2.1kb S

HBeAg-(88A010)

HBeAg+(A2A004)

HBeAg-• Majority of S transcripts are

fused at the 3’ end to chimp sequence

• Fusion points typically between DR2 and DR1as expected if transcripts arose from integrated HBV dslDNA

HBeAg+• Most S transcripts terminate

near HBV poly(A) signal as expected

HBV-aligning

HBV non-aligning

S ORFs

S transcripts in HBeAg- chimps often lack target sites for ARC-520

ARC-520 siRNAs

Wooddell, C et al (2016)

Novel findings: Predominant liver HBV DNA differs in HBeAg neg and HBeAg pos chimps

Liver biopsy at initiation of ARC-520 treatment revealed:• Most HBV DNA in liver of HBeAg pos is cccDNA• 500-fold less cccDNA in HBeAg neg

– Only 5% of total HBV DNA in liver in HBeAg neg was cccDNA and total HBV DNA levels were not affected by NUCs

• HBV DNA profile in HBeAg neg chimps is consistent with a high proportion of integrated HBV DNA

• Direct antiviral effect on serum HBsAg, HBeAg, and HBcrAg levels which are substantial

• No rebound to baseline from nadir

A phase 2a study evaluating the multi-dose activity of ARB-1467 in HBeAg-positive and negative virally suppressed patients with HBV

AASLD 2017

Significant reductions in HBsAg with single doses of ARB-1467 0.2 mg/kg and 0.4 mg/kg Stepwise, additive reductions with multiple doses (>1 log10 IU/mL in 5/11 patients with 0.4 mg/kg) No significant differences in serum HBsAg between HBeAg-negative and HBeAg-positive Well tolerated

Safety, n (%)

HBeAg–0.2 mg/kg

(n=6)

HBeAg–0.4 mg/kg

(n=6)

HBeAg+0.4 mg/kg

(n=6) Placebo

(n=6)

Any AE 5 (83) 5 (83) 2 (33) 5 (83)

Grade 3–4 AE 1 (17) 0 0 0

Serious AE 1 (17) 0 0 0

D/C due to AE 0 1 (17) 0 0

Grade 3 or 4 lab abnormalities

4 (67) 5 (83) 4 (67) 4 (67)

Isolated elevated glucose, decreased lymphocytes, and low phosphate seen across all groups, included placebo

0.50

0.00

–0.50

–1.00

–1.501* 29* 57* 85

Day

HBsA

g (lo

g10

IU/m

L)

HBeAg-negative ARB-1467 0.2 mg/kgHBeAg-negative ARB-1467 0.4 mg/kgHBeAg-postive ARB-1467 0.4 mg/kgPlacebo

Efficacy

*dosing

5. Targeting the HBV Nucleocapsid

Heteroaryldihydropyrimidine compound GLS4 regulates both assembly and disassembly of HBV

capsids to inhibit cccDNA formation

Wang J, et al. AASLD 2017, Washington DC. #937

Different classes of capsid assembly modulators

Heteroaryldipyrimidine derivatives(HAP)

Phenylpropenamide derivatives(AT series)

Compounds in evaluationBAY41-4109

HAP-12AT-130

NVR3-778JNJ-379

ABI-H0731ABI-H0808

GLS4JHSHAP_R01 SBA_R01AB-423

Multiple-dose study of GLS4JHS, interfering with the assembly of HBV core particles, in patients infected with HBV

Improved antiviral potency with RTV boosting but no effect on cccDNA markersDing Y, et al. AASLD 2017, Washington DC. #920

Changes in therapeutic effect factors at different time points after treatment

Compared with values at baseline

1

10 40

–3

–5

–1

0

Mea

n H

BV D

NA

chan

ge fr

om b

aslin

e (L

og10

IU/m

L)

0 20 30

–2

–4

1 400

3M

ean

HBe

Ag le

vel

(Log

10IU

/mL)

0 14 29

2

1

4

33

1 40

Time (day)

3

5

Mea

n H

BsAg

leve

l (L

og10

IU/m

L)

0 14 29

4

33 7 40

Time (day)

–40

40

Mea

n AL

T ch

ange

from

bas

elin

e (IU

/mL)

3 14 21

0

–20

60

33

20

29

GLS4JHS/r 240/100 mg QDETV 0.5 mg QD GLS4JHS/r 120/100 mg QD

GLS4JHS/r combination is generally well-tolerated

PK profiles and efficacy support QD dosing

In chronic HBV patients, GLS4JHS/r combination was demonstrated potent and rapid HBV DNA and HBeAg reduction

These findings support further clinical investigation of GLS4JHS/r combination for the treatment of chronic HBV infection

Cohort

Decline from baseline after 28 days

HBV DNA (log

IU/mL)

HBsAg (log

IU/mL)HBeAg

(PE IU/mL)ETV –3.5 –0.33 –0.43

GLS4JHS/r 120/100 –1.42 –0.06 –0.25

GLS4JHS/r 240/100 –2.13 –0.14 –0.30

6. HBsAg Targeting Strategies

• HBsAg clearance an endpoint of therapy• Decline in HBsAg levels may restore the

antiviral activity of exhausted T cells• Several strategies in evaluation

-RNA interference (SiRNA): « gene silencing »

-Nucleic acid polymers (NAPs): HBsAg release

-HBs antibodies

Immune Regulation by HBsAg

• HBsAg secreted in vast excess over virions (>103 fold)

• Circulate in blood 100-400 µg/ml(1% of total serum protein)

• Unique conformational structure (8 cysteines and 8 prolines )

• Associated with increased risk of HCC (Yuen, MF. et al 2008. Gastro;135:1192–1199)

• Plays a key role in HBV persistence

• Suppress both innate (TLR-2, TLR-9 and IFN-α) as well as adaptive (mDC) responses to infection

Wang, S et al 2013. J Immunol;190:5142.; Xu, Y et al 2009. Mol Immunol;46:2640.; Op den

Brouw, ML et al 2009. Immunol;126:280.

7. Putting It All TogetherPotent

NA

cccDNA Inhibitor

HBV Antigen Inhibitor

Immune Activator

⊕

⊕

⊕/

agent to prevent viral spread and cccDNA re-amplification

safe and selective agent to reduce or silence cccDNA

agent(s) to block/inhibit the HBV life-cycle [entry, cell-spread, capsid assembly, HBx, HBeAg, HBsAg]

agent(s) to activate specific antiviral immune responses or relieve repression/exhaustion of the system

• The medium-term aim for the field is to achieve “functional cure” − HBsAg seroconversion off treatment