Nouvelles approches thérapeutiques dans l’hépatite B - Pr Massimo LEVRERO (Université de Rome)

1. Nouvelles approches thérapeutiques
dans l’hépatite B
Massimo Levrero
Department of Internal Medicine (DMISM) - Sapienza University – Rome
IIT – Sapienza Center for Life-Nanosciences
INSERM U1052 - CRCL - Lyon

2. Disclosures
● BMS: Advisory Board and invited speaker; investigator
● Gilead: Advisory Board and invited speaker; investigator
● Janssen: Advisory Board and invited speaker, investigator
● MSD: Advisory Board and invited speaker; investigator
● Roche: Consultancy; invited speaker
● Tekmira: Advisory Board
● Medimmune: Advisory Board
● Galapagos: Advisory Board

3. - PEG-IFN + NAs
- PEG-IFN “add-on” on NAs
New approaches
- early clinical development
- pre-clinical studies
- target discovery
+ = ??
Licensed drugs
Off-label use of licensed drugs

4. •Functional cure
- off-therapy persistent HBV suppression
make all patients true ”inactive carriers”
- HBsAg loss as preferred endpoint
•cccDNA eradication
- HBsAg loss and anti-HBs seroconversion: surrogate endpoint
Long-term suppression
of viral replication
(DAA)
Viral Suppression
Functional cure (very few)
Eradication ?
Suppression
of viral replication
Immune control
(IFNα)
HBV: concepts about « cure »

5. Long-term suppression
of viral replication
(DAA)
HBV: concepts about « cure »
Viral Suppression
Functional cure (very few)
Eradication
Suppression
of viral replication
Immune control
(IFNα)
long-term to life long therapies
risk of HCC persistslong-term to life long therapies
risk of HCC persists

6. - PEG-IFN in combination with TDF (NIH, Gilead GS-149)
+ = ??
Current initiatives PEG-IFN treatment
- PEG-IFN “add on” in NAs suppressed patients
(ARES, NL; PEGON, Fudan; PEGAN, ANRS; HERMES, Roche IT)

7. NAs and PegIFN used in combination therapy
• should have additive or synergistic activity against HBV
• should have no added toxicity
• may induce cccDNA loss or control and higher rates of HBsAg loss

8. HBsAg Loss With Tenofovir Disoproxil Fumarate Plus Peginterferon Alfa-2a in
Chronic Hepatitis B: Results of a Global Randomized Controlled Trial (GS-149)
On-Treatment Changes in HBsAg Levels
(wk 48)
Marcellin AASLD 2014
HBsAg Loss Over Time (wk 72) HBsAg Loss by hBeAg status and genotype (wk

9. CD8+ T cell NK cell
Partial restoration
PEG-IFNPEG-IFN
HBV NAsHBV NAs
+
Boni, 2001; Boni, 2013
Pre-treatment with NAs

10. PEG-IFNPEG-IFN
HBV NAsHBV NAs
PEG-IFNPEG-IFN
HBV NAsHBV NAs
PEG-IFNPEG-IFN
HBV NAsHBV NAs
PEG-IFNPEG-IFN
HBV NAsHBV NAs
PEG-IFN add-on
ARES (HBe+, ETV) [Brouver, Hepatology 2014]
Increased HBs declineARES (HBe+, ETV) [Brouver, Hepatology 2014]
Increased HBs decline
PEGON (HBe+, NA) [Chi, AASLD 2014]
Increased HBe loss, and HBs decline
PEGON (HBe+, NA) [Chi, AASLD 2014]
Increased HBe loss, and HBs decline
PEGAN (HBe -, NA) [Bourliere, EASL 2015]
Low HBs loss (7.7% IT; 10.7% PP + full IFN)
Low BL HBs predictive
PEGAN (HBe -, NA) [Bourliere, EASL 2015]
Low HBs loss (7.7% IT; 10.7% PP + full IFN)
Low BL HBs predictive
HERMES (HBe -, NA) [Lampertico, AASLD 2014]
Increased HBs decline,
1 pt HBs loss; 2 pts qHBs < 10 UIHERMES (HBe -, NA) [Lampertico, AASLD 2014]
Increased HBs decline,
1 pt HBs loss; 2 pts qHBs < 10 UI

11. Better responses in HBeAg positive patients
Less convincing in anti-HBe positive (genotype D) patients
Better responses in patients with lower HBsAg
(and anamnesis of HBeAg seroconversion)
Better responses in HBeAg positive patients
Less convincing in anti-HBe positive (genotype D) patients
Better responses in patients with lower HBsAg
(and anamnesis of HBeAg seroconversion)

12. Long-term suppression
of viral replication
(DAA)
HBV: concepts about « cure »
Viral Suppression
Functional cure (very few)
Eradication
Suppression
of viral replication
Immune control
(IFNα)
Strategies
Viral targetsImmune system

13. HBV Cure
Strategies
Viral targetsImmune system
CD8+
T cell
B cell
Activate
antiviral
immunity
Deplete or
silence cccDNA

14. HBV cure landscape
Integrated
HBV DNA
Inhibitors of HBsAg
release, Replicor
RNA interference,
Arrowhead, Tekmira,
Alnylam, GSK
Polymerase inhibitors
•Nucleoside analogues, e.g. Gilead, BMS
•Non-nucleoside, e.g. LB80380
Targeting
cccDNA
Entry inhibitors
•Lipopeptides, e.g.
Myrcludex-B
Immune modulation
•Toll-like receptors agonists, Gilead, Roche
•Anti-PD-1 mAb, BMS, Merck
•Vaccine therapy: Transgene, Gilead, Roche Innovio, Medimmune, ITS
Inhibition of nucleocapsid
assembly
Novira, AssemblyPHARMA,
Gilead, Janssen

15. RNAi treatment for chronic hepatitis B

16. RNA-interference (RNAi) mechanism:
role of delivery

17. RNA-interference (RNAi) mechanism:
role of delivery
ARC-520: in phase 2; efficacy vs toxicity signals
Tekmira/Oncor: clinical developmet from Q1 2015ARC-520: in phase 2; efficacy vs toxicity signals
Tekmira/Oncor: clinical developmet from Q1 2015

18. Direct targeting of HBsAg
from Nassal et al, Virus Research 2008
Integrated
HBV DNA
ASSUMPTION: decrease of HBsAg ameliorates “immunologic” incompetence
Unknown: impact on other viral proteins
Interference with cccDNA regulatory loops
Inhibitors of HBsAg release,
Replicor Rep2139
RNA interference,
Arrowhead, Tekmira,
Alnylam, GSK
1
2
WARNING: fate of “unreleased” HBsAg2
1

19. CD4 CD4
CD8
CTL -/+
IFN-γ -/+
IL-2 -/+
Proliferation -/+
T CELL
DYSFUNCTION
NUC treatment and/or
Decline of antigen load
RECOVERY OF T CELL
RESPONSIVENESS
HBsAg CLEARANCE
ANTI-HBs
SEROCONVERSION
CD4 CD4
CD8
CTL ++
IFN-γ ++
IL-2 ++
Proliferation ++
Antigenload
Modified from: Ferrari C. Gastroenterology 2008
TLR7 / 8 AGONISTS
TLR-7 AGONISTS GS-9620GS-9620
Preclinical studies
•HBsAg and HBV-DNA reduction in
woodchucks and chimpanzees
Clinical Development
•Favorable safety profile in healthy
donors and CHB patients
•No clear cut virological responses in
CHB patients
MYD88
IRF7 NFkB
IRF7 IRF7 NFkB
Pro-inflammatoryIFN, ISGs

20. Modified from U. Protzer et al. Nature Reviews in Immunology 2012
BLOCKING INHIBITORY RECEPTORS ON T CELLS
RESTORATION OF THE T CELL FUNCTION BY COMBINED
MANIPULATION OF PD-1/PD-L1 AND CD137/CD137L PATHWAYS
Fisicaro P et al Gastroenterology 2012
PD-1 PATHWAY BLOCKADEPD-1 PATHWAY BLOCKADE
Proof of concept ofProof of concept of αα-PD-1 in Chronic HCV-PD-1 in Chronic HCV
Gardiner et al. 2013. PLoS ONE 8(5): e63818.

22. AAA
TCA
CYCLE
Mt
DNA
FATTY ACIDS
METABOLISM
I
IIIII
IV
V
HEME
BIOSINTHESIS
AMINOACIDS
METABOLISM
METABOLISMMt DNA
transcription
and translation
Electron
transport chain
&
oxidative phosphorylation
Inner membrane translocases (TIMM)
and other transmembrane transporters
Mitochondrial fission and fusion
Mitochondrial dysfunction of exhausted CD8 cells from
chronic patients and its correction by MitoQ
%DepolarizedCD8+cells
p = 0.015
Untreated
control
MitoQ
treated
20
Untreated
control
MitoQ
treated
p = 0.015
Mean%depolarizedcells
uponpeptidestimulation
HealthyChronic
%Depolarizeddextr+cells
-10
0
10
20
30
40
p < 0.001
Unstimulated Stimulated
0.6%2%
1.5%
Chronic
Healthy
JC1 FL-1
JC1FL-2
CD8
Dextramer
34%
Mytochondrial dysfunction of exuasted CD8 cells from
chronic HBV patients and its correction by mytoC
Fisicaro et al. 2015 (submitted)
Res
Chronic
Acute

23. HBV Cure
Strategies
Viral targetsImmune system
CD8+
T cell
B cell
Activate
antiviral
immunity
Deplete or
silence cccDNA

24. HBV cure landscape
Integrated
HBV DNA
Inhibitors of HBsAg
release, Replicor
RNA interference,
Arrowhead, Tekmira,
Alnylam, GSK
Polymerase inhibitors
•Nucleoside analogues, e.g. Gilead, BMS
•Non-nucleoside, e.g. LB80380
Targeting
cccDNA
Entry inhibitors
•Lipopeptides, e.g.
Myrcludex-B
Immune modulation
•Toll-like receptors agonists, Gilead, Roche
•Anti-PD-1 mAb, BMS, Merck
•Vaccine therapy: Transgene, Gilead, Roche Innovio, Medimmune, ITS

25. HBV cccDNA
from Nassal et al, Virus Research 2008
Integrated
HBV DNA
• template for all HBV mRNAs and the HBV pgRNA
• not directly targeted by NUCs
• may exist hepatic “latent” reservoire (non integrated functionally
competent HBV genomes): OBI and inactive carriers

26. Antivirals do not directly target cccDNA
Modified from Nassal et al, Virus Research 2008
?
1 yr of monotherapy with nucleos(t)ide analogues (ADV, LAM, ETV) reduced
median intrahepatic cccDNA amounts by 1 log
Zoulim,Petersen,Locarnini, Gastroenterology 2004,
Wong, Antivir Ther 2006, Sung, Gastroenterology 2005

27. Antivirals do not directly target cccDNA
Modified from Nassal et al, Virus Research 2008
?
1 yr of monotherapy with nucleos(t)ide analogues (ADV, LAM, ETV) reduced
median intrahepatic cccDNA amounts by 1 log
Zoulim,Petersen,Locarnini, Gastroenterology 2004,
Wong, Antivir Ther 2006, Sung, Gastroenterology 2005
Evidence for ongoing low level viremia in patients
with CHB receiving long term NUC therapy
Marcellin , AASLD 2014
Evidence for ongoing low level viremia in patients
with CHB receiving long term NUC therapy
Marcellin , AASLD 2014

28. Persistence of cccDNA
Belloni, Levrero, Gaeta HBV meeting 2010
B2B1 B3 B4 C2C1 N1 N2
pgRNAcp/ngcDNA
0
0.02
0.04
0.06
0.08
0.10
0
0.25
0.50
0.75
1.0
cccDNAcopies/cell
1149.4neg>250
HBsAg
Persistence of cccDNA in 3 out of 4 patients with long term HBV
suppression under lamivudine
In 2 out 3 patients cccDNA is inactive (no pgRNA)
• Detected in the liver of NUCs long-term suppressed patients after HBsAg to anti-HBs
seroconversion [Maynard, 2005; Belloni unpublished]
• Detected in the liver of HBsAg negative patients (occult HBV infection)
[Werle-Lapostolle, 2004; Pollicino unpublished]
• Present in 30 /30 patients with occult HBV infection and HCC [Pollicino, 2004]

29. • antivirals do not directly target cccDNA
• persists in NUC long-term suppressed patients, occult HBV
infection and subjects recovered from AVH
(Werle-Lapostolle 2004; Pollicino 2004; Maynard, 2005; Belloni, 2010)
cccDNA persistence
active cccDNA (pgRNA pos)
inactive cccDNA (pgRNA neg)
cccDNA persistence
active cccDNA (pgRNA pos)
inactive cccDNA (pgRNA neg)
Persistence of cccDNA

30. The HBV cccDNA as a “minichromosome”
• HBV cccDNA is organized as a minichromosome in the nucleus of
infected cells by histone and non-histone proteins
(Newbold 1995, Bock 2001, Pollicino 2006).
Bock, T. et al 1994.
Bock, T. et al 2001
High Replication
Phenotype
Transcriptionally Active
High Viraemia
Low Replication
Phenotype
Quiescent or active
Medium to Low Viraemia
Newbold et al, 1995
Histones
CBPPCAF
p300
CBP
PCAF
p300
High Replication
Pollicino, 2006; Belloni 2009)
Low Replication
Sirt1
Sirt1
HDAC1HDAC1
Histones
Ezh2

31. The HBV cccDNA as a “minichromosome”
• HBV cccDNA is organized as a minichromosome in the nucleus of
infected cells by histone and non-histone proteins
(Newbold 1995, Bock 2001, Pollicino 2006)
Bock, T. et al 1994.
Bock, T. et al 2001
High Replication
Phenotype
Transcriptionally Active
High Viraemia
Low Replication
Phenotype
Quiescent or active
Medium to Low Viraemia
Newbold et al, 1995
Histones
CBPPCAF
p300
CBP
PCAF
p300
High Replication
Pollicino, 2006; Belloni 2009)
Low Replication
Sirt1
Sirt1
HDAC1HDAC1
Histones
Ezh2
Distinct HBV populations of cccDNA
minichromosomes may exist
Distinct HBV populations of cccDNA
minichromosomes may exist

32. Cleavage of cccDNA
by targeted gene disruption strategies
 Zinc Finger [Weber, PlosOne, 2014]
 Talens [Chen, Mol Therapies, 2014]
 Bacterial CRISP / Cas
RNA-guided DNA endonucleases
[Seeger, Mol Therapy Nucl Acids, 2014;
[Lin, Mol Therapy Nucl Acids, 2014;
[Kennedy, Virology, 2015]
CRISP/CAS,
Talens, Zn-fingers

33. Cleavage of cccDNA
by targeted gene disruption strategies
 Zinc Finger [Weber, PlosOne, 2014]
 Talens [Chen, Mol Therapies, 2014]
 Bacterial CRISP / Cas
RNA-guided DNA endonucleases
[Seeger, Mol Therapy Nucl Acids, 2014;
[Lin, Mol Therapy Nucl Acids, 2014;
[Kennedy, Virology, 2015]
CRISP/CAS,
Talens, Zn-fingers
Even if efficacy issues are solved, delivery
may remain unpractical
Even if efficacy issues are solved, delivery
may remain unpractical

34. Lucifora et al. Science 343, 1221-8, 2014
- Interferon-α and lymphotoxin-β-receptor
activation up-regulated APOBEC3A and 3B
cytidine-deaminases, respectively, in HBV-
infected cells, primary hepatocytes and
human liver-needle biopsies.
- HBV-core protein mediates the interaction
with nuclear cccDNA resulting in cytidine-
deamination, apurinic/apyrimidinic site
formation and finally cccDNA degradation

35. Lucifora et al. Science 343, 1221-8, 2014
- Interferon-α and lymphotoxin-β-receptor
activation up-regulated APOBEC3A and 3B
cytidine-deaminases, respectively, in HBV-
infected cells, primary hepatocytes and
human liver-needle biopsies.
- HBV-core protein mediates the interaction
with nuclear cccDNA resulting in cytidine-
deamination, apurinic/apyrimidinic site
formation and finally cccDNA degradation
What about viral proteins ?
can we target cccDNA-bound viral proteins
(HBx and HBc) to modulate cccDNA function?What about viral proteins ?
can we target cccDNA-bound viral proteins
(HBx and HBc) to modulate cccDNA function?

36. HBc protein / capsid
HBV capsid
(120 HBc dimers)
Bock, 2001
HBc dimer
Input
aHBc
IgG
wt
moc
k
aHBc
5
10
1
5
20
ArbitraryUnits
Belloni 2009
 HBc binds the cccDNA and modifies cccDNA nucleosome spacing
Lupacchini (unpublished)
Ezh2
FI%Input
0
2
4
6
IL29
c-Src
E2F2
FI%Input
IL6
FI%Input
 HBc binds to cellular promoters and
regulates gene expression
(Guo, BMC genomics, 2013)
 HBc binds to (and represses) the IFN-b,
IL-29 and OAS1 cellular promoters
(Durantel D, AASLD 2013)
ChIP anti-HBc

37. HBV capsid
(120 HBc dimers)
HBc dimer
Assembly inhibitors
Core Protein Assembly Modulators (CpAMs)
Core inhibitors / Anti-capsid
• HAP12 and AT130 misdirect HBV capsid assembly and block HBV replication.
• AT130 capsids are more “normalish” but inactive; HAP12 leads to aberrant structures
HAP12 AT130
Hetero-aryl-dihydropyrimidines (HAPs) and the
Phenyl-Propenamide derivatives, AT130 are a
new class of antivirals that target the HBV
capsid and inhibit HBV replication in vitro
(Deres, Science 2003; Stray, PNAS 2005).
Allosteric modulation
and focused activity

38. • Core inhibitors (Hap12 and AT130) impact on Cp nuclear functions at multiple
levels:
- block new cccDNA accumulation (Rc-DNA delivery and/or core particles recycling)
- reduce the size of an established cccDNA pool
- inhibit HBc recruitment on the cccDNA
The effects on HBc recruitment on cellular genes remains to be determined
Anti-capsid drugs
Core
inhibitors
1
2, 3
4
1
2
3
4

39. • Core inhibitors (Hap12 and AT130) impact on Cp nuclear functions at multiple
levels:
- block new cccDNA accumulation (Rc-DNA delivery and/or core particles recycling)
- reduce the size of an established cccDNA pool
- inhibit HBc recruitment on the cccDNA
The effects on HBc recruitment on cellular genes remains to be determined
Anti-capsid drugs
Core
inhibitors
1
2, 3
4
1
2
3
4
Core inhibitors are the first “viral specific”
compounds capable to target the cccDNA
Core inhibitors are the first “viral specific”
compounds capable to target the cccDNA

40. Core inhibitors affects the cccDNA
formation/accumulation in HepG2-NTCP infected cells
nt
1μM HAP
NTCP-HepG2 T6
Capsid associated
HBV DNA
Foldinduction
cccDNA
Foldinduction
nt
1μM HAP
NTCP-HepG2 T0
cccDNA
Foldinduction
Foldinduction
Capsid associated
HBV DNA

41. Core inhibitors affects the cccDNA
formation/accumulation in HepG2-NTCP infected cells
nt
1μM HAP
NTCP-HepG2 T6
Capsid associated
HBV DNA
Foldinduction
cccDNA
Foldinduction
nt
1μM HAP
NTCP-HepG2 T0
cccDNA
Foldinduction
Foldinduction
Capsid associated
HBV DNA
In HepG2-NTCP infected cells HAP12 treatment blocks
viral replication, interferes with/prevents cccDNA
accumulation and might reduce the cccDNA pool

42. HBV capsid
(120 HBc dimers)
HBc dimer
Core inhibitors / Anti-capsid
A growing family
Phenylpropenamide derivatives (AT61, AT130) [Gilead]
Heteroaryldihydropyrimidines (HAP-1 and Bay 41-4109)
Sulfamoylbenzamide derivatives (DVR-23, DVR-56 and Novira
Therapeutics NVR-1221) [Novira]
BCM-599 [2-amino-N-(2,6-dychloropyridin-3-yl) [acetamide family]
Isothiafludine (pg-RNA packaging)
Preclinical and Early Clinical Profile of
NVR 3-778, a Potential First-In-Class
HBV Core Inhibitor
Gane, AASLD 2014
Preclinical and Early Clinical Profile of
NVR 3-778, a Potential First-In-Class
HBV Core Inhibitor
Gane, AASLD 2014

43. Klumpp et al. EASL 2015
human
hepatocytes
In vivo
HBV infection
PXB mice
Humanized liver
PXB mice
HBV-DNA
Mock PEG
IFN
NVR
IFN
NVR
ccc-DNA
Mock PEG
IFN
NVR
IFN
NVR
C/pg-DNA
Mock PEG
IFN
NVR IFN
NVR
Preclinical characterization of the antiviral activity of NVR 3-778, a
Potential First-In-Class HBV Core Inhibitor, in vivo

44. p300
P/CAF
TFn1TFn2
Ac Ac
hSirt1/2
Ac Ac
TFn1TFn2
Ac Ac
hSirt1/2
Ac
Ac
Active cccDNA
Suppressed cccDNA
Targeting cccDNA-bound HATs and HDACs
by “epigenetic” compounds
Foldinduction
IPαAcH4
IPαAcH4
Foldinduction
PCAF/p300 inhibitor
Sirt1/2 stimulator
IPαTriMeK27H3
Foldinduction
IPαPCAF IPαp300
EML264
MC2791
IPαSirt1 IPαSirt2
IPαEzh2
JMD3 inhibitor
MC3119
EMs modify chromatin
remodelling enzymes recruitment
onto HBV minichromosome

45. p300
P/CAF
TFn1TFn2
Ac Ac
hSirt1/2
Ac Ac
TFn1TFn2
Ac Ac
hSirt1/2
Ac
Ac
Active cccDNA
Suppressed cccDNA
Targeting cccDNA-bound HATs and HDACs
by “epigenetic” compounds
Foldinduction
IPαAcH4
IPαAcH4
Foldinduction
PCAF/p300 inhibitor
Sirt1/2 stimulator
IPαTriMeK27H3
Foldinduction
IPαPCAF IPαp300
EML264
MC2791
IPαSirt1 IPαSirt2
IPαEzh2
JMD3 inhibitor
MC3119
EMs modify chromatin
remodelling enzymes recruitment
onto HBV minichromosome
Pre-clinical proof of concept stage
Make active carriers „true“ inactive and, eventually, over time
„occult“ carriers by „locking“ the cccDNAPre-clinical proof of concept stage
Make active carriers „true“ inactive and, eventually, over time
„occult“ carriers by „locking“ the cccDNA

46. p300
P/CAF
TFn1TFn2
Ac Ac
hSirt1/2
Ac Ac
TFn1TFn2
Ac Ac
hSirt1/2
Ac
Ac
Active cccDNA
Suppressed cccDNA
Targeting cccDNA-bound HATs and HDACs
by “epigenetic” compounds
Foldinduction
IPαAcH4
IPαAcH4
Foldinduction
PCAF/p300 inhibitor
Sirt1/2 stimulator
IPαTriMeK27H3
Foldinduction
IPαPCAF IPαp300
EML264
MC2791
IPαSirt1 IPαSirt2
IPαEzh2
JMD3 inhibitor
MC3119
EMs modify chromatin
remodelling enzymes recruitment
onto HBV minichromosome

47. p300
P/CAF
TFn1TFn2
Ac Ac
hSirt1/2
Ac Ac
TFn1TFn2
Ac Ac
hSirt1/2
Ac
Ac
Active cccDNA
Suppressed cccDNA
Targeting cccDNA-bound HATs and HDACs
by “epigenetic” compounds
Foldinduction
IPαAcH4
IPαAcH4
Foldinduction
PCAF/p300 inhibitor
Sirt1/2 stimulator
IPαTriMeK27H3
Foldinduction
IPαPCAF IPαp300
EML264
MC2791
IPαSirt1 IPαSirt2
IPαEzh2
JMD3 inhibitor
MC3119
EMs modify chromatin
remodelling enzymes recruitment
onto HBV minichromosome
cccDNA silencing
=
functional cure
cccDNA silencing
=
functional cure

48. HBV cure landscape
Integrated
HBV DNA
Inhibitors of HBsAg
release, Replicor
RNA interference,
Arrowhead, Tekmira,
Alnylam, GSK
Polymerase inhibitors
•Nucleoside analogues, e.g. Gilead, BMS
•Non-nucleoside, e.g. LB80380
Targeting
cccDNA
Entry inhibitors
•Lipopeptides, e.g.
Myrcludex-B
Immune modulation
•Toll-like receptors agonists, Gilead, Roche
•Anti-PD-1 mAb, BMS, Merck
•Vaccine therapy: Transgene, Gilead, Roche Innovio, Medimmune, ITS
Inhibition of nucleocapsid
assembly
Novira, AssemblyPHARMA,
Gilead, Janssen

49. 1. recipient
HBV
2.
recipients
+ Myrcludex-B
d30
post Tx
d90
post Tx
n=4
n=5
no treatment
human
liver donor
HBV-inf.
PHHs
cccDNA copies / hu. hepatocyte
0 10 20 30 40 50 60 70 80 90 100 110
0.001
0.01
0.1
1
10
inkl. 1083!!!
untreat
Myrclud
days post tx
Viremia
50 60 70 80 90 100
10-2
10-1
100
101
102
experiment 2 +Myrc
experiment 2
days post tx
foldchange
[HBVDNA/mlserum]
pregenomic HBV RNA / hu-GAPDH
0 10 20 30 40 50 60 70 80 90 100 110
0.001
0.01
0.1
1
10
inkl. 1083!!!
untreated
Myrcludex-B
days post tx
Cell proliferation combined with antiviral treatment to block re-infection (Myrcludex B)
promoted cccDNA clearance in the majority of the human hepatocytes.
Allweiss, Petersen, Dandri et al, EASL 2014, O101
Entry inhitor plus cell proliferation support loss of cccDNA and HBsAg

50. 1. recipient
HBV
2.
recipients
+ Myrcludex-B
d30
post Tx
d90
post Tx
n=4
n=5
no treatment
human
liver donor
HBV-inf.
PHHs
cccDNA copies / hu. hepatocyte
0 10 20 30 40 50 60 70 80 90 100 110
0.001
0.01
0.1
1
10
inkl. 1083!!!
untreat
Myrclud
days post tx
Viremia
50 60 70 80 90 100
10-2
10-1
100
101
102
experiment 2 +Myrc
experiment 2
days post tx
foldchange
[HBVDNA/mlserum]
pregenomic HBV RNA / hu-GAPDH
0 10 20 30 40 50 60 70 80 90 100 110
0.001
0.01
0.1
1
10
inkl. 1083!!!
untreated
Myrcludex-B
days post tx
Cell proliferation combined with antiviral treatment to block re-infection (Myrcludex B)
promoted cccDNA clearance in the majority of the human hepatocytes.
Allweiss, Petersen, Dandri et al, EASL 2014, O101
Entry inhitor plus cell proliferation support loss of cccDNA and HBsAg
Evidence for ongoing low level viremia in patients
with CHB receiving long term NUC therapy
Marcellin , AASLD 2014
Evidence for ongoing low level viremia in patients
with CHB receiving long term NUC therapy
Marcellin , AASLD 2014

51. HBV Phase 2a Results
HDV Pilot Study

52. Viral
targets
Immune
modulation
Entry inhibition cccDNA
- formation
- stability / destruction
- epigenetic regulation
HBc functions
Other viral targets: HBx etc.
Stimulating innate responses
- Specific ligands
Stimulating adaptive responses
- Co-inhibitory signals
- Co-stimulatory signals
Therapeutic vaccination
Functional cure / control ?
Complete cure ?
What Might HBV Cure Will Look Like?
let’s keep an open mind
Modified from F Zoulim 6.2014

53. Laboratory of Gene Expression
Massimo Levrero
Laura Belloni
Gianna Aurora Palumbo
Leonardo Lupacchini
Francesca Guerrieri
Natalia Pediconi
Ludovica Calvo
Debora Salerno
Silvia di Cocco
Collaborations:
Fabien Zoulim
Barbara Testoni
David Durantel
INSERM U761 -Lyon
Thanks to
Adam Zlotnick
Lichun Li
Department of Molecular & Cellular Biochemistry,
Indiana University, Bloomington, USA
Uri Lopatin
Adam Zlotnick
Assembly Pharmaceuticals
Financial support