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29/09/2014
1
Sacrificing infected cells to promote
cures for chronic infections
Marc Pellegrini
Infection & Immunity Division
Disclosure
• The work that will be presented was conducted at the Walter and
Eliza Hall Institute of Medical Research (WEHI).
• WEHI has a license agreement with TetraLogic Pharmaceuticals
(USA) the manufacturers of birinapant.
• A PCT has been filed by TetraLogic on behalf of WEHI describing
IP relating to the use of birinapant and other drugs to treat chronic
human infections..
• Marc Pellegrini has no investments or interests in TetraLogic.
Promote clearance
of infected cells
Promoting death of infected cells may clear infection Hydrodynamic injection of HBV DNA causes chronic
HBV infection in mice
AAV cis vector
MCS 3’ITR
Promoterless
5’ITR
3’ITR 5’ITR HBV 1.2 overlength genome (3818bp)
MCS
Hydrodynamic injection
pAAV/HBV1.2
Hydrodynamic injection of HBV DNA causes chronic
HBV infection in mice
HBV core-protein is present in hepatocytes
Small surface protein
Medium surface protein
Large surface protein
DNA
Polymerase
Core
HBV-core
DAPI
Liver 3 wk post infection (100X)
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HBV virions are present in the serum
40 nm
Virion
(Dane particle)
Subviral particles
(Spheres)
40 nm
HBV infected mice produce HBsAg in serum
HBV infected mice produce HBeAg in serum
HBeAg (pre-core)
HBV preclinical model mimics chronic
human HBV infection
n = 10
Error bars = s.e.m. Repeated > 3 times
Human HBV lifecycle replicated in mouse
model
HBV control is associated with mild elevations in
serum liver enzymes levels
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Preclinical model recapitulates HBV replicative
lifecycle in human hepatocytes
The levels of serum cytokines are upregulated
during infection
Time post infection (d)
CD8 T cells infiltrate areas of HBV infection
CD8
Liver 4 w post infection 100x
HBV-core
DAP
I
CD4 T cells infiltrate areas of HBV infection
CD4
HBV-core
DAP
I
Liver 4 w post infection 100x
CD8+ T cells may be redundant for the control of
HBV infection
C57BL/6
NK cell
B cell
CD4+
T cell CD8+
T cell
CD8+ T cells may be redundant for the control of
HBV infection
C57BL/6
NK cell
B cell
CD4+
T cell CD8+
T cell
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CD8+ T cells may be redundant because MHC-I is
not upregulated on infected cells
Naive
Poly I:C
HBV-infected
LCMV-infected
MHC-I DAPI HBV Core
CD4+
T cell
CD4+ T cells are essential for the control of
HBV infection
OT–II
NK cell
B cell
CD8+
T cell
Type I interferons do not impact on
HBV viral set point
C57BL/6
Hepatocytes
Immune cell
(n=5)
(n=6)
Type II interferons are essential for early control of
HBV infection
NK cell
B cell
CD8+
T cell CD4+
T cell
IFN–γ KO
Type II interferons are essential for early control of
HBV infection
NK cell
B cell
CD8+
T cell CD4+
T cell
IFN–γ KO
Log-rank (Mantel-Cox) test
**p<0.01
TNF-α is required to control HBV infection
NK cell
B cell
CD8+
T cell CD4+
T cell
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The level of TNF-α is upregulated during infection
Time post infection (d)
Inhibitors of apoptosis (IAPs) may determine fate of
infected hepatocyte
TNF-α /
Fas / Trail TLR / NOD Inflammation
Death receptors Pattern recognition
receptors
IAPs NFκB
activation
Cell survival
Cellular
activity
Active IAPs
Inflammasome
TNF-α /
Fas / Trail TLR / NOD Necroptosis
Death receptors Pattern recognition
receptors
Suppressed IAPs Inflammasome
CELL DEATH
TNFR-1 expression changes during HBV infection Mice deficient in cIAP1/2 but not XIAP control
HBV infection with improved kinetics
cIAP2 deficiency promotes HBV control Enhanced clearance of HBV in cIAP1/2 deficient
mice is dependent on TNF-α
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Enhanced HBV seroconversion in cIAP1/2
deficient mice
C57BL/6 +
cIAP1ΔHep/ΔHepcIAP2-/- +
Analysis 2wks p.inf. (n=5)
Elevated transaminases and cell death in cIAP1/2
deficient mice during HBV clearance
C57BL/6 +
cIAP1ΔHep/ΔHepcIAP2-/- +
Analysis 2wks p.inf. (n=5)
Enhanced immune infiltrates in cIAP1/2 deficient
mice during HBV clearance
Analysis 2wks p.inf. (n=5)
Enhanced immune infiltrates in cIAP1/2 deficient
mice during HBV clearance
Analysis 2wks p.inf. (n=5)
cIAP1/2 deficient mice clear HBV genome in liver
TetraLogic Pharmaceuticals have developed a small
molecule IAP antagonist called birinapant
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Birinapant combined with TNF kills HBV
infected primary hepatocytes
Birinapant antagonises cIAPs in vivo
Birinapant clears HBV infection in vivo
Efficacy of birinapant is dose dependent
CD4+
T cell
CD4+ but not CD8+ T cells are required for
birinapant’s efficacy
OT–II or
MHC-I KO
NK cell
B cell
CD8+
T cell
TNF-α is required for birinapant’s efficacy
NK cell
B cell
CD8+
T cell CD4+
T cell
C57BL/6
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Birinapant mediated HBV clearance associated
with increased T cell responses
Birinapant mediated clearance of HBV infection is
associated with a transaminitis
C57BL/6, analysis 2wks p.inf.
Birinapant causes selective death of HBV infected
hepatocytes in vivo
C57BL/6, analysis 2wks p.inf. 12h p.treatment
Birinapant causes selective death of HBV infected
hepatocytes in vivo
Birinapant clears HBV genome from livers
Birinapant enhances efficacy of
polymerase inhibitors
** p<0.01
*** p<0.001
Log-rank (Mantel-Cox) test
n=6 each
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Birinapant clears infection but polymerase
inhibitors do not
CH3 mice cannot control HBV infection
Birinapant clears HBV infection in C3H mice
(n=5)
(n=5)
(n=5)
Birinapant clears HBsAg in C3H mice
Birinapant progresses to HBV clinical trials
• Double blind placebo control
– Dose escalation / multidose
– Multicentre (Melbourne / Adelaide / Perth / Auckland / Christchurch)
• 6 cohorts of 8 participants / 6 active drug + 2 placebo
• Established on nucleoside/tide analogues
• No more than Child-Pugh score of 5 and normal FibroScan®
• Confinement for 24-48 hours
Design
Inclusion / Exclusion
Birinapant progresses to HBV clinical trials
• Transaminitis / Quantitative HBsAg (HBeAg)
• HBsAb / Serum cytokines / differential cell count / cIAP levels
Primary endpoints
Secondary endpoints
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Birinapant efficacy may be augmented by co-
administration of Trail
TNF-α /
Fas / Trail TLR / NOD Necroptosis
Death receptors Pattern recognition
receptors
Suppressed IAPs Inflammasome
CELL DEATH
Birinapant efficacy may be augmented by co-
administration of Trail
Jesse Toe
Liana Mackiewicz
Gregor Ebert
Simon Preston
James Cooney
Cody Allison
Hamish Scott
Samar Ojaimi
Michael Stutz
Acknowledgements
Ding-Shinn Chen
Pei-Jer Chen
National Taiwan University
VIDRL
Stephen Locarnini
Peter Revill
Ray Czaijko
Joseph Toressi
Nadia Warner
Lynne Waddington CSIRO
Austin Health
Melbourne Health
WEHI
John Silke
Ueli Nachbur
Burnet Institute
Sharon Lewin
Danni Colledge
TetraLogic C. Glenn Begley Stephen Condon
Scott Bowden
Nikola Baschuk Peter McCallum Monash IMR
Paul Hertzog
Paul Cameron
Karey Cheong
Nitasha Kumar
Talia Mota