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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