Combination treatment of a TLR7 agonist RO7020531 and a core protein

allosteric modulator RO7049389 achieved sustainable viral load

suppression and HBsAg loss in an AAV-HBV mouse model

HBV Cure Workshop 2018, Toronto, Canada

Nov 8th, 2018

Yonghong Zhu1, MD, PhD, Translational Project Leader on behalf of the following authors:

Lue Dai1, Youjun Yu1, Xue Zhou1, Lili Gu1, Jie Zhao1, Ying Ji1, Hongying Yun1, Wei Zhu1, John A. T. Young2, and Lu Gao1

1. Roche Innovation Centre Shanghai, Shanghai, China; 2. Roche Innovation Centre Basel, Basel, Switzerland

Background

• 257 million people have chronic hepatitis B virus (HBV) infection which remains a leading cause

of cirrhosis and hepatocellular carcinoma

• While current oral antiviral therapies can suppress HBV DNA levels and prevent disease

progression and complications, most patients require life-long NUC therapy with issues of side

effects, resistance and cost

• Development of a finite HBV cure will likely require combinations of novel compounds which

inhibit HBV replication, reduce antigen production and enhance HBV-specific immune responses

2

Cure

Virus Targeted Approach

CpAM Class I RO7049389

VIRAL CYCLE

Immunoenhancers

TLR7 Agonist RO7020531

ActivationViral-mediated

suppression

Peg-IFN

Existing SoC

Virus Targeted asset

Immunoenhancer asset

Functional impairment of anti-HBV immune responses is a key feature of chronic HBV infection.

Development of a finite HBV cure will likely require combinations of novel compounds which inhibit HBV replication,

reduce antigen production and enhance HBV-specific immune responses3

HBV LNA

Nucleoside

Roche approach toward combination therapy for HBV cure

RO7049389 is an orally administered Class I HBV CpAM

HBV core protein dimers

Class II CpAM

Functional

nucleocapsids

Empty capsids

Class I CpAM

Phenylpropenamide derivatives

Sulfamoylbenzamide derivatives

Aberrant core protein

aggregates that are

subsequently degraded

RO7049389

RO7049389

EC50 (HBV DNA, n=3) 6.1 ± 0.9 nM

CC50 (n=3) > 100 µM

Selectivity Index > 10000

pgRNA·RT

Heteroarylpyrimidine derivatives

4

RO7049389 is highly potent and

selective against HBV

• Active against the most prevalent HBV

genotypes (A-D)

• No cross-resistance with nucleos(t)ide

analogue resistance variants

Antiviral activity and cytotoxicity

HepG2.2.15 cells

Zhou X et al. [poster]. EASL 2018; SAT-360

Roche TLR7 agonist is positioned to be differentiated as an oral

double prodrug selectively converted in the liver

• TLR7 agonist induces broad immuno-modulatory effects including:

– Activation of IRF7, NFκB, and AP-1 transcription factors

– Differentiation of plasmacytoid dendritic cells (pDCs), and upregulation of co-stimulatory molecules and secretion of type I IFN and other

cytokines/chemokines leading to activation of T-cells and NK cells

– Differentiation of B-cells to antibody-producing plasma cells

• RO7020531 is an orally available double pro-drug of a TLR7 agonist which also activates TLR8 with lower potency

• It was safe and well tolerated in healthy volunteers with a favorable PK profile in single and multiple QOD doses up to 170 mg

Hydrolysis

Esterase

Oxidation

Aldehyde Oxidase

hTLR7 reporter EC50: 55.2 ±22.5 M

hTLR8 reporter EC50: 296 ±45 M

PBMC MEC IFNα induction: 3-6 M

CC50: > 1000 M

RO7020531

Double pro-drugSingle pro-drug Active form

Dai, L. et al. [poster]. EASL 2018; SAT-345; Gane, E. et al. [poster]. EASL 2018; FRI-3375

AAV-HBV mouse model is suitable to examine the anti-HBV

activities of both immune modulators and direct antiviral agents

• rAAV8-1.3HBV infection/transduction through mouse

tail vain injection

– 1.3mer HBV genome

– AAV2 ITR

– AAV8 capsid (hepatotropic)

• Viral infection is fully established by ~ 4 weeks and can persist

for more than five months

• Anti-HBV efficacy can be monitored by standard biomarkers

– Serum HBV DNA, HBsAg, HBeAg, anti-HBs Ab

• The immune response can be monitored by:

– HBV-specific T/B cell ELISpot (spleen)

– Cytokine, interferon stimulated gene (ISG) mRNA

expression

• Other parameters include the mouse body weight, serum

ALT/AST and the pharmacokinetic profile of the investigational

agent

HBV 1.3

ITR ITR

AAV vectors

6

0

5

1 0

1 5

2 0

2 5

% o

f a

cti

va

ted

B

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

an

ti-H

Bs

-SC

(pe

r 2

X1

06 c

ell

s)

RO7020531 increases the number of germinal center B cells and

HBsAg-specific B and T cells in the spleen of AAV-HBV infected mice

• HBsAg-specific B cells were captured and measured by ELISPOT with HBsAg-coated plate.

• HBsAg-specific T cells were measured by IFN- γ ELISPOT in the presence of HBsAg peptides.

***

Weeks

-4 4-3 -2 0-1 1 2 3

AAV-HBV injection Oral treatment

Germinal Center B cells B ELISPOT (HBsAg-specific) T ELISPOT (HBsAg-peptide stimulated)

7

0

5 0

1 0 0

1 5 0

IFN

-g+

ce

lls

(pe

r 0

.5x

10

6 c

ell

s)

*** **

Vehicle RO7020531

100 mg/kg, QOD

Vehicle RO7020531

100 mg/kg, QOD

Vehicle RO7020531

100 mg/kg, QOD

The anti-HBV activity of RO7020531 in AAV-HBV infected mice relies on

functional adaptive immune response (B- and T- cells)

C57B/Bl6 (wild type mice) SCID (T- and B-cell deficient mice)

• Note: no change on HBeAg level was observed.

• Increasing dose levels of RO7020531 in SCID mice up to 300 mg/kg did not demonstrate anti-HBV activity.

• Plasma exposure of TLR7 agonist and innate immune responses (mRNA upregulation of interferon induced genes) in both

SCID and C57B/Bl6 mice were comparable8

In the AAV-HBV mouse model, oral combination treatment with the CpAM

and TLR7 agonist leads to sustained viral load suppression and HBsAg loss

9

• The combination of RO7049389 and RO7020531 reduced HBsAg level to below LLOQ at the end of treatment in 5 of 7 animals, reduced HBV DNA level

to below LLOQ in all animals, which sustained in 4 of 7 during 6-week off-treatment follow-up.

Results are presented as meanSEM (n = 7). LLOQ = lower limit of quantification; QD = once a day; QOD = every other day

Vehicle

TLR7 agonist

100 mg/kg QOD

CpAM

20 mg/kg QD

Combo

(n=7)

HBV DNA HBsAg

HBeAg

LLOQ

Anti-HBs antibody

5

4

3

2

0 7 14 21 28 35 42 49 56 63 70 77 84Days

Log 1

0N

CU

/ml s

eru

m Treatment end

Log 1

0co

pie

s/m

l ser

um

6

5

4

3

2

0 7 14 21 28 35 42 49 56 63 70 77 84Days

Log 1

0IU

/ml s

eru

m

Treatment end

200

150

100

50

14 21 28 35 42 49 56 63 70 77 84Days

mIU

/ml

Treatment end

0

10

9

8

7

6

5

0 7 14 21 28 35 42 49 56 63 70 77 84Days

Treatment end

LLOQ LLOQ *

In the AAV-HBV mouse model, combination treatment of the TLR7 agonist and entecavir

demonstrates similar level of HBV DNA suppression as entecavir alone, similar level of

HBsAg reduction as RO7020531 alone

No effect on HBeAg

• Note: on day 0, the HBsAg level in the combo group was already ~0.4-log lower than other groups.

Vehicle

TLR7 agonist 100

mg/kg QOD

Entecavir

0.03 mg/kg QD

Combo

(n=8)

Combo treatment is associated with an altered liver gene expression

profile compared to the two monotherapy arms in the AAV-HBV mouse

model

• TLR7 and CpAM induced distinct gene

signatures.

• Additional or enhanced gene upregulation may

be induced with the combination treatment.

• Gene enrichment analysis indicates broad

activation of a connected immune network

consisting of various immune cells and

responses.

• Further analysis in gene expression between

mono and combo therapies may reveal

potential early efficacy biomarkers.

Vehicle TLR7 CpAM Combo

11

Summary

• RO7049389 is a Class I HBV core protein allosteric modulator (CpAM)

• RO7020531 is a double pro-drug of TLR7 agonist

• In the AAV-HBV mouse model, the oral combination of the CpAM and TLR7 agonist

demonstrated robust suppression of both HBsAg and HBV DNA levels and with the additional

emergence of anti-HBs antibodies in several animals

• Both compounds are currently in Phase I clinical trials with early data presented in conferences

• These promising preclinical results and Phase 1 clinical data provide encouragement for further

exploring this combination drug therapy as a means to achieve a functional cure for CHB

infection

12

Acknowledgements

• RO7049389 Project team

• RO7020531 Project team

13

14