epitope mapping the hbsag to identify biomarkers...

Epitope Mapping the HBsAg to Identify Biomarkers Associated with

HBsAg Loss in a Treatment Naïve Cohort of Genotype A Chronic

Hepatitis B (CHB) Patients Receiving Nucleotide Analogue (NA) Therapy:

Implications for Functional Cure

Prof Stephen Locarnini, Dr Hans Netter & Dr Renae Walsh WHO Regional Reference Centre for Hepatitis B

Victorian Infectious Diseases Reference Laboratory, Doherty Institute

Melbourne, Victoria 3000, AUSTRALIA

What Would Hepatitis B Cure Look Like? Off Treatment: In the blood: HBV DNA/HBsAg negative anti-HBs positive In the liver: no HBV cccDNA no HBV RC/DSL DNA HBcAg staining negative ± HBsAg (occasional)

[reflecting integrated HBV DNA] Functional Cure: HBsAg loss/Seroconversion Absolute or Complete Cure: Maintenance of undetectable

serum HBV DNA off-treatment No cccDNA anywhere

Outline of Presentation: Steps to Functional Cure

1. Background: HBsAg and Anti-HBs 2. HBsAg Epitope Changes During

Functional Cure 3. Broadly Neutralizing Antibodies and

CHB 4. Modified HBsAg VLPs 5. Next Steps…….

Background

HBsAg Clearance & Functional Cure HBsAg clearance improves survival rates and reduces risk of HCC

Retrospective study of 309 cirrhotics, mean follow-up of 5.7 years

Fattovich et al. Am J Gastroenterol 1998

Need to achieve clearance < age 50 to reduce HCC risk

Yuen M-F, et al. Gastroenterology 2008; 135:1192

There is no biomarker to predict HBsAg loss/seroconversion

Cumulative risk of HCC

Background

HBsAg Clearance & Functional Cure

Rate of spontaneous HBsAg loss/seroconversion ~2% Rate of HBsAg loss/seroconversion on NA therapy (HBeAg+) at 1 year: • telbivudine 0% • adefovir 0% • lamivudine 1% • entecarvir 2% • tenofovir 3% EASL Clinical Practice Guidelines Panel. J Hepatol. 2009;50:227-42

Rate of HBsAg loss/seroconversion on NA therapy (HBeAg+) at 2 years:

Lok ASF & McMahon BJ. Hepatology 2007;45:507–539. Lau GKK et al. N Engl J Med 2005;352:2682–2695. Chang T-T et al. J Gastroenterol Hepatol 2004;19:1276–1282. Marcellin P et al. Hepatology 2008;48:750–758. Gish RG et al. Gastroenterology 2007;133:1437–1444. Gane E et al. Presented at: 59th Annual Meeting of the American Association for the Study of Liver Diseases, San Francisco, USA, 31 October–4 November 2008; Poster 942. Heathcote E et al. Presented at: 59th Annual Meeting of the American Association for the Study of Liver Diseases, San Francisco, USA, 31 October–4 November 2008; Abstract 158.

Median number of years to clear HBsAg on NA therapy 52.2 years

Chevaliez, et al. J Hepatol (2013) 58:676-683

HBsAg Burden • HBsAg secreted in vast excess

over virions (>103 fold)

• circulate in blood 100-400 µg/ml (1% of total serum protein) • half-life 1-38 days (multiple studies) • 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.

Detection of ‘Free’ v’s Complexed Anti-HBs ‘Free’ anti-HBs:

• ‘Free’ anti-HBs (concurrent HBsAg and anti-HBs) is more commonly described in HBeAg-positive CHB patients with a high viral load [Shiels M, et al (1987) Gastro 93:675; Zhang J, et al (2007) Clin Infect Dis 44:1161]

• Reduced detection rates of ‘free’ anti-HBs (co-existing with HBsAg) probably reflects ‘recent’ updates of diagnostic kits as required by the FDA (i.e. to recombinant Ag & mAbs). In comparison, more sensitive ‘non-commercial’ assays developed report the detection co-existing anti-HBs (with HBsAg) in ~50% of CHB patients [Maruyama T , et al (1993) J Clin Invest 91:2586]

Complexed anti-HBs:

Concurrent or complexed HBsAg and anti-HBs has been reported in most CHB patients [de Niet, et al (2014) AVT 19:259]

• Did not cause or result in significant changes to infection status

• Possibly represents a heterologous sub-type driven anti-HBs response; i.e. HBsAg serotype ‘ad’ subtype which is accompanied by an ‘ay’ anti-HBs response, & vice versa [Fouteh P, et al (1983) Ann Int Med 99:460; Sheils M, et al (1987) Gastro 93:675]

Clinical Significance of Concurrent Anti-HBs & HBsAg

• No apparent significant protective or pathogenic effect • Usually associated with high replicative activity (HBeAg-positive CHB)

• Conclusion: - production of anti-HBs is NOT completely blocked in CHB • B-cells encoding high affinity antibodies to the persons own HBsAg are somehow

ineffective or not measurable • Whereas: - B-cell clones encoding anti-HBs with low affinity to the

homologous HBsAg could expand and express their antibody • these anti-HBs do not bind person’s own HBsAg but to HBsAg with slightly different

antigenicity • unlikely due to super-infection with a second HBV strain of different

serotype/genotype

• Partly explained by sG145R-type VEM • 9.5% in anti-HBs positive CHB versus 2.4% in anti-HBs negative CHB (European

patients) (Lada, O et al 2006. J Virol;80:2968-75) • not seen in Asian patients (HBeAg-pos CHB) (Zhang, JM et al 2007. Clin Infect Dis;44:1161-9) • position sT/I126 most affected (? possible Polymorphic site) [sT/I126, N, A, I, L, S]

Rituximab • a chimeric monoclonal antibody against CD20 • CD20 cell surface marker for B-cells • selectively destroys B-cells by antibody-dependent cellular

cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)

• also induces apoptosis of CD20+ cells • binding to amino acids 170-173 and182-185 on CD20

Non-Rituximab regimen vs. Rituximab regimen Evens AM, et al. 2011

Rituximab: Risk of HBV Reactivation

• in the results of a meta-analysis, rituximab containing regimen has been demonstrated to be at a higher risk for HBV reactivation than non-rituximab regimen.

• a chimeric monoclonal antibody against CD20 (B-cells)

Frequency of HBV reactivation Non-Rituximab regimen 0.6% (3/453)

Rituximab regimen 8.2% (20/244)

Hazard ratio 5.64 (2.18-14.54) Non-R R

*14-fold*

Background

Hepatitis B Functional Cure: Role of B Cells & Anti-HBs

Hypothesis

A functional B cell response is essential to achieve a functional HBV cure = HBsAg loss/seroconversion

Challenges and Opportunities:

There are a range of B cell / anti-HBs responses, not all responses are equal in driving HBsAg clearance

• How do we identify and categorise the anti-HBs effectiveness (assays etc)?

• How can we apply this to drive B cell recovery in CHB & promote HBsAg clearance?

• How do we favour ‘clearance’ versus ‘non-clearance’ anti-HBs / B cell responses?

Major HBV Neutralisation Domain • the ‘a’ determinant (aa 99-169) is highly conformational, with a raft of

cysteine & proline residues forming discrete loops [loop1:aa107-138; loop2:aa139-147] containing the major immunogenic epitopes [Cooreman, et al., J Biomed Sci. 2001. 8:237-47]

• alterations HBsAg ‘a’ determinant topology directly influence the HBV neutralisation phenotype

• genotype/serotype variations

• clinical Vaccine Escape (VEM) sG145R

• selective therapeutic or immune (anti-HBs) pressures

can influence epitope availability and HBsAg profile

(loss or gain of binding)

• potentially a predictive biomarker for HBsAg response

• on-treatment measured as ‘shape shifting’ or epitope

availability (loss or gain) [Adapted from Torresi et al., Virology. 2002. 293:305-313]

BioPlex Multiplex HBsAg Mapping Assay

Multiplex assay to identify/predict HBs variants & VEM’s, and anti-HBs epitope profile by mapping the HBsAg fingerprint

Developed a 19plex panel of anti-HBs

mAbs covering HBsAg ‘a’ determinant

and C-terminal domain (residues 99-226)

HBsAg Profile Assay Design

mAb 7, 8, 11, 12, 16, 17, 19

LOOP 2

LOOP 1

mAb10

226

mAb 1

mAb5, 6

mAb3 mAb 4

mAb2

mAb 13, 14, 15

mAb15

mAb13, 14

Conformational: mAbs 9, 18

mAb 7, 8, 11, 12, 16, 17, 19

LOOP 2

LOOP 1

mAb10

226

mAb 1

mAb5, 6

mAb3 mAb 4

mAb2

mAb 13, 14, 15

mAb15

mAb13, 14

mAb 7, 8, 11, 12, 16, 17, 19

LOOP 2

LOOP 1

mAb10

226

mAb 1

mAb5, 6

mAb3 mAb 4

mAb2

mAb 13, 14, 15

mAb15

mAb13, 14

Conformational: mAbs 9, 18

N-terminal (1) Loop 1 (5,6,10) Loop 2 (7,8,11,12,16,17,19)

Combo loop1/2 (13,14,15)

C-terminal (2,3,4)

Conformational (9, 18)

[Adapted from Bio-Rad; www.bio-rad.com]

Anti-HBs mAbs sourced from industry & academic collaborators

VEM Fingerprint: sG145R in vitro

Aberrant sG145R VEM phenotype detected across 9/19 mAbs

VEM predictive mAb = 17 (loop2), increased epitope binding for sG145R VEM

VEM k/o phenotype across 3 domains: loop1 (6), loop2 (8,12,16), Conformational (9)

Loop 2 sG145R VEM alters HBsAg to confer an aberrant phenotype across multiple epitopes or ‘a’ determinant domains

HBsAg Variants Phenotype Analysis

95%

CI 0.5

- 0.5

A2-adw2 baseline

In vitro sn

C-term

Loop2

Loop2 (mAb17)

N-term

Conformational

gain

loss

k/o Loop1

HBsAg Profile: Detection of HBsAg Response Leading to Anti-HBs

Seroconversion on Therapy Hypothesis HBsAg epitope profiles are sensitive to therapeutic and immune (i.e. recovery of the B cell / Ab & innate immune responses on-treatment) pressure, and this can be predictive of HBsAg response (loss or seroconversion) on-treatment

HBsAg clearance profile (CP)

HBsAg epitope pressure (reduced recognition) at both

loop 1 AND loop 2 epitopes

- associated with HBsAg response/decline (>1log) and potentially HBsAg

loss/seroconversion

HBsAg non-clearance (or escape) profile (NCP)

No change in HBsAg epitope profile, OR reduced epitope binding at only one loop

- associated with no HBsAg response/decline (

Mapping HBsAg epitope profiles to predict HBsAg loss/seroconversion in a treatment naïve cohort of genotype A chronic hepatitis B (CHB)

patients receiving tenofovir disoproxil fumarate (TDF) therapy

GS-US-174-0103 (G103) study cohort: HBsAg epitope profile analysis of HBeAg+ patients on NA therapy:

• Treatment naive CHB patients in the immune clearance phase (HBeAg positive)

• Treated with tenofovir (TDF) for 48weeks, & continued/followed out to > 4 years

• 96% achieved viral suppression & 15% HBsAg loss (22/142) at 4 years We studied a subset of 25 HBV genotype A patients: • 14 achieved HBsAg loss/seroconversion • 11 had no change in HBsAg (

Pre-TDF HBsAg NCP in HBsAg Loss Patient Typical pre-treatment NCP for HBsAg loss/seroconversion (Pt ID 3058) [normalised to HBV genotype/serotype backbone]

A2 adw2 baseline

HBsAg loss/seroconversion patient with HBsAg NCP pre-TDF (switched to CP on-treatment)

On-treatment HBsAg Profile Analysis

On-TDF HBsAg CP in HBsAg Loss Patient Typical on-treatment CP for HBsAg loss/seroconversion (Pt ID 3058) [normalised to patient’s baseline profile]

Pre-TDF baseline

HBsAg loss/serconversion patient with HBsAg CP on-TDF: • Switched to CP from pre-treatment NCP • CP by wk12 [blue] &maintained at wk48 [orange] • HBsAg loss by wk180


Pre-TDF HBsAg CP in HBsAg Loss Patient Typical pre-treatment CP for HBsAg loss/seroconversion (Pt ID 7002) [normalised to HBV genotype/serotype backbone]


A2 adw2 baseline

HBsAg loss/seroconversion patient with HBsAg CP pre-TDF (CP maintained on-treatment)

On-TDF HBsAg CP in HBsAg Loss Patient Typical on-treatment CP for HBsAg loss/seroconversion (Pt ID 7002) [normalised to patient’s baseline profile]

Pre-TDF baseline

HBsAg loss/serconversion patient with HBsAg CP on-TDF: • CP maintained from pre-treatment at wk12 [blue] & wk48 [red] • HBsAg loss by wk168


Pre-TDF HBsAg NCP in HBsAg Non-Responder Typical pre-treatment NCP for HBsAg non-responder (Pt ID 7905) [normalised to HBV genotype/serotype backbone]

A2 adw2 baseline

HBsAg non-responder patient with HBsAg NCP pre-TDF (NCP maintained on-treatment)


On-TDF HBsAg NCP in HBsAg Non-Responder Typical on-treatment NCP for HBsAg non-responder (Pt ID 7905) [normalised to patient’s baseline profile]

Pre-TDF baseline

HBsAg non-responder patient with HBsAg NCP on-TDF: • NCP maintained from pre-treatment at wk24 [purple] & wk48 [blue]


Summary: G103 HBsAg CP There is a significant association between the development, maintenance or enhancement of a HBsAg CP and an outcome of HBsAg loss/seroconversion

p-value 0.02 PPV: 83% NPV: 69%

HBV Biology and ARC-520 MOA

Untreated ARC-520

Synopsis: ARC-520 Effect on HBsAg CP

Cohort HBeAg BL (pre-treat)

W1 W2 W3 W4 W6 W8 W12

1 (ARC, n=6) neg 4 1 1 1 1 2 nt 0

1 (placebo, n=2) neg 2 0 0 0 0 0 nt 0

2 (ARC, n=6) neg 3 1 3 3 3 1 0 0

2 (placebo, n=2) neg 1 0 0 0 0 0 0 0

3 (ARC, n=6) neg 3 3 5 4 4 4 1 1

3 (placebo, n=2) neg 2 0 0 0 1 0 0 1

4 (ARC, n=6) neg 2 4 2 4 4 4 2 2

4 (placebo, n=2) neg 0 0 0 0 0 0 1 0

5 (ARC, n=6) pos 1 2 1 4 3 3 2 2

5 (placebo, n=2) pos 0 0 0 0 1 0 0 0

Cohorts 1-5 BL (pre-treat)

W1 W2 W3 W4 W6 W8 W12

ARC (n=30) 13 11 12 16 15 14 5 (of 24) 5

placebo (n=10) 5 0 0 0 2 0 1 (of 8) 1

p-value 0.730 0.038 0.019 0.003 0.145 0.007 1.000 1.000

Identification of an HBsAg CP during the ARC-520 treatment cohorts trials 1-5:

There is a significant association between development of a HBsAg CP and ARC-520 RNAi treatment at multiple timepoints

HBsAg Profiling ARC-520 Assumption: At pre-treatment / study baseline (day1) HBsAg & anti-HBs co-exist in equilibrium

Treatment: ARC-520: RNAi Therapy

Primarily induces HBsAg response / drop

Secondarily facilitates partial recovery of the B cell (Ab) & innate immune responses

Expected Outcomes: To observe a temporal relationship between treatment and an observed effect on the epitope profile. Thus a two phase profile:

1) initial reduction in HBsAg (approx. weeks 2-3)

2) followed by a shift in epitope profile (approx. weeks 3-8+)

Complexed Anti-HBs: Hypothesis Aim: To characterise the anti-HBs ‘recovery’ response during and following ARC-520

Effect ARC-520 Treatment:

Induces HBsAg response (reduction), potentially facilitating partial recovery of the B cell (Ab) & innate immune responses

Predicted On-treatment Outcomes:

‘Recovery’ of the anti-HBs response (complexed anti-HBs/HBsAg) observed as:

1) Initial reduction in HBsAg & a shift in HBsAg epitope profile (possibly linked to host anti-HBs response)

2) Followed by detection of complexed anti-HBs (with HBsAg) due to antibody response recovery and/or improved ratio of complexed anti-HBs/HBsAg compared to ‘free’ HBsAg

Analysis qHBsAg: Strong HBsAg response to week2, relapse from week3 to EOS HBsAg epitope profile: Early weak clearance profile at loop1&2 epitopes at week1, not maintained ‘Free’ anti-HBs: No ‘free’ anti-HBs detected at any sample timepoint Complexed anti-HBs: Complexed anti-HBs detected early at week1, & weak at week4, not maintained

Conclusions: 1x1mg induced early HBsAg response & Clearance profile, and early anti-HBs recovery, not maintained

Walsh, R et al. 2016. J Hepatol

Therapy Associated HBsAg & Anti-HBs Responses

Mechanisms of HBsAg reduction • direct HBsAg load reduction effected by ARC-520 therapy, and/or

• ‘free’ HBsAg reduced as complexed HBsAg/anti-HBs increases

HBsAg Clearance Profile (loss of recognition at loop 1 & 2 epitopes) • structural or folding change less likely with virus replication controlled to limit

variant HBsAg

• anti-HBs specific for loop1 AND 2 HBsAg epitopes (either ‘freed up’ or newly synthesised) drive a Clearance Profile, via:

1) increased anti-HBs complexing with HBsAg, or

2) treatment-targeted load reduction of ‘free’ HBsAg to increase the ratio of complexed HBsAg/anti-HBs to result in HBsAg clearance profile

Complexed anti-HBs • direct anti-HBs recovery to increase complexed anti-HBs, and/or

• Reduction of ‘free’ HBsAg increases the ratio of complexed HBsAg/anti-HBs to detectable levels

ARC-520 treatment drives down HBsAg load & causes epitope profile changes as a consequence of direct antiviral activity AND enhanced anti-HBs recovery

Walsh, R et al. 2016. J Hepatol. EASL submitted

Are Broadly Neutralising Anti-HBs Responses Needed to Develop

Functional Cures in CHB?

Study Design

Distribution of the Occurrences of HBeAg Seroconversion over

the Study Period

Vandepapeliere, P et al 2007. Vaccine;25:8585-8597

Median HBV-DNA Over Time

All patients remained HBsAg positive In Vaccinated Arm: • 83% seroconverted to anti-HBs • Anti-HBs typically 10-100 IU/L • Several patients > 1,000 IU/L

Anti-HBs Responses

HBV in vitro Neutralisation Assay

Soppe, S. 2015

HepG2-NTCP

AD-38 virus MOI 500

Serially diluted sera/antibody

PEG

Cells supplied from Prof Stefan Urban

HBV XTL Antibodies

Chart1

10000.00084852810.0008485281

1000.00226274170.0022627417

100.03606244580.0360624458

10.2616295090.261629509

0.10.39597979750.3959797975

0.010.45961940780.4596194078

IU/L

PEIU's

XTL-17 Antibody

0.0361

0.0314

0.1675

1.165

4.22

4.125

Sheet1

NTCP

AntibodyIU/LDilution 1/xDay 6Day 6Av.St.Dev.Day 9Day 9Av.St.Dev.Day 3Day 3Av.St.Dev.

No Ab0.320.372

XTL-17100010^30.070.060.060.000.040.040.040.00

10010^40.050.050.050.000.030.030.030.00

1010^50.240.180.210.040.190.140.170.04

110^61.341.891.620.390.981.351.170.26

0.110^74.954.274.610.484.503.944.220.40

0.0110^84.383.804.090.414.453.804.130.46

07.068.637.851.116.937.887.410.67

XTL-19100010^30.640.560.600.050.550.440.490.08

10010^41.100.720.910.270.970.630.800.24

1010^51.991.651.820.241.721.501.610.16

110^63.164.413.790.883.063.773.420.50

0.110^73.383.603.490.163.273.953.610.48

0.0110^84.706.495.601.274.315.795.051.05

07.068.637.851.116.937.887.410.67

XTL 17/19100010^30.050.050.050.000.040.030.030.01

10010^40.070.050.060.010.030.030.030.00

1010^50.350.270.310.060.280.210.240.05

110^64.132.993.560.813.602.503.050.78

0.110^76.124.705.411.005.464.655.060.57

0.0110^88.427.958.190.338.157.347.750.57

07.068.637.851.116.937.887.410.67

CHO 6510^35.865.675.770.134.915.255.080.24

10^44.405.454.930.744.515.244.880.52

10^54.765.755.260.704.495.835.160.95

10^64.265.985.121.224.195.164.680.69

10^75.045.775.410.524.615.755.180.81

Sheet1

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL-17

0.283

0.9375

0.291

0.559

1.0585

0.534

2.72

3.89

2.52

24.15

40.2

28.3

18.9

37.3

24.5

11.475

19.95

14.45

Sheet2

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL-19

1.342

2.47

1.6665

0.676

1.31

0.96

1.8065

3.185

2.44

1.735

3

2.26

2.46

4.135

3.225

4.145

7.38

5.685

Sheet3

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL 17/19

0.2555

0.567

0.3955

0.4725

0.8335

0.5725

3.635

10.545

3.51

18.55

25.3

19.55

29.7

50.35

34.5

35.9

60.55

46.65

XTL-17

XTL-19

XTL 17/19

CHO 65

IU/L

PEIU

Neutralisation Assay #4 Day 6

XTL-17

XTL-19

XTL 17/19

CHO 65

IU/L

PEIU


0.00084852810.0008485281

0.00226274170.0022627417

0.03606244580.0360624458

0.2616295090.261629509

0.39597979750.3959797975

0.45961940780.4596194078

IU/L

PEIU's

XTL-17 Antibody

Chart1

10000.07707463910.0770746391

1000.23758787850.2375878785

100.15556349190.1555634919

10.50204581460.5020458146

0.10.48083261120.4808326112

0.011.04651803621.0465180362

IU/L

PEIU's

XTL-19 Antibody

0.4905

0.802

1.61

3.415

3.61

5.05

Sheet1

NTCP


No Ab0.320.372

XTL-17100010^30.070.060.060.000.040.040.040.00

10010^40.050.050.050.000.030.030.030.00

1010^50.240.180.210.040.190.140.170.04

110^61.341.891.620.390.981.351.170.26

0.110^74.954.274.610.484.503.944.220.40

0.0110^84.383.804.090.414.453.804.130.46

07.068.637.851.116.937.887.410.67

XTL-19100010^30.640.560.600.050.550.440.490.08

10010^41.100.720.910.270.970.630.800.24

1010^51.991.651.820.241.721.501.610.16

110^63.164.413.790.883.063.773.420.50

0.110^73.383.603.490.163.273.953.610.48

0.0110^84.706.495.601.274.315.795.051.05

07.068.637.851.116.937.887.410.67

XTL 17/19100010^30.050.050.050.000.040.030.030.01

10010^40.070.050.060.010.030.030.030.00

1010^50.350.270.310.060.280.210.240.05

110^64.132.993.560.813.602.503.050.78

0.110^76.124.705.411.005.464.655.060.57

0.0110^88.427.958.190.338.157.347.750.57

07.068.637.851.116.937.887.410.67

CHO 6510^35.865.675.770.134.915.255.080.24

10^44.405.454.930.744.515.244.880.52

10^54.765.755.260.704.495.835.160.95

10^64.265.985.121.224.195.164.680.69

10^75.045.775.410.524.615.755.180.81

Sheet1

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL-17

0.283

0.9375

0.291

0.559

1.0585

0.534

2.72

3.89

2.52

24.15

40.2

28.3

18.9

37.3

24.5

11.475

19.95

14.45

Sheet2

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL-19

1.342

2.47

1.6665

0.676

1.31

0.96

1.8065

3.185

2.44

1.735

3

2.26

2.46

4.135

3.225

4.145

7.38

5.685

Sheet3

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL 17/19

0.2555

0.567

0.3955

0.4725

0.8335

0.5725

3.635

10.545

3.51

18.55

25.3

19.55

29.7

50.35

34.5

35.9

60.55

46.65

XTL-17

XTL-19

XTL 17/19

CHO 65

IU/L

PEIU


XTL-17

XTL-19

XTL 17/19

CHO 65

IU/L

PEIU


0.00084852810.0008485281

0.00226274170.0022627417

0.03606244580.0360624458

0.2616295090.261629509

0.39597979750.3959797975

0.45961940780.4596194078

IU/L

PEIU's

XTL-17 Antibody

0.07707463910.0770746391

0.23758787850.2375878785

0.15556349190.1555634919

0.50204581460.5020458146

0.48083261120.4808326112

1.04651803621.0465180362

IU/L

PEIU's

XTL-19 Antibody

Chart1

10000.00883883480.0088388348

1000.00247487370.0024748737

100.04666904760.0466690476

10.77781745930.7778174593

0.10.57275649280.5727564928

0.010.57275649280.5727564928

IU/L

PEIU's

XTL-17/19 mix

0.03365

0.03265

0.244

3.05

5.055

7.745

Sheet1

NTCP


No Ab0.320.372

XTL-17100010^30.070.060.060.000.040.040.040.00

10010^40.050.050.050.000.030.030.030.00

1010^50.240.180.210.040.190.140.170.04

110^61.341.891.620.390.981.351.170.26

0.110^74.954.274.610.484.503.944.220.40

0.0110^84.383.804.090.414.453.804.130.46

07.068.637.851.116.937.887.410.67

XTL-19100010^30.640.560.600.050.550.440.490.08

10010^41.100.720.910.270.970.630.800.24

1010^51.991.651.820.241.721.501.610.16

110^63.164.413.790.883.063.773.420.50

0.110^73.383.603.490.163.273.953.610.48

0.0110^84.706.495.601.274.315.795.051.05

07.068.637.851.116.937.887.410.67

XTL 17/19100010^30.050.050.050.000.040.030.030.01

10010^40.070.050.060.010.030.030.030.00

1010^50.350.270.310.060.280.210.240.05

110^64.132.993.560.813.602.503.050.78

0.110^76.124.705.411.005.464.655.060.57

0.0110^88.427.958.190.338.157.347.750.57

07.068.637.851.116.937.887.410.67

CHO 6510^35.865.675.770.134.915.255.080.24

10^44.405.454.930.744.515.244.880.52

10^54.765.755.260.704.495.835.160.95

10^64.265.985.121.224.195.164.680.69

10^75.045.775.410.524.615.755.180.81

Sheet1

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL-17

0.283

0.9375

0.291

0.559

1.0585

0.534

2.72

3.89

2.52

24.15

40.2

28.3

18.9

37.3

24.5

11.475

19.95

14.45

Sheet2

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL-19

1.342

2.47

1.6665

0.676

1.31

0.96

1.8065

3.185

2.44

1.735

3

2.26

2.46

4.135

3.225

4.145

7.38

5.685

Sheet3

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL 17/19

0.2555

0.567

0.3955

0.4725

0.8335

0.5725

3.635

10.545

3.51

18.55

25.3

19.55

29.7

50.35

34.5

35.9

60.55

46.65

XTL-17

XTL-19

XTL 17/19

CHO 65

IU/L

PEIU


XTL-17

XTL-19

XTL 17/19

CHO 65

IU/L

PEIU


0.00084852810.0008485281

0.00226274170.0022627417

0.03606244580.0360624458

0.2616295090.261629509

0.39597979750.3959797975

0.45961940780.4596194078

IU/L

PEIU's

XTL-17 Antibody

0.07707463910.0770746391

0.23758787850.2375878785

0.15556349190.1555634919

0.50204581460.5020458146

0.48083261120.4808326112

1.04651803621.0465180362

IU/L

PEIU's

XTL-19 Antibody

0.00883883480.0088388348

0.00247487370.0024748737

0.04666904760.0466690476

0.77781745930.7778174593

0.57275649280.5727564928

0.57275649280.5727564928

IU/L

PEIU's

XTL-17/19 mix

Chart1

10^30.24041630560.2404163056

10^40.51618795030.5161879503

10^50.94752308680.9475230868

10^60.68589357780.6858935778

10^70.80610173060.8061017306

Dilution 1/x

PEIU's

CHO 65 control Ab

5.08

4.875

5.16

4.675

5.18

Sheet1

NTCP


No Ab0.320.372

XTL-17100010^30.070.060.060.000.040.040.040.00

10010^40.050.050.050.000.030.030.030.00

1010^50.240.180.210.040.190.140.170.04

110^61.341.891.620.390.981.351.170.26

0.110^74.954.274.610.484.503.944.220.40

0.0110^84.383.804.090.414.453.804.130.46

07.068.637.851.116.937.887.410.67

XTL-19100010^30.640.560.600.050.550.440.490.08

10010^41.100.720.910.270.970.630.800.24

1010^51.991.651.820.241.721.501.610.16

110^63.164.413.790.883.063.773.420.50

0.110^73.383.603.490.163.273.953.610.48

0.0110^84.706.495.601.274.315.795.051.05

07.068.637.851.116.937.887.410.67

XTL 17/19100010^30.050.050.050.000.040.030.030.01

10010^40.070.050.060.010.030.030.030.00

1010^50.350.270.310.060.280.210.240.05

110^64.132.993.560.813.602.503.050.78

0.110^76.124.705.411.005.464.655.060.57

0.0110^88.427.958.190.338.157.347.750.57

07.068.637.851.116.937.887.410.67

CHO 6510^35.865.675.770.134.915.255.080.24

10^44.405.454.930.744.515.244.880.52

10^54.765.755.260.704.495.835.160.95

10^64.265.985.121.224.195.164.680.69

10^75.045.775.410.524.615.755.180.81

Sheet1

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL-17

0.283

0.9375

0.291

0.559

1.0585

0.534

2.72

3.89

2.52

24.15

40.2

28.3

18.9

37.3

24.5

11.475

19.95

14.45

Sheet2

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL-19

1.342

2.47

1.6665

0.676

1.31

0.96

1.8065

3.185

2.44

1.735

3

2.26

2.46

4.135

3.225

4.145

7.38

5.685

Sheet3

100010001000

100100100

101010

111

0.10.10.1

0.010.010.01

Day 5

Day 8

Day 10

IU/L

PEIU

XTL 17/19

0.2555

0.567

0.3955

0.4725

0.8335

0.5725

3.635

10.545

3.51

18.55

25.3

19.55

29.7

50.35

34.5

35.9

60.55

46.65

XTL-17

XTL-19

XTL 17/19

CHO 65

IU/L

PEIU


XTL-17

XTL-19

XTL 17/19

CHO 65

IU/L

PEIU


0.00084852810.0008485281

0.00226274170.0022627417

0.03606244580.0360624458

0.2616295090.261629509

0.39597979750.3959797975

0.45961940780.4596194078

IU/L

PEIU's

XTL-17 Antibody

0.07707463910.0770746391

0.23758787850.2375878785

0.15556349190.1555634919

0.50204581460.5020458146

0.48083261120.4808326112

1.04651803621.0465180362

IU/L

PEIU's

XTL-19 Antibody

0.00883883480.0088388348

0.00247487370.0024748737

0.04666904760.0466690476

0.77781745930.7778174593

0.57275649280.5727564928

0.57275649280.5727564928

IU/L

PEIU's

XTL-17/19 mix

Dilution 1/x

PEIU's

CHO 65 control Ab

Prof M Mondelli HBV S MAbs

PLOS ONE DOI:10.1371/journal.pone.0125704 April 29, 2015. Pages 1-10

Hepatitis B Vaccinees: High Responders

[anti-HBs: >1000 IU/L]

Chart1

5000.45961940780.4596194078

501.2020815281.202081528

58.3438600188.343860018

0.52.82842712472.8284271247

0.056.22253967446.2225396744

07.70746391497.7074639149

RE

IU/L

HBeAg

RE- 5000 IU/L

2.455

22.35

30.7

28.5

30

28.55

Sheet1

NTCP

AntibodyIU/LDilution 1/xDay 6Day 6Av.St.DevDay 9Day 9Av.St.Dev.Day 3Day 3Av.

No Ab

XTL-19100010^33.202.092.650.783.282.122.700.82

10010^44.803.254.031.105.763.074.421.90

1010^58.336.677.501.179.076.747.911.65

110^611.209.8210.510.9813.509.6111.562.75

0.110^714.6012.3013.451.6317.9011.9014.904.24

0.0110^820.5014.9017.703.9621.8015.4018.604.53

DD380010^10.060.050.050.000.080.060.070.01

38010^22.451.952.200.352.522.062.290.33

3810^326.3019.4022.854.8830.4021.4025.906.36

3.810^431.8022.6027.206.5138.4024.6031.509.76

0.3810^531.1023.1027.105.6639.2027.2033.208.49

0.03810^630.3023.5026.904.8138.2023.8031.0010.18

RE50010^12.591.922.260.472.132.782.460.46

5010^219.6018.7019.150.6423.2021.5022.351.20

510^323.0027.8025.403.3924.8036.6030.708.34

0.510^423.8027.1025.452.3326.5030.5028.502.83

0.0510^522.9026.9024.902.8325.6034.4030.006.22

No Ab022.3025.2023.752.0523.1034.0028.557.713.903.51

ML1710^149.3029.3039.3014.1457.5036.0046.7515.20

1.710^240.2031.4035.806.2248.6035.9042.258.98

0.1710^339.0025.9032.459.2647.7029.9038.8012.59

0.01710^433.1023.1028.107.0739.7024.6032.1510.68

0.001710^529.8021.4025.605.9440.9023.0031.9512.66

No Ab044.9030.7037.8010.0456.5033.5045.0016.26

CL0.210^138.7043.6041.153.4652.0054.7053.351.91

0.0210^228.3036.6032.455.8731.8048.2040.0011.60

0.00210^325.7032.3029.004.6730.0038.4034.205.94

0.000210^422.7027.8725.293.6631.6023.9027.755.44

No Ab025.3034.4029.856.4327.5042.0034.7510.25

no virus0.470.230.350.170.580.420.500.12

Sheet1

0.78488852710.78488852710.82024386620.8202438662

1.09601551081.09601551081.90211724141.9021172414

1.17379725681.17379725681.64755880021.6475588002

0.9758073580.9758073582.75064537882.7506453788

1.62634559671.62634559674.24264068714.2426406871

3.95979797463.95979797464.52548339964.5254833996

Day 6

Day 9

IU/L

PEIU

#3 XTL-19

Sheet2

0.00353553390.00353553390.0101116270.010111627

0.35355339060.35355339060.32526911930.3252691193

4.87903679024.87903679026.36396103076.3639610307

6.50538238696.50538238699.75807358049.7580735804

5.65685424955.65685424958.48528137428.4852813742

4.80832611214.808326112110.182337649110.1823376491

Day 6

Day 9

IU/L

PEIU

#3 DoDa

Sheet3

0.47376154340.47376154340.45961940780.4596194078

0.63639610310.63639610311.2020815281.202081528

3.39411254973.39411254978.3438600188.343860018

2.33345237792.33345237792.82842712472.8284271247

2.82842712472.82842712476.22253967446.2225396744

2.05060966542.05060966547.70746391497.7074639149

Day 6

Day 9

IU/L

PEIU

#3 EdRo

14.142135623714.142135623715.202795795515.2027957955

6.22253967446.22253967448.98025612118.9802561211

9.26309883359.263098833512.586500705112.5865007051

7.07106781197.071067811910.677312395910.6773123959

5.9396969625.93969696212.657211383212.6572113832

10.040916292810.040916292816.263455967316.2634559673

Day 6

Day 9

IU/L

PEIU

#3 LiMa

3.46482322783.46482322781.90918830921.9091883092

5.86898628385.868986283811.596551211511.5965512115

4.66690475584.66690475585.9396969625.939696962

3.65574205873.65574205875.44472221515.4447222151

6.43467170886.434671708810.253048327210.2530483272

Day 6

Day 9

IU/L

PEIU

#3 LaCa

38000.0101116270.010111627

3800.32526911930.3252691193

386.36396103076.3639610307

3.89.75807358049.7580735804

0.388.48528137428.4852813742

0.03810.182337649110.1823376491

DD

IU/ml

HBeAg

0.07065

2.29

25.9

31.5

33.2

31

5000.45961940780.4596194078

501.2020815281.202081528

58.3438600188.343860018

0.52.82842712472.8284271247

0.056.22253967446.2225396744

07.70746391497.7074639149

RE

IU/L

HBeAg

RE- 5000 IU/L

2.455

22.35

30.7

28.5

30

28.55

1715.202795795515.2027957955

1.78.98025612118.9802561211

0.1712.586500705112.5865007051

0.01710.677312395910.6773123959

0.001712.657211383212.6572113832

016.263455967316.2634559673

ML

IU/ml

HBeAg

46.75

42.25

38.8

32.15

31.95

45

0.21.90918830921.9091883092

0.0211.596551211511.5965512115

0.0025.9396969625.939696962

0.00025.44472221515.4447222151

010.253048327210.2530483272

CL

IU/ml

HBeAg

CL < 2 IU/L

53.35

40

34.2

27.75

34.75

Chart1

15900.36133156520.3613315652

1590.63639610310.6363961031

15.91.00409162931.0040916293

1.591.59806132551.5980613255

0.1591.77483802081.7748380208

0.01592.24859956422.2485995642

BC

IU/L

PEIU's

BC - 15900 IU/L

0.7945

2.37

7.85

8.97

8.015

9.31

Sheet1

NTCP

AntibodyIU/LDay 9Day 9Av.St.Dev

No Ab

BT1325.444.374.910.76

13.212.808.3010.553.18

1.329.378.078.720.92

0.13214.108.4011.254.03

0.01329.347.868.601.05

0.0013210.907.739.322.24

RW89.65.276.185.730.64

8.967.319.278.291.39

0.8968.288.838.560.39

0.08968.939.809.370.62

0.008966.328.227.271.34

010.109.689.890.30

BC15901.050.540.790.36

1592.821.922.370.64

15.98.567.147.851.00

1.5910.107.848.971.60

0.1599.276.768.021.77

0.015910.907.729.312.25

RaDi42.47.669.948.801.61

4.248.3311.8010.072.45

0.4246.618.857.731.58

0.04248.6510.009.330.95

08.009.108.550.78

Sheet1

0.75660425590.7566042559

3.18198051533.1819805153

0.91923881550.9192388155

4.03050865284.0305086528

1.04651803621.0465180362

2.24152849642.2415284964

BT

HBeAg

Sheet2

0.64346717090.6434671709

1.38592929111.3859292911

0.38890872970.3889087297

0.61518289960.6151828996

1.34350288431.3435028843

0.29698484810.2969848481

RW

HBeAg

Sheet3

0.36133156520.3613315652

0.63639610310.6363961031

1.00409162931.0040916293

1.59806132551.5980613255

1.77483802081.7748380208

2.24859956422.2485995642

BC

IU/L

PEIU's

BC - 15900 IU/L

RaDi

HBeAg

HBV Entry into Hepatocytes

Urban, S et al 2014. Gastroenterology;147:48-64

Inhibitors of HBV Attachment and Entry

Sodium taurocholate cotransporting polypeptide (NTCP) identified as HBV and HDV receptor in 2012

Yan H, Elife 2012; 1:e00049 Lempp RA, Urban S. Intervirol 2014’; 57: 151

Myrcludex in phase 2 trials in chronic HBV and chronic HDV decrease in HBV DNA and HDV RNA

Incubation o/n at 37°C

Collection of supernatant of days 8-12p.i.

Measurement of secreted HBsAg/HBeAg etc.

Infection of HepaRG cells or PHH

Gripon et al., PNAS, 99 (24) 2002 Urban et al., J. Virol, 79 (3), 2005 Glebe et al., Gastroenterology, 129, 2005 Engelke et al., Hepatology, 43, 2006 Schulze et al., Hepatology, 46, 2007

A Synthetic Peptide Derived from the Large Envelope Protein of HBV Blocks HBV Infection in Susceptible Cells….

Modified HBsAg VLPs (Bio-Nano-Particles

[BNP]), Anti-HBs Immunogenicity, Broadly

Neutralising Antibody and Functional Cure

Journal of Virology 2015;89(22);11312-11322

VLPs : WT : sN146Q : sT116N : sG130N

Schematic Diagram of HBsAg

Hyakumura, M et al 2015. J Virol;89:11312-11322

Secreted BNPs: WT, sN146Q, sT116N, sG130N


N = non-glycosylated (24 kD) M = mono-glycosylated (27 kD) D = di-glycosylated (30-31 kD)

Electron Micrograph of BNP


WT

sT116N

sN146Q

sG130N

Immunogenicity in BALB/c Mice of BNPs


Week 4 Week 6

Week 14 Week 20

? Neutralisability the Same as Immunogenicity?

Predicting HBsAg Loss On-Treatment: Summary Summary:

• Mapping the change in epitope recognition on TDF therapy revealed selection pressures directed against

the HBsAg

• The HBsAg CP is a viral biomarker for HBsAg loss/response on-treatment, possibly reflecting emerging

anti-HBs selection pressure

• Confirmed with the ARC-520 RNAi inhibitor studies

• Role in:

• Facilitating individualised patient therapy

• Predicting HBsAg loss / anti-HBs seroconversion

• Therapeutic applications (e.g. identification of novel B cell epitopes)

Future Studies:

• Understand the mechanism of immune pressure on HBsAg

• Analysis of the anti-HBs response (quant anti-HBs, complex HBs/anti-HBs, serotype specific anti-HBs,

anti-HBs isotype, broadly neutralising versus non-neutralising responses)

• Novel MOA : 1.Block Entry; 2.Accelerate Clearance; 3.Block HBV/HBsAg Release; 4.Fc-Dependent

Processes (Opsono-phagocytosis;virolysis;ADCC)

• Therapeutic BNPs/peptide vaccine to mimic and effect an anti-HBs ‘clearance profile’ (CP) response?

Epitope Mapping the HBsAg to Identify Biomarkers Associated with HBsAg Loss in a Treatment Naïve Cohort of Genotype A Chronic Hepatitis B (CHB) Patients Receiving Nucleotide Analogue (NA) Therapy: Implications for Functional CureWhat Would Hepatitis B Cure Look Like?Outline of Presentation: Steps to Functional CureSlide Number 4Slide Number 5Slide Number 6Detection of ‘Free’ v’s Complexed Anti-HBsClinical Significance of Concurrent Anti-HBs & HBsAgRituximab Rituximab: Risk of HBV ReactivationHepatitis B Functional Cure: Role of B Cells & Anti-HBsMajor HBV Neutralisation DomainSlide Number 13Slide Number 14HBsAg Profile: Detection of HBsAg Response Leading to Anti-HBs Seroconversion on Therapy Mapping HBsAg epitope profiles to predict HBsAg loss/seroconversion in a treatment naïve cohort of genotype A chronic hepatitis B (CHB) patients receiving tenofovir disoproxil fumarate (TDF) therapySlide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Summary: G103 HBsAg CPHBV Biology and ARC-520 MOASynopsis: ARC-520 Effect on HBsAg CPHBsAg Profiling ARC-520Complexed Anti-HBs: HypothesisSlide Number 28Therapy Associated HBsAg & Anti-HBs ResponsesAre Broadly Neutralising Anti-HBs Responses Needed to Develop Functional Cures in CHB?Study DesignDistribution of the Occurrences of HBeAg Seroconversion over the Study PeriodHBV in vitro Neutralisation AssayHBV XTL AntibodiesProf M Mondelli HBV S MAbsHepatitis B Vaccinees: �High Responders�[anti-HBs: >1000 IU/L]HBV Entry into HepatocytesInhibitors of HBV Attachment and EntrySlide Number 39Modified HBsAg VLPs (Bio-Nano-Particles [BNP]), Anti-HBs Immunogenicity, Broadly Neutralising Antibody and Functional Cure Slide Number 41Schematic Diagram of HBsAgSecreted BNPs: WT, sN146Q, sT116N, sG130NElectron Micrograph of BNPImmunogenicity in BALB/c Mice of BNPsPredicting HBsAg Loss On-Treatment: Summary

epitope mapping the hbsag to identify biomarkers...

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