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
On-treatment HBsAg Profile Analysis
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]
On-treatment HBsAg Profile Analysis
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
On-treatment HBsAg Profile Analysis
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-treatment HBsAg Profile Analysis
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]
On-treatment HBsAg Profile Analysis
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
Neutralisation Assay #4 Day 9
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
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
Neutralisation Assay #4 Day 9
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
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
Neutralisation Assay #4 Day 9
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
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
Neutralisation Assay #4 Day 9
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
Hyakumura, M et al 2015. J Virol;89:11312-11322
N = non-glycosylated (24 kD) M = mono-glycosylated (27 kD) D = di-glycosylated (30-31 kD)
Electron Micrograph of BNP
Hyakumura, M et al 2015. J Virol;89:11312-11322
WT
sT116N
sN146Q
sG130N
Immunogenicity in BALB/c Mice of BNPs
Hyakumura, M et al 2015. J Virol;89:11312-11322
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