hcv ns5a and ns5b inhibitor...
TRANSCRIPT
HCV NS5A AND NS5B INHIBITOR RESISTANCE
13TH EUROPEAN HIV & HEPATITIS MEETING JUNE 04, 2015
Christos J Petropoulos, PhD
Disclosures
• Christos J Petropoulos is:
– The Chief Scientific Officer of Monogram Biosciences, a member of the LabCorp Specialty Testing Group • Monogram Biosciences markets services for the diagnosis
and treatment of viral diseases including HIV and HCV infection
– An Officer (Vice President) of Laboratory Corporation of America
– A shareholder of Laboratory Corporation Holdings (LH)
Acknowledgements
• Monogram R&D
– Special thanks to Jackie Reeves and Wei Huang
– Alicia Newton, Jen Cook, Arne Frantzell, Elizabeth Anton, Kristi Strommen, Sam Jauregui,
• Monogram PDO (Yuping Tan, Jeff Larson)
• Monogram Bioinformatics (Agnes Paquet, Mojgan Haddad)
• Monogram Clinical Reference Laboratory Personnel
– Special thanks to Jeannette Whitcomb (GM, VP Ops)
• Our many industry, government and academic collaborators
• Patients that have donated samples for testing
Outline
• HCV DAA resistance perspective • Landscape of NS5A/B DAA susceptibility
– NS5A – NS5B NNI – NS5B NI (RBV, IFN)
• Preliminary observations from NS5A and NS5B inhibitor
resistance testing
5
HIV Drug Development and Resistance Testing
5
6 6
HCV Drug Development and Resistance Testing
If everything seems under control,
you're just not going fast enough.
Mario Andretti, 02/28/1940 -
Italian automobile racer
8
Routine HCV NS3/4A Testing Ju
l 11
Sep
11
Nov
11
Jan
12
Mar
12
May
12
Jul 1
2
Sep
12
Nov
12
Jan
13
Mar
13
May
13
Jul 1
3
Sep
13
Nov
13
Jan
14
Mar
14
May
14
Jul 1
4
Sep
14
Nov
14
Jan
15
Mar
15
BOC TVR
SMV
SOF
SOF/LDV
SOF+SMV
OMV/PTV/r +DSV
FDA approval
NS5A Inhibitor Resistance
5844
36
106
104
103
109
101
Con1
108
102
9199 96
97 92 8773 78
84
85
107
8177
90
83
88 95
98 89 8074
9472105 82
9386100
7679
75
26
2823 411231574647
2219
1
53
5661
65
43
251121
662068
NS5
A 1A
H77
7155
62
6340
592934518
67
54
1497
352
3510
49 2769
1539
386
42
17
4
37
3360
18
5
24832
50 7016
2430
1364
0.01
1a with ≥1 RAM
1a Wildtype
1b Wildtype
1b with ≥1 RAM
Phylogeny of HCV NS5A Sequences Derived from DAA Naïve Subjects
GT-1a sequences
GT-1b sequences
J Cook et al., CROI 2015
Replication of HCV Replicons Containing Patient-derived NS5A Sequences
RC
(% re
fere
nce)
Median (range) RC (% of Con1) 1a+1b= 20% (1-164%) 1a= 11% (1-56%) 1b= 57% (19-164%)
1a+1b N=109
1a N=71
1b N=38
0.1
1
10
100
1000
Susceptibility of HCV Replicons Containing Patient-derived NS5A Sequences
Fold
Cha
nge
Samples with NS5A DRMs (red) 1 (1a). M28T 2 (1a). Q30H 3 (1a). Q30H 4 (1a). Q30H, Y93H 5 (1a) Q30Q/H, Y93Y/H/N 6 (1a). L31L/M 7 (1b). L31L/M, Y93Y/H 8 (1b). Y93Y/H
0.1
1
10
100
1a-IC50 1a-IC95 1b-IC50 1b-IC95
Median FC (range) relative to Con1 1a IC50 FC = 1.38 (0.52->200) 1a IC95 FC = 2.90 (1.12->200) 1b IC50 = 0.71 (0.49-2.50) 1b IC95 = 0.95 (0.54->200)
NS5A Inhibitor Resistance Phenotype: IC95 is a more sensitive discriminator than IC50
Sample 7 Pool (1b): L31M + Y93Y/H
IFN Drug 2 Drug 1
IFN Drug 2 Drug 1
Sample 7 clone-2: L31M + Y93H (9/39)
IFN Drug 2 Drug 1
Sample 7 clone-1: L31M + Y93 (30/39)
Sample 8 Pool (1b): Y93Y/H
IFN Drug 1 Drug 2
IFN Drug 2 Drug 1
Sample 8 clone-1: Y93 (24/41)
Sample 8 clone-2: Y93H (17/41)
IFN Drug 2 Drug 1
GT1a GT1b DRM Distinctions
J Cook et al., CROI 2015
NS5AI
IFN
1B 1A
NS5A Resistance Summary
• Phenotype: • Replicons containing GT1 NS5A sequences exhibit variability in replication
capacity and NS5A inhibitor susceptibility (compared to NI susceptibility) • Pre-existing NS5A DRM s in DAA naïve viruses confer large reductions in
NS5A susceptibility. • Reductions in NS5A inhibitor susceptibility may manifest as reductions in the
% inhibitory maximum rather than increases in IC50/IC95. • NS5A DRMs introduced onto an GT1a backbone generally conferred larger
reductions in susceptibility than DRMs introduced into an GT1b backbone
• Genotype (sequence): • GT1 NS5A sequences exhibit a relatively large degree of sequence diversity
• Including two distinct clusters within GT1a • The prevalence and diversity of NS5A RAV/DRM was higher in GT1a viruses
versus GT1b viruses
• Implication: The combination of multiple resistance pathways, lower genetic and resistance barriers may provide advantages for GT1a viruses to escape NS5A inhibition
NS5B Non-nucleoside Inhibitor Resistance
21G
T1a
H771
238
241310617
15113
101418 9
10519
5 22
1612 6
7 20
3829 33
242645 31
32 35
GT1b Con134
3940 43
28 2744 37
30 41 3625 4292 99 97 10
113
1 104
96 100
102
130
9398 91 GT4 ED43
103132
9594133
127
125
128
77 GT3 S
52
87123
8086121124838912012285126
90788479129
8288
50110
4854 56 115107109
5247
114112
4951
11155
46113
108G
T2a JFH-1
53
73
7275
76
11971
5911858
6869
57
636174606462 117
116 GT2b JPUT971017
7067 65 66
0.02
GT1b
GT1a
GT4a
GT4dGT4? GT4n
GT3
GT2aGT2b
GT2k
Phylogeny of HCV NS5B Sequences Derived from DAA Naïve Subjects
18
• The vast majority of recombinant replicons exhibited a replication capacity sufficient for evaluating inhibitor susceptibility
Replication Capacity of Replicons Containing GT1, 2, 3, 4 NS5B Regions
# 49 27 22 41 21 20 23 8 Median 34 16 73 9 5 21 11 5
Max 174 78 174 111 31 111 82 12 Min 1 1 21 0.06 0.06 4 0.03 0.3
Range 174 78 8.3 1850 517 28 2733 40
RC threshold for susceptibility testing
Replication 1>2,3,4 1b>1a 2b>2a
1 1 a 1 b 2 2 a 2 b 3 40 .0 1
0 .1
1
1 0
1 0 0
1 0 0 0
RC
(%
of
Co
n1
)
19
NNI Susceptibility of Replicons Containing GT1, 2, 3, 4 NS5B Regions
• Replicons exhibit large variation in NNI susceptibilities • NNI-A inhibitors have GT1,3 and 4 activity • NNI-D inhibitors have pan-genotypic (GT1-4) activity
IC FC Range NNI-A: 827 NNI-B: >8.2 NNI-D: 152
Susceptibility NNI-A: 1,3,4>2
NNI-B: (1)>4>2,3 NNI-D: 1,3>2,4
IC within 2-fold of Con1 reference
Sig. diff. from GT1 Sig. diff. between subtypes (Wilcoxon rank sum test ) * *
IC FC > maximum drug concentration evaluated
>
*
1IF N
1N N I-A
1 aN N I-A
1 bN N I-A
2N N I-A
2 aN N I-A
2 bN N I-A
3N N I-A
4N N I-A
2N N I-B
2 aN N I-B
2 bN N I-B
3N N I-B
4N N I-B
1N N I-D
1 aN N I-D
1 bN N I-D
2N N I-D
2 aN N I-D
2 bN N I-D
3N N I-D
4N N I-D
0 .1
1
1 0
1 0 0
1 0 0 0
G T
IC5
0 F
C
IF N N N I-A N N I- B N N I- D
> >
>
* * * * * * * * *
• NNI susceptibility can vary based on GT1 subtype (H77-1a vs Con1-1b) • NNI susceptibility can vary across NNI binding site groups (NNI-C vs NNI-D)
DRM Distinctions: Subtype and Binding Site Group
NS5B NNI Resistance Summary
• Phenotype: • Variation in replication capacity and NS5B NNI susceptibility across GT1-4
NS5B replicons was comparable to the variability within GT1 NS5A replicons. • Pre-existing NS5A DRM s in DAA naïve viruses confer large reductions in
NS5B NNI susceptibility. • NS5B NNI susceptibility varied significantly across HCV genotypes based on
the NNI-binding site group. • The NNI-D inhibitor exhibited pan-inhibitory activity across GT1,2,3,4
• NS5B NNI DRMs introduced into GT1 replicons exhibited subtype and NNI-binding site group differences
• Genotype (sequence):
• GT1 NS5B sequences exhibit a relatively large degree of sequence diversity • Less diversity than NS5A
• The prevalence and diversity of NS5B RAV/DRM was equivalent in GT1a and GT1b viruses, however sample numbers were small.
• Implication: Accurate determination of HCV genotype-subtype may become important for the appropriate use of NS5B NNI inhibitors.
NS5B Nucleoside Inhibitor Resistance
E SOF 2’CMeA_1 2’CMeA_2
A IFN
C RBV
IFN, RBV and NI Susceptibility Replicons Containing GT1, 2, 3, 4 NS5B Regions
NS5B NI Resistance Summary
• Phenotype: • IFN susceptibility was equivalent across replicons containing GT1-4 NS5B • RBV susceptibility was greater for replicons containing GT2,3,4 NS5B
sequences compared to GT1 NS5B sequences • NS5B NI (SOF) susceptibility was greater for replicons containing GT1,2
NS5B sequences compared to GT3,4 NS5B sequences • Replicons containing the S282T mutations exhibits severely impaired
replication capacity….and increased susceptibility to RBV (data not show) • IC95 or inhibition slope are more accurate determinants of NS5B NI resistance
(data not shown)
• Genotype (sequence): • The prevalence of NS5B NI DRM is low DAA naïve and routine resistance
testing samples
• Implication: Genotype specific differences in NI and/or RBV susceptibility may contribute to improved SVR rates with NI and/or RBV containing regimens
Preliminary Observations from NS5A and NS5B Inhibitor Resistance Testing
Routine HCV NS5A/B Testing
3/29 4/5 4/12 4/19 4/26 5/3 5/10 5/17
sam
ple
volu
me
date
Series1
Series2
NS5A
NS5B
NS5A-I RAV Prevalence: NGS 10% Threshold
27
Comparison of DAA naïve viruses and routine testing virusesAA positions K24R M28T/V Q30H/R/L/S L31M/V H58D A92T Y93C/H/N/R/SDAA naïve 1a = 15/71 (21%) 2 6 5 1 3routine testing 1a = 26/164 (16%) 4 11 8 5 1 8
AA positions L31M A92T Y93HDAA naïve 1b = 2/38 (5%) 1 2routine testing 1b = 10/39 (26%) 2 1 8
15.6
21.1
5.3
0.0
5.0
10.0
15.0
20.0
25.0
1a+1b viruses 1a viruses 1b viruses
NS5A-I RAVs of DAA naive viruses (%)
17.715.9
25.6
0.0
5.0
10.0
15.0
20.0
25.0
30.0
1a+1b viruses 1a viruses 1b viruses
NS5A-I RAVs of routine testing viruses (%)
routine testing viruses No. RAVs %1a+1b viruses 203 36 17.7
1a viruses 164 26 15.91b viruses 39 10 25.6
DAA naïve viruses No. RAVs %1a+1b viruses 109 17 15.6
1a viruses 71 15 21.11b viruses 38 2 5.3
DSV/SOF RAV Prevalence: NGS 10% Threshold
28
Comparison of DAA naïve and routine testing virusesAA position G307E/K/R C316Y S556G/NDAA naïve 1a = 1/25 (4.0%) 1 1routine testing 1a = 10/122 (8.2%) 7 3
AA position L159F C316H/N V321I S556G/NDAA naïve 1b = 2/25 (8.0%) 1 2 1 1routine testing 1b = 1/26 (3.8%) 1
7.48.2
3.8
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
1a+1b viruses 1a viruses 1b viruses
DSV/SOF RAVs of routine testing viruses (%)
routine testing viruses No. RAVs %1a+1b viruses 148 11 7.4
1a viruses 122 10 8.21b viruses 26 1 3.8
DAA naïve viruses No. RAVs %1a+1b viruses 50 3 6.0
1a viruses 25 1 4.01b viruses 25 2 8.0
6.0
4.0
8.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
1a+1b viruses 1a viruses 1b viruses
DSV/SOF RAVs of DAA naive viruses (%)
29
Future Considerations
• Patient group: – Genotype/subtype – Disease stage – Co-morbidities – Co-infection – Re-infection
• Treatment: – Tx duration – Rx composition – Baseline RAV – RAV persistence – 1st vs 2nd line
Acknowledgements
• Monogram R&D
– Special thanks to Jackie Reeves and Wei Huang
– Alicia Newton, Jen Cook, Arne Frantzell, Elizabeth Anton, Kristi Strommen, Sam Jauregui,
• Monogram PDO (Yuping Tan, Jeff Larson)
• Monogram Bioinformatics (Agnes Paquet, Mojgan Haddad)
• Monogram Clinical Reference Laboratory Personnel
– Special thanks to Jeannette Whitcomb (GM, VP Ops)
• Our many industry, government and academic collaborators
• Patients that have donated samples for testing