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