HCV NS5A AND NS5B INHIBITOR RESISTANCE 13 TH 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
HIV Drug Development and Resistance Testing 5 5
HCV Drug Development and Resistance Testing 6 6
If everything seems under control, you're just not going fast enough. Mario Andretti, 02/28/1940 - Italian automobile racer
Routine HCV NS3/4A Testing SOF/LDV FDA approval SOF+SMV SOF OMV/PTV/r +DSV SMV BOC TVR Jul 11 Sep 11 Nov 11 Jan 12 Mar 12 May 12 Jul 12 Sep 12 Nov 12 Jan 13 Mar 13 May 13 Jul 13 Sep 13 Nov 13 Jan 14 Mar 14 May 14 Jul 14 Sep 14 Nov 14 Jan 15 Mar 15 8
NS5A Inhibitor Resistance
55 Phylogeny of HCV NS5A Sequences Derived from DAA Naïve Subjects 91 1a Wildtype 1a with 1 RAM 1b Wildtype GT-1b sequences 104 103 106 Con1 109 101 108 102 99 96 97 92 87 84 73 78 90 81 77 85 107 100 83 98 74 72 75 88 95 89 80 86 94 105 76 79 82 93 1b with 1 RAM 58 15 42 17 6 38 18 33 2 48 32 5 50 70 24 30 13 44 64 36 26 16 GT-1a sequences 49 10 7 35 9 39 52 69 27 67 3 8 4 14 51 40 60 37 63 62 NS5A 1A H77 71 66 20 68 19 43 25 11 21 47 22 56 61 28 23 12 31 46 65 57 1 53 41 J Cook et al., CROI 2015 54 0.01 34 29 59
Replication of HCV Replicons Containing Patient-derived NS5A Sequences 1000 RC (% reference) 100 10 1 Median (range) RC (% of Con1) 1a+1b= 20% (1-164%) 1a= 11% (1-56%) 1b= 57% (19-164%) 0.1 1a+1b N=109 1a N=71 1b N=38
Susceptibility of HCV Replicons Containing Patient-derived NS5A Sequences Fold Change 100 10 1 0.1 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) 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
NS5A Inhibitor Resistance Phenotype: IC95 is a more sensitive discriminator than IC50 Sample 7 Pool (1b): L31M + Y93Y/H Sample 8 Pool (1b): Y93Y/H IFN Drug 1 Drug 2 IFN Drug 1 Drug 2 Sample 7 clone-1: L31M + Y93 (30/39) Sample 8 clone-1: Y93 (24/41) IFN Drug 1 Drug 2 IFN Drug 1 Drug 2 Sample 7 clone-2: L31M + Y93H (9/39) Sample 8 clone-2: Y93H (17/41) IFN Drug 1 Drug 2 IFN Drug 1 Drug 2
GT1a GT1b DRM Distinctions 1B 1A NS5AI IFN J Cook et al., CROI 2015
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 IC 50 /IC 95. 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
6462 113 Phylogeny of HCV NS5B Sequences Derived from DAA Naïve Subjects 7 20 29 38 GT1b Con1 26 45 24 33 32 GT1b 34 35 31 39 40 43 28 27 37 44 36 25 30 41 42 97 99 92 101 131 GT4a 104 102 96 100 93 98 130 GT4? 91 GT4 ED43 103 94 132 95 133 GT4n GT4d 105 14 11 18 10 5 9 17 13 106 4 12 16 22 19 3 15 2 8 GT1a 6 23 1 GT1a H77 21 127 125 128 88 82 129 79 120 90 84 78 87 77 GT3 S52 123 80 86 121 83 89 122 85 126 124 GT3 0.02 GT2b JPUT971017 70 67 116 GT2b 65 66 117 61 57 74 60 63 69 68 58 59 71 118 119 73 72 75 76 GT2k GT2a JFH-1 53 108 46 55 52 47 49 51 111 114 112 48 54 50 110 56 107 109 115 GT2a
Replication Capacity of Replicons Containing GT1, 2, 3, 4 NS5B Regions 1000 RC threshold for susceptibility testing R C (% of C on1) 100 10 1 0.1 Replication 1>2,3,4 1b>1a 2b>2a 0.01 1 1a 1b 2 2a 2b 3 4 # 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 The vast majority of recombinant replicons exhibited a replication capacity sufficient for evaluating inhibitor susceptibility 18
NNI Susceptibility of Replicons Containing GT1, 2, 3, 4 NS5B Regions IC within 2-fold of Con1 reference > IC FC > maximum drug concentration evaluated IC 50 FC 1000 100 10 1 > * * * * * * * * * * > > Sig. diff. from GT1 * Sig. diff. between subtypes (Wilcoxon rank sum test ) IC FC Range NNI-A: 827 NNI-B: >8.2 NNI-D: 152 0.1 1 1 1a 1b 2 2a 2b 3 4 2 2a 2b 3 4 1 1a 1b 2 2a 2b 3 4 IFN NNNI-A NNI-A NNI-A NNI-A NNI-A NNI-A NNI-A NNI-A NNI-A NNI-B NNI-B GT NNI-B NNI-B NNI-B NNI-D NNI-D NNI-D NNI-D NNI-D NNI-D NNI-D NNI-D NNI-D Susceptibility NNI-A: 1,3,4>2 NNI-B: (1)>4>2,3 NNI-D: 1,3>2,4 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 19
DRM Distinctions: Subtype and Binding Site Group 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)
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 NNIbinding 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
IFN, RBV and NI Susceptibility Replicons Containing GT1, 2, 3, 4 NS5B Regions A IFN C RBV E SOF 2 CMeA_1 2 CMeA_2
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) IC 95 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 sample volume Series1 NS5A Series2 NS5B 3/29 4/5 4/12 4/19 4/26 5/3 5/10 5/17 date
NS5A-I RAV Prevalence: NGS 10% Threshold NS5A-I RAVs of DAA naive viruses (%) NS5A-I RAVs of routine testing viruses (%) 25.0 20.0 21.1 30.0 25.0 25.6 15.0 10.0 5.0 15.6 5.3 20.0 15.0 10.0 5.0 17.7 15.9 0.0 1a+1b viruses 1a viruses 1b viruses 0.0 1a+1b viruses 1a viruses 1b viruses DAA naïve viruses No. RAVs % 1a+1b viruses 109 17 15.6 1a viruses 71 15 21.1 1b viruses 38 2 5.3 routine testing viruses No. RAVs % 1a+1b viruses 203 36 17.7 1a viruses 164 26 15.9 1b viruses 39 10 25.6 Comparison of DAA naïve viruses and routine testing viruses AA positions K24R M28T/V Q30H/R/L/S L31M/V H58D A92T Y93C/H/N/R/S DAA naïve 1a = 15/71 (21%) 2 6 5 1 3 routine testing 1a = 26/164 (16%) 4 11 8 5 1 8 AA positions L31M A92T Y93H DAA naïve 1b = 2/38 (5%) 1 2 routine testing 1b = 10/39 (26%) 2 1 8 27
DSV/SOF RAV Prevalence: NGS 10% Threshold DSV/SOF RAVs of DAA naive viruses (%) DSV/SOF RAVs of routine testing viruses (%) 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 8.0 6.0 4.0 1a+1b viruses 1a viruses 1b viruses 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 8.2 7.4 3.8 1a+1b viruses 1a viruses 1b viruses DAA naïve viruses No. RAVs % 1a+1b viruses 50 3 6.0 1a viruses 25 1 4.0 1b viruses 25 2 8.0 routine testing viruses No. RAVs % 1a+1b viruses 148 11 7.4 1a viruses 122 10 8.2 1b viruses 26 1 3.8 Comparison of DAA naïve and routine testing viruses AA position G307E/K/R C316Y S556G/N DAA naïve 1a = 1/25 (4.0%) 1 1 routine testing 1a = 10/122 (8.2%) 7 3 AA position L159F C316H/N V321I S556G/N DAA naïve 1b = 2/25 (8.0%) 1 2 1 1 routine testing 1b = 1/26 (3.8%) 1 28
Future Considerations Patient group: Genotype/subtype Disease stage Co-morbidities Co-infection Re-infection Treatment: Tx duration Rx composition Baseline RAV RAV persistence 1 st vs 2 nd line 29
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