Antiviral Activity of Tenofovir Alafenamide Against HIV-1 With

Antiviral Activity of Tenofovir Alafenamide Against HIV-1 with Thymidine Analog Mutation(s) and M184V Poster 560 NA Margot, RR Ram, ME Abram, R Haubrich, C Callebaut Gilead Sciences, Inc. 333 Lakeside Drive Foster City, CA 94404 Gilead Sciences, Inc., Foster City, CA 1-800-445-3235

Introduction Methods Results

♦♦ Tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) are prodrugs of ♦♦ Site-directed mutants (SDM) containing combinations of TAMs (M41L, D67N, K70R, the HIV-1 nucleotide reverse transcriptase (RT) inhibitor tenofovir (TFV) (Figure 1)1 L210W, T215Y, and/or K219Q) with or without M184V were generated Figure 5. Phenotypic Susceptibility of AZT and TAF in TAM- Figure 6. Phenotypic Assay Correlations Table 3. Isolates with Viral Breakthrough: Time to VB ♦♦ In vivo, TAF achieves ~90% lower plasma TFV levels and ~4-fold higher intracellular ♦♦ Antiviral drug susceptibilities (fold change [FC] EC50 relative to wild-type) were Containing Mutants With or Without M184V 6 2 6 2 6 2 levels of TFV diphosphate (TFV-DP) compared to TDF, which results in improved determined in MT-2 cells using either a 2-day Single-Cycle (SC; n=96) or Single-Cycle Assay Multi-Cycle Assay R = 0.63 R = 0.67 R = 0.69 Time to VB Isolate TAF Mutant Mutant 2 5 5 (days)

) 5 renal and bone safety 5-day Multi-Cycle (MC; n=96) HIV assay (Figure 3) from at least 3 independent + M184V - M184V Mutant RT Sequence ) ) ID FC Category Type TAF TAF ♦♦ Thymidine analog mutations (TAMs) in HIV-1 RT that emerged in the HIV-infected experiments 6 6 4 4 4 TFV TAF

0 0 onogram In-House) In-House) In-House) 5 5 5 5 (In-house population in the early days of AZT monotherapy still persist; TAM frequencies in the (In-house C –– Single-cycle HIV-1 was generated by co-transfection of the replication-deficient 3 3 (M 3 EC F FC F FC F FC e e WT 1.0 No mutations WT WT >28 >28 e cl cl eE 5 4 4 cl TA HIV+ population are not negligible to this day (Figure 2) proviral HIV plasmid pKS13 and pHCMV-G in HEK293T cells TA TA 2 2 Cy 2 3 3 hange ♦♦ TAMs confer incremental reduced susceptibility to TFV, and patients with TAM- hang Multi-Cycle( 1 1.9 M41L L210W T215Y 3 TAMs PD 13 > 28 Multi-Cycle( Multi-Cycle( Multi-Cy –– Multi-cycle HIV-1 was generated by transfection of the replication competent Multi-Cy

2 2 1 1 Single- 1 containing HIV-1 may benefit from higher levels of TFV-DP delivered by TAF, 6 Full set Subset Subset proviral HIV pXXLAI plasmid in HEK293T cells n=95 n=30

Fol dC n=30 Fol dC 1 1 D67N T69N K70R T215V K219Q 3 TAMs + compared to TDF 0 0 0 2 2.4 PD 22 > 28 ♦♦ Additional patient-derived (PD; n=14) mutants with TAMs were tested using the MC 0 0 M184V M184V ♦♦ In addition, the presence of the M184V mutation increases susceptibility to TFV 0 TAMs 1 TAMs 2 TAMs 3 TAMs 4 TAMs 5 TAMs 6 TAMs 0 TAMs 1 TAMs 2 TAMs 3 TAMs 4 TAMs 5 TAMs 6 TAMs Single-CycleTAF FC (In-house) Single-CycleTAF FC (In-house) TAF FC 3 assay Single-Cycle (In-house) Single-Cycle (In-house) Single-Cycle (In-house) 3 2.7 M41L D67N L210W T215Y 4 TAMs SDM 14 > 28 during TDF-based (and presumably TAF) therapy AZT AZT –– Samples from treatment-experienced patients with virologic failure7 400 400 ♦♦ Virologic outcome of subjects harboring HIV with M184V/I ± 1 to 2 TAMs are 300 300 4 2.7 D67N T69T/A K70R T215F K219Q 4 TAMs PD 4 > 28 0 0 200 200 5 5 100 100 2 EC ♦♦ Comparison of TAF and TFV resistance profiles were further assessed in viral EC currently being studied clinically (GS-US-292-1824) 10 10 ♦♦ Good correlation between assays (R >0.63) 5 3.0 M41L D67N K70R L210W T215Y 5 TAMs SDM 19 > 28 4 –– Preliminary Week 12 data showed virologic success in all 37 patients treated breakthrough (VB) experiments using clinically relevant drug concentrations using 8 8 ♦♦ A random subset of 30 mutants was analyzed using Monogram SC assay for 7 6 3.0 M41L D67N K70R T215Y K219Q 5 TAMs SDM 13 > 28

Change 6 TAM-containing viruses (SDM and PD) as previously described (Figure 4) Change 6 comparison ♦♦ Here, the in vitro activity of TAF was evaluated in a large set of TAM-containing site- M41L D67N K70R T215F K219Q 5 TAMs + 4 4 ♦♦ Correlation between single-cycle and multi-cycle suggests that replication capacity 7 3.3 PD 20 > 28 Fold directed mutants and patient-derived HIV-1, with or without M184V –– Assay mimicked the physiological 4-fold higher intracellular levels of TFV-DP Fold M184V M184V 2 2 has little impact on resistance delivered by TAF compared to TDF in vivo 0 0 0 TAMs 1 TAMs 2 TAMs 3 TAMs 4 TAMs 5 TAMs 6 TAMs 0 TAMs 1 TAMs 2 TAMs 3 TAMs 4 TAMs 5 TAMs 6 TAMs 8 3.3 M41L D67N L210W T215Y K219Q 5 TAMs SDM 18 > 28 Figure 1. Tenofovir Prodrugs –– TFV is the in vitro equivalent of TDF M41L D67N K70R L210W T215Y ♦♦ List of Abbreviations ♦♦ High level of AZT resistance in the presence of TAMs (up to 315 fold) Figure 7. Differential Viral Breakthrough with TAF & TFV* (n=68 Mutants) 9 4.0 6 TAMs SDM 19 > 28 NH2 K219Q N N ♦♦ Intermediate level of TAF resistance seen in presence of TAMs (up to 5 fold) Physiological Conditions UT LASMA –– DP: Diphosphate; FC: Fold change; MC: Multi-cycle; MP: Monophosphate; PD: Patient- Tenofovir O N N G P LYMPHOID CELL P O ♦♦ Presence of M184V increases the sensitivity to both AZT and TAF M41L D67N T69N K70R T215F 5 TAMs + (TFV) HO derived; RT: Reverse transcriptase; SC: Single-cycle; SD: Standard deviation; SDM: OH No Viral Breakthrough Viral Breakthrough 10 4.2 PD 10 > 28 TFV X Site directed mutant; TAF: Tenofovir alafenamide; TAM: Thymidine analog mutation; K219E M184V M184V NH 2 T = 0.41 min Table 1. TAF Susceptibility and M184V Effect Relative N N TDF ½ TFV TDF: Tenofovir disoproxil fumarate; TE: Treatment-experienced; TFV: Tenofovir; VB: 11 4.3 M41L L210W T215Y 3 TAMs PD 4 > 28 Tenofovir O O N N TFV Loading P O Viral breakthrough; VF: Virologic failure Site-Directed Mutants ^ disoproxil fumarate O O O T = 90 min O O TAF ½ TFV [TFV-DP] 12 6.6 M41L D67N L210W T215Y K219R 5 TAMs PD 10 20 (TDF) O O Mean TAF EC50 Fold Change Compared to Wild-Type TFV-MP Single-Cycle (SC) Assay Multi-Cycle (MC) Assay NH2 13 8.1 M41L D67N L210W T215Y 4 TAMs PD 10 > 28 O Figure 3. Phenotypic Assay Workflows Mutant Class N N TAF 4x O Without With M184V b Without With M184V b O N Tenofovir HN N a P-Value a P-Value P O M184V M184V Effect M184V M184V Effect M41L D67N T69D L74I L210W T215Y alafenamide (TAF) O TFV-DP Single-Cycle 14 10.7 5 TAMs PD 4 13 No TAMs (n=2) 1.0 1.3 0.8 n/a 1.0 0.4 2.3 n/a K219R Replication- MT2 cells Drug Plate Readout 1 TAMs (n=12) 1.4 1.3 1.0 0.81 0.5 0.5 1.0 1.0 TDF 1x M41L D67N T69D L210W T215Y HIV Deficient Virus 15 12.6 5 TAMs PD 5 10 2 TAMs (n=30) 1.8 1.5 1.2 0.58 1.3 0.8 1.7 0.002 (TFV*) ^ ∆env K219R 3 TAMs (n=24) 2.3 1.2 2.0 0.002 1.3 0.9 1.4 0.02 ♦♦ Upon treatment with TDF, cells are loaded with TFV Transfection Infection Plating 2 days Luciferase FC: EC fold-change; PD: patient-derived; SDM: site-directed mutant; VB: viral breakthrough; TFV and TAF –– TFV is the in vitro equivalent of TDF HEK293T 37°C BrightGlo™ 4 TAMs (n=14) 3.5 1.8 1.9 0.02 2.3 1.6 1.5 0.02 TAF Fold-Change 50 concentrations were 50 µM and 0.8 µM, respectively 5% CO2 ♦♦ TAF: VSV-G 5 TAMs (n=12) 3.9 2.0 2.0 0.03 2.7 1.8 1.5 0.06 (*) TFV is the in vitro equivalent of TDF (*) TFV is the in vitro equivalent of TDF –– Novel tenofovir prodrug with >200-fold increase in plasma stability (vs. TDF) env 6 TAMs (n=2) 5.5 2.3 2.4 n/a 4.0 2.1 1.9 n/a –– Improves the delivery of intracellular TFV by ~ 4-fold while reducing plasma All Mutants (N=96) 2.4 1.5 1.6 <0.0001 1.6 1.0 1.5 <0.0001 HIV ∆env: NL4.3-based vector; vpr- and env-deleted; reporter gene in place of nef (firefly luciferase) ♦♦ Virus breakthrough was observed as follows: ♦♦ At physiological concentration, viral breakthrough was observed in 15 mutants [TFV] by ~90% (vs. TDF) a. Mean values calculated from ≥3 independent triplicate experiments for each mutant –– For TFV, with viruses with ~2-fold reduced susceptibility to TAF (n=15) –– 10-fold lower dose for same or higher efficacy (vs. TDF) b. Ratio of “without M184V” to “with M184V” with TFV compared to only 3 mutants with TAF Multi-Cycle c. Wilcoxon signed rank test –– For TAF, with viruses with >6-fold reduced susceptibility to TAF (n=3) TAF wild-type EC was 5.1 nM in the SC assay and 15 nM in the MC assay Replicating MT2 cells Drug Plate Readout 50 Virus Figure 2. TAMs in Treatment-Naïve and -Experienced Subjects ♦♦ Gradual increase in resistance to TAF observed with increasing # of TAMs HIV Transfection Infection Plating 5 days Cell Viability ♦♦ Significant M184V effect in both assays CellTiterGlo™ Conclusions HEK293T 37°C ♦♦ TAF and TFV FC in MC assay were highly correlated and virtually identical (not shown) 2000 to 2013 1999 2008 2015 5% CO2 ♦♦ Low and gradual reduction in TAF susceptibility was observed with increasing number of TAMs (Table 1 and Figure 5) Naïve Studies Studies 902/907: Study 183-0145: Study 292-0119: Table 2. Susceptibility to NRTIs TAMs at Screening TAMs at Entry TAMs at Entry TAMs at Entry ♦♦ In the presence of M184V, the antiviral activity of TAF was increased in HIV-1 mutants harboring TAMs, similarly to TFV (Table 1 (n=6704) (n=566) (n=702) (n=135) Patient-Derived Mutants (n=14) 25% 25% 25% 25% and Table 2) AM s Figure 4. Viral Breakthrough Assay Mean EC50 Fold-Change Compared to Wild-Type (SD) 20% 20% 20% 20% Mutant Class N th T TAF TFV AZT –– M184V effect was most pronounced in mutants with ≥3 TAMs 15% 15% 15% 15% CPE Scoring 3 TAMs 3 2.9 (1.3) 3.0 (1.4) >72 (26) ♦♦ In viral breakthrough assays, TAF inhibited the breakthrough of most TAMs-containing HIV-1 (Table 3) 10% 10% 10% 10% Replicating ubjects wi MT2 cells 3 TAMs + M184V 2 2.1 (0.4) 2.1 (0.9) 14.6 (13)

S Virus 5% 5% 5% 5% 4 TAMs 2 5.4 (3.8) 6.4 (4.4) >94 (n/a) –– Breakthrough with TAF observed in only 3 mutants compared to 15 mutants with TFV (Table 3) % of 0% 0% 0% 0% Drug Loading Plating 4 TAMs + M184V 2 1.6 (1.0) 1.8 (1.1) 51.2 (21) –– Viruses that showed breakthrough with TAF also had breakthrough with TFV #TAMs 4-5 days 1 day 37°C 5 TAMs 3 10.0 (3.1) 9.3 (4.6) >83 (18) TAMs (thymidine analog mutations): Treatment-naïve ♦♦ These results suggest that TAF has a higher resistance barrier than TDF (Figure 7) M41L, D67N, K70R, L210W, T215Y/F, K219Q/E/N/R in RT TAF or TFV at 5% CO2 5 TAMs + M184V 2 3.8 (0.6) 4.3 (0.8) >84 (13) Treatment-experienced physiological Wild-type EC s were 0.015 µM, 3 µM, and 0.16 µM, for TAF, TFV, and AZT, respectively concentration 50 Split culture SD: standard deviation; n/a: not applicable ♦♦ 1999: 67% TE subjects (viremic with VF) had TAMs at enrollment (Gilead studies 902 & 907) into new plate (*) TFV is the in vitro equivalent of TDF References ♦♦ 2008: 31% TE subjects (viremic with VF) had TAMs at enrollment (Gilead study 183-0145) with fresh CPE: cytopathic effect media/drug ♦♦ Higher level of resistance to TAF observed in patient-derived mutants compared to SDMs ♦♦ 2015: 42% TE subjects (suppressed) had TAMs at enrollment (Gilead study 292-0119) ♦♦ Similar M184V effect in both mutant types 1. Callebaut et al, 2015 Antimicrobial Agents and Chemotherapy 4. Perez-Valero et al, 2017 XXVI International Workshop on HIV Drug 6. Shi and Mellors, 1997 Antimicrobial Agents and Chemotherapy 2. Sax et al, 2015 Lancet Resistance and Treatment Strategies; Johannesburg, South Africa 7. Margot et al, 2016 Antiviral Research 8 ♦♦ Similar FC for TAF and TFV as expected 3. Miller et al, 2004 Journal of Infectious Diseases 5. Balakrishnan et al, 2013 PLoS One 8. Margot et al, 2015 Antimicrobial Agents and Chemotherapy