In vitro susceptibility of CRF02_AG to fostemsavir Francesco Saladini, PhD Department of Medical Biotechnologies University of Siena, Italy AREVIR MEETING, KÖLN MAY 4-5, 2018
Fostemsavir - overview Fostemsavir (BMS-663068) Temsavir (BMS-626529) Gastrointestinal Blood lumen plasma Alcaline phosphatase Prodrug Active moiety
Fostemsavir - overview Langley et al., Proteins 2014
Fostemsavir - In vitro antiviral activity Susceptibility to BMS-626529 of clinical isolates in PBMCs n. 11 n. 8 n. 16 n. 13 Subtype B, n. 40 Nowicka-Sans et al., AAC 2012
Fostemsavir - In vitro antiviral activity Susceptibility to BMS-626529 of pseudotyped viruses in Phenosense Entry Assay Median IC 50 IC 50 range Subtype (n) (nM) (nM) A (33) 2.26 0.38 - >100 B (133) 0.34 0.05 - >100 C (36) 1.3 0.07 - >100 >100 >100 CRF01_AE (5) F (3) 0.1 0.06 – 0.4 F1 (4) 3.75 0.84 – 16 CRF_BF (15) 3.09 0.26 - >100 1.77 1.67 – 2.98 CRF02_AG (3) Due to natural polymorphisms S375H – M475I? Nowicka-Sans et al., AAC 2012
Fostemsavir - In vitro antiviral activity Susceptibility to BMS-626529 of clinical isolates in Susceptibility to BMS-626529 of pseudotyped viruses in PBMCs Phenosense Entry Assay Nowicka-Sans et al., AAC 2012
In vitro susceptibility of CRF02_AG to fostemsavir CRF43_02G CRF28_BF CRF36_cpx CRF31_BC A1 C 1% 1% 1% 1% 1% 2% Other 4% CRF01_AE F1 2% 4% CRF02_AG is the CRF02_AG second most 5% prevalent subtype circulating in Italy (ARCA database, www.dbarca.net) B 78% A1 CRF28_BF CRF43_02G CRF36_cpx CRF31_BC C CRF01_AE F1 CRF02_AG B Altri
In vitro susceptibility of CRF02_AG to fostemsavir https://spread.crp-sante.lu/public/subtype
In vitro susceptibility of CRF02_AG to fostemsavir Production of pseudotyped viruses Generation of CMV-ENV PCR fragments Production of pseudotyped viral particles from 14 CRF02_AG strains + NL4-3 (X4- tropic) and AD8 (R5-tropic) Determination of coreceptor usage and measurement of susceptibility to Temsavir in U87-CXCR4 and/or -CCR5 cells Lin et al., JVM 2010
In vitro susceptibility of CRF02_AG to fostemsavir Detection of Temsavir RAMs in CRF02_AG Env sequences In vitro/vivo RAMs selected by Temsavir * : L116P, A204D, S375I/N, M426L, M434I, M475I, V506M ID sample L116P A204D S375I/N M426L M434I M475I V506M Rare variants not selected by Temsavir: 147290 L A S P M M V 150235 L A S M M M V 204T/V e 426P in 0/187 LANL • 146886 L T S M M M V CRF02_AG sequences 146733 L T S M M M V M434K in 1/187 LANL CRF02_AG • sequences (0.5%) 138460 L A S M M M V 142972 L A S M I M V 139179 L A S M M M V Temsavir RAMs: 134352 L A S M M M/I V 434I in 31/187 LANL CRF02_AG 146996 L A/V S M M M V • sequences (16.6%) 139188 L A S M M M V 475I in 2/187 LANL CRF02_AG • 148738 L A S M M M V sequences (1.1%) 134039 L A S M M/K M V 149856 L A S M M M V 135576 Not available * Ray et al. JAIDS 2013; Zhou et al. JAC 2014
In vitro susceptibility of CRF02_AG to fostemsavir Determination of coreceptor usage ID Infection Infection Temsavir susceptibility testing on: Tropism FPR sample U87-CXCR4 U87-CCR5 147290 YES YES DUAL 0 Both U87-CXCR4/U87- • 150235 YES YES DUAL 0.8 CCR5 cell lines 134352 YES YES DUAL 0.1 146886 NO YES R5 11.4 146733 NO YES R5 12.0 138460 NO YES R5 84.9 142972 NO YES R5 19.1 139179 NO YES R5 9.0 146996 NO YES R5 23.6 U87-CCR5 cell line only • 139188 NO YES R5 9.0 148738 NO YES R5 26.9 149856 NO YES R5 92.9 135576 NO YES R5 83.0 134039 NO YES R5 99.4 AD8 NO YES R5 35.3 U87-CXCR4 cell line only NL4-3 YES NO X4 0.5 •
In vitro susceptibility of CRF02_AG to fostemsavir Determination of susceptibility to Temsavir CRF02_AG ID sample Tropism Mean IC50 (nM) ± SD Median IC 50 : 11.9 nM (IQR 2.4-22.6 nM) 147290 DUAL 267.1 ± 162.0 (X4: 152.5; R5: 381.6) Range IC 50 : 0.6 – 381.6 nM 150235 DUAL 24.1 ± 3.2 (X4: 26.3; R5: 21.8) Median FC : 6.4 (IQR 1.4-11.0) 134352 DUAL 16.7 ± 6.9 (X4: 11.8; R5: 21.5) 0.9 ± 0.5 146886 R5 R5 5.4 ± 6.8 146733 R5 3.4 ± 0.9 138460 R5 0.6 ± 0.4 142972 16.6 ± 4.1 139179 R5 31.6 ± 19.3 146996 R5 R5 15.7 ± 12.5 139188 R5 8.1 ± 4.1 148738 R5 33.6 ± 9.2 149856 R5 2.1 ± 2.8 135576 1.7 ± 1.8 134039 R5 AD8 R5 2.4 ± 1.9 2.4 ± 1.2 NL4-3 X4
In vitro susceptibility of CRF02_AG to fostemsavir Determination of susceptibility to Temsavir CRF02_AG Median IC 50 : 11.9 nM - Median FC : 6.4 ID sample Tropism Substitution Mean IC 50 (nM) ± SD FC 147290 DUAL M426P 267.1 ± 162.0 (X4: 152.5; R5: 381.6) 111.3 Strain/clone LANL frequency % IC 50 ± SD nM (FC) FC HIV-1 LAI Site-directed NL4-3 (426M) 93.6 2.4 ± 1.2 *Lataillade et al. mutagenesis on NL4-3 + 426P 0 9.9 ± 0.8 (4.1) 6.1* # Zhou et al. NL4-3 strain NL4-3 + 426L 4.8 44.2 ± 6.3 (18.4) 81 # NL4-3 + 426K 0.5 3.7 ± 0.3 (1.5) / NL4-3 + 426R 0.5 1.1 ± 0.3 (0.5) 0.9* ID sample L116P A204D M426L M434I M475I V506M Mean IC 50 (nM) ± SD FC 146886 L T M M M V 0.9 ± 0.5 0.4 Samples with 146733 L T M M M V 5.4 ± 6.8 2.3 variants at 142972 L A M I M V 0.6 ± 0.4 0.3 Temsavir RAMs 134352 L A M M M/I V 16.7 ± 6.9 (X4: 11.8; R5: 21.5) 7.0 146996 L A/V M M M V 31.6 ± 19.3 13.2 134039 L A M M/K M V 1.7 ± 1.8 0.7
Conclusions The distribution of IC 50 values for CRF02_AG is similar to other subtypes • – Wide range of IC 50 values (0.6 – 381.6 nM) – Median FC 6.4 close to previous data obtained with only 3 isolates (FC 5.2, Nowicka-Sans et al.) One strain with IC 50 > 100 nM: what is the impact in vivo? • – Phase 2 AI438-006 study: 4/10 patients with IC 50 >100 nM have <1 log HIV-1 RNA decrease during fostemsavir monotherapy (Nettles et al., JID 2012) – By contrast, one patient with IC 50 6.6 μM has >1 log HIV -1 RNA decrease Is it necessary to predict/measure fostemsavir activity? • – Is it possible to predict fostemsavir susceptibility through genotypic assays? – Will there be renewed interest in phenotypic testing? • Phase 2b clinical trial AI438-011 excluded patients with virus showing IC 50 >100 nM (Lataillade et al., JAIDS 2017) • Phase 3 clinical trial AI438-047 (BRIGHTE Study, NCT02362503) had no cutoff IC 50 value for inclusion
THANK YOU FOR YOUR ATTENTION! Prof. Maurizio Zazzi research team: Ilaria Vicenti Alessia Giannini Adele Boccuto Filippo Dragoni
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