Co-downregulated by Enza and EPI (74) Strongly downregulated by EPI only(22) Strongly downregulated by Enza only (25) Strongly downregulated by Enza + EPI combo (11) HALLMARK_Androgen response (p adj=3.25x10 -70 ) GO_Reproduction (p adj=3.1x10 -10 ) GO_Regulation of Cell population Proliferation (p adj=3.1x10 -10 ) GO_Cell Cycle Phase Transition (p adj=2.22x10 -16 ) GO_DNA Replication Initiation (p adj=3.95x10 -16 ) HALLMARK_TNFa Signaling via NFkB (p adj=3.82x10 -7 ) HALLMARK_EMT (p adj=38.21x10 -4 ) HALLMARK_Androgen Response (p adj=3.82x10 -7 ) Pathway Implication Gene R1881-activated genes ORC6, CDC45, MCM4, CCNA2, E2F1, NDRG1, CDC6, AKAP12, CCNE2, POLA2, HIST2H2AB, FANCD2, TYMS, CREB3L4, CDK2, BRCA1, ERCC6L, CAMKK2, PLK1, ORC1, UBE2C, HIST1H2BN STK17B, PTPN21, SEC24D, ID2, SAT1, SLC16A6, KLF4, TNFAIP8, WIPI1, DNAJB9, HERPUD1, B4GALT1, CALU, ZBTB10, ITGAV, LMAN1, B2M, PGM3, GHR, ABHD3, ERRFI1, SNAI2, CENPN, STK39, HERC3, IQGAP2, ALDH1A3, UAP1 KLK3, NKX3-1, KLK2, TMPRSS2, GUCY1A3, ZBTB16, RRP9, HES6, KLK4, ENDOD1, ABHD2, SMAGP The preclinical characterization and development of EPI-7386, an N-terminal domain androgen receptor inhibitor for the treatment of prostate cancer Ronan Le Moigne1, Nan Hyung Hong1, C. Adriana Banuelos2, Nasrin R Mawji2, Teresa Tam2, Jun Wang2, Kunzhong Jian3, Raymond J. Andersen3, Alessandra Cesano1, Marianne D. Sadar2, Han-Jie Zhou1, Peter Virsik1 1ESSA Pharma Inc., Houston, TX, and South San Francisco, CA, USA. 2Department of Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada. 3Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada. BACKGROUND The androgen receptor (AR) pathway continues to drive most castration-resistant prostate cancers (CRPC) even in late stages of the disease through resistance mechanisms, including gain-of-function mutations in the C-terminal li- gand-binding domain (LBD), AR amplification and expres- sion of constitutively active truncated AR splice variants lacking the LBD, such as AR-V7. A new method of inhibiting the androgen pathway is needed to overcome these AR-based mechanisms of re- sistance. One possibility is through selective inhibition of the N-terminal domain (NTD) of the AR which can inhibit its transcriptional activity even in the presence of LBD-driven anti-androgen resistance. EPI-7386 represents a new generation of NTD inhibitors (Anitens) and is designed to inhibit transcriptional activity of the AR by interacting with the NTD. In doing so, EPI-7386 is active against both full-length AR and splice-variant AR. A phase 1 clinical trial of EPI-7386 is beginning and its pre- clinical efficacy, selectivity, and safety profile are present- ed. CONCLUSION • Clinical candidate EPI-7386 displays preclinically: a. Similar potency in vitro to the ‘lutamides in full length AR models b. LBD independent inhibition of AR demonstrated by activity on AR-V7 driven gene expression c. Specific activity on the AR transcriptome, similar but different to enzalutamide, while displaying broader and deeper AR inhibition when combined together with en- zalutamide d. Activity in several in vitro and in vivo CRPC cell lines, including enzalutamide resistant models e. Dose response activity with a minimal active exposure ~ 80,000 ng*h/mL in mouse VCaP xenograft models f. Tolerability in 28-days tox studies in rats and dogs at AUC ≤ 2,000,000 ng*h/mL, with activity seen on andro- gen-sensitive target organs g. Favorable human PK parameters supporting QD dosing h. Initial clinical starting dose of 200mg • EPI-7386 IND allowed by the FDA with the first-pa- tient-in planned for June 2020 EPI-7386 inhibits androgen-induced transcrip- tional activity A B AR inhibition is on target, LBD independent and effective in AR-V7 driven models Figure 2: Effect against androgen-induced PSA-luciferase activity in LNCaP cells (A) A dose-dependent decrease in AR-transcriptional activity was demonstrated in LNCaP cells transfected with the PSA reporter gene and incubated with compounds in the pres- ence of androgen (R1881). (B) Summary of IC50s calculated across multiple independent ex- periments. LNCaP cells bear a T877A mutation on the AR gene that decreases affinity of apalutamide and darolutamide. A C B D EPI-7386 was well tolerated in rat and dog tox studies and is predicted to achieve high exposures in humans A B C E Figure 6: Toxicology overview, human projected exposure and solid form of EPI-7386 (A) 28-day GLP TK and tox conclusion in male Sprague-Dawley rats. (B) 28-day GLP TK and tox conclu- sion in male Beagle dogs. (C-F) TK graph of EPI-7386 concentration in plasma over time, in D1 in rats (C), D23 in rats (D), D1 in dogs (E) and D27 in dogs (F). (G) Estimated human maximum recommended starting dose for phase 1 calculated from tox studies. (H) Human estimated PK curves and (I) PK pa- rameters at steady state, from 50-800 mg per day, based on in vitro in vivo correlation . Figure 3: EPI-7386 activity and selectivity in full-length AR and AR-V7 models (A-B) Androgen-induced proliferation of LNCaP and viability of PC-3 cells was measured with Alamar blue while BrdU was used for LNCaP95 cells. (C) The transcriptional activity of endogenous AR-FL was measured in LNCaP cells using a PSA(6.1kb)-luciferase reporter plasmid which encodes the promoter and enhancer region of the human PSA/KLK3 gene. (D) The transcriptional activity of ectopic AR-V7 was measured in LNCaP cells transiently transfected with a plasmid encoding AR-V7 (pARV7) and the V7BS3-luciferase reporter (driv- en by AR-V7). (E) Expression levels of AR-FL regulated genes measured by qPCR in LNCaP cells treated +/- R1881 (1nM) and exposed 24h to 10 uM EPI-7386 or 5 uM enzalutamide. (F) Expression levels of AR-V7 regulated genes measured by qPCR in LNCaP95 cells grown in castrated conditions and exposed 24h to 10uM EPI-7386 or 5 uM enzalutamide. EPI-7386 is active in a variety of castrate-sensitive and resistant prostate cancer xenograft models Figure 5: In vivo activity in CRPC xenograft models (A) Summary of in vivo activity of EPI-7386 in a subset of prostate cancer xenograft models bearing various AR-dependent and independent tumors. (B) Example of dose response activity obtained in castrated male SCID Beige mice bearing VCaP tumors. (C) EPI-7386 PK parameters in different strains of mice. (D) Tumor growth of EPI-7386 in combination with enzalut- amide in castrated male SCID Beige mice bearing VCaP tumors. (E) Summary of preclinical responses in the VCaP study. A C B Figure 1: Anitens are first-in-class NTD inhibitors of the androgen receptor Structure of the androgen receptor (AR) and mechanism of inhibition. The AR is orga- nized in 3 distinct domains: the LBD, involved in binding with androgens, the DBD, and the NTD, which orchestrate the transactivation of the receptor. It was previously demonstrated that the first-generation aniten EPI-002, and its stereoisomeres, specifi- cally binds to the transactivation unit 5 (Tau5) of AR NTD to block essential protein–pro- tein interactions required for transcriptional activity of AR 1-4 . 1 De Mol et al., ACS Chem Biol, 2016. 2 Andersen et al., Cancer cell, 2010. 3 Sadar , Cancer Res, 2011. 4 De Mol et al., Structure, 2018. N-Terminal Domain (NTD) DNA Binding Domain (DBD) Ligand Binding Domain (LBD) Resistance to current anti-AR therapy occurs predominantly in the LBD and can cause the AR pathway to remain active Androgen Androgen Deprivation CYP17A inhibition Anti androgens Reduce level of androgen Inhibit synthesis of androgen Block androgen binding to LBD Current anti-AR Therapies Anitens D E E D 0 10 20 30 40 50 0 200 400 600 800 VCaP Castrated Dosing Days Mean Tumor Volume (mm 3 ) ± SEM Vehicle (DMSO/NMP/Solutol/PEG400) Enzalutamide 15 mg/kg- PO qd EPI-7386 30 mg/kg- PO qd Combo Enza - 15 mg/kg + EPI-7386 - 30/60 mg/kg p=0.0005 p<0.0001 Compound IC50 (nM) n EPI-002 9,580 2 EPI-7386 421 5 Enzalutamide 154 5 Bicalutamide 242 7 Apalutamide 4,540 3 Darolutamide 616 3 Compound LNCaP PC-3 LNCaP95 EPI-002 9.0 >10 ~20 EPI-7386 0.56 >10 3.7 Enzalutamide 0.35 >10 >10 Cellular prolifera �on IC50 (uM) First in human clinical study 0.1 1 10 0 25 50 75 100 125 Conc (uM) % Relative to DMSO Control ± SEM EPI-7386 LNCaP95 LNCaP PC-3 F Arm Progressive Disease (PD) Stable Disease (SD) Par �al Response (PR) Complete Response (CR) Enza (15 mg/kg) 4 (57%) 3 (43%) 0 (0%) 0 (0%) EPI-7386 (30 mg/kg) 1 (14%) 5 (71%) 0 (0%) 1 (14%) COMBO (Enza+EPI-7386) 0 (0%) 1 (20%) 4 (80%) 0 (0%) pc D N A DM SO AR - V7 D M S O 5 u M - E n za 3 5uM - EP I -0 02 5 u M - E P I -73 86 0 25 50 75 100 125 150 Percent activity Relative to DMSO Control ± SEM LNCaP expressing AR-V7 and V7SB3-Luc reporter Abstract MP79-04 Contact: [email protected] F 0.01 0.1 1 10 100 0 25 50 75 100 125 Concentration (uM) LNCaP PSA-Luc % activity ± SD EPI-002 EPI-7386 Enzalutamide Darolutamide Apalutamide Bicalutamide B4GALT1 SLC30A7 SNX14 HIF1A BIRC5 CCNA2 PLK1 UBE2C 0 2 4 6 5.4 2.5 1.2 0.5 0.2 0.2 0.3 0.6 1.0 1.0 1.1 0.8 1.1 1.0 1.0 0.9 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Fold change Vehicle Enza EPI-7386 V7-repressed V7-activated STEAP4 KLK3 FKBP5 TMPRSS2 NDRG1 NKX3.1 0 10 20 30 40 50 5000 7500 10000 4.2 0.1 1.1 0.7 2.5 1.3 4.2 0.1 1.1 0.7 2.5 1.3 2.6 3.7 1.1 3.4 1.0 4.3 2.6 3.7 1.1 3.4 1.0 4.3 17.3 40.0 40.4 20.3 24.2 17.3 40.0 40.4 20.3 24.2 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Fold change No stim R1881 R1881+Enza R1881+EPI-7386 8155.0 DM S O 5 uM En za luta mi d e 3 5uM EPI-002 5 u M E PI-7386 DM S O 5 uM En za luta mi d e 3 5uM EPI-002 5 u M E PI-7386 DM S O 5 uM En za luta mi d e 3 5uM EPI-002 5 u M E PI-7386 DM S O 5 uM En za luta mi d e 3 5uM EPI-002 5 u M E PI-7386 0 50 100 150 LNCaP expressing PSA(6.1kb)-Luc reporter Percent activity Relative to DMSO Control ± SEM pCDNA ETOH pCDNA-R1881 ARV-7 ETOH ARV-7 R1881 LNCaP LNCaP95 Male SD rat 28-days GLP tox Male Beagle dog 28-days GLP Tox 0 4 8 12 16 20 24 100 1,000 10,000 100,000 Time (hr) EPI-7386 (ng/mL) EPI-7386 Human PK Simulations 800 mg QD 400 mg QD 200 mg QD 100 mg QD 50 mg QD Dose (mg) Cmax (ng/mL) AUC0-24 (ng*hr/mL) 50 1,729 34,320 100 3,458 68,639 200 6,915 137,278 400 13,830 274,556 800 27,659 549,113 NOAEL: No-observed -adverse-event level; HNSTD: Highest non-severe toxic dose. Repeat dose showed no accumulation between D1 and D23. NOAEL: No-observed -adverse-event level; HNSTD: Highest non-severe toxic dose. Repeat dose showed minimal accumulation between D1 and D27 . Anti-androgen effects observed on target organs. 0 5 10 15 20 25 0 200 400 600 800 VCaP Castrated Dosing Days Mean Tumor Volume (mm 3 ) ± SEM Vehicle Enzalutamide - 15 mg/kg- PO qd EPI-7386 - 3 mg/kg- PO qd EPI-7386 - 10 mg/kg- PO qd EPI-7386 - 30 mg/kg- PO qd AUC and Cmax calculated at steady state in vivo model AR status Male Mice Condi �on Compound Dose (mg/kg) Formula �on Regimen TGI (%) p value Enzalutamide 30 >100% p<0.0001 EPI-7386 60 93% p<0.0001 Enzalutamide 15 66% p<0.05 EPI-7386 3 54% p<0.01 EPI-7386 10 95% p<0.0001 EPI-7386 30 >100% p<0.0001 Enzalutamide 15 <0% NS EPI-7386 30 54% p<0.0001 High level AR-V7 Other oncogenic pathways AR + , PSA + Enzalutamide 15 Solu� on <0% NS AR-V7 unknown EPI-7386 60 Suspension 58% p<0.05 Non func � onal AR Enzalutamide 15 0% NS Other oncogenic pathways EPI-7386 30 0% NS Sta� s� cs: 2-way ANOVA with Dunne � correc � on for mul � ple comparisons. NS: Non signi fi cant. TGI: Tumor Growth Inhibi � on AR FL High level AR-V7 Other oncogenic pathways EPI-7386 p<0.0001 30 48% Castrated Castrated Castrated Castrated qd24 Solu� on Ampli fi ed AR Some AR-V7 PC-3 Intact Solu� on qd28 Castrated qd24 qd24 qd28 qd28 Solu� on Solu� on Solu� on LNCaP VCaP LNCaP95 22Rv1 HID28 Study design - 3+3 design - n = ~ 18 patients for dose escalation - n = ~ 10 patients for dose expansion Patient population mCRPC patients progressing on standard of care (including the latest antiandrogens) Study endpoints - Recommended Phase 2 dose (RP2D) - Safety and PK - PSA response Correlative studies - CTC conversion - CTC AR-V7 - ctDNA Timeline - FPI anticipated of 2Q 2020 Dose (mg/kg/day) AUC0–24h D23 (ng·h/mL) Major Findings Conclusion 60 1,119,000 Well tolerated and non-adverse Below NOAEL 120 1,640,000 Well tolerated and non-adverse NOAEL 240 2,350,000 Adverse body weight and food consump� on loss. No other signi fi cant clinical or anatomical pathology fi ndings HNSTD 0 4 8 12 16 20 24 100 1,000 10,000 100,000 1,000,000 Mean plasma conc of EPI-7386 in male SD rats - D23 Time (hr) [plasma] in ng/mL ± SD EPI-7386-60 mg/kg/day D23 (30 mg/kg/dose bid) EPI-7386-120 mg/kg/day D23 (60 mg/kg/dose bid) EPI-7386-240 mg/kg/day D23 (120 mg/kg/dose bid) 0 4 8 12 16 20 24 100 1,000 10,000 100,000 1,000,000 Mean plasma conc of EPI-7386 in male SD rats - D1 Time (hr) [plasma] in ng/mL ± SD EPI-7386-60 mg/kg/day D1 (30 mg/kg/dose bid) EPI-7386-120 mg/kg/day D1 (60 mg/kg/dose bid) EPI-7386-240 mg/kg/day D1 (120 mg/kg/dose bid) 0 4 8 12 16 20 24 100 1,000 10,000 100,000 1,000,000 Mean plasma conc of EPI-7386 in male Beagle dogs - Day 1 Time (hr) [plasma] in ng/mL ± SD EPI-7386-20 mg/kg/day D1 (10 mg/kg/dose bid) EPI-7386-50 mg/kg/day D1 (25 mg/kg/dose bid) EPI-7386-90 mg/kg/day D1 (45 mg/kg/dose bid) 0 4 8 12 16 20 24 100 1,000 10,000 100,000 1,000,000 Mean plasma conc of EPI-7386 in male Beagle dogs - Day 27 Time (hr) [plasma] in ng/mL ± SD EPI-7386-50 mg/kg/day D27 (25 mg/kg/dose bid) EPI-7386-70 mg/kg/day D27 (35 mg/kg/dose bid) EPI-7386-20 mg/kg/day D27 (10 mg/kg/dose bid) GLP Toxicology Study Reference Dose Converted to BSA (mg/kg/day) (mg/m2) mg/m � mg/day Rat 240 1,440 10 144 233 Dog 70 1,400 6 233 378 Es�mated Human MRSD Species Applied Safety Factor Abbreviations: BSA = body surface area; MRSD = maximum recommended starting dose. Conversion for mg/kg to mg/m2: rat (mg/kg) × 6; dog (mg/kg) × 20 . MRSD = 1/10 of STD 10 (mg/m2) in rodents or 1/6 high dose (mg/m2) in non-rodents. Conversion factor from mg/m2 to mg/day in humans is 1.62 m2 for a standard adult body surface area Dose (mg/kg) Mouse strain Day of dosing Cmax (ng/mL) AUC0-last (ng·h/mL) 3 CD-1 1 6,610 87,200 10 CD-1 1 16,300 220,000 30 CD-1 1 45,300 534,000 30 Nude / SCID / SCID Beige / NOG 1 38,750 475,000 30 Nude / SCID / SCID Beige / NOG 7 31,000 282,000 KLK2 FKBP5 TMPRSS2 KLK3 NCAPD3 NKX3-1 NDRG1 STEAP4 FAM105A -8 -6 -4 -2 0 Log 2 Fold Change Enza EPI-7386 Enza+EPI-7386 42 59 69 7.5 uM Enza 7.5 uM EPI-7386 5/5 uM Combo 0 20 40 60 80 Number of genes Fold change > 4 Gene Log2 Fold Change 7.5 Enza 7.5 EPI 5/5 Combo 1 KLK2 -1.71 -2.71 -6.63 2 FKBP5 -5.01 -4.47 -6.09 3 TMPRSS2 -2.66 -3.32 -5.68 4 KLK3 -1.24 -2.42 -5.40 5 NCAPD3 -4.32 -4.40 -5.03 6 NKX3-1 -1.72 -2.28 -4.54 7 NDRG1 -2.04 -4.17 -4.37 8 STEAP4 -4.22 -4.22 -4.22 9 FAM105A -3.37 -2.78 -4.18 10 AKAP12 -1.91 -3.84 -4.10 11 PMEPA1 -3.05 -2.16 -4.05 12 PLPP1 -2.48 -3.53 -3.97 13 SNAI2 -3.97 -1.79 -3.97 14 ACSL3 -3.43 -3.62 -3.90 15 ERRFI1 -4.51 -2.76 -3.90 16 CDC6 -1.20 -3.60 -3.86 17 ELL2 -3.39 -3.05 -3.81 18 CENPN -3.31 -2.13 -3.79 19 RHOU -3.94 -3.03 -3.78 20 EAF2 -3.32 -3.81 -3.52 Figure 4: Transcriptomic analysis from Nanostring androgen receptor panel (585 custom AR dependent genes) in LNCaP cells (A) Heat map showing expression of R1881-activated genes (> 2 fold) in LNCaP cells treated for 24 hrs, in conditions described in Fig. 3E. (B) Table summarizing pathways and genes specfic to each treatment arms. (C) 20 most downregulated genes in the EPI-7386 5 uM / enzalutamide 5 uM combination treated group compared to single agent treated group at 7.5 uM. All conditions were compared to R1881-stimulated conditions only. (D) Graphical representation of Log2 fold change in the 9 most down regulated genes in combination arm (5/5 uM) vs each single agent (7.5 uM). (E) Number of genes showing > 4 fold change in the EPI-7386 / enzalutamide combination treated group compared to single agents. H G I R1881 EPI-7386 7.5 uM Vehicle Enza 7.5 uM EPI-7386 inhibition of the AR pathway is similar but slightly different to enzalutamide while the combina- tion with enzalutamide exhibits a broader and deeper response on AR dependent genes A C B D E Enza + EPI-7386 5 / 5uM Dose (mg/kg/day) AUC0–24h D27 (ng·h/mL) Major Findings Conclusion 20 529,000 Well tolerated and non-adverse NOAEL 50 1,350,000 Well tolerated and non-adverse Below HNSTD 90/70 1,850,000 Adverse body weight and food consump� on loss. No other signi fi cant clinical or anatomical pathology fi ndings HNSTD