IR/PR IASLC 2016: YH25448, a highly selective 3rd generation EGFR TKI, exhibits superior survival benefit over osimertinib in mice with brain metastases of NSCLC

YH25448, a highly selective 3rd generation EGFR TKI, exhibits superior survival benefit over osimertinib in mice with brain metastases of NSCLC Min Hee Hong1, In Yong Lee1, Jong Sung Koh3, Jaekyoo Lee3, Byung-Chul Suh3, Ho-Juhn Song3, Paresh Salgaonkar3, Jaesang Lee3, Young- Sung Lee2, Se-Woong Oh2, Jong Gyun Kim2, Su Youn Nam2, and Byoung-Chul Cho1 1Division of Medical Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, South Korea, 2Yuhan R&D Institute, Yuhan Corporation, Seoul, South Korea, 3Genosco Inc., Cambridge, MA02138, USA BACKGROUND & OBJECTIVES METHODS Fig. 5. Dose response efficacy of YH25448 in H1975 subcutaneous and intracranial dual implantation tumor model in BALB/c nude mice Fig. 7. Pharmacodynamic activity of YH25448 in subcutaneous tumor tissue YH25448, 10 mg/kg YH25448, 3 mg/kgVehicle AZD9291, 10 mg/kg Vehicle Fig. 9. Histological improvement of brain metastases by YH25448 and live imaging Vehicle-treated control group YH25448-3 mg/kg treated group Kinase IC50, nM AZD9291 YH25448 EGFR (L858R/T790M) 8 2 EGFR (T790M) 2.2 1.7 EGFR (L858R) 12.2 20.6 EGFR (del19) 8.6 5.3 ErbB2 44 364 ErbB4 54 1,017 Wild type EGFR 54 76 IGF1R 1,023 >3,000 RESULTS Cell lines EGFR genotype GI50, nM Gefitinib AZD9291 YH25448 PC9 del19 70.6 15.3 2.2 PC9-GR del19+T790M by Gefitinib > 15000.0 4.5 0.9 PC9-GRER del19+T790M by Gefitinib & EGF816 5321.0 11.9 2.9 HCC827 del19 53.4 1.8 0.7 H1975 T790M+L858R > 15000.0 18.7 6.3 YU-1003* del19 > 15000.0 13.1 1.3 YU-1005* Wild type 13162.0 4011.1 5128.5 H1666 Wild type 1838.0 333.9 377.5 H2291 Wild type > 15000.0 4345.0 5506.0 * NSCLC patient-derived cell line -5 -4 -3 -2 -1 0 1 2 0 25 50 75 100 125 Gefitinib AZD9291 YH25448 Drug concentration (log10 M) Relative Cell Viability (%) -5 -4 -3 -2 -1 0 1 2 0 25 50 75 100 125 Gefitinib AZD9291 YH25448 Drug concentration (log10 M) Relative Cell Viability (%) PC9-GR PC9-GRER -5 -4 -3 -2 -1 0 1 2 0 25 50 75 100 125 Gefitinib AZD9291 YH25448 Drug concentration (log10 M) Relative Cell Viability (%) YU-1003* Fig. 6. Dose response efficacy of YH25448A (a salt form) in H1975 intracranial tumor model in BALB/c nude mice YH25448 YH25448 Fig. 2-2. In vitro anti-cancer activity of YH25448 0 2 4 6 8 10 12 14 16 18 20 22 24 26 0.1 1 10 0 20 40 60 80 100 120 Unbound plasma conc. at 3 mg/kg IC50, p-EGFR (0.6 ng/ml) % inhibition of tumor p-EGFR Time (hours) Unbound plasma conc. (ng/ml) % inhibition of tumor p-EGFR PK/PD correlation 0.1 1.0 10.0 100.0 0 4 8 12 16 20 24 Conc. (ng/mL or ng/g) Time (hr) YH25448, 10 mg/kg YH25448 CSF (10 mg/kg) YH25448 Plasma (10 mg/kg) CSF Plasma (unbound) 0.1 1.0 10.0 100.0 1000.0 10000.0 0 4 8 12 16 20 24 Conc. (ng/mL or ng/g) Time (hr) YH25448, 10 mg/kg YH25448 Brain (10 mg/kg) YH25448 Brain tumor (10 mg/kg) YH25448 Plasma (10 mg/kg) IC50 for p-EGFR in H1975 Brain PK in intracranial tumor bearing mice Currently-available EGFR tyrosine kinase inhibitors (TKIs) are ineffective for the treatment and prevention of brain metastases (BrM) due to the limited blood-brain barrier (BBB) penetration. YH25448 is a potent, highly mutant-selective and irreversible 3rd generation EGFR TKI that is able to penetrate the BBB, and targets both the T790M mutation and activating EGFR mutations while sparing wild type (wt). In vitro biochemical, functional experiments, in vivo PK and pharmacology/pharmacodynamic studies were performed to characterize the non-clinical profile of YH25448 using relevant assays, cell lines and animal models. CONCLUSION Fig. 8. PK/PD correlation and tumor/brain tissue distribution properties of YH25448 Fig. 3. Apoptosis inducing activity [In Vitro Pharmacology]  Kinase assay  Cell-free kinase assays were conducted using time-resolved fluorescence resonance energy transfer (FRETTM) technology. The ATP concentration corresponding to the Km of each enzyme was used. Kinase reactions were incubated at room temperature for 1 hour and the IC50 values were determined using GraphPad PrismTM software.  Anti-proliferation assay  Human NSCLC cell lines or patients-derived cells with various EGFR mutant status were incubated with test compounds. After 72 hours, cell viability was measured by quantifying the total amount of ATP using an ATPLiteTM assay kit.  p-EGFR assay  To determine whether its cellular anti-tumor activity against NSCLC cells correlated with molecular potency as assessed by change in phosphorylation of EGFR mutant protein, YH25448 or the reference inhibitors were incubated for 2 hours in representative NSCLC cells, followed by Western blot analysis.  Apoptosis assay  To understand the mechanism responsible for the anti-cancer activity of YH25448, we investigated cell death signaling induced by EGFR inhibition. Since caspase-3/7 are the key regulators of apoptosis, caspase-3/7 activity was determined in H1975 and PC9 cells after 48 hr incubation with each inhibitor. [In Vivo Efficacy and Pharmacokinetics/Pharmacodynamics]  H1975 subcutaneous implantation and brain metastases model  Luciferase-transfected H1975 human NSCLC cells were implanted into right flank and/or brain in female BALB/c nude mice.  Tumor burden of intracranial lesions and tumor size of right flank were measured using a bioluminescence imaging (BLI) technique and a digital caliper, respectively.  Established tumor bearing mice were treated with once daily oral dose of YH25448 or AZD9291 from 13 days post implantation (n=7-8/group).  In vivo pharmacodynamic study  In H1975 human NSCLC subcutaneous xenograft model, tumor tissue were collected at 2hr, 8hr and 24hr after 14 days treatment and p-EGFR and total EGFR were evaluated by Western blotting.  Pharmacokinetics  Blood, tumor, brain, or CSF samples were collected from the animals at the designated time points and concentration of YH25448 was determined using a validated LC-MS/MS method.  Statistics  All data are presented as mean ± SEM. Data were analyzed by using one-way ANOVA followed by Dunnett’s test or Student’s t-test. The survival analysis was performed using Kaplan-Meier survival curve and log-rank test comparing each group. *:p<0.05, **:p<0.01, ***:p<0.001 vs. vehicle, #:p<0.05, ##:p<0.01, ###:p<0.001 vs. AZD9291 YH25448 is a potent and highly mutant selective EGFR inhibitor and it induces apoptosis of NSCLC cells through induction of Bim Fig. 1. In vitro kinase inhibition activity Fig. 4. Down-regulation of p-EGFR and down-stream p-AKT and p-ERK signaling by YH25448 leads to induction of Bim in EGFR mutant NSCLC cells YH25448 showed excellent efficacy in a brain metastases model with NSCLC cells carrying the T790M mutation, and provided superior survival benefit over AZD9291 with good tolerability High BBB penetration profile and clear PK/PD correlation was well translated into excellent efficacy in brain metastases model Survival YH25448A 50 mg/kg AZD9291 50 mg/kg Cell lines EGFR genotype GI50, nM Afatinib AZD9291 YH25448 H2073 (+EGF) Wild type 6.4 244.4 318.1 PC9 del19 2.3 4.8 2.7 H1975 L858R/T790M 6.9 11.8 3.6 Fig. 2-1. In vitro anti-cancer activity IASLC 17th World Congress of Lung Cancer, December 4-7, 2016, Vienna, Austria  YH25448 shows dose dependent tumor growth inhibition and more potent and complete anti-cancer efficacy in both subcutaneous and intracranial tumor growth than AZD9291 with better tolerability at high dose level.  High potency and high BBB penetration profile of YH25448 well translates into the greater survival benefit in brain metastases model of NSCLC.  These findings provide rationale for the further development of YH25448 as a novel therapeutic for the treatment of EGFR mutant-positive patients. In particular, YH25448 will be able to address the urgent unmet needs for EGFR mutant-positive patients with brain metastases. YH25448 YH25448 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 Vehicle YH25448 1 mg/kg YH25448 3 mg/kg YH25448 10 mg/kg AZD9291 1 mg/kg AZD9291 3 mg/kg AZD9291 10 mg/kg ** *** ****** ****** Days after tumor implantation Percent survival 50 60 70 80 90 100 110 120 12 17 22 27 32 37 42 47 52 57 62 Body weight (%) Days after tumor implantation Body Weight Vehicle YH25448A 10 mg/kg YH25448A 25 mg/kg YH25448A 50 mg/kg AZD9291 50 mg/kg *** *** *** *** *** *** ** *** *** *** *** *** *** *** *** *** ****** *** ### ### 50 60 70 80 90 100 110 120 130 140 12 18 24 30 36 42 48 54 60 66 72 78 84 Body weight (%) Days after tumor implantation Body Weight Vehicle YH25448 1 mg/kg YH25448 3 mg/kg YH25448 10 mg/kg AZD9291 1 mg/kg AZD9291 3 mg/kg AZD9291 10 mg/kg (n=6) (n=3) ###### ## # # # # # 1.E+06 1.E+07 1.E+08 1.E+09 1.E+10 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 Log total flux (p/s) Days after tumor implantation Brain Tumor Growth Vehicle YH25448 1 mg/kg YH25448 3 mg/kg YH25448 10 mg/kg AZD9291 1 mg/kg AZD9291 3 mg/kg AZD9291 10 mg/kg ** *** *** *** *** *** * ** *** *** *** *** *** *** *** *** ** *** *** *** *** *** *** *** *** *** *** *** *** *** ** * * ** ** ** (n=0) (n=0) (n=1)(n=0) (n=4) (n=6) (n=6) 0.1 1 10 100 1000 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 Log tumor size (mm 3) Days after tumor implantation Subcutaneous Tumor Growth Vehicle YH25448 1 mg/kg YH25448 3 mg/kg YH25448 10 mg/kg AZD9291 1 mg/kg AZD9291 3 mg/kg AZD9291 10 mg/kg*** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** * * * * * * * (n=5) (n=6) (n=3) (n=6) 1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08 1.0E+09 1.0E+10 12 17 22 27 32 37 42 47 52 57 62 Log total flux (p/s) Days after tumor implantation Brain Tumor Growth Vehicle YH25448A 10 mg/kg YH25448A 25 mg/kg YH25448A 50 mg/kg AZD9291 50 mg/kg *** *** *** *** *** *** *** *** *** *** *** *** YH25448 * Tumor tissues were collected after 13 days once daily repeated oral administration of test drugs. (n=8/group) (n=7/group) * Bioluminescence imaging post 121 days drug-treatment * Histological examination post 13 days drug-treatment (n=3/group) For more information, please contact us at: [email protected] , +82-31-899-4203, or visit www.yuhan.co.kr P3-02B-119

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