San Antonio Breast Cancer Symposium – December 6-10, 2016 P1-12-05 Etirinotecan Pegol: Survival Advantage Over Standard of Care Drugs in a Model of Brain Metastases of Breast Cancer Shah N1, Mohammad AS1, Adkins CE1, Dolan EL1, Grif th J1, Jagannathan R1, Ute Hoch2, and Lockman PR1 1Department of Pharmaceutical Sciences, School of Pharmacy, HSC, West Virginia University, 1 Medical Center Drive, Morgantown, WV, 26506-9050, USA; 2Nektar Therapeutics, 455 Mission Bay Blvd South, San Francisco, CA 94158, USA.

Background Methods Results

Novel Pharmacology of Etirinotecan Pegol • Ef cacy Figure 2. Blood-Brain Barrier is Dif cult to Penetrate 5 Table 1. Physical-Chemical Properties of Small Molecule Chemotherapies Figure 8. Etirinotecan Pegol Concentrates in Brain Tumor Tissue and Allows (NKTR-102) – Female athymic nude mice were inoculated with 1.75 x 10 MDA-MB-231-BrLuc for Metastatic Breast Cancer Compared to Etirinotecan Pegol Accumulation of SN38 Capillary in Healthy Brain Capillary in Brain with Tumor cells via intracardiac injection Molecular Efux Etirinotecan Pegol and SN38 Concentrations in Orthotopically (Brain) Implanted MDA-MB-231 • Compared to irinotecan, Irinotecan Basement Mitochondria Basement membrane membrane Mitochondria – Starting on day 21, gemcitabine (60 mg/kg) and eribulin (1.5 mg/kg) were dosed Weight (Da) Hydrophilic Transport Tumors After IV Administration of Etirinotecan Pegol etirinotecan pegol prolongs Ester-based Pinocytic Astrocyte More Astrocyte foot pinocytic IP every 4 days; etirinotecan pegol (50 mg/kg), irinotecan (50 mg/kg), paclitaxel 1 vessels foot Taxanes (paclitaxel, docetaxel) ~800 Hydrophobic Yes ) Single Dose

Releasable vessels g elimination half-life of SN38 from Nucleus / 100 (6 mg/kg), vinorelbine (10 mg/kg), docetaxel (10 mg/kg), and vehicle (saline or 0.1

1 Linker (n g

Eribulin 730 Hydrophobic Yes 2 days to 40 days in patients . 10 dextrose) were dosed weekly via tail vein c Tumor: Continuous Release n 0.01 Vinorelbine 779 Hydrophobic Yes o 1 Tumor: Accumulation and Accumulation of SN38 Fenestra C

• Etirinotecan pegol penetrates 20 kDa, – Ef cacy was measured by tumor burden and survival l of Etirinotecan Pegol o 0.001 30x 4-arm PEG Ixabepilone 507 Hydrophobic Yes g 0.1 the leaky tumor vasculature, Tight e 170x Plasma P

Pericyte SN38 Conc. (ng/g) junction - Tumor burden was determined based on twice-weekly bioluminescence Less tight junction Etirinotecan Pegol 20000 Hydrophilic No n 0.01 0.0001 concentrating the active a Plasma c

2 Brain tumor cell measurements e 0 1 2 3 4 5 6 7 t 0.001

metabolite in tumor Efux pump o Time (d) n i

Hydrolysis r – At the end of the survival study, brains from select animals (n=4/treatment group) Etirinotecan Pegol physical-chemical properties favor accumulation and retention in i 0.0001 t

• In a murine model of brain E were harvested, sectioned, and mounted onto slides. Slides were stained with CNS tumors 0 1 2 3 4 5 6 7 metastases, etirinotecan pegol Time (d) results in 100-fold greater H&E to visualize brain metastases. Brain sections were evaluated using an BEACON Phase 3 Trial Olympus MVX10 microscope with a 2X objective (NA=0.5) and an optical zoom of Figure 5. Etirinotecan Pegol (EP) Prolongs Survival of Animals With Median concentration of SN38 in brain Survival Irinotecan and SN38 Concentrations in Orthotopically (Brain) Implanted MDA-MB-231 Tumors After (0.63-6.3X) to determine size and number of brain metastases Triple-Negative Breast Cancer Brain Metastases (Days) IV Administration of Irinotecan lesions compared to irinotecan, Hydrolysis • The randomized phase 3 BEACON trial compared etirinotecan Vehicle 37 prolonging survival3 EP 50 mg/kg IV 74 1 pegol to treatment of physician’s choice (TPC; one of seven • Tumor distribution EP 10 mg/kg IV 35 Single Dose 100 Treatment Irinotecan 50 mg/kg IV 35 0.1 Vehicle 5 Treatment • Etirinotecan pegol prolongs SN38 single-agent regimens) in women with advanced breast cancer – MDA-MB-231-BrLuc cells were implanted into brain and allowed to grow to 1×10 started ) started Vehicle g 4 100 / 10 /sr) Irinotecan 2 EP 50 mg/kg survival in patients with breast (Figure 3) 20-30 mg in size, prior to IV administration of 50 mg/kg irinotecan or etirinotecan 4 0.01 (n g 1×10 EP 10 mg/kg 50 mg/kg . 1

4 c Irinotecan Plasma cancer having stable brain metastases – Grade 3 adverse events were signi cantly less common with pegol. Blood (2 mL) was harvested and processed to plasma (5000 xg, 5 min, n 0.001 EP o 0.1 3 C Tumor: Short Exposure to SN38 10 mg/kg SN38 Conc. (ng/g) EP (48% vs 63% with TPC, P<0.0001) 2-8 ˚C). Brain tumors were surgically resected, weighed, added to 2 mL 1×10 50 Tumor: No Accumulation • Etirinotecan pegol design results in low initial peak and sustained 0.0001 preservation solution. Plasma and brain tumor samples were assayed for EP 0.01 of Irinotecan concentrations of active topoisomerase-1 inhibitor (Figure 1) – EP prolonged median overall survival (OS) by 2.1 months 50 mg/kg 2 3 out of 10 survived 0 1 2 3 4 5 6 7 1×10 0.001 Plasma Time (d) (12.4 vs 10.3 months; HR 0.87, P=0.08) etirinotecan pegol, irinotecan, and SN38 using liquid chromatography–tandem 3 5 7 13 Irinotecan Percent Survival (%) Percent n=10-18/group Weeks After BLI Signal (p/sec/cm 0 0.0001 mass spectrometry (LC/MS/MS) methods 1×101 – Quality of life is improved with EP compared with TPC Intracardiac Injection 5 out of 10 survived 0 21 28 35 42 49 56 63 70 77 84 91 0 1 2 3 4 5 6 7 0 21 28 35 42 49 56 63 70 77 84 91 Days After Intracardiac Injection Time (d) Figure 1. Comparative Pharmacokinetics of SN38: • Although OS did not reach statistical signi cance, there was a • Animals were euthanized according to international animal care guidelines Days After Intracardiac Injection Irinotecan vs Etirinotecan Pegol clinically meaningful and statistically signi cant improvement in = Irinotecan5 OS for a prede ned subgroup of patients with a history of brain Figure 6. Common Treatments for Metastatic Breast Cancers 100 6 = Etirinotecan pegol metastases at baseline (Figure 4) Median Lack Ef cacy in the Brain Metastasis Model Survival Treatment (Days) Conclusions Plasma Active Results Vehicle Vehicle started Vehicle 38 10 1×104 Treatment Metabolite SN38 started EP 50 mg/kg 100 EP 50mg/kg 86

Figure 3. BEACON Phase 3 Study Design Eribulin /sr) Docetaxel Docetaxel 39 Concentration 2 3 • Etirinotecan pegol penetrates into brain metastases, is retained and serves as a • Etirinotecan pegol physical-chemical properties favor accumulation and retention in 1.5 mg/kg 1×10 Vinorelbine Vinorelbine 43 (ng/mL) Single-Agent Etirinotecan Pegol Eribulin Eribulin 40 Locally recurrent or metastatic Docetaxel reservoir for continued release of SN38 in CNS tumors 1 2 1×102 Gemcitabine Gemcitabine 48 breast cancer (n=852) 145 mg/m every 3 weeks Primary Endpoint CNS tumors (Table 1) 10 mg/kg (n=429) • Overall survival 50 – Etirinotecan pegol generates 100x greater SN38 brain tumor exposure compared to • Prior treatment with anthracycline, 1 a taxane, and capecitabine Vinorelbine 1×10 R Secondary Endpoints • Etirinotecan pegol prolongs survival in murine model of breast cancer brain 10 mg/kg EP signi cantly Single-Agent Treatment of SN38 from irinotecan 0.1 • ECOG PS 0-1 • PFS, ORR, CBR, DoR, HRQoL 0 different from TPC Physician’s Choice (TPC) metastases (Figure 5) 1×10 survival Percent 0 3 6 9 12 • 2-5 prior chemotherapies for Exploratory Endpoints Gemcitabine (P<0.05) Docetaxel, eribulin, gemcitabine, n=10 • Etirinotecan pegol prolongs survival of animals due to higher brain tumor SN38 advanced disease 60 mg/kg BLI Signal (p/sec/cm 1 week Dose Schedule (weeks) ixabepilone, nab-paclitaxel, paclitaxel • PD Markers in CTC, others – Survival with 50 mg/kg EP is signi cantly longer compared to vehicle 1×10-1 0 • Stable brain mets allowed or vinorelbine 3 4 5 6 7 0 21 28 35 42 49 56 63 70 77 84 91 0 21 28 35 42 49 56 63 70 77 84 91 exposure (n=423) – Survival rate is 30% and 40-50% at 90 days, the trial endpoint, for 10 mg/kg and Weeks Days After Intracardiac Injection Days After Intracardiac Injection Strati cation: 135 centers in US, Canada, Belgium, France, Germany, • Standard of care chemotherapy agents for metastatic breast cancer used as TPC in • Geographic region Italy, Korea, Russia, Spain, The Netherlands, UK 50 mg/kg EP, respectively • Prior eribulin use BEACON lack ef cacy in this murine model of breast cancer brain metastases Enrollment: Dec 2011 – Aug 2013 Challenge of Brain Metastases • Receptor status Event cutoff: Dec 2014 – Surviving animals show decrease in tumor burden Figure 7. Etirinotecan Pegol Has Higher Tumor Permeability Compared to Irinotecan in Brain Metastases • Pharmacokinetic and ef cacy results in the murine model of breast cancer brain • Breast cancer is the second most common solid tumor • BEACON TPC agents lack ef cacy in murine model of breast cancer brain metastases provide biologic rationale for doubling of overall survival observed in a Distribution of Radioactivity at Cmax associated with brain metastases (15% of MBC) Figure 4. Preplanned Overall Survival in Patients With metastases (Figure 6) subset of BEACON patients with history of brain metastases 2 hr After Irinotecan 6 hr After Etirinotecan Pegol Comparison of Irinotecan and EP History of Brain Metastases (n=67) – Up to 50% of HER2+ subtype – Median survivals were not signi cantly different from vehicle control median 800 • Etirinotecan pegol is currently under review in the EU for conditional approval in 2000 2000 ****P < 0.0001 1.0 72.2% (54.5-84.0) Events OS (95% CI) survival (p>0.05) s s **** – From 25-46% of TNBC subtype 45.2% (27.4-61.4) i i patients with breast cancer and a history of brain metastases Etirinotecan Pegol (n=36) 31 10.0 mo (7.8-15.7) s s a a

t 1500 t 1500 600 0.8 TPC (n= 31) 29 4.8 mo (3.7-7.3) s s

– Tumor burden increased nearly 100-fold in all TPC and vehicle groups a a • A con rmatory phase 3 trial, ATTAIN, is ongoing in this population (NCT02915744) • Limited survival upon diagnosis of brain metastases 44.4% (28.0-59.6) t t 19.4% (7.9-34.6) e e M M 0.6 1000 1000 400 **** n n – 10-23 months for HER2+ and 3-7 months for TNBC HR (95% CI): 0.511 (0.304-0.858) • Quantitative autoradiography showed signi cantly higher concentrations in brain i i a a 0.4 Log-rank P-value = 0.0099 metastases compared to irinotecan at respective Tmax (2 hr for irinotecan and 6 hr r r • Management of brain metastases includes local therapy, and 500 13.87 ± 0.83 500 200

Concentration (ng/g) Concentration (ng/g) References Concentration (ng/g) Survival Probability 389.2 ± 8.02 BDT/ B BDT/ B there are no approved cytotoxic or molecularly targeted agents 0.2 for EP) (Figure 7) BDT/Brain Metastasis 0 0 0 1. Jameson et al. Clin Cancer Res. 2013;19:268-78. 4. Perez et al. Lancet Oncol. 2015;16:1556–1568. • The blood-brain barrier prevents many chemotherapeutic agents 0.0 • Improved ef cacy of etirinotecan pegol in this murine model of breast cancer brain 0 1 2 3 0 1 2 3 BDT Brain 0 3 6 9 12 15 18 21 24 27 30 Brain Metastasis Size (mm2) Brain Metastasis Size (mm2) Metastases 2. Hoch et al. Cancer Chemother Pharmacol. 5. Xie et al. J Clin Oncol. 2002;20:3293-3301. Months from Randomization metastases correlates with accumulation of SN38 in brain tumors, secondary to Irinotecan (2 hr) used to control systemic disease from reaching effective 2014;74:1125-1137. 6. Jameson et al. Clin Cancer Res. 2013;19:268-78. Number at Risk: 36 33 26 22 16 13 4 3 2 1 0 Brain tumor distant tissue (BDT) Etirinotecan Pegol (6 hr) concentrations in brain metastases (Figure 2) 31 27 14 7 6 4 2 2 1 0 EPR-mediated accumulation and retention of etirinotecan pegol (Figure 8) 3. Adkins et al. BMC Cancer. 2015;15:685. 7. Lin et al. Clin Cancer Res. 2013;19:6404-18.

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