Poster - RNAi Therapeutics by Silence Therapeutics - American Society of Clinical Oncology

First-in-human Phase I study of Atu027, a liposomal siRNA formulation,targeting protein kinase N3 (PKN3) in patients with advanced solid tumorsDirk Strumberg1, Beate Schultheis1, Ulrich Traugott2, Christiane Vank2, Ansgar Santel2, Oliver Keil2, Klaus Giese2, Jörg Kaufmann2, Joachim Drevs31University of Bochum, Marienhospital Herne, Department of Hematology and Oncology, Germany; 2Silence Therapeutics AG, Berlin, Germany; 3Tumorzentrum UniSantus, Cologne, Germany Abstract # 3057 Pharmacokinetic/-dynamic of Atu027 in Non-Human Primates (Cynomolgus)BACKGROUND: A Protein knockdown in lung tissue B mRNA knockdown in lung tissue C siRNA concentration in plasmaAtu027 is a novel RNAi therapeutic based on cationic lipoplexes containing chemically stabilized siRNAs, which targets PKN3 gene (Western blot) (bDNA assay) samplesexpression in the vascular endothelium when administered via the systemic route. The lipoplexes (AtuPLEX) form particles of chemically infusionstabilized siRNA molecules (AtuRNAi) interspersed between lipid bilayers comprised of a novel cationic lipid, a PEGylated lipid, and a 120 Vehicle 1,2 hPKN3/hPPIBmRNA 1.2 0.3 mg/kgneutral helper lipid. Cell culture experiments revealed that PKN3 acts as a downstream effector of the PI3K-signaling pathway and is control 0.3 1.0 3.0 mg/kgimplicated in controlling cellular morphology and locomotion in endothelial and cancer cells. Preclinical data obtained in various cancer 0.1 mg/kg 0,8 0.8 siRNA (A-strand) [ng/ml] animal # 1 2 3 4 5 6 7 8mouse models revealed target-specific, RNAi-mediated silencing of PKN3 expression and significant inhibition of tumor progression and 0.03 mg/kgmetastasis formation, affecting both lymphatic and hematogenous dissemination. 0,4 0.4 80 PKN3METHODS: 0,0 0.0 vehicle 0.03 0.1 0.3 1.0 3.0 p110α 0.03 0.1 0.3 1.0 3.0 vehicle controlPatients (pts) (ECOG PS 0-2) received Atu027 as a single 4h-infusion with 3wks follow-up, and were thereafter treated twice weekly over control mg/kg ATU027/23Hanother four week period. Upon SD, pts were given the opportunity to continue until PD. Dose escalation was accompanied by assessment Actin mg/kg Atu027 40of data related to toxicity and pharmacokinetics (PK).RESULTS:The study design comprises 11 escalating doses. To date, 24 pts have received Atu027 of eight dose levels up to 0.180 mg/kg. Mean age = D RNAi in Cynomolgus lung tissue verified by 5‘-RACE 060.9 years (range 29-81), 14 female, 10 male. No pre-medication was required. No cytokine activation (TNF-α, IL-1β, IFN-γ, IL-6) wasobserved. In some subjects transient activation of the complement system (C3a, Bb, sC5b-9) was found, but without any clinical relevance. 0 4 8 12 16 20 24 Vehicle 0.3Preliminary PK-data showed dose-dependent increase in plasma siRNA as well as lipid levels. Across the dose levels tested, Atu027 was control mg/kg cleavage site verified Time [hours]generally well-tolerated. Adverse events possibly related to Atu027 were fatigue grade G1 (8pts), hair loss G1 (2pts), sweating G1 (1pt),nightmare G1 (1pt), mood alteration G2 (1pt) and abdominal pain G2 (1pt). G3 AEs not considered as DLTs were elevated lipase (1 pt, DL2) Cynomolgus In vivo (0.3 mg/kg):and diarrhea (1 pt, DL5). No dose limiting toxicities (DLTs) were seen so far. Stable disease after repeated treatment was observed in 9 pts. Exposure levels siRNA (A-strand - Day 29)At end of study (3 months after treatment start) stable disease has been confirmed in 6 pts (according to RECIST). Two pts with 166 bpneuroendocrine cancer had disease stabilization for 9 months, and partial regression of pulmonary metastases, respectively. Another cmax: 114  17 ng/mL [15 nM] PKN3 RNAipatient with breast cancer had slight regression of liver metastases. 5´-RACE AUC0-24h:999  432 ng•h/mLCONCLUSIONS:Atu027 is well-tolerated and antitumor activity has been observed. Accrual is ongoing to determine the MTD of Atu027. Systemic administration of Atu027 by repeated infusions in non-human primates results in dose-dependent specific RNAi-mediated Atu027 – Components, Structure and Delivery Properties silencing of PKN3 expression. 0.3 mg/kg siRNA was observed as the minimal active dose.Atu027 (a liposomal siRNA formulation targeting human PKN3) is composed of the blunt ended negatively charged 23mer siRNA Cynomolgus monkeys were treated with Atu027 twice weekly (every 4th day) for four weeks up to a total of 8 infusions.olignucleotide (AtuRNAi) and three lipids: the positively charged AtuFect01, the neutral, fusogenic DPhyPE helperlipid and the PEGylated Lung tissue samples were collected 24 hours after last treatment.lipid MPEG-2000-DSPE (molar ratio: 50/49/1). A: Reduction of PKN3 protein level in lung extracts from treated Cynomolgus monkeys is shown by immunoblot analysis for two individualAtuPLEX O O animals per treatment group; p110a and actin served as loading controls. siRNA-Cy3 AtuFect01 N N H NH B: A branched-chain DNA assay (bDNA assay) was carried out for showing dose dependent PKN3 knockdown in Cynomolgus lung tissue. NH3 NH3 Tumor cell nuclei H2N NH2 Cl Cl C: Dose-dependency of siRNA antisense strand (A-strand) plasma concentration shown by sandwich hybridization assay. Cynomolgus data are Cl predictive for humans. Figure shows mean values of dose groups (n= 6) for last treatment. O O O DPhyPE O O P O NH3 D: Verification of PKN3-mRNA cleavage site detected by 5´-RACE in lung tissue samples from Cynomolgus (dose level 0.3 mg/kg). O O O O O H Phase I Study Design & Enrollment Schedule DSPE-PEG O O P O N O O 45 45 O O O Na+ Na patients within each dose level Patient 3 Baseline FU-1 FU-2 End of Study Regular review by Sequential enrollment of AtuRNAi drug safety board AtuPLEX delivers to vascular endothelial cells (arrows) prior to dose Lamellar structure Shown: vasculature of mouse tumor xenograft Patient 2 Baseline FU-1 FU-2 End of Study escalation. Atu027 targets PKN3 expression Single Repeated FU: Follow Up phase Treatment Treatment Growth factor receptor extracellular Patient 1 Baseline FU-1 FU-2 End of Study Atu027 treatment = 4h iv infusion PKN3 intracellular Key regulator during angiogenesis/lymphangiogenesis Weeks -2 -1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 PI3-K p110a PTEN  Compassionate Use* Months 0 1 2 3 4 p110b Major regulator of metastasis/motility during pathological processes Ras  Myc Tumor staging (CT/MRT) Akt-1 Hif-1 Akt-2 Inhibition of PKN3 leads to Biomarkers PTB-1B PKN3 p53  reduced nutrient/oxygen supply to the tumor Blood Cytokines & Bcl-2 Redd1 sampling Complement  inhibited tumor/endothelial cell motility/ metastasis formation PK * Option to continue treatment Glucose Survival Tumor Metastases Vital Signs, ECGs & in sinlge patients until PD uptake progression Motility safety blood sampling (compassionate use/individual treatment attempt following Adverse events (AEs/SAEs) German medicines law).

Key Inclusion and Exclusion Criteria Patient OverviewINCLUSION CRITERIA: Stable disease – according to RECIST and compared to the results of the baseline staging examination - has been observed in 9 patients- Histologically and/or cytologically proven advanced, recurrent or metastatic solid malignancy for which standard curative or palliative measures do not exist, are no longer effective, or are unlikely to be effective measured 1 week after the repeated treatment. At end of study, 6 stable disease cases were confirmed.- Age ≥ 18 years / ECOG 0-2 / Life expectancy ≥ 3 months / weight ≥ 50 kg- Recovered from the acute reversible effects of previous anti-cancer chemotherapy, endocrine therapy, immunotherapy, radiotherapy or DOSE OUTCOME DOSE OUTCOME surgery. At least 30 days since major surgery and at least 5 half-lives (t½) must have elapsed since treatment with any investigational LEVEL AGE GENDER TYPES OF CANCER PRIOR THERAPIES AT EoS LEVEL AGE GENDER TYPES OF CANCER PRIOR THERAPIES AT EoS agent. # (3 MONTHS) # (3 MONTHS)- Written informed consent 52 Male Leiomyosarcoma 1x C/1x R/1x I PT (NA) 66 Male Pancreas CA 2x C (ps) PDEXCLUSION CRITERIA: 54 Male Esophagus 4x C/2x R/3x O PT (PD) 05 65 Female Cholangio CA 1x C/2x O PD 01- Evidence of central nervous system (CNS) metastases (Squamous cell CA) 68 Female Pancreas CA 4x C (1x adj) PT (PD)- Abnormal hematologic, renal or hepatic parameters as defined: Neutrophil count < 1.500/mm3 (=1.5x109/l); Platelet count < 100.00/mm3 69 Male Melanoma 2x C PT (PD) Neuroendocrine (=100x109/l); WBC < 3x109/l; HB < 9.0 g/l; ASAT (SGOT), ALAT (SGPT) ≥ 1,5x upper limit of normal (ULN) or ≥ 2.0x ULN in case of liver Neuroendocrine 58 Female 1x C/1xR SD  CU 29 Male 1x C SD  CU (lung) metastases; Total bilirubin ≥ 1.5x ULN; Creatinine clearance < 50 ml/min (calc. by Cockroft-Gault formula) (primacy unknown) 06 Sarcoma- Any concurrent disease, medical or social condition that could affect compliance with the protocol or interpretation of results as judged 02 Oropharynx 81 Male 1x R PT (PD) 53 Male 1x MAB/1x O PD (testicular cord) by the investigator (e.g.: seizures, poorly controlled diabetes, poorly controlled hypertension, severe dyspnea or severe pulmonary (Squamous cell CA) 73 Female Breast CA 4xC /1x R/4x H&O PD dysfunction, autoimmune and inflammatory disease, active infection or known bacteremia, HIV or chronic infection with hepatitis B or C, 68 Male Colon CA 5x C (1x adj)/2x O PD history of acute or chronic pancreatitis, substance abuse, pregnancy) 66 Female Rectum CA 1x C&R (ps)/2x C PT (PD) 62 Female Breast CA 1x C/1x R/1x H SD  CU 07 70 Female Colon CA 2x C PD 03 66 Female Breast CA 3x C (1x adj)/1x R/2x H PD Enrollment overview 73 Male Rectum CA 4x C/1x R/1x O SD 64 Male Prostate CA 2x C/1x R/2x H SD 39 Female Melanoma 1x C/1x I PD 24 patients (female: 14 / male: 10) have been enrolled in 60 Female Cholangio CA 2x C PD ALL FEMALE MALE 08 59 Female Breast CA 3x C(ps)/2x MAB/2x C PT (PD) dose level #01 to #08 so far. Mean age is 61 (female: 39-74 / 04 51 Female Breast & Ovarial CA 7x C (1x adj) SD NeuroendocrinePATIENTS ENROLLED male: 29-81) and mean weight is 70 kg (female: 66kg (50-81) 46 Female Cervix CA 1x C/2x O PD 71 Female (lung) 2x C/1x R/1x I ongoing 24 14 10(LEVEL #01-08: á 3 PATIENTS) / male: 77kg (50-133)). 20 patients (female: 11 / male: 9)AGE (YEARS) 29-81 39-74 29-81 completed treatment, for 4 patients (female: 3 / male: 1) study participation was terminated prematurely after the adj: Adjuvant / C: Chemotherapy / CA: Carcinoma / CU: Compassionate use / H: Hormone therapy / I: Immunotherapy / MAB: Therapy with monoclonal antibodies / NA: Not applicable /PATIENTS COMPLETED single treatment or during the repeated treatment phase, O: Other therapy / PD: Progressive disease / ps: Pre surgery / PT: Premature termination / R: Radiotherapy / SD: Stable disease 20 11 9TREATMENT (LEVEL #01-#08) respectively, due to disease progression. 15 patients (female:PATIENTS COMPLETED STUDY 15 9 6 9 / male: 6) completed the study so far, 3 male patientsPREMATURE TERMINATON (PT) 7 3 4 dropped out of study during the post treatment follow up Hints for Efficacy - Case Report phase (FU-2). Phase I Study Dose Escalation & Human PK (siRNA) Female patient with neuroendocrine cancer, BEFORE AFTER manifested in the lung. Stable disease was DOSE Atu027 siRNA concentration in human plasma confirmed at End of study. Patient received 2 Dose-dependent concentration of siRNA LEVEL # (mg/kg – siRNA) - first treatment - antisense strand shown in human plasma additional repeated treatment courses 01 0.001 infusion samples. (2x 8 additional treatments). 02 0.003 120 8 0.180 mg/kg 7 0.120 mg/kg Graphs show means of raw data of 3 patients This patient shows disease stabilisation for 03 0.009 siRNA (A-strand) [ng/ml] 6 0.072 mg/kg per dose group. more than 7 months since start of Atu027 5 0.036 mg/kg 04 0.018 80 4 0.018 mg/kg treatment. 3 0.009 mg/kg Baseline (pre-treatment) 1 week after last repeated treatment 05 0.036 2 0.003 mg/kg Plasma samples are frequently taken on 06 0.072 1 0.001 mg/kg treament days and during follow up phases. 07 0.120 40 Summary Sampling time points on first treatment day: 08 0.180 pre-dose, 0.5h, 1h, 2h, 4h, 4.5h, 5h, 6h, 8h, Atu027 is a liposomal siRNA formulation containing the human specific siRNA (AtuRNAi) directed against PKN3. Therapeutic efficacy had 09 0.253 0 12h and 24h, 0h: start of 4h iv infusion. been demonstrated in different animal tumor models (see literature). Pharmacological activity of Atu027 on PKN3 expression was 10 0.336 0 4 8 12 16 20 24 demonstrated in Cynomolgus monkeys by Western blot, bDNA assay and 5´RACE for treatment doses ≥0.3 mg/kg siRNA. Time [hours] 11 0.447 Phase I-Study: • Atu027 is well tolerated (dose level #08: 0.18mg/kg) - no dose-dependent trends in clinical or laboratory adverse events Safety Summary • MTD is not reached, yetAdverse Events - possibly or probably releated to Atu027 treatment: Serious Adverse Events: • No pre-medication to suppress immune response required • 14 SAEs in 10 different patients • Human plasma PK shows dose dependent increase DOSE (3x dose level #01*/ 1x dose level #04/ 2x dose • 6 patients with confirmed stable disease (RECIST) at end of study PATIENT ADVERSE EVENTLEVEL # level #05/ 1x dose level #06/ 2x dose level #07/ 1x dose level #08) • 3 patients continued treatment after study completion -001 Sweating G1, Mood alteration G2, Hair loss G1, Fatigue G1 01 -002 Nightmares G1 • All SAEs judged unrelated to Atu027 treatment  Dose escalation is continuing -003 Fatigue G1 (except *mood alteration (hospitalisation)) 02 -001 Lipase increase G3 -002 Fatigue G1 Cytokines: Literature on Atu027 03 • isolated high values at different time points -003 Fatigue G1 04 -001 Fatigue G1 without sytematic 1) Leenders, F., et al. 2004. PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase. EMBO J. 23:3303-13. 2) Santel, A., et al. 2006a. RNA interference in the mouse vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy. Gene Ther. 13:1360-70. 05 -003 Abdominal pain G2, diarrhea G3 3) Santel, A., et al. 2006b. A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium. Gene Ther. 13:1222-34. Complement Activation: 07 -003 Fatigue G1 4) Aleku, M., et al. 2008a. Intracellular localization of lipoplexed siRNA in vascular endothelial cells of different mouse tissues. Microvasc Res. 76:31-41. -002 Fatigue G1 • Atu027-related transient activation of the 5) Aleku, M., et al. 2008b. Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression. Cancer Res. 68:9788-98. 08 alternative pathway of the complement system 6) Kaufmann, J., et al. 2010. RNA interference for therapy in the vascular endothelium. Microvasc Res. 80:286-93. -003 Fatigue G1 7) Santel, A., et al. 2010. Atu027 prevents pulmonary metastasis in experimental and spontaneous mouse metastasis models. Clin Cancer Res. 16:5469-80.

Poster - RNAi Therapeutics by Silence Therapeutics - American Society of Clinical Oncology

by Silence Therapeutics on Dec 05, 2011

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Poster - RNAi Therapeutics by Silence Therapeutics - American Society of Clinical Oncology — Presentation Transcript