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 Drevs3

1University of Bochum, Marienhospital Herne, Department of Hematology and Oncology, Germany; 2Silence Therapeutics AG, Berlin, Germany; 3Tumorzentrum UniSantus, Cologne, Germany

Abstract # 3057

BACKGROUND:

Atu027 is a novel RNAi therapeutic based on cationic lipoplexes containing chemically stabilized siRNAs, which targets PKN3 gene

expression in the vascular endothelium when administered via the systemic route. The lipoplexes (AtuPLEX) form particles of chemically

stabilized siRNA molecules (AtuRNAi) interspersed between lipid bilayers comprised of a novel cationic lipid, a PEGylated lipid, and a

neutral helper lipid. Cell culture experiments revealed that PKN3 acts as a downstream effector of the PI3K-signaling pathway and is

implicated in controlling cellular morphology and locomotion in endothelial and cancer cells. Preclinical data obtained in various cancer

mouse models revealed target-specific, RNAi-mediated silencing of PKN3 expression and significant inhibition of tumor progression and

metastasis formation, affecting both lymphatic and hematogenous dissemination.

METHODS:

Patients (pts) (ECOG PS 0-2) received Atu027 as a single 4h-infusion with 3wks follow-up, and were thereafter treated twice weekly over

another four week period. Upon SD, pts were given the opportunity to continue until PD. Dose escalation was accompanied by assessment

of 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 =

60.9 years (range 29-81), 14 female, 10 male. No pre-medication was required. No cytokine activation (TNF-α, IL-1β, IFN-γ, IL-6) was

observed. In some subjects transient activation of the complement system (C3a, Bb, sC5b-9) was found, but without any clinical relevance.

Preliminary PK-data showed dose-dependent increase in plasma siRNA as well as lipid levels. Across the dose levels tested, Atu027 was

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)

and diarrhea (1 pt, DL5). No dose limiting toxicities (DLTs) were seen so far. Stable disease after repeated treatment was observed in 9 pts.

At end of study (3 months after treatment start) stable disease has been confirmed in 6 pts (according to RECIST). Two pts with

neuroendocrine cancer had disease stabilization for 9 months, and partial regression of pulmonary metastases, respectively. Another

patient with breast cancer had slight regression of liver metastases.

CONCLUSIONS:

Atu027 is well-tolerated and antitumor activity has been observed. Accrual is ongoing to determine the MTD of Atu027.

Atu027 targets PKN3 expression

PKN3

Key regulator during angiogenesis/lymphangiogenesis

Major regulator of metastasis/motility during pathological processes

Inhibition of PKN3 leads to

reduced nutrient/oxygen supply to the tumor

inhibited tumor/endothelial cell motility/ metastasis formation

Akt-1

PTEN

Growth factor receptor

intracellular

Glucoseuptake

Tumor progression

Metastases

MotilitySurvival

Akt-2

Bcl-2p53

Redd1

Hif-1

PTB-1B PKN3

RasMyc

p110a

p110b

PI3-K

extracellular

Atu027 – Components, Structure and Delivery Properties

Pharmacokinetic/-dynamic of Atu027 in Non-Human Primates (Cynomolgus)

animal # 1 2 3 4 5 6 7 8

Vehiclecontrol 0.3 1.0 3.0 mg/kg

Protein knockdown in lung tissue(Western blot)

Cynomolgus In vivo (0.3 mg/kg):

Exposure levels siRNA (A-strand - Day 29)

cmax: 114 17 ng/mL [15 nM]

AUC0-24h:999 432 ng•h/mL

0,0

0,4

0,8

1,2

vehicle 0.03 0.1 0.3 1.0 3.0

control mg/kg ATU027/23H

mRNA knockdown in lung tissue(bDNA assay)

hPKN

3/hPPIB

mRN

A

1.2

0.8

0.4

0.0

vehic

le

contr

ol 0.03

mg/kg Atu027

3.00.1 0.3 1.0

siRNA concentration in plasmasamples

cleavage site verified

RNAi in Cynomolgus lung tissue verified by 5‘-RACE

Atu027 (a liposomal siRNA formulation targeting human PKN3) is composed of the blunt ended negatively charged 23mer siRNA

olignucleotide (AtuRNAi) and three lipids: the positively charged AtuFect01, the neutral, fusogenic DPhyPE helperlipid and the PEGylated

lipid MPEG-2000-DSPE (molar ratio: 50/49/1).

Lamellar structure

AtuPLEX siRNA-Cy3

Tumor cell nuclei

AtuPLEX delivers to vascular endothelial cells (arrows)

Shown: vasculature of mouse tumor xenograft

Phase I Study Design & Enrollment Schedule

Compassionate Use*

* Option to continue treatment

in sinlge patients until PD

(compassionate use/individual

treatment attempt following

German medicines law).Adverse events (AEs/SAEs)

Vital Signs, ECGs &

safety blood sampling

Tumor staging (CT/MRT)

Cytokines &

Complement

PK

Blood

sampling

Biomarkers

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16-2 -1

0 1 2 3 4Months

Weeks

Single

Treatment

Repeated

Treatment

Baseline FU-2FU-1 End of StudyPatient 1

Sequenti

alenro

llm

ent

of

pati

ents

wit

hin

each

dose

level

Baseline FU-2FU-1 End of Study

Patient 2

Patient 3

Baseline FU-2FU-1 End of Study

Atu027 treatment

= 4h iv infusion

FU: Follow Up phase

166 bp

PKN3 RNAi

5´-RACE

Vehicle

control

0.3

mg/kg

PKN3

p110α

Actin

Systemic administration of Atu027 by repeated infusions in non-human primates results in dose-dependent specific RNAi-mediated

silencing of PKN3 expression. 0.3 mg/kg siRNA was observed as the minimal active dose.

Cynomolgus monkeys were treated with Atu027 twice weekly (every 4th day) for four weeks up to a total of 8 infusions.

Lung tissue samples were collected 24 hours after last treatment.

A: Reduction of PKN3 protein level in lung extracts from treated Cynomolgus monkeys is shown by immunoblot analysis for two individual

animals per treatment group; p110a and actin served as loading controls.

B: A branched-chain DNA assay (bDNA assay) was carried out for showing dose dependent PKN3 knockdown in Cynomolgus lung tissue.

C: Dose-dependency of siRNA antisense strand (A-strand) plasma concentration shown by sandwich hybridization assay. Cynomolgus data are

predictive for humans. Figure shows mean values of dose groups (n= 6) for last treatment.

D: Verification of PKN3-mRNA cleavage site detected by 5´-RACE in lung tissue samples from Cynomolgus (dose level 0.3 mg/kg).

infusion

0

40

80

120

0 4 8 12 16 20 24

0.3 mg/kg

0.1 mg/kg

0.03 mg/kg

siRN

A(A

-str

and)

[ng/m

l]

Time [hours]

A B C

D

AtuFect01 N NH

O

NH3

O

NH

NH3

H2N NH2Cl ClCl

DPhyPE

O

O O

O

O

P

O

O

O

NH3

AtuRNAi

O

O O

O

O

P

O

O

O

HN

Na

O

O

O45

DSPE-PEG

Na+

45

Regular review by

drug safety board

prior to dose

escalation.

Hints for Efficacy - Case Report

Patient Overview

Literature on Atu027

Summary

DOSE

LEVEL

#

AGE GENDER TYPES OF CANCER PRIOR THERAPIES

OUTCOME

AT EoS

(3 MONTHS)

01

52 Male Leiomyosarcoma 1x C/1x R/1x I PT (NA)

54 Male Esophagus

(Squamous cell CA)

4x C/2x R/3x O PT (PD)

69 Male Melanoma 2x C PT (PD)

02

29 MaleNeuroendocrine

(primacy unknown)1x C SD CU

53 MaleOropharynx

(Squamous cell CA)1x MAB/1x O PD

68 Male Colon CA 5x C (1x adj)/2x O PD

03

62 Female Breast CA 1x C/1x R/1x H SD CU

66 Female Breast CA 3x C (1x adj)/1x R/2x H PD

73 Male Rectum CA 4x C/1x R/1x O SD

04

60 Female Cholangio CA 2x C PD

51 Female Breast & Ovarial CA 7x C (1x adj) SD

46 Female Cervix CA 1x C/2x O PD

adj: Adjuvant / C: Chemotherapy / CA: Carcinoma / CU: Compassionate use / H: Hormone therapy / I: Immunotherapy / MAB: Therapy with monoclonal antibodies / NA: Not applicable /

O: Other therapy / PD: Progressive disease / ps: Pre surgery / PT: Premature termination / R: Radiotherapy / SD: Stable disease

Safety Summary

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.

3) Santel, A., et al. 2006b. A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium. Gene Ther. 13:1222-34.

4) Aleku, M., et al. 2008a. Intracellular localization of lipoplexed siRNA in vascular endothelial cells of different mouse tissues. Microvasc Res. 76:31-41.

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.

6) Kaufmann, J., et al. 2010. RNA interference for therapy in the vascular endothelium. Microvasc Res. 80:286-93.

7) Santel, A., et al. 2010. Atu027 prevents pulmonary metastasis in experimental and spontaneous mouse metastasis models. Clin Cancer Res. 16:5469-80.

DOSE

LEVEL #PATIENT ADVERSE EVENT

01

-001

-002

-003

Sweating G1, Mood alteration G2, Hair loss G1, Fatigue G1

Nightmares G1

Fatigue G1

02 -001 Lipase increase G3

03-002

-003

Fatigue G1

Fatigue G1

04 -001 Fatigue G1

05 -003 Abdominal pain G2, diarrhea G3

07 -003 Fatigue G1

08-002

-003

Fatigue G1

Fatigue G1

Adverse Events - possibly or probably releated to Atu027 treatment:

Key Inclusion and Exclusion Criteria

Serious Adverse Events:

• 14 SAEs in 10 different patients

(3x dose level #01*/ 1x dose level #04/ 2x dose

level #05/ 1x dose level #06/ 2x dose level #07/

1x dose level #08)

• All SAEs judged unrelated to Atu027 treatment

(except *mood alteration (hospitalisation))

Cytokines:

• isolated high values at different time points

without sytematic

Complement Activation:

• Atu027-related transient activation of the

alternative pathway of the complement system

DOSE

LEVEL

#

AGE GENDER TYPES OF CANCER PRIOR THERAPIES

OUTCOME

AT EoS

(3 MONTHS)

05

66 Male Pancreas CA 2x C (ps) PD

65 Female Cholangio CA 1x C/2x O PD

68 Female Pancreas CA 4x C (1x adj) PT (PD)

06

58 FemaleNeuroendocrine

(lung)1x C/1xR SD CU

81 MaleSarcoma

(testicular cord)1x R PT (PD)

73 Female Breast CA 4xC /1x R/4x H&O PD

07

66 Female Rectum CA 1x C&R (ps)/2x C PT (PD)

70 Female Colon CA 2x C PD

64 Male Prostate CA 2x C/1x R/2x H SD

08

39 Female Melanoma 1x C/1x I PD

59 Female Breast CA 3x C(ps)/2x MAB/2x C PT (PD)

71 FemaleNeuroendocrine

(lung)2x C/1x R/1x I ongoing

ALL FEMALE MALE

PATIENTS ENROLLED

(LEVEL #01-08: á 3 PATIENTS)24 14 10

AGE (YEARS) 29-81 39-74 29-81

PATIENTS COMPLETED

TREATMENT (LEVEL #01-#08)20 11 9

PATIENTS COMPLETED STUDY 15 9 6

PREMATURE TERMINATON (PT) 7 3 4

24 patients (female: 14 / male: 10) have been enrolled in

dose level #01 to #08 so far. Mean age is 61 (female: 39-74 /

male: 29-81) and mean weight is 70 kg (female: 66kg (50-81)

/ male: 77kg (50-133)). 20 patients (female: 11 / male: 9)

completed treatment, for 4 patients (female: 3 / male: 1)

study participation was terminated prematurely after the

single treatment or during the repeated treatment phase,

respectively, due to disease progression. 15 patients (female:

9 / male: 6) completed the study so far, 3 male patients

dropped out of study during the post treatment follow up

phase (FU-2).

Stable disease – according to RECIST and compared to the results of the baseline staging examination - has been observed in 9 patients

measured 1 week after the repeated treatment. At end of study, 6 stable disease cases were confirmed.

Baseline (pre-treatment)

BEFORE

1 week after last repeated treatment

AFTERFemale patient with neuroendocrine cancer,

manifested in the lung. Stable disease was

confirmed at End of study. Patient received 2

additional repeated treatment courses

(2x 8 additional treatments).

This patient shows disease stabilisation for

more than 7 months since start of Atu027

treatment.

Enrollment overview

siRNA concentration in human plasma

- first treatment -

Phase I Study Dose Escalation & Human PK (siRNA)

INCLUSION CRITERIA:

- 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

- 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

surgery. At least 30 days since major surgery and at least 5 half-lives (t½) must have elapsed since treatment with any investigational

agent.

- Written informed consent

EXCLUSION CRITERIA:

- Evidence of central nervous system (CNS) metastases

- Abnormal hematologic, renal or hepatic parameters as defined: Neutrophil count < 1.500/mm3 (=1.5x109/l); Platelet count < 100.00/mm3

(=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

metastases; Total bilirubin ≥ 1.5x ULN; Creatinine clearance < 50 ml/min (calc. by Cockroft-Gault formula)

- Any concurrent disease, medical or social condition that could affect compliance with the protocol or interpretation of results as judged

by the investigator (e.g.: seizures, poorly controlled diabetes, poorly controlled hypertension, severe dyspnea or severe pulmonary

dysfunction, autoimmune and inflammatory disease, active infection or known bacteremia, HIV or chronic infection with hepatitis B or C,

history of acute or chronic pancreatitis, substance abuse, pregnancy)

DOSE

LEVEL #

Atu027

(mg/kg – siRNA)

01 0.001

02 0.003

03 0.009

04 0.018

05 0.036

06 0.072

07 0.120

08 0.180

09 0.253

10 0.336

11 0.447

Dose-dependent concentration of siRNA

antisense strand shown in human plasma

samples.

Graphs show means of raw data of 3 patients

per dose group.

Plasma samples are frequently taken on

treament days and during follow up phases.

Sampling time points on first treatment day:

pre-dose, 0.5h, 1h, 2h, 4h, 4.5h, 5h, 6h, 8h,

12h and 24h, 0h: start of 4h iv infusion.0

40

80

120

0 4 8 12 16 20 24

8

7

6

5

4

3

2

1

0.180 mg/kg

0.120 mg/kg

0.072 mg/kg

0.036 mg/kg

0.018 mg/kg

0.009 mg/kg

0.003 mg/kg

0.001 mg/kg

siRN

A(A

-str

and)

[ng/m

l]

Time [hours]

infusion

Atu027 is a liposomal siRNA formulation containing the human specific siRNA (AtuRNAi) directed against PKN3. Therapeutic efficacy had

been demonstrated in different animal tumor models (see literature). Pharmacological activity of Atu027 on PKN3 expression was

demonstrated in Cynomolgus monkeys by Western blot, bDNA assay and 5´RACE for treatment doses ≥0.3 mg/kg siRNA.

Phase I-Study:

• Atu027 is well tolerated (dose level #08: 0.18mg/kg) - no dose-dependent trends in clinical or laboratory adverse events

• MTD is not reached, yet

• No pre-medication to suppress immune response required

• Human plasma PK shows dose dependent increase

• 6 patients with confirmed stable disease (RECIST) at end of study

• 3 patients continued treatment after study completion

Dose escalation is continuing