ALN-VSP02 Phase 1 Trial UpdateDemonstrating RNAi in Man

Jared A. Gollob, M.D.

January 4, 2011

Dana-Farber Cancer Institute

Agenda

LNPs and ALN-VSP02: Background

Phase 1 Trial Safety Data

Pharmacodynamic Data

» 5’ RACE

» DCE-MRI

Summary

2

Lipid Nanoparticles for Systemic Delivery

3

Liver TargetingIn Vivo Silencing of ApoB in Non-Human Primates

4

Nature, 441, 111-114, Mar 2006

0

20

40

60

80

100

1mg/kg 2.5 mg/kg 1 mg/kg 2.5 mg/kg

2 day 11 day

% C

on

tro

l

Efficacy in primates with Systemic RNAi after single IV injection Rapid, potent, dose-dependent and durable effects

RNAi specific and leads to measurable therapeutic benefit

RNAi mechanism proven in vivo

8.911.7

*

9.2

**23.2

**

31.7

60.5

**72.4

Protein

mRNA** P < .005* P <.05

**

79.0

% C

on

tro

l

Day 11 Post-Dose (2.5 mg/kg)

Cholesterol LDL HDL

20

40

60

80

100109.8

34.1

*

14.2

**

>65%

Inhibition>85%

Inhibition

** P < .005* P <.05

0

Tumor TargetingMurine Liver Cancer Model

Orthotopic tumor model with intrahepatic Hep3B seeding in SCID miceSingle IV bolus injection of ALN-VSP or control siRNA

Mitotic arrest (monoasters) clearly detected in VSP-treated animals

KSP and VEGF target mRNAs cleaved in tumors confirming RNAi mechanism

5

Control siRNA

ALN-VSP

Keystone: RNAi, Feb 2009

hKSP mRNA

hKSP mRNA 3’ cleavage productRNA adapter

hKSP mRNA 3’ cleavage product

GR5N

cDNARev2Rev3

hVEGF mRNA

hVEGF mRNA 3’ cleavage product

hVEGF mRNA 3’ cleavage product

GR5N

cDNARev3Rev4

400

300200

100

4 2 1 mg/kg

VSP 02

400

300200

100

4 2 1 mg/kg

VSP 02

RNA adapter

PCR product 380 nt PCR product 210 nt

Does Animal Pharmacology Translate to Man?VSP02: First Opportunity to Show RNAi POC/POM in Man with LNP

VSP02 TTR01

Target kd in humans •VEGF: DCE-MRI and/or

angiogenesis biomarker

changes

•TTR lowering in blood

Clinical effect of target

kd

•Tumor response on CT

scans

•Improvement in neuropathy

score

•Improvement in cardiac

imaging

Improved clinical

outcomes with target

kd

•Improved PFS •Improved PFS

NDA •Improved PFS

•Improved survival

•Improved PFS

•Improved QOL

5’-RACE •Tumor biopsies • N/A

Target mRNA kd •Tumor biopsies • N/A

Specificity of target kd •Tumor biopsies • N/A

6

POC

POM

Liver Cancer ProgramALN-VSP02

RNAi to treat primary and secondary liver cancersPrevalent solid tumor and common site of

metastatic disease» ~700,000/yr: Incidence of HCC worldwide

» ~500,000/yr: Patients with liver metastasis

ALN-VSP is first dual-targeted RNAi drug» Targeting 2 pathways with 2 different siRNAs

increases potential therapeutic impact– Proliferation: Kinesin Spindle Protein (KSP)

– Angiogenesis: VEGF

» Lipid nanoparticle (LNP) formulation– From Tekmira Pharmaceuticals

Preferential biodistribution of LNPs to liver,

spleen, tumors » May be able to avoid dose-limiting on-target

toxicities associated with systemic delivery of small

molecules and antibodies:– KSP: myelosuppression, gastro-intestinal toxicity

– VEGF: Hypertension, bleeding, thrombosis,

proteinuria, bowel perforation

7

ALN-VSP02

VEGF

KSP

ALN-VSP02 Phase I Study Design

Dose levels and dosing schedule0.1, 0.2, 0.4, 0.7, 1.0, 1.25, 1.5, 1.7 mg/kg

3 + 3 cohort design, planned expansion phase of up to 20 pts at MTD

15-min IV infusion q2 wks; premed with steroids, H1 and H2 blockers, acetaminophen

Cycle = 2 doses (1 month), tumor measurements after every 2 cycles, treat until disease

progression» ALN-VSP02-002 extension study for pts remaining on study beyond 4 cycles

8

Cycle 1 Cycle 4Cycle 3Cycle 2

W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4

Screening

Rx Rx Rx Rx Rx Rx Rx Rx

Adverse events

DCE-MRI

Tumor biopsy

Plasma samples

for VEGF/PLGF

d3-5 d8-10

d3-6

CT scan for tumor

measurement

Agenda

LNPs and ALN-VSP02: Background

Phase 1 Trial Safety Data

Pharmacodynamic Data

» 5’ RACE

» DCE-MRI

Summary

9

ALN-VSP02 Phase I Study StatusAs Reported at Chemotherapy Foundation Symposium

N=28 Enrolled to date; Cohorts 1-6

Total of 127 ALN-VSP02 doses administered

» Range of doses per patient: 2-13

Dose escalation ongoing

» Current dose 1.25 mg/kg

» MTD not yet reached

Key demographics

» Median age 56 yrs (range 34-78)

» 13 males, 15 females

» Tumor types

– Colorectal cancer (N=16)

– Pancreatic neuroendocrine tumor (N=1)

– Papillary renal cell cancer (N=1)

– Squamous cell cancer of head and neck (N=1)

– Pancreatic cancer (N=1)

– Esophageal cancer (N=1)

– Endometrial cancer (N=2)

– Angiosarcoma (N=1)

– Ovarian cancer (N=2)

– Synovial sarcoma (N=1)

– Mullerian stromal tumor (N=1)

All patients treated with multiple prior anti-angiogenic and/or chemotherapy regimens

10

Chemotherapy Foundation Symposium, November 2010

ALN-VSP02 Phase I Safety Summary

ALN-VSP02 generally well tolerated to date

» 127 doses administered to 28 patients across 6 dose levels

» Up to 13 doses given to single patient

No dose-dependent trends in clinical or laboratory adverse events

No dose-dependent changes in LFTs

Human plasma PK showed dose-proportional Cmax and AUC with no evidence

of drug accumulation

» Animal PK studies accurately predicted for human

Two dose-limiting toxicities

» 0.7 mg/kg: Liver failure and death after 2 doses (possibly related to study drug) in patient with

near complete replacement of liver by tumor and prior partial hepatectomy and splenectomy

» 1.25 mg/kg: Grade 3 thrombocytopenia after dose 1 (related to study drug), resolved within 5

days

Three grade 2 infusion reactions (one each at 0.4, 0.7 and 1.25 mg/kg), all 3

tolerated further treatment with prolongation of infusion duration

MTD not yet reached, dose escalation continuing

11

Chemotherapy Foundation Symposium, November 2010

Agenda

LNPs and ALN-VSP02: Background

Phase 1 Trial Safety Data

Pharmacodynamic Data

» 5’ RACE

» DCE-MRI

Summary

12

ALN-VSP02 PharmacologyTumor Biopsies

17 Tumor biopsies obtained from 9 patients

» 3 at 0.4 mg/kg

» 2 each at 0.7, 1.0 and 1.25 mg/kg

» Liver tumor biopsies in 6 patients

» Extrahepatic tumor biopsies in 3 patients

CT-guided core needle biopsies obtained pre-

and post-dose 1

» Analyses ongoing:

– 5’ RACE

– qPCR

– Drug levels

13

Drug Levels for All Biopsies to Date

Pt #

Dose

mg/kg

Tumor Type

(site of metastasis)

siRNA Levels (ng/g)

VEGF KSP

007 0.4 Colorectal (liver) 25.7 12.5

016 0.4 SCC H&N (liver) N/A N/A

017 0.4 Ovarian

(liver)

28.9 17.2

018 0.7 Pancreatic (liver) N/A§ N/A§

019 0.7 Colorectal (liver) 142 73.3

022 1.0 Colorectal (adrenal) 9.8 3.8

025 1.0 Sarcoma (gluteal

muscle)

0.45 0.4

026 1.25 Colorectal

(liver)

0.32 <LLOQ

031 1.25 Ovarian

(peri-umbilical)

4.9 3.6

14

N/A: Assay not performed§Very little tissue available for analysis

LLOQ: lower limit of quantitation

ALN-VSP02 PharmacologyTumor Biopsies

17 Tumor biopsies obtained from 9 patients

» 3 at 0.4 mg/kg

» 2 each at 0.7, 1.0 and 1.25 mg/kg

» Liver tumor biopsies in 6 patients

» Extrahepatic tumor biopsies in 3 patients

CT-guided core needle biopsies obtained pre-

and post-dose 1

» Analyses ongoing:

– 5’ RACE

– qPCR

– Drug levels

15

RNAi Produces Precise Cleavage of Target

mRNA

16

mRNA

mRNAdegradation

dsRNAdicer

Cleavage

Strand separation

Complementary pairing

Cleavage

(A)n

(A)n

Synthetic siRNA

Targeted Gene

Silencing

RISC

5’ RACE

5’ RACE AssayMethod for Demonstrating RNAi

17

Sequence Analysis

Factors Influencing Ability to Show RNAi By

5’ RACE in Tumor Biopsies

Expression level of target mRNA

Amount of tissue in biopsy that expresses target

18

VEGF-A and KSP mRNA Levels in Normal Liver and Tumors

Very low expression of KSP mRNA in normal liver and tumors

Relatively abundant expression of VEGF-A mRNA in both

normal liver and tumor

19

1

10

100

1000

10000

liver hcc crc

Mo

lecu

les

VEGF KSP

Tumor Core Biopsies

20

Fibrosis

Tumor necrosis

Viable tumor

Viable tumor

Core biopsy

CT-guided Core Needle Biopsy

Tumor Core Biopsies

21

Liver

Viable Tumor

Liver

Tissue Components of Biopsies Analyzed to Date

Tumor biopsies from liver very heterogeneous with regards to amounts of

tumor, normal liver, and dead tissue

Post-treatment biopsies from 3 patients at 0.4 mg/kg:» Little to no tumor, abundant normal liver

» Can be used for VEGF 5’ RACE, since robust VEGF expression in liver

» Not informative for KSP 5’ RACE, as very little expression in normal liver

Post-treatment biopsies from 2 patients at 0.7 mg/kg:» Little tumor, very little normal liver

» Not informative for either VEGF or KSP 5’ RACE

22

Pt#

Dose

mg/kg

Tumor Type

site of metastasis

Pre-Treatment Biopsy (%) Post-Treatment Biopsy (%)

Tumor Liver Necrosis Tumor Liver Necrosis

007 0.4 Colorectal (liver) 14 0 86 17 80 3

016 0.4 SCC H&N (liver) 10 0 90 0 100 0

017 0.4 Ovarian (liver) N/A N/A N/A 0 95 0

018 0.7 Pancreatic (liver) 40 0 0 8 5 52

019 0.7 Colorectal (liver) 0 100 0 20 5 60

N/A: Biopsy not performed

Dominant Band Seen in 5’ RACE for VEGF in Two Post-Dose Clinical Samples

23

200

300

007 017

pre post

016

pre post post

0.4 mg/kg

150

400

100

Non-clin

crchcc liver

Predicted VEGF mRNA

cleavage product band

Human RNAi Proof of MechanismResults from Blinded Molecular Analysis of Human Biopsy Samples

24

Patient 007-016

Pre-treatment

Patient 006-017

Post-treatment (2 days)

Patient 007-016

Post-treatment (2 days)

Post-treatment

Pre-treatment

27.9%

29.2%

1.4%

rea

ds/t

ota

l rea

ds m

ap

pin

g to

th

e tra

nscrip

t

% Specific cleavage

as proportion of all

sequence readsUntreated controls

Evidence

of RNAi

NO

YES

YES

Untreated control

(colorectal cancer-crc)

Untreated control

(hepatocellular cancer-hcc)

Untreated control

(normal liver)

0.64%

0.26%

0.1%

5’ --- Location along VEGF Transcript ---3’

NO

NO

NO

as.factor(start)

pe

r_

ob

s

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

0.000.050.100.150.200.25

12

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694

9007

pre

007po

s0

16

pre

01

6p

os

01

7p

os

01

8pre

018

po

st

01

9pre

019

po

st

sample

nc

post

pre

Patient 002-007 Pre-treatment

Patient 002-007 Post-treatment (7 days)

0.55%

0.66%

RNAi

Cleavage

NO

NO

p<0.0001*

p<0.0001

*T-test

5’ RACE Tumor Biopsy DataConclusions

In first 5 patients analyzed, 3 had abundant

normal liver/total mRNA that permitted VEGF 5’

RACE analysis

Predicted VEGF mRNA cleavage product seen

post-treatment in livers of 2/3 patients

» p<0.0001

» Biopsy from negative patient was obtained 7 days post

treatment

First demonstration of RNAi in man with LNP-

formulated siRNA

25

Agenda

LNPs and ALN-VSP02: Background

Phase 1 Trial Safety Data

Pharmacodynamic Data

» 5’ RACE

» DCE-MRI

Summary

26

ALN-VSP02 PharmacologyDCE-MRI

DCE-MRI (dynamic contrast-enhanced MRI) established as

radiologic test for assessing anti-VEGF effect of novel drugs in

clinical trials

Ktrans is key parameter

» Measure of blood flow and blood vessel permeability in tumors

» 40% or greater drop in Ktrans post-treatment considered a significant

drop in tumor blood flow

27

Example of Ktrans drop in patient treated

with oral anti-VEGFR drug AG-013736Liu G et al. JCO 2005;23:5464-5473

KtransBaseline (BL) Pre-Dose Day 4 Post-Dose 1 Day 7 Post-Dose 1

DCE-MRI Result at 0.7 mg/kgPatient 012: Pancreatic Neuroendocrine Tumor

28

T: liver tumor number

Ktrans2/IAUGC2: Δ DCE-MRI #1 (BL) to DCE-MRI #2 (Day 4)

Ktrans3/IAUGC3: Δ DCE-MRI #1 (BL) to DCE-MRI #3 (Day 7)

T1 T2 T3

Baseline MRI, coronal view Patient 003-012

-80

-60

-40

-20

0Ktrans2

IAUGC2

Ktrans3

IAUGC3

Ktr

an

s a

nd

IA

UG

C C

ha

ng

e

fro

m B

aselin

e (

%)

T1

T2

T3

T1

T2

T3

T1

T2

T3

T1

T2

T3

ASCO, June 2010

DCE-MRI ResultsSummary of Cohorts 1-4

29

» 21 evaluable liver tumors in

12 patients

» 19/21 tumors (90%)

showed decline in Ktrans

» 13 of 21 tumors (62%) had

↓Ktrans of ≥40%

» 8 of 12 patients (67%) had

average ↓Ktrans of ≥40%

# of evaluable

liver tumors:

0.1

mg/kg

0.2

mg/kg

0.4

mg/kg

0.7

mg/kgDose level:

Patient Number

Ave

rag

e K

tra

ns C

han

ge

Fro

m B

as

eli

ne (

%)

40% ↓

001 003 004 007 008 009 015 012 013 018 019 021-100

-80

-60

-40

-20

0

1 3 1 1 2 3 1 3 1 2 2 1

ASCO, June 2010

Agenda

LNPs and ALN-VSP02: Background

Phase 1 Trial Safety Data

Pharmacodynamic Data

» 5’ RACE

» DCE-MRI

Summary

30

Summary

Safety of LNP delivery to liver established in ALN-VSP02

Phase 1 liver cancer trial

Liver delivery and VEGF mRNA target engagement with

LNP-formulated siRNA demonstrated through 5’ RACE

assay on liver tumor biopsies

» Clear proof of RNAi activity in man

Preliminary DCE-MRI data from imaged liver tumors

further supportive of anti-VEGF pharmacology with ALN-

VSP02

Translatability of safety and pharmacology from NHP to

man greatly de-risks the LNP delivery platform

» Also highlights potential of liver-directed programs currently in

development including ALN-TTR and ALN-PCS

31

Acknowledgements

ALN-VSP02 Principal InvestigatorsSarah Cannon Cancer Center» Howard “Skip” Burris

Karmanos Cancer Institute» Pat LoRusso

Dana-Farber Cancer Institute» Geoffrey Shapiro

Beth Israel Deaconess Medical Center» Daniel Cho

Vall d’Hebron University Hospital (Barcelona)» Josep Tabernero

Hospital Clinico Universitario (Valencia)» Andres Cervantes

Hospital Universitario Virgen del Rocio (Seville)» Luis Paz-Ares

Alnylam PharmaceuticalsTekmira

32