1

Disclosures

Susan Lee

Sr. Research Associate

Employee of Onyx Pharmaceuticals

2

Potent Inhibition of Multiple Proteasome Subunits by

Carfilzomib in Solid Tumor and Multiple Myeloma Patients

Lee, S.J., Woo, T.M., Vallone, M., Arastu-Kapur, S.,

Chan, E.T., Wong, A.F., Le, M.H., Hannah, A.L., Kirk, C.J.

Onyx Pharmaceuticals, South San Francisco, CA

3

The Ubiquitin Proteasome Pathway

Adapted from Orlowski RZ, et al. Clin Cancer Res. 2008;14:1649-1657.

Meiners S, et al. J Biol Chem. 2003;21517-21525.

26S Proteasome

Poly-Ubiquitin Tag

Cellular Proteins

Ubiquitin

+ 20S Core

ligases

Proteasome inhibition results in:

Impaired cell proliferation

Activation of apoptotic pathways

Upregulation of de novo proteasome

production

4

HN

NH

OHN

O

O

NH

O O

O

PR-171 (carfilzomib)

N

O

Carfilzomib: A Novel Agent Designed to Promote Selective and Sustained Proteasome Inhibition

• Carfilzomib is a next generation, highly selective, irreversible proteasome inhibitor– Potent and sustained target suppression

– Improved antitumor activity

– Minimal off-target activity with low neurotoxicity

EpoxyketoneTetrapeptide

Adapted from Kuhn DJ, et al. Blood. 2007;110:3281-3290.

5

Clinical Program OverviewTrial N Design/Population Status

003-A0 46 Ph 2 Single-Agent (RR / MM) Completed

003-A1 266 Ph 2b Single-Agent (RR / MM) Completed

004 165 Ph 2 Single-Agent (R / MM) Enrolled

005 50 Ph 2 Single-Agent (RR MM with Renal Impairment) Enrolled

011 84 FOCUS: Single Agent vs. Best Supportive Care (R&R MM) Ongoing

006 84 Ph1/2 Combination with Len/Dex (Relapsed MM) Enrolled

009 700 ASPIRE: Combination CRd vs. Rd (Relapsed MM) Ongoing

007 145Ph 1b/2 Single-Agent Dose-Escalation

(Relapsed Solid Tumors &MM)Ongoing

6

Carfilzomib Active in MM

0

20

40

60

80

100

6% 9%

29%40%

18%19% 12%

25%

38%

8%10% 12%

9%

41%35% 35%

19%

8%

SD

MR

PR

≥VGPR% o

f su

bje

cts

003-A0 003-A1 004(BTZ-treated)

004(BTZ-naïve)

006

N 39 257 34 64 50

# Prior Therapies

5 (median)

5 (median)

1-32

(median)1-3

ORR (≥PR)

18% 25% 21% 54% 78%

Dosing 20 mg/m2 20/27 mg/m2 20 mg/m2 20/27 mg/m2

15-27 mg/m2

+ Len (10-25 mg) + Dex (40 mg qw)

Jagannath S, et al. ASH 2009. ASH/ASCO joint session oral presentation;Stewart AK, et al. EHA 2010. Abstract 1099 (oral presentation).

Siegel D, et al. ASH 2010. Abstract 985 (oral presentation).Vij R, et al. ASH 2010. Abstract 1938 (poster presentation).

Wang M, et al. Lymphoma and Myeloma 2010. Poster presentation; Martin T, et al. Lymphoma and Myeloma 2010. Poster presentation.

IMWG

Response

7

Carfilzomib is Well Tolerated as a Single Agent

003 (A0)(N=46)

003 (A1)(N=266)

004(N=143)

005(N=50)

All Studies(N=505)

Hematologic

ThrombocytopeniaAnemiaLymphopeniaNeutropenia

26%37%28%4.3%

27%22%19%10%

15%12%12%13%

20%28%18%6%

23%21%18%10%

Non-hematologic

PneumoniaFatigueHyponatremia

11%8.7%13%

8.3%7.1%7.5%

13%5.6%2.1%

12%14%4%

10%7.5%6.1%

Singhal SB, et al. ASH 2010. Abstract 1954 (poster presentation).

All treatment-emergent adverse events of Grade ≥ 3 (≥10%)

8

Two Classes of ProteasomesConstitutive Proteasome

b6

b7

b1

b5 b2

b3b4

b6

b7

LMP2

LMP7 MECL1

b3b4

Immunoproteasome

abba20S proteasome

b ring

Unique N-terminal Threonine active sites

Adapted from Groettrup M, Kirk C, and Basler M. Nat Rev Immunol. 2010;10(1):73-78.

Carfilzomib • 1o Target: chymotrypsin-like active sites (ß5 / LMP7); 2o Target: LMP2 & MECL1

Bortezomib • 1o Target: chymotrypsin-like active sites (ß5 / LMP7); 2o Target: LMP2

9Standard Assays for Measuring Proteasome Activity Involve Substrate Cleavage

• Inhibition of both Beta5 & LMP7 necessary to induce

Myeloma tumor cell death1

• MM cells express both classes of proteasome

• Substrates for other active sites are poorly defined

• Role of individual active sites in drug response is currently

unknown1Parlati, F., et al. Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like

activity of the proteasome. Blood, Vol. 114, Issue 16, 3439-3447, October 15, 2009

b5/L7

AMCLLVYAMC

LLVY

Chymotrypsin-Like (CT-L)

Subunits

10

+1. Activation Denaturation

step

Capture on

beads

b5

b5

b2

b2b1

b1

1. Wash

2. Add 1° Abb5 b2b1 1. Wash

2. Add 2° Ab

Luminescence

Signal

2. Probe

ProteasomeProbe

ProCISE (Proteasome Constitutive-Immuno Subunit ELISA)

Measures All 6 Proteasome Subunits

b5 b2b1

ProCISE Measures:

1. Active Site Inhibition

2. Proteasome Levels

11

0

5

10

15

20

25

y = 1.123x - 0.06

R2 = 0.99

Beta 5

0

5

10

15

20

y = 0.988x - 0.20

R2 = 0.98

LMP 7

0

5

10

15

20

y = 1.083x - 0.17

R2 = 0.97

Beta 1

0

5

10

15

20

y = 1.095x - 0.27

R2 = 0.98

LMP 2

0 5 10 15 200

5

10

15

y = 1.007x - 0.60

R2 = 0.96

Beta 2

0 5 10 15 200

5

10

15

y = 0.858x - 0.85

R2 = 0.95

MECL 1

Expected proteasome concentration (g/mL)

Me

asu

red

pro

tea

so

me

co

nc

en

tra

tio

n (

g

/mL

)

Whole Blood PBMC

ProCISE ValidationRecovery Experiment

• Low day-to-day variability

• Beta5 & LMP7 activity is equivalent to LLVY activity

• Dynamic range allows for detection of up to 90% inhibition

12

% p

rote

aso

me

inh

ibitio

n 0

Time (weeks): 1 2 3

80

D1 D2

4

D8 D9 D15 D16

Rest period (12 d)

QDx2, weekly for 3 weeks (28 day cycle)

CD138+(Bone Marrow)

Blood PBMC

LLVY CT-L CT-L CT-L

ProCISEBeta5LMP7

MECL1

Beta5 Beta2 Beta1

LMP7LMP2

MECL1

# Patients Analyzed 40 74 71

Carfilzomib Dosing Schedule & PD Analysis

13

Kinetics of Proteasome Inhibition in Carfilzomib Treated Patients

Whole Blood

0

20

40

60

80

100

120 LLVYBeta5Beta1Beta2

% A

cti

vit

y (

rela

tiv

e t

o p

red

os

e)

PBMC

Pre

1h

r

Pre

1h

r

Pre

1h

r

Pre

1h

r0

20

40

60

80

100

120 LLVYLMP7MECL1LMP2

Day 1 Day 2 Day 8 Day 1

Cycle 1 Cycle 2

% A

cti

vit

y (

rela

tiv

e t

o p

red

os

e)

•Potent Inhibition After 1st Dose

•>80% for CT-L

• Cumulative Inhibition in Blood

due to irreversible nature of CFZ

•Prolonged Inhibition in PBMCs

for 48 hr

•Recovery of activity in PBMC

following rest period

Dose: 15 mg/m2

14

LMP7

0

20

40

60

80

100 LLVY

0

20

40

60

80

100

%

Ac

tivit

y(R

ela

tive t

o P

re-d

ose)

Renal Status or Co-Administration of other Agents

Does Not Alter Carfilzomib Pharmacodynamics

LMP7

Norm

al

Mild

/ M

oderat

e

Sev

ere

/ Dia

lysi

s0

20

40

60

80

100Normal

Mild / Moderate Renal Impairment

Severe Renal Impairment / Dialysis

PX-171-005

15 mg/m2

LMP7

0

20

40

60

80

100 LLVY

0

20

40

60

80

100

%A

cti

vit

y(R

ela

tive t

o P

re-d

ose)

PX-171-006

LMP7

0

20

40

60

80

10015mg/m2 CFZ

15mg/m2 CFZ + 40mg Dex + 10mg Len

15mg/m2 CFZ + 40mg Dex + 15mg Len

15

20 mg/m2

Pre Post0

20

40

60

80

100

% A

cti

vit

y (

rela

tiv

e t

o p

red

os

e)

36 mg/m2

Pre Post0

20

40

60

80

100

56 mg/m2

Pre Post0

20

40

60

80

100LLVY

LMP7

MECL1

LMP2

51% 64% 76%

High Dose Carfilzomib Results in Near Complete

Inhibition of Immunoproteasome Subunits

• Immunoproteasome subunits show dose-dependant inhibition by CFZ

• At 56mg/m2, CT-L activity is below the limit of detection (≤10% activity) & total immunoproteasome inhibition reaches 76%

• Only inhibition of Beta5 is seen in Whole Blood

16

Differences in proteasome inhibition are observed between responding &

non-responding patients

Patient Response May Correlate to

Proteasome Subunit Inhibition

0

25

50

75

100

125

150LLVY

% A

ctiv

ity

(%

of

C1D

1 p

red

ose)

LMP7

PR

Pre

1h

r

Pre

1h

r

Pre

1h

r

Pre 1h0

25

50

75

100

125

150MECL1

C1 C2

D1 D2 D8 D1

% A

ctiv

ity

(%

of

C1D

1 p

red

ose)

Pre

1h

r

Pre

1h

r

Pre

1h

r

Pre 1h

LMP2

C1 C2

D1 D2 D8 D1

PD

N = 10-20 (15 – 27mg/m2)

N = 3-5 (15 – 56mg/m2)

17

Baseline CD138+ Bone Marrow Proteasome Activity

Trial

Ave # Prior

Regimens

(range)

LLVY

(M AMC /

g Protein)

(N = 25)

b5

(ng/g

Protein

(N = 17)

LMP7

(ng/g

Protein)

(N = 19)

% LMP7

003, 004, 005 5 (1 -13) 10.2 ± 3 0.9 ± 0.1 2.5 ± 0.4 73.7%

Proteasome Activity in Cell Lines1

MM1.S 3.9 ± 0.5 2.9 ± 0.2 45%

8226 4.0 ± 0.5 2.4 ± 0.2 37%

Arh77 4.0 ± 0.2 3.4 ± 0.2 46%

1Parlati, F., et al. Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like

activity of the proteasome. Blood, Vol. 114, Issue 16, 3439-3447, October 15, 2009

Proteasome Content in

Patient-Derived Tumor Cells

18

Pre Post0.0

0.5

1.0

1.5b5

20

s u

g/m

L

Pre Post0

1

2

3

4

5

6LMP7

20

s u

g/m

L

Pre Post0

1

2

3

4

5

6MECL1

20

s u

g/m

L

PX-171-004 (20 mg/m2)

CD138+ Cells

47.5%

66.2%68.1%

Pre Post0

2

4

6

8LLVY

M

AM

C /

g

Pro

tein

88.1%

Carfilzomib Administration Results in

Proteasome Inhibition in Tumor Cells

Levels of

inhibition similar

to Whole Blood

& PBMC

2 of 3 patients show

inhibition in Bone

Marrow Tumor Cells

Pre

Pos

t

0

1

2

3

0.0

0.5

1.0

1.5

20s u

g/m

L

uM

AM

C / u

g P

rote

in

Pre

Pos

t

0

2

4

6

0

2

4

6

Beta5

LMP7

MECL1

LLVY

20s u

g/m

L

uM

AM

C / u

g P

rote

in

Pre

Pos

t

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.5

1.0

1.5

20s u

g/m

L

uM

AM

C / u

g P

rote

in

19

LMP2

Pre

Post

MECL1

Pre

Post

Beta1Beta2

LMP7

Pre

Post

020406080

100120

%

Ac

tivit

y

Beta5

020406080

100120

% A

cti

vit

yComparison of the Proteasome Inhibition Profile

of Carfilzomib and Bortezomib

LMP2

Pre Post

27 mg/m2 CFZ

1.3 mg/m2 BTZ

20 mg/m2 CFZ

20

Conclusions

• ProCISE is the first assay to measure all 6 proteasome

subunits in patient samples

• Carfilzomib administration results in ≥ 85% inhibition of

CT-L subunits (Beta5 / LMP7)

• Inhibition in MM tumor cells similar to Blood & PBMC

• Levels of inhibition with carfilzomib compare favorably

to that of other classes of proteasome inhibitors

• Near complete inhibition of the immunoproteasome is

achieved at well-tolerated high-dose CFZ and is

currently under further clinical investigation

• Potent immunoproteasome inhibition may correlate with

response in MM patients

21

Multiple Myeloma Research Consortium (MMRC)

Susan Kelley, MD

Sandra Wear, BSN

Onyx Pharmaceuticals

(Biostatistics)

Felicia Fong

Lauren Bray

David Eber

All of the patients and families who contributed to this study

Mayo Clinic

Gregory Ahmann

All of the participating research investigators, doctors,

nurses and data coordinators

Experimental Oncology and Clinical Trials

Unit, Kantonsspital, St. Gallen, Switzerland

Christoph Driessen

Acknowledgements