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