Mechanistic characterization of MM-131, a bispecific antibody that blocks c-Met signaling
through concurrent targeting of EpCAM
ABSTRACT #1690
Jessica B. Casaletto*, Adnan Abu-Yousif*, Kristina Masson, Aaron Fulgham, Melissa Geddie, Birgit Schoeberl, Ulrik B. Nielsen, Gavin MacBeath
Merrimack Pharmaceuticals, Cambridge, MA, USA *These authors contributed equally
P
GAB1
Y1349
Y1356
GRB2
FAK
MEK
PI3K
SOS
RAS
RAF
P
STAT3/5
SHC
RAC1
P
P
P
P
P
P
P
P
P
AKT
The c-Met signaling pathway can be activated by autocrine- or paracrine-mediated HGF binding
to c-Met, or by HGF-independent mechanisms such as c-Met gene amplification,
overexpression, or genetic mutation.
A549
104
4
10
10
1000−fold
5
10
6
MM-131
better
-8
-9
-7
-6
NCI-H1993
MKN45
Molecule concentration (log10 M)
Molecule concentration (log10 M)
-9
-8
-7
shControl + MM-131
shEpCAM + MM-131
-6
Molecule concentration (log10 M)
shControl + MM-131EpCAMmut
300
200
100
20
15
25
0.5
-9
-10
-8
-7
1.0
0.5
0.0
-6
-11
1.2
1.0
0.8
0.6
0.4
-11
-10
-9
-8
-7
-6
-8
-7
-6
1.2
1.0
0.8
0.6
0.4
0.2
-11
-10
-9
-8
-7
-6
-8
-7
-6
1.4
MM-131
OA-5D5/
“MetMab”
1.2
1.0
0.8
0.6
0.2
-5
-11
-10
-9
-8
-7
-6
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-11
Molecule concentration (log10 M)
shControl EpCAM:c-Met ratio ~30
shEpCAM EpCAM:c-Met ratio ~3
-10
-9
-8
-7
-6
Molecule concentration (log10 M)
-5
1.2
1.0
shControl + MM-131
shEpCAM + MM-131
shControl + OA-5D5/“MetMab”
0.8
0.6
0.4
MM-131 inhibits HGF-independent migration in
c-Met-amplified HCC827-GR5 cells
1.5
1.0
0.5
0.0
-9
-10
-9
-8
-7
-8
-7
-6
Molecule concentration (log10 M)
-5
200
100
0
30
10
20
15
shControl + Vehicle
shControl + MM-131
shControl + “MetMab”
shEpCAM + Vehicle
shEpCAM + MM-131
600
400
200
0
10
25
Time post-implantation (days)
20
15
25
1.0
0.5
0.0
-9
* as determined by a
decrease in IC50 or EC50
-8
-7
-6
Molecule concentration (log10 M)
shEpCAM + MM-131
30
Time post-implantation (days)
OA-5D5/“MetMab” (10 mg/kg q7d)
2.5
MKN45
NCI-H441
1.5
2.0
1.5
1.0
0.5
0.0
c-Met/actin
1.5
1.0
0.5
1.0
0.5
0.0
0.0
c-Met, HGF, AND EpCAM ARE CO-EXPRESSED IN TUMORS
Lung
Colorectal
c-Met
Gastric
c-Met
c-Met
HGF
HGF
33%
35%
1.5
EpCAM
Lung Cancer
0.5
0.0
-9
-8
-7
c-Met
EpCAM
HGF
-6
Molecule concentration (log10 M)
1.5
Positive Score
n
(%)
46
47%
88
96%
25
26%
c-Met & EpCAM
c-Met & HGF
EpCAM & HGF
c-Met & EpCAM & HGF
42
15
23
15
Total
Evaluable
97
92
95
Colorectal Cancer
Positive Score
n
(%)
72
77%
83
89%
40
44%
Total
Evaluable
93
93
91
Positive Score
n
(%) Cancer
Gastric
Positive Score
n
(%)
67
68%
89
92%
51
52%
72
32
36
32
Total
Evaluable
98
97
99
64
34
45
32
Primary tumor samples were collected from patients with lung, colorectal, and gastroesophageal cancers. Samples
positive for c-Met (blue), EpCAM (red), and HGF (yellow) are included in the Venn diagrams above.
1.0
0.5
0.0
EpCAM
EpCAM
1.0
-6
1.5
shControl + MM-131
0.2
0.0
-11
MM-131 inhibits HGF-dependent
migration in NCI-H441 cells
-5
NCI-H441
shControl EpCAM:c-Met ratio ~90
shEpCAM EpCAM:c-Met ratio ~6
HGF-independent migration
Molecule concentration (log10 M)
D EpCAM knock-down decreases potency
HT29
HGF-dependent migration
0.4
Molecule concentration (log10 M)
Molecule concentration (log10 M)
-9
Molecule concentration (log10 M)
1.4
-5
-10
300
800
16%
0.5
0.0
-11
400
MM-131 INHIBITS CANCER CELL MIGRATION IN VITRO
1.0
Molecule concentration (log10 M)
1.4
0.2
-9
-10
NCI-H747
High EpCAM:c-Met
1.5
ratio ~65
Normalized migration rate
pAkt
(Normalized to 1 nM HGF)
1.0
0.0
NCI-H441
Medium EpCAM:c-Met
ratio ~30
1.5
500
HCC827-HGF
HGF
A549
Low EpCAM:c-Met
1.5
ratio <1
600
MM-131 (12 mg/kg q7d)
c-Met level (molecules/cell)
B
c-Met High/EpCAM High/HGF-
Tumor volume (mm3)
400
shControl + OA-5D5/“MetMab”
MM-131 inhibits proliferation in HGF-dependent c-Met activated and HGF-independent c-Met activated (c-Met amplified) cancer
cell lines in vitro. Consistent with the decrease in potency observed in EpCAM knock-down cell lines, MM-131 is less effective
at inhibiting proliferation in EpCAM knock-down cells (shEpCAM). Similarly, a variant of MM-131 in which the anti-EpCAM scFv is
mutated to prevent EpCAM targeting (MM-131EpCAMmut) is less effective at inhibiting proliferation.
c-Met High/EpCAM High/HGF-
C MM-131 induces downregulation of c-Met in vivo
0.6
-10
Tumor volume (mm3)
NCI-H358-HGF
Vehicle
0.8
-11
40
Time post-implantation (days)
500
1.0
0.4
35
O
“M A et 5D5
M /
ab
”
-10
-5
30
M
-1
31
-6
25
M
-7
20
e
-8
25
cl
-9
20
15
0
hi
-10
10
200
Ve
-11
0.4
5
400
(Normalized to control)
0.6
0.6
25
c-Met/actin
(Normalized to control)
0.8
0.8
0
600
O
A
“M -5
et D5
M /
ab
”
100−fold
1.0
100
Time post-implantation (days)
1.2
1.0
200
M
-1
31
5
1.2
300
shControl + Vehicle
shControl + MM-131
shControl + “MetMab”
shEpCAM + Vehicle
shEpCAM + MM-131
Time post-implantation (days)
10
MM-131 inhibits HGF-independent proliferation in
c-Met-amplified MKN45 cells (left) and HCC827-GR5 cells (right)
1.2
400
c-Met High/EpCAM High/HGF+
HGF-independent c-Met activation
MM-131 inhibits HGF-dependent
proliferation in NCI-H441 cells
500
800
Time post-implantation (days)
0
HGF-dependent c-Met activation
600
M
10
MM-131 induces downregulation of c-Met more potently in cells
with a high ratio of EpCAM:c-Met (NCI-H441) than in cells with a
low ratio of EpCAM:c-Met (A549), suggesting that EpCAMtargeting leads to more potent downregulation of c-Met by MM-131.
20
15
e
10−fold
NCI-H441
cl
10
equal
100
0
10
MM-131 INHIBITS CANCER CELL PROLIFERATION IN VITRO
Anti-EpCAM scFv
KD = 15 nM
200
700
e
nm
Bio-layer interferometry (ForteBio) was used to measure ligand
blocking of MM-131. Antibodies were loaded onto anti-human
IgG Fc capture biosensors, incubated in 200 nM human c-Met for
two minutes, followed by the addition of 200 nM HGF.
300
c-Met High/EpCAM High/HGF-
cl
A549
400
400
c-Met High/EpCAM High/HGF-
hi
300
500
NCI-H441
Ve
200
100
Tumor volume (mm3)
Normalized signal
Anti-c-Met Fab
KD = 2 nM
HGF competitive
Cell surface levels of c-Met and EpCAM were determined in a panel of
cancer cell lines using quantitative fluorescence activated cell sorting
(qFACS). We then determined how potently MM-131 (or OA-5D5/“MetMab”)
inhibits HGF-dependent c-Met signaling via pAkt ELISA in each cell line.
Using these data, we built a computational model to demonstrate the effect
of EpCAM targeting. (A) Heatmap of cell lines tested reveals that many
cell lines have an EpCAM:c-Met ratio in which MM-131 is predicted to be
more potent than OA-5D5/“MetMab”. (B) We tested this model by comparing
the IC50 of MM-131 and OA-5D5 in cell lines with low, medium, and high
EpCAM:c-Met ratios. As predicted, MM-131 inhibits HGF-dependent c-Met
signaling more effectively in cell lines with higher EpCAM:c-Met ratios. (C)
Phenotypic assays (CTG, 96 h) show a correlation between inhibition of pAkt
and inhibition of cell viability. (D) Consistent with model predictions, MM-131
is less potent in cell lines in which EpCAM is knocked-down by RNAi.
10−fold
6
PAK
Cell proliferation, migration, invasion, survival
0
0
600
hi
NCI-H441
NCI-H747
mTOR
MAPK
0.0
Normalized migration rate
P
P
−6
Normalized migration rate
P
c-Src
P
−7
25
Relative viability
(Normalized to control)
107
OA-5D5/
“MetMab”
1000−fold
better
pAkt
(Normalized to 1 nM HGF)
P
P
SHP2
−8
Molecule concentration (log10 M)
30-100-fold increase
in potency
Relative viability
(Normalized to 1 nM HGF)
P
PLCγ
−9
Relative viability
(Normalized to 1 nM HGF)
MM−131 efficacy over
OA-5D5/“MetMab”
A
Relative viability
(Normalized to 1 nM HGF)
P
Y1234
Y1235
−10
EpCAM-TARGETING INCREASES POTENCY* OF MM-131
pAkt
(Normalized to 1 nM HGF)
Y1349
Y1356
P
P
-6
−11
Relative viability
(Normalized to control)
Less potent c-Met
inhibition
C
P
−12
Add
targeting arm
Molecule concentration (log10 M)
Y1234
Y1235
-7
0.5
Molecule concentration (log10 M)
Remove pathway
activation
-11
P
-8
-9
0
50
Time (sec)
c-Met
P
-10
0.2
1.0
75
700
Tumor volume (mm3)
Molecule concentration (log10 M)
-11
Avidity
0.4
MM-131
Positive control
Negative control
PBS
OA-5D5/
“MetMab”
MM-131
EpCAM-targeting
increases efficacy in vivo
MKN45
O
“M A-5
et D5
M /
ab
”
-12
-5
100
1.5
M
-1
31
-6
0.8
c-Met High/EpCAM High/HGF+
M
-7
0.0
HGF
0.6
HCC827-HGF
125
Ve
-8
0.5
2.0
c-Met/actin
(Normalized to control)
-9
1.0
B
HGF-independent
c-Met activation
c-Met activation
Tumor volume (mm3)
-10
1.0
Bispecific
Monospecific
A HGF-dependent
MM-131 induces
downregulation of c-Met
Relative c-Met expression
(24 h post incubation)
0.0
-11
HGF
c-Met
p-c-Met
(Normalized to 10 nM HGF)
0.5
Monovalent anti-Met
Bivalent anti-Met
HGF
Media
Normalized migration rate
HGF-independent
c-Met activation
1.0
1.5
pAkt
(Normalized to 1 nM HGF)
HGF-dependent
c-Met activation
Monovalent anti-c-Met
Bivalent anti-c-Met
MM-131 INHIBITS TUMOR GROWTH IN VIVO
MM-131 blocks
HGF binding to c-Met
Avidity drives anti-c-Met
inhibitor potency
1.2
Relative viability
(Normalized to 1 nM HGF)
HGF-DEPENDENT AND -INDEPENDENT ACTIVATION OF c-MET
Bivalent anti-c-Met molecules
activate c-Met signaling
pAkt
(Normalized to 1 nM HGF)
Consistent with its design, we found that MM-131 is more potent at inhibiting HGF-dependent
c-Met signaling, cell proliferation, and cell migration in EpCAM-high cells than in EpCAM-low cells.
In addition, MM-131 potency is reduced when EpCAM levels are knocked down by RNA
interference. Similarly, by mutating the EpCAM-targeting arm of MM-131, the observed potency in
EpCAM-high cells is noticeably reduced. Further cell biological characterization of MM-131
revealed two distinct mechanisms of action: (1) MM-131 blocks HGF binding to c-Met; and (2)
MM-131 induces downregulation of c-Met. In side-by-side comparison studies, MM-131 was found
to be more potent at inhibiting HGF-dependent signaling, cell proliferation, and cell migration than
one-armed-5D5 (OA-5D5/“MetMab”), and uniquely effective at inhibiting HGF-independent
signaling through downregulation of c-Met. Consistent with the design criteria, MM-131 did not
exhibit any discernable agonistic activity characteristic of bivalent anti-c-Met antibodies. The
molecular effects of MM-131 observed in vitro were also observed in vivo: MM-131 inhibited tumor
growth in models of ligand-dependent and ligand-independent c-Met signaling, induced
downregulation of c-Met, and was more effective at inhibiting tumor growth in EpCAM-high cells.
These findings support the clinical development of MM-131 in c-Met-driven epithelial tumors that
also express EpCAM.
1.5
p-c-Met
(Normalized to 1 nM HGF)
To assess the role of EpCAM in mediating avid binding of MM-131 to c-Met, we quantified the
cell surface levels of c-Met and EpCAM in a panel of cancer cell lines using flow cytometry and
determined how potently MM-131 inhibits HGF-dependent c-Met signaling in each cell line. Using
these data, we built a computational model to explain and quantify the effect of EpCAM targeting.
We then tested this model by (1) predicting the activity of MM-131 in other cell lines, based on
their EpCAM:c-Met ratios; (2) knocking down EpCAM in cell lines by RNA interference; and (3)
comparing the potency of MM-131 to a variant of MM-131 in which its EpCAM-targeting arm was
mutated to impair binding. To uncover the mechanism by which MM-131 inhibits HGF-independent
c-Met signaling, we monitored the effect of MM-131 on c-Met levels.
Bivalent anti-c-Met molecules
potently inhibit c-Met signaling
EpCAM level (molecules/cell)
MM-131 is a purely antagonistic, bispecific antibody that potently inhibits HGF/c-Met signaling
by co-targeting c-Met and the widely expressed tumor antigen EpCAM. The purpose of these
studies is to uncover the mechanism by which MM-131 exhibits potent inhibition of both
HGF-dependent and HGF-independent c-Met signaling in EpCAM positive tumor cells.
DUAL MECHANISM OF ACTION
Tumor volume (mm3)
SYSTEMS BIOLOGY-GUIDED DESIGN CRITERIA
ABSTRACT
CONCLUSIONS
-9
-8
-7
-6
•
MM-131 potently inhibits HGF-dependent and HGF-independent c-Met signaling in vitro
and in vivo by antagonizing HGF-binding to c-Met and inducing downregulation of c-Met.
•
Unlike bivalent c-Met antibodies, MM-131 does not activate c-Met signaling.
•
The EpCAM-targeting arm of MM-131 increases avidity and potency in pre-clinical models.
Molecule concentration (log10 M)
shControl + MM-131EpCAMmut
shControl + OA-5D5/“MetMab”
In this migration/wound healing assay, the relative wound density (density of cells inside the wound relative to that outside the wound)
was quantified at two-hour intervals. The migration rate was then calculated from these time-course data. MM-131 is less effective at
inhibiting migration in EpCAM knock-down cells (shEpCAM) than in parental cells (shControl). Similarly, a molecule in which the
anti-EpCAM scFv is mutated to prevent EpCAM targeting (MM-131EpCAMmut) is less effective at inhibiting migration than MM-131.