#3638
MM-302, HER2-targeted liposomal doxorubicin, does not impair cardiomyocyte function in vitro
Joe Reynolds , Elena Geretti, Stephan Klinz, Chris Espelin, Shannon Leonard, Helen Lee, Isabelle Eckelhofer, Daniel Gaddy, Ken Olivier, Sam Agresta, Bart Hendriks, Tom Wickham
Merrimack Pharmaceuticals, Cambridge, MA USA
ABSTRACT
In HER2+ (ErbB2+) breast cancer, the ability to combine
anthracyclines, such as doxorubicin, with anti-HER2 targeted
therapies is limited clinically due to potential synergistic
cardiotoxicity: doxorubicin directly damages heart cells and HER2
is an important mediator of cardiac repair signaling. Herein we
detail findings for MM-302, a novel HER2-targeted liposomal
doxorubicin formulation. The liposomal encapsulation is
designed to protect the heart from doxorubicin exposure and the
HER2-targeting component mediates uptake in HER2-positive
cells without blocking HER2 signaling. In order to study the
potential effects of MM-302 on the heart, we performed studies
using Embryonic Stem Cell-derived (ESCd) and induced
pluripotent stem cell derived (iPSd) cardiac cells. Total cell uptake
of free doxorubicin and MM-302 in both models of human cardiomyocytes was measured using HPLC. We found
that, despite expressing low levels of HER2, the human cardiac cells do not take up MM-302, whereas
doxorubicin freely enters the cells in a time and dose-dependent manner over a 3 hour time course. Subsequent
to delivery, cell viability was assessed in response to free doxorubicin or MM-302 at 24 hours post 3-hour
exposure. Doxorubicin resulted in cell death in a dose-dependent manner, whereas MM-302 had no effect on
cardiac cell viability. To understand the mechanism underlying the cardiotoxicity associated with doxorubicin
exposure, and to differentiate MM-302 from existing therapies, we measured cell functional responses in ESCd
cardiac cells, including cell stress (phopsho-p53 and pHSP27), DNA damage (gamma-H2AX) and apoptosis
(cleaved-PARP) with high-content microscopy. We demonstrated that, while doxorubicin treatment results in
DNA damage, cell stress, apoptosis, and ultimately cell death in a dose-dependent manner, MM-302 does not.
Furthermore, the interruption of the HER2-mediated signaling pathway in cardiomyocytes has been associated
with cardiac damage following stress. We demonstrate that the HER2-signaling pathway remains active and
uninterrupted in iPSd cardiomyocytes following treatment with components of MM-302. Our findings
demonstrate that MM-302 provides a means to deliver doxorubicin to high HER2-expressing cancer cells, while
sparing exposure, accumulation, and toxicity in non-target (low HER2-expressing) cells, such as cardiomyocytes.
This opens an exciting avenue for potentially introducing a novel anthracycline product for the treatment of
HER2+ breast cancer.
0
0
24
48
72
96
Time (hr)
120
144
0
0
168
24
D.
48
72
96
Time (hr)
120
144
0.5h
168
bound doxorubicin
NUCLEAR DOX: 3 mg/kg; HEART MOUSE v2p3.xls
0.25
0.5h
4h
24h
IMAGE ANALYSIS
MM-302 3mg/kg
Free dox 3mg/kg
UT-PLD 3mg/kg
50
40
Nuclei
0
24
48
72
96
Time (hr)
120
144
HRG stimulated pERK
basal pAKT
10
0
TIme after injection (h)
TISSUE ANALYSIS
Figure 5. To address if pretreatment with MM-302 affects HER2-mediated signaling (an essential pathway in cardiomyocytes), iPS-derived cardiomyocytes
were exposed to components of MM-302 and the levels of phospho-AKT (pAKT) and phospho-ERK (pERK) were assessed. Cells were pretreated for 24
hours with either trastuzumab, lapatinib, or the MM-302 antibody (F5-scFv) and a MM-302 molecule not containing doxorubicin (F5-lipo) at an
equivalent concentration to 5.0ug/ml of MM-302. Cells were stained and imaged using high content microscopy for (A) pAKT and (B) pERK following a 10
minute stimulation with 10nM and 5nM of Heregulin, respectively. Single cell signal intensity for each stain was quantified and represented as the mean
relative intensity of individual cells.
SUMMARY
SAFETY: Unlike free doxorubicin, MM-302 is not effectively taken up into human cardiomyocytes and had little or no effect on
168
Figure 1. Computational modeling is used to quantify the competing kinetic processes in doxorubicin
delivery via MM-302. The model was developed and calibrated on literature and in-house data for free
(A) and liposomal doxorubicin (B) in a mouse heart. The rate of liposome deposition into heart tissue is
1/10th of that of the tumor and 1/1000th the rate of free doxorubicin (C). The model predicts that MM302 dramatically reduces peak and total exposure of nuclear bound doxorubicin in the heart (D).
phospho-ERK
basal pERK
20
0.1
0
B.
HRG stimulated pAKT
0.15
0.05
CARDIOMYOCYTE SIGNALING
A.
phospho-AKT
30
Merge
ug doxorubicin/g tissue
24h
60
C.
Doxorubicin
MM-302
0.2
4h
MM-302 3mg/kg
30
5
Free Doxorubicin 3mg/kg
20
Bound
10
CTL
10
5
3 mg/kg, [3]H-CHE (PMID: 15867261)
19.4 mg/kg, [111]In (PMID: 10901379)
10 mg/kg, [111]In (PMID: 11395251)
10 mg/kg, [111]In (PMID: 11395251)
10 mg/kg, [111]In (PMID: 11395251)
3 mg/kg, doxorubicin (PMID: 15867261)
5 mg/kg, doxorubicin (PMID: 15272215)
5 mg/kg, doxorubicin (PMID: 19429296)
5 mg/kg, doxorubicin (PMID: 19429296)
3 mg/kg, doxorubicin (PMID: in-house BD-07312010)
3 mg/kg, doxorubicin (PMID: in-house BD-07312010)
Model Fit
B.
0
10
Heart Total Liposome (%ID/g)
Heart Total Doxorubicin (%ID/g)
15
5 mg/kg (PMID: 19429296)
7.5 mg/kg (PMID: 2440568)
12 mg/kg (PMID: 7261254)
2.8 mg/kg (PMID: 7136849)
5 mg/kg (PMID: 15272215)
6 mg/kg (PMID: 12115848)
10 mg/kg (PMID: 12115848)
20 mg/kg (PMID: 3484382)
0.5 mg/kg (PMID: 621570)
6 mg/kg (PMID: 15766591)
cPARP
Figure 4. ESCd human cardiomyocytes were treated for 3h with MM-302, UT-PLD, and free doxorubicin at the
indicated concentrations and then incubated for an additional 24h with fresh media. Cells were stained and
imaged using high content microscopy for the DNA damage marker gammaH2AX (A), the cell stress proteins
phospho-p53 (B) and phospho-HSP27 (C), and the apoptosis protein cPARP (D), shown in red. Images were
analyzed and individual cells were segmented using Hoechst 33342 staining (blue). Single cell intensity for each
stain was quantified and represented as the mean relative intensity of individual cells. Cells positive for high
gammaH2AX signal (A) were gated and presented as percent of cells contributing to that population.
A.
MOUSE Liposome Heart Deposition (3 mg/kg)
PeIN-heart-lipo = 1.13e-8 dm/min; PeOUT-heart-lipo = 1.27e-7 dm/min
D.
Cell Viability
MICRODISTRIBUTION IN MOUSE CARDIAC TISSUE
liposomal doxorubicin
C. phospho-HSP27
Zandstra Laboratory, Univ. of Toronto
(Toronto, Canada)
Figure 2. HER2-overexpressing BT474-M3 cells (A) and induced pluripotent stem cell (iPS)derived and embryonic stem cell-derived (ESCd) human cardiomyocytes (B) were treated with
15ug/ml of MM-302, UT-PLD, and free doxorubicin for the indicated times. Total cellular
doxorubicin was quantified by HPLC. Free doxorubicin accumulates in all cell types tested.
MM-302 accumulates exclusively in the HER2-overexpressing cancer cells, while remaining
absent in both models of human cardiomyocytes. (C) ESCd human cardiomyocytes were
treated for 3h with drug at the indicated concentrations and incubated for an additional 24h
with fresh media and cell viability was assessed.
Vasculature
MOUSE; 5 mg/kg free doxorubicin
15
C.
B.
free doxorubicin
Cell Dynamics International
(Wisconsin, USA)
C.
Doxorubicin
A.
(high Her2-expressing cells)
Doxorubicin positive nuclei (%)
KINETIC MODELING OF DRUG DELIVERY
CARDIOMYOCYTE AND TUMOR CELL UPTAKE AND VIABILITY
CARDIOMYOCYTE CELLULAR RESPONSES
CANCER CELLS
HUMAN STEM CELL DERIVED CARDIOMYOCYTES
A. gammaH2AX
B. phospho-P53
BT474-M3 cells
A.
B. Induced Pluripotent Stem Cell (iPS)
Embryonic Stem Cell Derived (ESCd)
Figure 3. Nu/nu mice were injected intravenously with MM-302, UT-PLD, and free doxorubicin at 3 mg/kg (dox equiv.). At the designated time
points, hearts were collected for the preparation of cryosections to analyze the microdistribution of liposomes and doxorubicin (A). FITC-lectin
was injected to visualize functional/perfused blood vessels. Heart sections were counterstained with Hoechst and imaged by confocal
fluorescence microscopy. Doxorubicin positive nuclei are shown in purple. Doxorubicin positive nuclei are visible in free doxorubicin treated
samples at 0.5h and not MM-302 treated samples (compare red boxes). (B) Quantification of doxorubicin positive nuclei from mouse heart
confocal images analyzed using Definiens®. (C) Hearts were collected at the designated time point for doxorubicin quantification by HPLC.
cellular markers associated with doxorubicin-mediated change. MM-302 does not inhibit HER2-mediated signaling in stem
cell-derived cardiomyocytes.
EFFICACY: MM-302 is selectively taken up into cancer cells expressing greater than 200,000 HER2 receptors per cell.
ACKNOWLEDGMENTS
We would like to thank Cell Dynamics International (Wisconsin, USA) and the Laboratory of Peter Zandstra at the University
of Toronto (Toronto, Canada) for providing us with stem cell-derived human cardiomyocytes.