Trastuzumab is the standard of care for HER2-positive breast cancer patients, markedly improving disease-free and overall survival. Combined with chemotherapy, it enhances patient outcomes, but cardiotoxicity due to the trastuzumab treatment poses a serious adverse effect. MM-302 is a HER2-targeted PEGylated liposome that encapsulates doxorubicin to facilitate its delivery to HER2-overexpressing tumor cells while limiting exposure to nontarget tissues, including the heart. In this study, we evaluated the feasibility and preclinical activity of combining MM-302 with trastuzumab. MM-302 and trastuzumab target different domains of the HER2 receptor and thus could simultaneously bind HER2-overexpressing tumor cells in vitro and in vivo. Furthermore, trastuzumab did not disrupt the mechanism of action of MM-302 in delivering doxorubicin to the n0ucleus and inducing DNA damage. Reciprocally, MM-302 did not interfere with the ability of trastuzumab to block prosurvival p-Akt signaling. Interestingly, coadministration of the two agents acutely increased the deposition of MM-302 in human xenograft tumors and subsequently increased the expression of the DNA damage marker p-p53. Finally, the combination of MM-302 and trastuzumab induced synergistic antitumor activity in HER2-overexpressing xenograft models of breast and gastric cancer. Collectively, our findings highlight a novel combination therapy that efficiently targets HER2-overexpressing cells through multiple mechanisms and support the ongoing investigation of combined MM-302/trastuzumab therapy for HER2-positive metastatic breast cancer in a randomized phase II clinical trial. Cancer Res; 76(6); 1517–27. ©2016 AACR.

An estimated 25% of breast cancer patients are defined as HER2-positive, characterized by tumor overexpression of the HER2 receptor protein and amplification of the HER2 gene. Other cancers including gastric, ovarian, and bladder also exhibit HER2 overexpression to varying degrees (1). Trastuzumab is a mAb developed to bind the HER2 receptor and proposed to function through multiple mechanisms including decreased PI3K/Akt signaling, increased degradation of the HER2 receptor protein following endocytosis, and antibody-dependent cellular cytotoxicity (ADCC; ref. 2). By identifying those patients whose tumors overexpress the necessary molecular target (HER2), breast cancer cells can be targeted directly while sparing healthy cells, thus mitigating potential adverse events and toxicities. Trastuzumab has thus become a key component in the oncologist's arsenal against HER2-positive cancer (3). Despite these advances, overall response rate to trastuzumab as a single agent is modest (15%–30%), while combination with various chemotherapies can increase response to 50%–75% in HER2-positive breast cancer patients (2, 4). However, among those who do respond, nearly all eventually progress following initial benefit and acquire resistance over time (4, 5). Thus, continued development of novel therapies for HER2-positive breast cancer and their use in combination with established targeted therapies such as trastuzumab remains a necessary goal to improve patient outcomes.

Anthracyclines such as doxorubicin are well-established chemotherapeutics used to treat a variety of cancers including leukemia, Hodgkin's lymphoma, bladder, multiple myeloma, and breast cancer among others. Doxorubicin has proven particularly effective in treating HER2-positive breast cancer patients, possibly as a result of the proximity between the TOP2A gene (a target of doxorubicin) and the HER2 gene on chromosome 17 (6, 7). However, administration of doxorubicin is associated with its own serious side effects including hematologic alterations and life-threatening cardiotoxicity, thus limiting cumulative dosage and clinical utility (8, 9). In the pivotal trial leading to approval of trastuzumab for HER2-positive breast cancer, clinical benefit was identified in combination with doxorubicin, but significant cardiotoxicity was also observed, resulting in an accompanying black box warning (4). As a result of these safety concerns, use of doxorubicin for treatment of HER2-positive breast cancer has decreased in recent years despite its efficacy (10, 11).

MM-302 is a HER2-targeted liposome encapsulating approximately 20,000 molecules of doxorubicin in its core and 45 single-chain anti-HER2 antibodies (scFv) conjugated to its surface. This agent is currently in development for the treatment of HER2-positive metastatic breast cancer and is under consideration for additional oncology indications. Liposomal encapsulation dramatically alters the pharmacokinetics and biodistribution of doxorubicin. By virtue of their comparatively large size, liposomes deposit in areas with leaky or functionally porous vasculature including tumors, areas of inflammation, the liver and spleen. This phenomenon is known as enhanced permeability and retention (EPR) effect (12). The normal vasculatures of healthy organs such as the heart typically prevent leakage and significant accumulation of liposomes. Clinical studies have demonstrated cardiosafety advantages of pegylated liposomal doxorubicin (PLD/Doxil) relative to free doxorubicin, but lack of risk–benefit ratio has precluded approval of Doxil for the treatment of breast cancer in the United States (13, 14). MM-302 shares the extended pharmacokinetics and EPR-mediated deposition in tumors with PLD. However, unlike PLD, following deposition in the tumor microenvironment, anti-HER2 antibodies on the surface of MM-302 specifically increase targeting to HER2-overexpressing tumor cells with a resultant increase in antitumor activity relative to PLD in multiple preclinical models (15, 16). Additional work has elucidated a critical threshold of surface HER2 expression, approximately 200,000 HER2 receptors per cell for efficient binding and uptake of MM-302 (17). Importantly, cardiomyocytes do not possess the requisite number of HER2 receptors required to efficiently mediate the uptake of MM-302, potentially mitigating safety concerns (18, 19).

In this article, we demonstrate the feasibility and enhanced antitumor activity of dual HER2-targeting with MM-302 and trastuzumab. MM-302 and trastuzumab each target different domains of the HER2 receptor (J. Marks; manuscript in preparation; ref. 20) and we show that both agents are able to simultaneously bind to HER2-positive cells. The mechanism of action of MM-302 (delivery of doxorubicin and DNA damage) is not altered by the presence of trastuzumab, while the ability of trastuzumab to decrease intracellular signaling (p-Akt) is not affected by the presence of MM-302. Interestingly, coadministration of trastuzumab with MM-302 acutely increased deposition of MM-302 to BT474-M3 and NCI-N87 xenograft tumors in vivo, with a resultant increase in DNA damage (p-p53). Finally, we demonstrate synergistic antitumor activity of the MM-302 and trastuzumab combination in human xenograft models of breast and gastric cancer. This work provides the preclinical foundation for the combination of MM-302 plus trastuzumab, which is currently being investigated in the ongoing phase II HERMIONE trial in metastatic breast cancer patients (ClinicalTrials.gov identifier NCT02213744).

Cell culture

BT474-M3 is a HER2-overexpressing cell line received as a gift from Hermes Biosciences. NCI-N87 cells were purchased from ATCC and authenticated before receipt. Cell lines were obtained between 2008 and 2011 and propagated for less than 6 months after resuscitation. Both cell lines were grown at 37°C in RPMI/10% FBS supplemented with penicillin–streptomycin and used at P5 or less.

Fluorescence microscopy

Liposome preparation.

MM-302 liposomes were prepared as previously described (16). Labeled liposomes were prepared containing far red-fluorescent carbocyanine tracer DiIC18(5)-DS (D12730, abbreviated DiI5; Life Technologies), which intercalates into the lipid bilayer of the liposome. Corresponding liposomes without doxorubicin were also prepared (“empty”).

Fluorescent-labeled trastuzumab.

Trastuzumab (Herceptin) was purchased through CuraScript, Inc. and labeled per vendor's instructions with Alexa Fluor-488 or Alexa Fluor-555 (Life Technologies).

In vitro fluorescence microscopy.

A total of 10,000 cells were plated per chamber (Nunc Lab Tek II Slides) and simultaneously treated with 1 μmol/L DiI5-MM-302 and 1 μmol/L Alexa 488–trastuzumab for 1 hour on ice at 4°C, fixed with 3.7% formaldaldehyde for 15 minutes at room temperature, and stained with Hoechst 33342 (Life Technologies). Images (20×) and (40×) were captured with a Nikon Eclipse TE2000U microscope.

Nuclear doxorubicin/liposome imaging by high content microscopy

BT474-M3 and NCI-N87 cells were plated in 96-well tissue culture plates at 10,000 cells per well and treated with DiI5-MM-302, trastuzumab, or DiI5-MM-302 and trastuzumab at the indicated final concentrations. Twenty-four hours later, cells were fixed with 3.7% formaldehyde (15 minutes at room temperature), incubated with 1:1,000 Hoechst 33342 (Life Technologies) and 1:10,000 Whole Cell Blue Dye (8403501; Life Technologies). Plates were scanned using an ArrayWorx High Content Scanner (Applied Precision) with a 10× objective for Hoechst 33342 (460 nm), doxorubicin (595 nm), and APC/DiI5 (657 nm). Identification of nuclei, cell segmentation, and signal quantitation were performed using ImageRail software (21). Data are presented as the mean pixel intensity for all cells in a given well for the indicated channel (approx. 4,000 cells evaluated per well).

In vitro viability

BT474-M3 and NCI-N87 cells were plated in 96-well tissue culture plates at 5,000 cells per well, treated with MM-302, trastuzumab, or MM-302 and trastuzumab at the indicated final concentrations (each in triplicate) at 37°C for 72 hours. Cell Titer-Glo assay was performed per vendor instructions (Promega). Luminescence was measured using an Envision 2103 Multilabel plate reader (Perkin Elmer). Percent viability for each treatment is calculated relative to untreated.

Animal studies: tumor growth inhibition and liposome delivery

Seven-week-old female NCR/nu mice were purchased from Taconic and 7-week-old female nu/nu mice were purchased from Charles River Laboratories. The care and treatment of experimental animals was in accordance with Institutional Animal Care and Use Committee guidelines.

Establishment of xenografts.

NCR/nu mice were implanted with a 17β-estradiol pellet (0.74 mg; 60-day release; Innovative Research of America) two to three days before inoculation with 10 × 106 BT474-M3 cells into the mammary fat pad (m.f.p.). Nu/nu mice were inoculated with 7.5 × 106 NCI-N87 cells subcutaneously into the right flank of the mouse.

Tumor growth inhibition.

Once tumors reached a volume of 200 to 300 mm3, mice were randomized into groups of 8 to 10 mice/group of equal average tumor volume and dosed intravenously with: PBS (control), MM-302 [3 mg/kg (doxorubicin), i.v. every 7 days for 3 doses], trastuzumab (7 mg/kg loading dose, then 3.5 mg/kg every 3 days for duration of study), or the combination of MM-302 and trastuzumab. Tumors were measured twice/week with a caliper. Tumor volumes were calculated using the formula: width2 × length × 0.52. Bliss (synergy) analysis was performed as follows: Tumor growth inhibition (TGI) in each treatment group was calculated using the following formula:

formula

where Vtreated and Vcontrol represent the median volumes of tumor at a given time point in mice treated with drug (MM-302 and/or trastuzumab) or PBS (control). By using TGI values from the monotherapy groups, the Bliss additivity model was used to predict the additive effect in the combination group:

TGIBliss Additivity = TGIMM-302 + TGITrastuzumab − TGIMM-302*TGITrastuzumab

Values less than additive indicate antagonism, while values above additive indicate synergy (22–26).

Delivery studies.

Once tumors reached a volume of 200 to 300 mm3, mice were dosed intravenously with PBS (control), trastuzumab (7 mg/kg), DiI5-MM-302 (3 mg/kg), or trastuzumab (7 mg/kg) and DiI5-MM-302 (3 mg/kg); or purified human IgG (7 mg/kg; 1-001-A, R&D Systems) or trastuzumab-DM1 (7 mg/kg; CuraScript). Alternatively, mice were dosed with Alexa-555-trastuzumab (7 mg/kg) and DiI5-MM-302 (3 mg/kg). Tumors were collected 4, 24 and 72 hours postinjection of agents. Five minutes prior to sacrifice, mice were dosed intravenously with 200 μL of FITC-lectin (Vector Laboratories, Inc.) to label perfused vessels, then perfused with PBS. Tumors were collected and processed in accordance with each assay described below.

Quantification of doxorubicin within tumors

After collection, tumors were immediately frozen on dry ice and stored at −80°C until processing. Quantification of doxorubicin in tumors by high-performance liquid chromatography (HPLC) was performed as described previously (18).

Histologic analysis of tumor liposome deposition

Tumors were embedded in OCT compound (TissueTek), frozen in liquid nitrogen immediately after collection, and prepared for cryosection (10-μm thickness). Slides were air-dried for 30 minutes at room temperature and counterstained with Hoechst 33342 diluted 1:5,000 in ProLong Gold mounting media (Molecular Probes). Slides were imaged on an Aperio ScanScope FL scanner (Leica Biosystems) at 20× magnification as described in Onsum and colleagues (19). Images were analyzed using custom rule sets written in Definiens Developer XD (Definiens). Briefly, individual tumors were identified using the Hoechst layer and the liposome mean fluorescence intensity (MFI) determined for each tumor. Distribution of the liposome MFI within the tumor was determined as follows: after defining the tumor region, the area was subdivided into 1-pixel subobjects and each subobject was assigned into different classes based on the liposome MFI: <1,000, 1,000–2,000, 2,000–3,000, 3,000–4,000, 4,000–6,000 MFI, and >6,000. The relative percentage of area occupied by different MFI classes was then determined.

Confocal microscopy of BT474-M3 xenografts

Slides with 10-μm thick frozen tumor sections were air-dried at room temperature for 20 minutes, counterstained, and mounted with Hoechst 33342 diluted 1:50,00 into ProLong Gold anti-fade reagent (Molecular Probes). Slides were imaged on a Leica SP8 X inverted confocal system at 405 nm (solid-state diode laser), 487 nm, 555 nm and 647 nm (WhiteLight lasers) with a 40X/1.3 oil objective (Harvard Medical School, Boston, MA). Images were visualized with open source Fiji software (http://fiji.sc/Fiji).

Phosphoprotein analysis: p-p53 (Ser15) and p-Akt (Ser473)

p-p53 (K15113D-1) or p-Akt (K150MND-1) assays were performed as per manufacturer's instructions (Meso Scale Discovery).

MM-302 and trastuzumab can bind cells simultaneously

The single-chain F5 antibody on the surface of MM-302 specifically targets domain I of the ErbB2/HER2 receptor protein while trastuzumab binds domain IV (Fig. 1A). Despite targeting different epitopes on the HER2 receptor, it was essential to determine whether both MM-302 and trastuzumab can bind to the same cells given the unique physical characteristics of a liposome and therapeutic antibody (Fig. 1B). First, we simultaneously incubated fluorescent DiI5-MM-302 liposomes and Alexa Fluor 488–trastuzumab with HER2-overexpressing BT474-M3 cells (breast) and evaluated membrane-bound signal using fluorescence microscopy. Empty liposomes without doxorubicin were used to minimize autofluorescence and experiments were performed on ice to minimize internalization. BT474-M3 cells tend to grow together in clumps or patches in vitro and when inspected, the MM-302 signal is clearly evident on the periphery of groups of cells (Fig. 1C, MM-302, top and middle). Trastuzumab is present in the same peripheral locations as well as between individual cells (Fig. 1C, trastuzumab, top and middle). These observations are consistent with the physical size differences between the antibody trastuzumab and MM-302. When individual BT474-M3 cells are located, the signal from MM-302 directly overlaps with trastuzumab, consistent with both drugs binding to the HER2 receptor on the same cell (Fig. 1C, bottom). Altering the ratio of either drug up to 50-fold in excess or adding the drugs sequentially in either order (with time intervals) did not affect binding (data not shown).

Figure 1.

MM-302 and trastuzumab colocalize on HER2-overexpressing BT474-M3 cells in vitro. A, cartoon depicting the ErbB2/HER2 receptor, including extracellular domain I bound by MM-302 and domain IV bound by trastuzumab. B, size comparison between liposome, antibody, and doxorubicin; drawn to scale. C, BT474-M3 cells were treated simultaneously with DiI5-MM-302 and Alexa 488-trastuzumab (trast.), fixed, stained with Hoechst, and visualized by fluorescence microscopy. Shown are MM-302 (red), trastuzumab (green), Hoechst (blue), and merged images (yellow) of multiple clusters of cells (top row), an individual cell cluster (middle row) and a single cell (bottom row).

Figure 1.

MM-302 and trastuzumab colocalize on HER2-overexpressing BT474-M3 cells in vitro. A, cartoon depicting the ErbB2/HER2 receptor, including extracellular domain I bound by MM-302 and domain IV bound by trastuzumab. B, size comparison between liposome, antibody, and doxorubicin; drawn to scale. C, BT474-M3 cells were treated simultaneously with DiI5-MM-302 and Alexa 488-trastuzumab (trast.), fixed, stained with Hoechst, and visualized by fluorescence microscopy. Shown are MM-302 (red), trastuzumab (green), Hoechst (blue), and merged images (yellow) of multiple clusters of cells (top row), an individual cell cluster (middle row) and a single cell (bottom row).

Close modal

We next quantitatively evaluated binding of MM-302 and delivery of doxorubicin to the cell nucleus in the presence of trastuzumab. BT474-M3 and HER2-overexpressing NCI-N87 cells (gastric) were simultaneously incubated with DiI5-MM-302 (0–2 μmol/L) and trastuzumab (0, 1, or 10 μg/mL) for 24, 48, and 72 hours and liposome signal (DiI5) and nuclear doxorubicin then evaluated by high-throughput fluorescence microscopy. A dose-dependent increase in liposome signal (Fig. 2A) and nuclear doxorubicin (Fig. 2B) is observed with increasing MM-302 concentration in both cell lines at 24 hours (blue lines). The addition of trastuzumab at 1 μg/mL (light green) or 10 μg/mL (dark green) did not alter the amount of MM-302 liposome bound or nuclear doxorubicin in either BT474-M3 or NCI-N87 cells (Fig. 2A and B). Similar results were observed at 48 and 72 hours (data not shown).

Figure 2.

MM-302 and trastuzumab simultaneously bind and do not interfere with each other's activity in vitro. A and B, BT474-M3 and NCI-N87 cells were treated for 24 hours with the indicated concentrations of DiI5-MM-302 alone (blue line) or in combination with trastuzumab at 1 μg/mL (light green line) or 10 μg/mL (dark green line). Bound liposome (A) or nuclear doxorubicin (B) were detected by high-throughput fluorescence microscopy. C and D, BT474-M3 and NCI-N87 cells were treated with 1 μmol/L MM-302 alone (blue line), 1 μmol/L MM-302 in combination with trastuzumab at 1 μg/mL (light green line) or 10 μg/mL (dark green line), trastuzumab alone (pink line), or untreated (black line) for the indicated length of time. p-p53 (Ser15; C) and p-Akt (Ser473; D) were evaluated by electrochemiluminescent assays (Meso Scale Discovery).

Figure 2.

MM-302 and trastuzumab simultaneously bind and do not interfere with each other's activity in vitro. A and B, BT474-M3 and NCI-N87 cells were treated for 24 hours with the indicated concentrations of DiI5-MM-302 alone (blue line) or in combination with trastuzumab at 1 μg/mL (light green line) or 10 μg/mL (dark green line). Bound liposome (A) or nuclear doxorubicin (B) were detected by high-throughput fluorescence microscopy. C and D, BT474-M3 and NCI-N87 cells were treated with 1 μmol/L MM-302 alone (blue line), 1 μmol/L MM-302 in combination with trastuzumab at 1 μg/mL (light green line) or 10 μg/mL (dark green line), trastuzumab alone (pink line), or untreated (black line) for the indicated length of time. p-p53 (Ser15; C) and p-Akt (Ser473; D) were evaluated by electrochemiluminescent assays (Meso Scale Discovery).

Close modal

MM-302, but not trastuzumab, activates p53

p53 protein levels and phosphorylation/activation are tightly controlled under normal conditions by the cell. However, in response to DNA damage (induced by UV, IR, or chemical agents including doxorubicin), p53 can become phosphorylated in the N-terminal domain, leading to its activation and accumulation (27). To investigate this response, BT474-M3 and NCI-N87 cells were treated with 1 μmol/L MM-302 for 0–24 hours and p-p53 (Ser15) evaluated by electrochemiluminescent MSD assay. Consistent with the accumulation of doxorubicin in the nucleus and resultant DNA damage, an increase in p-p53 is observed when cells are treated with MM-302 (Fig. 2C, blue line). The increase in p-p53 observed with MM-302 is not altered by cotreatment with trastuzumab at 1 or 10 μg/mL (Fig. 2C, light/dark green lines). Trastuzumab alone has no effect on p-p53 under the conditions tested (Fig. 2C, pink lines).

Trastuzumab, but not MM-302, reduces p-Akt signaling

One of the reported mechanisms by which trastuzumab halts tumor growth is by decreasing signaling cascades downstream of the HER2 receptor, including the PI3K/Akt pathway (28). Treatment of BT474-M3 and NCI-N87 cells with trastuzumab alone (1 μg/mL) results in a significant decrease in basal p-Akt (Ser473) signal, with this reduction maintained at least until 24 hours (Fig. 2D, pink line). Cotreatment with MM-302 did not alter the decrease in p-Akt signal attributed to trastuzumab (Fig. 2D, green line), while MM-302 alone has no effect on p-Akt signal at the same timepoints (Fig. 2D, blue line).

Combining MM-302 and trastuzumab increases cell death in vitro

To determine the effect of combining MM-302 and trastuzumab on cell growth in vitro, BT474-M3 and NCI-N87 cells were treated with trastuzumab alone (10 μg/mL), MM-302 alone (0.5 μmol/L), or MM-302 and trastuzumab. Viability was evaluated 72 hours following treatment and calculated relative to untreated cells. Trastuzumab alone reduced viability in both cell lines by approximately 15% (Fig. 3, pink) while MM-302 alone reduced BT474-M3 viability by 45% and NCI-N87 by 65%. In both BT474-M3 and NCI-N87 cells, the combination of MM-302 with trastuzumab significantly reduces in vitro viability relative to either treatment alone (58 and 80%, respectively; Fig. 3, green), consistent with an additive effect based on percent viability.

Figure 3.

The combination of MM-302 and trastuzumab is more effective than either single agent in decreasing viability of BT474-M3 and NCI-N87 cells in vitro. BT474-M3 and NCI-N87 cells were treated with trastuzumab alone (10 μg/mL, pink), MM-302 alone (0.5 μmol/L, blue), or MM-302 and trastuzumab (green) for 72 hours. Viability was evaluated using Cell Titer-Glo assay and expressed as percent viability relative to untreated cells. Statistics indicate Student unpaired t test.

Figure 3.

The combination of MM-302 and trastuzumab is more effective than either single agent in decreasing viability of BT474-M3 and NCI-N87 cells in vitro. BT474-M3 and NCI-N87 cells were treated with trastuzumab alone (10 μg/mL, pink), MM-302 alone (0.5 μmol/L, blue), or MM-302 and trastuzumab (green) for 72 hours. Viability was evaluated using Cell Titer-Glo assay and expressed as percent viability relative to untreated cells. Statistics indicate Student unpaired t test.

Close modal

MM-302 and trastuzumab can bind the same cells in human xenograft tumors

We next sought to determine whether both MM-302 and trastuzumab could bind to the same cells in vivo, as we observed in vitro. Mice bearing BT474-M3 tumors were injected with fluorescent DiI5-MM-302 (“empty”; 3 mg/kg) and Alexa-555-trastuzumab (7 mg/kg) for 4 or 24 hours and tumor sections then imaged by confocal fluorescence microscopy. MM-302 is present within and proximal to perfused blood vessels (visualized by FITC-lectin) as well as on the cell membranes of multiple nearby cells at 24 hours (Fig. 4, MM-302/red). Trastuzumab is widely distributed and observed on the surface of most tumor cells within the section (Fig. 4, trastuzumab/green), but is not as evident within vessels as MM-302 (consistent with faster distribution of an antibody). Colocalization of MM-302 and trastuzumab is readily apparent on multiple cells within proximity to the blood vessels (Fig. 4, merge-yellow). Similar co-localization is observed at 4 hours (additional representative images are shown in Supplementary Fig. S1).

Figure 4.

MM-302 and trastuzumab colocalize in BT474-M3 human xenografts. NCR/nu mice bearing BT474-M3 tumors were simultaneously administered DiI5-MM-302 (“empty”, no doxorubicin) and Alexa-555–trastuzumab. Five minutes prior to sacrifice, mice were injected with FITC-lectin to identify perfused blood vessels. Twenty-four hours after administration of DiI5-MM-302 and Alexa-555–trastuzumab, tumors were excised, immediately frozen, and sectioned. Slides were imaged on a Leica SP8 X inverted confocal system with a 40×/1.3 oil objective. Representative images of MM-302 (red), trastuzumab (green), vessels (magenta), and DNA (Hoechst; blue) are shown. Yellow, colocalization of MM-302 and trastuzumab. Bottom row, close-up of selected region from upper row.

Figure 4.

MM-302 and trastuzumab colocalize in BT474-M3 human xenografts. NCR/nu mice bearing BT474-M3 tumors were simultaneously administered DiI5-MM-302 (“empty”, no doxorubicin) and Alexa-555–trastuzumab. Five minutes prior to sacrifice, mice were injected with FITC-lectin to identify perfused blood vessels. Twenty-four hours after administration of DiI5-MM-302 and Alexa-555–trastuzumab, tumors were excised, immediately frozen, and sectioned. Slides were imaged on a Leica SP8 X inverted confocal system with a 40×/1.3 oil objective. Representative images of MM-302 (red), trastuzumab (green), vessels (magenta), and DNA (Hoechst; blue) are shown. Yellow, colocalization of MM-302 and trastuzumab. Bottom row, close-up of selected region from upper row.

Close modal

Combining MM-302 and trastuzumab increases DNA damage signaling in vivo

Building on our in vitro findings, we next evaluated whether we could also detect changes in p-p53 levels within tumor xenografts following treatment with MM-302. Mice bearing BT474-M3 or NCI-N87 human xenograft tumors were treated with trastuzumab alone, MM-302 alone or MM-302 and trastuzumab for 4, 24, or 72 hours. Tumors were collected and levels of p-p53 (Ser15) evaluated. No changes in p-p53 were observed in the untreated or trastuzumab-treated lysates at any timepoint (Fig. 5A, gray/pink). Significant p-p53 was observed 24 hours after MM-302 treatment (P < 0.0001), with a comparable increase in signal from 4 to 24 hours with or without trastuzumab coadministration (Fig. 5A, blue/green). Interestingly, while both treatments increase p-p53 signal at 72 hours relative to the 24-hour timepoint, the signal is greater with trastuzumab coadministration (P = 0.0009). Similar trends are observed in both xenograft models although the absolute values and variability are much greater in the NCI-N87 model, resulting in significance only at the 72-hour timepoint (P = 0.0112).

Figure 5.

Coadministration of MM-302 with trastuzumab increases p53 phosphorylation and MM-302 deposition. A, BT474-M3 or NCI-N87 tumor-bearing mice (n = 5–8/group) were dosed with trastuzumab alone (7 mg/kg; pink), MM-302 alone (3 mg/kg; blue), or MM-302 (3 mg/kg) and trastuzumab (trast; 7 mg/kg; green); untreated (U; gray). Tumors were collected 4, 24 or 72 hours after treatment, frozen, and lysates prepared. p-p53 (Ser15) signal was evaluated by electrochemiluminescent assay (Meso Scale Discovery). B, mice were treated as in A and tumors analyzed by HPLC to determine the total doxorubicin in the tumor expressed as percent of injected dose per gram of tissue (% i.d./g). MM-302 alone, blue; MM-302 + trastuzumab, green. Untreated tumor produced no doxorubicin signal by HPLC. Horizontal bar, the mean for each group. Statistics represent Student unpaired t test.

Figure 5.

Coadministration of MM-302 with trastuzumab increases p53 phosphorylation and MM-302 deposition. A, BT474-M3 or NCI-N87 tumor-bearing mice (n = 5–8/group) were dosed with trastuzumab alone (7 mg/kg; pink), MM-302 alone (3 mg/kg; blue), or MM-302 (3 mg/kg) and trastuzumab (trast; 7 mg/kg; green); untreated (U; gray). Tumors were collected 4, 24 or 72 hours after treatment, frozen, and lysates prepared. p-p53 (Ser15) signal was evaluated by electrochemiluminescent assay (Meso Scale Discovery). B, mice were treated as in A and tumors analyzed by HPLC to determine the total doxorubicin in the tumor expressed as percent of injected dose per gram of tissue (% i.d./g). MM-302 alone, blue; MM-302 + trastuzumab, green. Untreated tumor produced no doxorubicin signal by HPLC. Horizontal bar, the mean for each group. Statistics represent Student unpaired t test.

Close modal

Trastuzumab acutely increases deposition of MM-302 in human xenograft tumors

Our in vitro combination experiments showed that trastuzumab did not increase p-p53 in combination with MM-302, yet increased levels of p-p53 were observed in tumor xenografts treated with the same combination. This led us to question whether trastuzumab was influencing the amount of MM-302 reaching the tumor in vivo. Mice bearing human BT474-M3 or NCI-N87 xenograft tumors were treated with MM-302 alone or simultaneously with trastuzumab, tumors collected 4, 24, or 72 hours after drug administration and doxorubicin levels analyzed by HPLC. When MM-302 was administered as a monotherapy to BT474-M3 tumors, approximately 5% of the injected doxorubicin/gram tissue (i.d./g) was detected in tumors at 4 hours (Fig. 5B left, blue). Consistent with the p-p53 results, when trastuzumab was injected simultaneously with MM-302, there was a greater than 2-fold increase in the amount of doxorubicin detected in the BT474-M3 tumor at 4 hours relative to MM-302 alone (P = 0.0076; Fig. 5B left, green). These results are consistent with a mechanism wherein trastuzumab increases MM-302 delivery, which in turn results in the increased p-p53 that was experimentally observed. Doxorubicin levels increase from 4 to 24 hours with MM-302 alone, consistent with continued deposition of liposome at the tumor site as a result of the long-circulating pharmacokinetics of PEGylated liposomes. There continues to be greater doxorubicin in the BT474-M3 cells at 24 hours with coadministration of trastuzumab relative to MM-302 alone, although the values were not statistically significant. By 72 hours, there is little difference between the amounts of doxorubicin in BT474-M3 tumors treated with MM-302 or MM-302 and trastuzumab. In the gastric NCI-N87 xenograft model, a nearly 2-fold increase in doxorubicin levels was observed at 4 hours when trastuzumab is coadministered with MM-302 (Fig. 5B right; P = 0.0449), while a significant increase is also observed at 24 hours with the combination (P = 0.0024). Changes in MM-302 deposition were not observed with coinjection of a nonspecific human IgG, a monoclonal human antibody against EGFR, or PBS, but an increase was seen with trastuzumab-DM1 (T-DM1; Supplementary Fig. S2 and data not shown). In addition, the pharmacokinetics of MM-302 was not affected by trastuzumab, indicating that changes in systemic exposure were not responsible for the differences in tumor uptake (Supplementary Fig. S3).

As further confirmation, frozen sections from BT474-M3 tumors treated with DiI5-MM-302 alone or in combination with trastuzumab were collected 4, 24, or 72 hours post-injection, then imaged and analyzed to quantify mean liposome fluorescence intensity (MFI) per tumor. Consistent with the doxorubicin results, coadministration of trastuzumab increased DiI5-MM-302 signal in the tumor at both 4 and 24 hours relative to MM-302 alone (Fig. 6A; P = 0.0159 and P = 0.0317, respectively). A trend towards increased MM-302 deposition was also observed at 72 hours with trastuzumab (P = 0.0517, n.s.).

Figure 6.

Coadministration of MM-302 with trastuzumab influences MM-302 liposome distribution. A, mice bearing BT474-M3 or NCI-N87 tumors (n = 5/group) were dosed with DiI5-MM-302 alone (3 mg/kg; blue circles) or DiI5-MM-302 (3 mg/kg) and trastuzumab (7 mg/kg; green triangles); untreated (U). Tumors were collected 4, 24, or 72 hours postdrug injection, prepared for cryosection, counterstained with Hoechst, and scanned on an Aperio FL scanner. Images were analyzed to quantify liposome MFI. B, variability of liposome MFI within the individual tumor sections. Representative tumors for the DiI5-MM-302 alone (left) and DiI5-MM-302 and trastuzumab (Trast.; right) groups are shown (24 hours). Scale bar, 1 mm. C, quantification of the tumor areas (% of total) with different liposome MFIs. T, trastuzumab.

Figure 6.

Coadministration of MM-302 with trastuzumab influences MM-302 liposome distribution. A, mice bearing BT474-M3 or NCI-N87 tumors (n = 5/group) were dosed with DiI5-MM-302 alone (3 mg/kg; blue circles) or DiI5-MM-302 (3 mg/kg) and trastuzumab (7 mg/kg; green triangles); untreated (U). Tumors were collected 4, 24, or 72 hours postdrug injection, prepared for cryosection, counterstained with Hoechst, and scanned on an Aperio FL scanner. Images were analyzed to quantify liposome MFI. B, variability of liposome MFI within the individual tumor sections. Representative tumors for the DiI5-MM-302 alone (left) and DiI5-MM-302 and trastuzumab (Trast.; right) groups are shown (24 hours). Scale bar, 1 mm. C, quantification of the tumor areas (% of total) with different liposome MFIs. T, trastuzumab.

Close modal

Because of their large size, nanoparticles are known to have challenges effectively penetrating into tumors. Therefore, we sought to investigate whether the effect on deposition seen with trastuzumab also improved liposome penetration. Changes in liposome spatial distribution were evaluated by assigning each individual pixel within the tumor to a distinct MFI class (from 0—1,000 up to >6,000 MFI). This allowed us to generate a “heatmap” of liposome distribution within the tumor; representative tumors for DiI5-MM-302 alone (left) and DiI5-MM-302 with trastuzumab (right) are shown for the 24-hour time point (Fig. 6B). The relative percentages of tumor area belonging to the defined MFI classes were quantified for each tumor. Coadministration with trastuzumab resulted in greater overall delivery of MM-302 as indicated by an increase in tumor areas (%) with higher DiI5 fluorescence intensity (Fig. 6C, yellow-orange-red). The percent tumor area with the highest liposome intensity (>6,000 MFI) increased from 0.2% to 0.7% at 4 hours (P = 0.0159), from 0.2% to 2.2% at 24 hours (P = 0.0317), and from 0.2% to 2.5% at 72 hours (n.s.), indicating that areas of the tumor are exposed to higher liposome concentrations (as opposed to a general overall increase). Similar trends were observed in the NCI-N87 model; however, the results were not statistically significant.

Combining MM-302 and trastuzumab improves antitumor activity

We observed DNA damage from MM-302 and reduced p-Akt signaling from trastuzumab when combined in vitro, as well as increased liposome deposition when MM-302 and trastuzumab were coadministered in vivo. We next investigated whether these observations would translate into increased antitumor activity. Mice bearing either BT474-M3 or NCI-N87 xenograft tumors were treated with MM-302 alone, trastuzumab alone, or coadministration of both drugs. Each monotherapy was effective at reducing tumor volume relative to control in both models (Fig. 7A and B, blue and pink). In the BT474-M3 model, the combination of MM-302 and trastuzumab had significantly greater antitumor activity than either drug alone (P < 0.0001), including 6 of 10 tumors with greater than 50% reduction in tumor volume and 2 complete regressions (Fig. 7A, green). Similarly, in the NCI-N87 model, the combination had greater antitumor activity than either trastuzumab (P < 0.0001) or MM-302 alone (P = 0.0002), including 2 of 9 tumors with greater than 50% reduction in tumor volume (Fig. 7B, green). Bliss additivity analysis of both models demonstrates that the benefit of the combination is synergistic, or greater than the predicted sum of each drug alone (Supplementary Fig. S4). In the case of the BT474-M3 xenograft tumors, synergy is indicated at every time point tested despite the magnitude likely being restricted or minimized due to the occurrence of complete regressions (Fig. 7 and Supplementary Fig. S4).

Figure 7.

Coadministration of MM-302 with trastuzumab enhances antitumor activity. Mice were inoculated with BT474-M3 breast (A) or NCI-N87 gastric cells (B) and at a tumor volume of 200 to 300 mm3 treated with either PBS (control; black), MM-302 (blue), trastuzumab (pink), or the combination of MM-302 and trastuzumab (green); n = 8–10/group. Changes in tumor volume over time are shown postinoculation. Data were analyzed by repeated measures ANOVA.

Figure 7.

Coadministration of MM-302 with trastuzumab enhances antitumor activity. Mice were inoculated with BT474-M3 breast (A) or NCI-N87 gastric cells (B) and at a tumor volume of 200 to 300 mm3 treated with either PBS (control; black), MM-302 (blue), trastuzumab (pink), or the combination of MM-302 and trastuzumab (green); n = 8–10/group. Changes in tumor volume over time are shown postinoculation. Data were analyzed by repeated measures ANOVA.

Close modal

Chemotherapies such as anthracyclines remain a primary option for treating cancer patients because of their broad antitumor activity. However, these agents are relatively indiscriminate with regard to their activity against healthy or cancerous cells, resulting in side effects that can impact quality of life and limit their use. Among cancer survivors, the use of conventional anthracyclines are associated with long-term secondary effects including the development of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS; refs. 9, 29–32). Cardiotoxicity associated with the use of anthracyclines has also been well documented and is often exacerbated in combination with drugs including trastuzumab and taxanes (4, 33). Recent studies have shown the benefit of continuing trastuzumab treatment in HER2-positive patients even following disease progression, making it likely that this drug will remain in use through all lines of therapy (34). For this reason, it is increasingly important to find suitable combination partners for trastuzumab. Targeted liposomes such as MM-302 provide an attractive option for incorporating anthracycline therapy with trastuzumab, while moderating potential side effects.

Liposomes are significantly larger than therapeutic antibodies such as trastuzumab and the target HER2 receptor. Answering the basic question whether both drugs could bind to the same HER2-overexpressing cells was a necessary starting point. On the basis of fluorescence microscopy, both agents are present on the same BT474-M3 cells both in vitro and in vivo. Some differences in the pattern of distribution of each agent are observed in vitro, but when single cells were identified, colocalization was readily apparent. We also evaluated the HER2-overexpressing SUM-190 cell line, which tends to grow as individual cells, and colocalization of trastuzumab and MM-302 on single cells was readily observed by fluorescence microscopy (data not shown). We were further able to capture binding of MM-302 and trastuzumab to the same tumor cells in vivo by injecting fluorescent DiI5-MM-302 and Alexa-555–trastuzumab into mice carrying BT474-M3 xenografts. By 4 hours, MM-302 is observed within tumor blood vessels as well as on the membranes of numerous proximal cells. Trastuzumab is observed on the surface of multiple cells throughout the tumor. As trastuzumab is likely to travel further and faster than MM-302, the colocalization is not absolute but is clearly evident in locations where both drugs are present. Future studies could include a more extensive time course (both earlier and later) to shed light on the relative kinetic distribution of each drug in vivo.

Beyond simultaneously binding to HER2-overexpressing cells, MM-302 and trastuzumab did not inhibit each other's activity. Delivery of doxorubicin to the nucleus by MM-302 and activation of the DNA damage pathway (as indicated by p-p53) was nearly identical whether trastuzumab was present or not. Stimulation of the HER2 receptor results in downstream activation of the PI3K–Akt pathway, which in turn leads to activation of transcription factors leading to increased proliferation, angiogenesis, metastases, and survival (35). Treatment of BT474-M3 or NCI-N87 cells with trastuzumab reduced basal pAkt signaling in vitro and this inhibition was unchanged with coadministration of MM-302. The additive killing effect when both drugs are coadministered in vitro is consistent with each drug performing its intended function. Thus, by multiple measurements, HER2-positive cells are impacted by the mechanisms of action of both MM-302 and trastuzumab. As antibody-dependent cell-mediated cytotoxicity (ADCC) is believed to be another significant mechanism of action for trastuzumab, we can speculate that even greater combinatorial benefit would be observed in the presence of a functional immune system (36, 37).

Tumor vasculature is known to be irregular and tortuous, with walls of varying thickness, typically favoring deposition of liposomes as they extravasate through leaky vessels, resulting in increased tumor accumulation relative to organs possessing normal vasculature (38). Multiple reports have described effects of trastuzumab on tumor vasculature and angiogenesis, including reduced rates of VEGF production, changes in microvessel density (MVD) and remodeling of tumor vessel organization (39–41). The increase in MM-302 deposition we observed when trastuzumab was coadministered was unexpected, especially so rapidly after injection of the drugs. Most published studies describe the effects of trastuzumab on tumor vessels days to weeks after treatment (rather than hours), although McCormack and colleagues describe changes in VEGF and CD31 in BT474 xenograft tumors 48 hours after administration of trastuzumab (39, 41, 42). We labeled CD31 to evaluate blood vessels in BT474-M3 tumors following treatment with trastuzumab and observed multiple subtle, yet significant, effects including reductions in vessel length, width and area (data not shown). It remains to be determined whether these changes are responsible for the increased liposome deposition. Normalization of the vessels would seem to oppose increased delivery of liposomes and is unlikely to occur in such a short time. However, brief, transient changes in vessel shape, size, diameter, etc. (before or during transition to normalization) could affect pressure or velocity of blood flow, forcing more liposomes through an already leaky tumor vasculature. Increased blood pressure has been shown to increase tumor delivery of nanoparticles clinically and possibly enhance the EPR effect, although we have not evaluated this effect in our preclinical models (12, 43). Another consideration is the release of cytokines or growth factors mediated by the interaction between trastuzumab and the HER2 receptor on the tumor. Such a response might alter the vessels or tumor microenvironment, making the tumor more amenable to MM-302 deposition. VEGF is one such factor known to influence many aspects of the vasculature including blood pressure, angiogenesis and vascular homeostasis among other biologic functions and it is a possibility we continue to explore (44). We did not observe increased deposition when we coadministered MM-302 with a nonspecific human IgG or antibodies specific for other targets. Notably, coadministration of T-DM1 (same antibody component as trastuzumab; refs. 45, 46) increased MM-302 deposition, further supporting the notion that the response is HER2-specific (Supplementary Fig. S2).

The use of anthracyclines in HER2-positive breast cancer has been declining for years due to concerns about cardiac risk (11). A retrospective analysis by Montemurro and colleagues (10) found that up to a third of eligible HER2-positive patients never received an anthracycline at any point during their treatment before dying. This is despite specific efficacy of anthracyclines against HER2-positive tumors (4, 47). We demonstrate significant benefit of combining a HER2-targeted liposomal anthracycline and trastuzumab against HER2-overexpressing breast (BT474-M3) and gastric (NCI-N87) xenograft models. MM-302 provides the benefit of a liposome, which avoids deposition in healthy tissues including the heart, combined with targeting capability to specifically bind HER2-overexpressing tumor cells and minimize interaction with low HER2-expressing cells. MM-302 had a manageable safety profile and demonstrated promising activity in heavily pretreated HER2-positive metastatic breast cancer patients in a phase I study (ClinicalTrials.gov Identifier: NCT01304797; ASCO 2012, Abstract TPS663, http://meetinglibrary.asco.org/content/98902-114; AACR 2015, Abstract #CT234, http://cancerres.aacrjournals.org/content/75/15_Supplement/CT234?cited-by=yes&legid=canres;75/15_Supplement/CT234). The recently initiated HERMIONE trial will investigate the efficacy and safety of MM-302 and trastuzumab in advanced HER2-positive breast cancer patients (ClinicalTrials.gov Identifier: NCT02213744). The opportunity to provide a targeted anthracycline such as MM-302 combined with trastuzumab using a dual HER2-targeting strategy may offer additional treatment options for HER2-positive cancer patients.

T.J. Wickham is a VP, Development, reports receiving a commercial research grant from, and has ownership interest (including patents) in Merrimack Pharma. B.S. Hendriks has ownership interest (including patents) in Merrimack Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.

Conception and design: C.W. Espelin, S.C. Leonard, T.J. Wickham

Development of methodology: C.W. Espelin, S.C. Leonard, E. Geretti

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.W. Espelin, S.C. Leonard, E. Geretti

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.W. Espelin, S.C. Leonard, E. Geretti, T.J. Wickham, B.S. Hendriks

Writing, review, and/or revision of the manuscript: C.W. Espelin, S.C. Leonard, T.J. Wickham, B.S. Hendriks

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.W. Espelin, S.C. Leonard, E. Geretti

Study supervision: C.W. Espelin, E. Geretti, T.J. Wickham, B.S. Hendriks

The authors thank Jaeyeon Kim, Joe Reynolds, Nancy Dumont, Maura Waxwood, Istvan Molnar, Violette Paragas, Defne Yarar, Helen Lee, Daniel Gaddy, Stephan Klinz, Paul Kopesky, Emily Florine, Ty McClure, Andrew Giansiracusa, Margo Bossom, Shinji Oyama, Daryl Drummond, Dmitri Kirpotin, Jim Marks, Lai Ding and Daniel Tom (Enhanced Neuroimaging Core, Harvard Medical School, Boston, MA), Ulrik Nielsen, Victor Moyo, and Birgit Schoeberl.

This study was sponsored by Merrimack Pharmaceuticals.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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