WO2017136770A1 - Treatment of her2-intermediate cancer - Google Patents

Abstract

Patients with gastric cancer or breast cancer, for example, metastatic breast cancer can be treated with doxorubicin encapsulated in a MM-302 HER-2 targeted immunoliposome, including patients with breast cancer characterized as HER-2 intermediate (e.g., having breast cancer or metastatic breast cancer, wherein each of the breast cancer or metastatic breast cancers are characterized by a HER2 IHC score of 1+ or 2+ with a FISH negative score, or alternatively are FISH negative, or alternatively, are characterized by HER2 IHC score of 1+ and FISH negative, or alternatively, the breast cancer or metastatic breast cancers are characterized by HER2 IHC score of 2+ and FISH negative, or alternatively, the breast cancer or metastatic breast cancers are characterized by a HER2 IHC score of 1+ and are FISH positive, or characterized by HER2 IHC score of 2+ and are FISH positive.

Description

TREATMENT OF HER2-INTERMEDIATE CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U. S. Provisional Application No. 62/291,802, filed February 5, 2016, U.S. Provisional Application No. 62/323,545, filed April 15, 2016, U.S. Provisional Application No. 62/362,253, filed July 14, 2016, and U. S. Provisional

Application No. 62/405,626, filed October 7, 2016, the disclosures of which are incorporated by reference herein in their entireties.

TECHNICAL FIELD

Pharmaceutical preparations comprising HER-2 targeted immunoliposomes encapsulating doxorubicin can be used to treat cancer cells expressing HER2, including treatment of breast cancer cells and gastric cancer cells expressing intermediate levels of HER2.

BACKGROUND

Breast cancer is the most frequently diagnosed life-threatening cancer in women and the leading cause of cancer death in women aged 20 to 59 years. 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. HER2 is one of the main oncogenes driving the growth and metastasis of breast cancer cells. The ErbB family of growth factor receptors is comprised of four receptors (ErbB 1-4) which are critical for biological processes including cell growth and survival. Epidermal growth factor receptor (EGFR) and HER2 are the targets of established anticancer agents.

Chemotherapies such as anthracyclines remain a primary option for treating cancer patients because of their broad anti-tumor activity; for example, anthracyclines such as doxorubicin are well-established chemotherapeutics used to treat a variety of cancers including leukemia, Hodgkin's lymphoma, bladder, gastric, multiple myeloma and breast cancer among others.. However, these agents are relatively indiscriminate with regard to their activity against healthy or cancerous cells, resulting in side effects, which can impact quality of life and limit their use. Among cancer survivors, the use of conventional anthracyclines is associated with long-term secondary effects, including the development of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Cardiotoxicity associated with the use of anthracyclines has also been well documented and is often exacerbated in combination with drugs including trastuzumab and taxanes. 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. 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.

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. However, administration of doxorubicin is associated with its own serious side effects including hematological alterations, and life-threatening and irreversible cardiotoxicity, thus limiting cumulative dosage and clinical utility. While emerging data suggest that the risk for cardiotoxicity can be reduced with newer formulations such as DOXIL®, a PEGylated liposomal doxorubicin (PLD), the use of anthracyclines in HER2 -positive breast cancer has been declining for years due to continued concerns about cardiac risk.

Trastuzumab is the standard of care for HER2-positive breast cancer patients, markedly improving disease-free and overall survival. Trastuzumab is a monoclonal antibody 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). 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. 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. However, among those who do respond, nearly all eventually progress following initial benefit and acquire resistance over time. Combined with chemotherapy, trastuzumab can enhance patient outcomes, but cardiotoxicity due to the trastuzumab treatment poses a serious adverse effect. 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. 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. In 1998, trastuzumab (HERCEPTIN®) was approved by the Food and Drug Administration (FDA) for the treatment of HER2 positive metastatic breast cancer (MBC) with subsequent line extensions into earlier breast cancer. Lapatinib (TYKERB®) in combination with capecitabine (XELODA®) was also approved by FDA in 2007 for refractory HER2 positive breast cancer. In 2012, pertuzumab (PERJETA®) was approved in combination with trastuzumab and docetaxel for the treatment of first line HER2-positive breast cancer. In 2013, the antibody drug conjugate ado-trastuzumab emtansine/T-DMl (KADCYLA®) was approved in HER2 -positive MBC patients who have previously received trastuzumab and a taxane. Most recently, trastuzumab has been approved for the treatment of HER2 over expressing metastatic gastric cancer. While the number of agents to treat HER2-positive

MBC has increased as well as the potential benefit, most patients unfortunately still progress; therefore, new safe and effective therapies are necessary for these patients.

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.

Despite advances in treatment using newly approved HER2 -targeted therapies, safe and effective treatments are still needed, not only for HER2 -positive metastatic breast cancer (MBC) patients, but also for patients with tumors expressing intermediate levels of HER2 that are considered HER2 -negative or HER2-equivocal (i.e., a HER2 score of 1+ or 2+ by immunohistochemistry (IHC) and/or FISH-negative). These "HER2 intermediate" MBC patients may not have high enough detectable levels of breast cancer cell HER2 receptor expression to benefit from HER2 targeted therapy targeted to HER2 3+ breast cancer cells. Thus, there remains a need for therapies for HER2-positive breast cancer remains a necessary goal to improve patient outcomes.

SUMMARY

Disclosed herein are methods of treating a HER2-expressing cancer using a HER2- targeted liposomal anthracycline. The HER2-targeted antibody-liposomal doxorubicin conjugate MM-302 provides the benefit of a liposome, which minimizes deposition in healthy tissues such as the heart, with the capability of binding specifically (targeting) HER2- expressing tumor cells and minimize interaction with low HER2-expressing cells. MM-302 demonstrated a manageable safety profile and promising activity in heavily pretreated HER2- positive MBC patients in a Phase I study (ClinicalTrials.gov Identifier: NCT01304797; ASCO 2012, Abstract TPS663; AACR 2015, Abstract #CT234). The opportunity to provide a targeted anthracycline such as MM-302 alone or combined with trastuzumab using a dual HER2 -targeting strategy may offer additional treatment options for HER2 -positive cancer patients. In Example 1 described infra, the feasibility and preclinical activity of combining MM-302 with trastuzumab was evaluated. 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. MM-302 and trastuzumab demonstrate synergistic anti-tumor activity of the combination in HER2-overexpressing xenograft models of breast and gastric cancer. Trastuzumab did not disrupt the mechanism of action of MM-302 in delivering doxorubicin to the nucleus and inducing DNA damage. 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. Reciprocally, MM-302 did not interfere with the ability of

trastuzumab to block pro-survival p-Akt signaling. Interestingly, co-administration of trastuzumab with MM-302 acutely increased deposition of MM-302 to BT-474-M3 and NCI- N87 xenograft tumors in vivo, with a resultant increase in DNA damage (phosphorylated p53, p-p53). These findings highlight a combination therapy that efficiently targets HER2- overexpressing cells through multiple mechanisms.

Additionally, MM-302 effectively targets and reduces pulmonary metastatic burden in HER2 intermediate breast cancer models. This is based in part on the discovery that the benefit of HER2 targeting in reducing metastatic burden is not due to differential attrition of primary tumor burden as MM-302 and PLD inhibited primary tumor growth to the same degree in both tumor models analyzed. One model uses the mouse 4T1 breast cancer cell line (ATCC® CRL-2539™) expressing the green fluorescent protein/luciferase (GFP-Luc2) construct and overexpressing HER2 (4Tl-GFPluc2-HER2), a cell line having about 100,000 HER2 receptors/cell. Another model uses MDA-MB-453 cells ((ATCC® # HTB-131™, derived from breast carcinoma cells): a HER2 -positive, FISH-positive cell line

overexpressing HER 2 (about 400,000 to 500,000 HER2 receptors/cell). Liposome distribution within 4Tl-GFPluc2-HER2 metastatic lesions varied depending on the size and nature of the lesion, i.e., distribution was poor and mainly peripheral in large nodular lesions but high and uniform in small nodules or sheet-like lesions. In metastatic lesions, MM-302 accumulation was higher than PLD. In Example 1, MM-302 was more effective than PLD at reducing total pulmonary metastatic burden in both HER2-intermediate models as evidenced by the lower number of surface metastases in the 4T1-HER2 model and the lower percent of human cytokeratin positive cells per lung in the MDA-MB-453 model. MM-302 and PLD were equally effective at slowing (4T1 -HER2) and inhibiting (MDA-MB-453) primary tumor growth. Mechanisms responsible for differences in efficacy are being explored. To date, better distribution of liposomes in metastatic lesions than in primary tumors has been observed where liposome delivery appears to be restricted to the tumor periphery in the 4T1- HER2 model.

Thus, in one aspect is provided a method of treating breast or gastric cancer in a human patient having HER2-intermediate MBC cancer or HER2-intermediate gastric cancer, the method comprising administering to the patient once on day 1 of a 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome for one or more treatment cycles. In some embodiments, the present disclosure provides a method of treating breast or gastric cancer in a human patient having HER2 -intermediate MBC cancer or HER2 -intermediate gastric cancer, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate MBC or HER2-intermediate gastric cancer in the patient. In another aspect is provided a method of treating breast or gastric cancer in a human patient having HER2- intermediate gastric cancer, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate gastric cancer in the patient.

Thus, in another aspect is provided a method of treating breast cancer in a human patient having HER2-intermediate MBC cancer, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome, for one or more treatment cycles to treat the HER-2 intermediate MBC in the patient.

Disclosed herein are methods of treating a human patient having an intermediate HER2 cancer comprising combining a HER2 -targeted liposomal anthracycline against HER2-overexpressing breast cancer. Doxorubicin encapsulated HER2-targeted liposomal MM-302 provides the benefit of a liposome, which avoids deposition in healthy tissues such as the heart, combined with targeting capability to specifically bind HER2-expressing tumor cells while minimizing interaction with low HER2-expressing cells. MM-302 demonstrated a manageable safety profile and promising activity in heavily pretreated HER2 -positive MBC patients in a Phase I study (ClinicalTrials.gov Identifier: NCT01304797; ASCO 2012, Abstract TPS663; AACR 2015, Abstract #CT234).

The HER2-intermediate MBC or gastric cancer can be characterized by a score of 1+ or 2+ by immunohistochemistry (IHC). In another embodiment, the HER2-intermediate MBC or gastric cancer is Fluorescence In-Situ Hybridization (FISH) negative. In some embodiments, the HER2-intermediate MBC or gastric cancer is characterized by a score of 2+ by IHC. In another embodiment, the HER2 intermediate MBC or gastric cancer is characterized by a HER2 IHC score of 1+ with a FISH negative score, or a HER2 IHC score of 1+ with a FISH positive score. In one embodiment, the method comprises treating the patient with subsequent treatment 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle. In another embodiment, treatment of the patient results in a reduction of the number of metastatic lesions in the patient.

The treatment of HER2 -intermediate cancer in a human can include administration of a HER2-targeted antibody-liposomal doxorubicin conjugate such as MM302 once every three weeks to a patient in an amount providing a doxorubicin dose of 30 mg/m² (doxorubicin HC1 basis). The HER2 -targeted antibody-liposomal doxorubicin conjugate can encapsulate the doxorubicin in a pharmaceutically acceptable salt, such as a doxorubicin sulfate salt. Preferably, at least 90%, 95%, 98%, 99% or more of the doxorubicin administered to the human diagnosed with a HER2-intermediate cancer in the HER2 -targeted antibody- liposomal doxorubicin conjugate is encapsulated in a liposome. As used herein "mg/m²" indicates mg of doxorubicin (formulated as MM-302 HER2-targeted antibody-liposomal doxorubicin conjugate) per square meter of body surface area of the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a cartoon depicting the ErbB2/HER2 receptor including extracellular domain I bound by MM-302 and domain IV bound by trastuzumab.

Figure IB is a cartoon showing a comparison of the relative sizes of a liposome, antibody and doxorubicin; drawn to scale.

Figure 1C shows immunofluorescence images of BT-474-M3 cells treated simultaneously with DiI5-MM-302 (liposome containing the far red-fluorescent carbocyanine tracer DiIC18(5)-DS (D 12730— abbreviated DiI5; Life Technologies) and Alexa-488- trastuzumab (trastuzumab with AlexaFluor® 546 dye), 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 1C shows that MM-302 and trastuzumab can co-localize on HER2-overexpressing BT-474-M3 cells in vitro.

Figures 2A-2D show that MM-302 and trastuzumab can simultaneously bind a cell and do not interfere with each other's activity in vitro. In Figures 2A and 2B, BT-474-M3 (breast) and NCI-N87 (gastric) cells were treated for 24h with the indicated concentrations of DiI5-MM-302 alone (dotted line) or in combination with trastuzumab at 1 μg/mL (dashed line) or trastuzumab at 10μg/mL (solid line). Bound liposome (Figure 2A) or nuclear doxorubicin (Figure 2B) were detected by high throughput fluorescence microscopy and expressed as Mean Fluorescence Intensity (MIF). In Figures 2C and 2D, BT-474-M3 and NCI-N87 cells were treated with 1 μΜ MM-302 alone (dotted line, circles), 1 μΜ MM-302 in combination with trastuzumab at ^g/mL (dashed line, squares) or 10μg/mL (solid line, squares), trastuzumab alone (solid line, triangles) or untreated (solid line, circles) for the indicated length of time. Phosphorylated p53 (p-p53; Serl5; Figure 2C) and p-Akt (Ser473; Figure 2D) were evaluated by electrochemiluminescent assays (Meso Scale Discovery).

Figure 3 shows that the combination of MM-302 and trastuzumab is more effective than either single agent in decreasing viability of BT-474-M3 and NCI-N87 cells in vitro. BT-474-M3 and NCI-N87 cells were treated with trastuzumab alone (10μg/mL, black bars), MM-302 alone (0.5μΜ, light gray, middle bars) or MM-302 and trastuzumab (dark gray bars) for 72h. Viability was evaluated using CellTiter-Glo® (CTG) Assay and expressed as percent viability relative to untreated cells. Statistics indicate student's unpaired t-test.

Figure 4 shows that MM-302 and trastuzumab co-localize in BT-474-M3 human xenografts. NCR/nu mice bearing BT-474-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. 24h following 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 40X/1.3 oil objective. Representative images of MM-302 (red), trastuzumab (green), vessels (magenta) and DNA (Hoechst; blue) are shown. Yellow indicates co- localization of MM-302 and trastuzumab. Lower row represents close-up of selected region from upper row.

Figures 5A-5B depict graphs showing that co-administration of MM-302 with trastuzumab increases p53 phosphorylation and MM- 302 deposition. Figure 5A shows BT- 474-M3 or NCI-N87 tumor-bearing mice (n = 5-8/group) were dosed with trastuzumab alone (7mg/kg; diamonds), MM-302 alone (3mg/kg; circles) or MM-302 (3mg/kg) and trastuzumab (7mg/kg; triangles); untreated (U; squares). Tumors were collected 4, 24 or 72h following treatment, frozen and lysates prepared. p-p53 (Serl 5) signal was evaluated by

electrochemiluminescent assay (Meso Scale Discovery®). In Figure 5B, mice were treated as in (Figure 5A) and tumors analyzed by high-performance liquid chromatography (HPLC) to determine the total doxorubicin in the tumor expressed as percent of injected dose per gram of tissue (% i.d./g). Untreated tumor produces no dox signal by HPLC. The left hand bars of each pair (hatched bars) represent data from mice treated with MM-302 alone, and the right had bars of each pair (striped bars) represent data from mice treated with MM-302 and trastuzumab. Horizontal bar indicates the mean for each group. Statistics represent student's unpaired t-test.

Figures 6A-6C show that co-administration of MM-302 with trastuzumab influences MM-302 liposome distribution. As shown in Figure 6A, mice bearing BT-474-M3 or NCI- N87 tumors (n = 5/group) were dosed with DiI5-MM-302 alone (3mg/kg; circles) or DiI5- MM-302 (3mg/kg) and trastuzumab (7mg/kg; triangles); untreated (U). Tumors were collected 4, 24 or 72h post-drug inj ection, prepared for cryosection, counterstained with

Hoechst and scanned on an Aperio FL scanner. Images were analyzed to quantify liposome Mean Fluorescence Intensity (MFI). Figure 6B shows images of tumor sections illustrating the variability of liposome MFI within the individual tumor sections: representative tumors for the DiI5-MM-302 alone (left) and DiI5-MM-302 and trastuzumab (right) groups are shown (24 hours). Scale bar = 1mm. Figure 6C is a graph showing quantification of the tumor areas (% of total) with different liposome MFIs.

Figures 7A-7B shows that co-administration of MM-302 with trastuzumab enhances anti-tumor activity. Mice were inoculated with BT-474-M3 breast (Figure 7A) or NCI-N87 gastric cells (Figure 7B) and at a tumor volume of 200-300 mm³ treated with either PBS (control; open circles), MM-302 (circles), trastuzumab (triangles) or the combination of MM- 302 and trastuzumab (diamonds); n=8-10/group. Changes in tumor volume over time (days) are shown post- inoculation. Data was analyzed by repeated measures ANOVA.

Figures 8A-8D show in vivo efficacy of MM-302 in 4T1 xenograft mouse model. Figure 8A is a schematic of the experimental design: 4Tl-GFPluc2-HER2 cells are inoculated into the right and left #4 mammary fat pad (mfp) of 6- week old nude mice that were then randomized and treated with vehicle control (PBS), 3 mg/kg non-targeted

PEGylated liposomal doxorubicin (PLD) or 3 mg/kg MM-302 once weekly for 3 weeks starting at day 20. Thirty- nine days following inoculation, the mice were sacrificed and their lungs were harvested for enumeration of surface metastases. Figure 8B shows gross images of Bouin solution (a fixative comprising picric acid, acetic acid, and formaldehyde in an aqueous solution) fixed lungs illustrating surface metastases (white nodules, arrows). Figure 8C is two graphs depicting the total number of surface metastases per lung (left) and the lung weight (right) for each mouse in each group. The number of surface lung metastases was 2. lx lower in the MM-302-treated mice than the PLD-treated mice while the lung weights were 1.3x lower. P value corresponds to Student's t-test comparing the number of surface metastases and lung weight in the PLD vs MM-302-treated groups. Figure 8D is a graph showing tumor growth curves indicating that treatment with free doxorubicin had no effect on primary tumor growth while MM-302 and PLD both slowed tumor growth slightly and to the same degree. The tumor growth curves represent the mean tumor volume +/- SEM. The days the mice were treated are indicated by triangles on the x-axis.

Figures 9A-9E show in vivo efficacy of MM-302 in MDA-MB-453 (breast) xenograft mouse model. Figure 9A is a schematic of the experimental design: MDA-MB-453 cells were inoculated into the right and left #4 mammary fat pad (mfp) of 7 to 10-week CIEA BALB/c- Rag2null-IL2Rgnull (BRG) mice that were then randomized and treated with vehicle control (PBS), 6 mg/kg non- targeted PEGylated liposomal doxorubicin (PLD) or 6 mg/kg MM-302 once weekly for 3 weeks starting at day 79. One hundred days following inoculation, the mice were sacrificed and their lungs were harvested for quantification of lung metastases. In Figure 9B (top) is shown gross images of Bouin solution fixed lungs illustrating a blanket of surface micro-metastases in control and PLD-treated mice but not in MM-302 -treated mice. The bottom panel is a graph depicting the percentage of mice with surface micro- metastases in each group. In Figure 9C (top) is shown representative photomicrograph images of immunohistochemical human cytokeratin (CK) stain segmentation in fixed lung sections. The bottom panel is a graph showing quantification of human CK positive cells in lung sections indicating that the lung area occupied by CK positive cells was approximately 10-fold lower in the MM-302 -treated group than in the PLD group. P value corresponds to Student's t-test comparing metastatic burden in the PLD vs MM-302 -treated groups. Figure 9D is a graph with tumor growth curves indicating that treatment with either MM-302 or PLD significantly inhibited primary tumor growth. The data represent the mean tumor volume +/- SEM. Mice were treated on days indicated by triangles on the x-axis. Figure 9E shows the delivery and anti-tumor activity of MM-302 vs. PLD to HER-2+ tumors in vivo. Mice that received MM- 302 had a 16x higher liposome delivery and a 12x higher anti -tumor activity as compared to mice that received PLD.

Figures 10A-10D depict the distribution of MM-302 in lungs, primary tumor and liver of tumor bearing mice. Figure 1 OA is an image of a hematoxylin and eosin (H&E) stain of frozen lung, matching primary tumor and liver tissues harvested from a 4Tl-GFPluc2-HER2 tumor-bearing mouse 24h after injection of fluorescently -labeled MM-302-DiI5. Figure 10B is an image of fluorescent serial section illustrating that in lungs harboring sheet-like metastases, liposome distribution is uniformly high throughout the entire lung while in primary tumors, liposome distribution is low and limited to the periphery of the tumor. Figure IOC is an image of an H&E stain of lungs harboring nodular metastatic lesions (arrows). Figure 10D is an image of a fluorescent serial section illustrating that in lungs harboring large nodular metastatic lesions, liposome distribution is peripheral while in small lesions, it is uniformly high. Note that there is no measurable liposome accumulation in normal lung tissue. Liposomes are shown in cyan and nuclei in blue.

Figure 11 shows photomicrographs of fluorescent images of primary tumors harvested from a 4Tl-GFPluc2-HER2 tumor-bearing mouse 36h after co-injection of both PLD and MM- 302 each fluorescently-labeled with a different fluorescent probe (DiR and DiI5, respectively). The images illustrate preferential accumulation of both PLD-DiR and MM- 302-DiI5 on the periphery of the tumors which is consistent with them being equally efficacious at slowing tumor growth.

Figures 12A-12C show distribution of liposomes in lung metastases from tumor bearing mice. Figure 12A depicts two images of H&E stains of frozen lungs harvested from 4Tl-GFPluc2-HER2 metastasis-bearing mice 24h after injection of fluorescently-labeled

PLD-DiI5 (left) or MM-302-DiI5 (right). Figure 12B depict two photomicrograph images of fluorescent serial sections illustrating the distribution of fluorescently-labeled liposomes (cyan) in lung metastases. Figure 12C is a graph showing quantification of mean liposome (PLD and MM-302) fluorescence intensity in lung metastases. The data represent the mean PLD and MM-302 fluorescence intensity per pixel +/- SEM (p<0.05).

Figures 13A and 13B depict the correlation between receptor number of various cell lines and cell viability following treatment with MM-302. Figure 13A shows the results of an in vitro binding assay showing the binding of MM-302 and PLD to various cell lines. Cells were collected, pelleted at 2000 rpm, and divided into four parts, each incubated (50 x 10⁶ cells/ml) for 1 h at 37°C under rotation with the either fluorescently labeled MM-302 (MM- 302-DiI5; 5μg/ml), PEGylated liposomal doxorubicin (PLD-DiI5; 5ug/ml), T-DM1 (T-DM1- DiI5; 27.8 μg/ml). At the end of the incubation, cells were pelleted at 2000 rpm, an aliquot of supernatant and cell pellet was collected to determine the unbound and bound drug fraction by means of DiI5 fluorescence measurement with an EnVision® Plate Reader. Figure 13B is an in vitro viability assay in the same cell lines. Here, cells were plated in 96-well plates at a density of 5,000 cells/well. The following day, media was replaced with fresh media alone (untreated), media containing MM-302 (0.08 to 10 μg/ml) or T-DM1 (0.08 to 10 μg/ml). Each treatment was performed in duplicate. Plates were incubated at 37°C for 72 hours. Media was then removed, replaced with 200 μΐ/well of a 1 : 1 mixture of RPMI and CellTiter- Glo® reagent, followed by incubation at RT for 30 min. Luminescence was measured using an EnVision plate reader.

Figures 14A-14E describe the PEG-HER2 assay that enables detection of HER2- mediated MM-302 internalization on clinical samples. Figure 14A is a pictographical representation of the PEG-HER2 assay. The assay uses a HER2 standard cell pellet array shown on the left, a PEG Standard Cell Array shown on the right, and the experimental tissue harvested in the center. The arrays are used to determine a standard curve for HER2 or PEG staining on the tumor tissue from animals or patients. For the HER2 standard cell array, MDA-MB-231, MCF7, T47D, DU145, IGROV1, MDA-MB-175-VII, MDA-MB-453, MDA- MB-361, MCF-7 clone 18 and SKOV3 cells were grown to -80% confluence (total of 2-4 x 10⁸ cells), rinsed with PBS and dissociated with Accutase™. Cells were then collected, pelleted at 2000 rpm and fixed with 10% neutral buffered formalin (NBF) for 4 h at 4°C. Following this, the cells were pelleted at 2000 rpm, washed with PBS, and packed by centrifugation at 2000 rpm. The cell pellets were resuspended with Histogel and transferred into a cloning ring until polymerization was accomplished. The resulting pellets were subsequently placed into cassettes, surrounded by O.C.T. (Optimal Cutting Temperature) compound, and frozen in an isopentane-dry-ice slurry. Frozen cell pellets were stored at - 80°C until processing. For the MM-302 standard cell pellet array, BT474-M3 were grown to -80% confluence (total of 1.6 x 10⁹ cells), rinsed with PBS and dissociated with Accutase™. Cells were collected, pelleted at 2000 rpm and divided into 10 parts, each incubated (50 x 10^λ6 cells/ml) for 1 h at 37°C under rotation with the following MM-302 concentrations: 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320 μg/ml. At the end of the incubation, cells were pelleted at 2000 rpm, an aliquot of supernatant and cell pellet was collected to determine the unbound and bound doxorubicin fraction of each reaction by HPLC. HPLC quantification of doxorubicin was performed as previously described (Reynolds JG, Toxicol Appl Pharmacol 2012) and allowed to determine the bound ng dox/cell and the # liposomes/cell for the individual reactions. The remaining cell pellet from each reaction mix was fixed with 10% NBF for 4 h at 4°C. Cells were pelleted at 2000 rpm, washed with PBS, and packed by centrifugation at 2000 rpm. The cell pellets were resuspended with Histogel™ and transferred into a cloning ring until polymerization was accomplished. The resulting pellets were subsequently placed into cassettes, surrounded by O.C.T. compound and frozen in an isopentane-dry-ice slurry. Frozen cell pellets were stored at -80°C until processing. For the generation of tumor xenografts, 7-week-old female NCR/nu, CIEA BRG nude mice, and CB17.Cg-PrkdcscidLyst bg/Cr were used to establish tumor xenografts using BT474-M3, SUM190 and MDA-MB-361 cells as previously described (Geretti E Mol Cancer Ther 2015, the disclosure of which is incorporated herein by reference in its entirety). MCF7 (7.5x 10⁶) tumor cells were inoculated into the right mammary fat pad #2 of NCR/nu mice. MDA-MB- 231 (1 x 10⁶) into the right mammary fat pad #4 of CB17.Cg-PrkdcscidLyst bg/Cr. IGROV1 (5xl0⁶) were inoculated subcutaneously (s.c.) into the right flank of CIEA BRG mice. When tumors reached a volume of about 300 mm³, mice were dosed intravenously (i.v.) with fluorescently labeled MM-302 (DiI5 -labeled) (3 mg/kg) or fluorescently labeled PLD (PEGylated liposomal doxorubicin) (3 mg/kg), or PBS as indicated. Mice were sacrificed 24 h or 72 h following liposome injection. Immediately after sacrificing, mice were perfused with PBS to remove liposomes still in circulation. Tumors were collected immediately after sacrificing. One part of each tumor was fixed for 24 h at RT in 10% NBF, cryoprotected for 24 h at 4°C in 15% sucrose, and subsequently frozen after being embedded in O.C.T.

compound. Frozen blocks were stored at -80°C until further processing to cryosections (7-um thick) (Figure 14B). Tumors were stained to detect HER2. PEG and cytokeratin and images acquired using an Aperio ScanScope® FL. To determine γ-Η2ΑΧ (or cleaved caspase 3 or cleaved PARP)-PEG-cytokeratin stained images, tumors were stained with anti phospho- histone H2AX, anti-human cleaved caspase 3, and mouse anti-human cytokeratin. Cell nuclei were detected using Hoescht® staining, followed by classification of the cells into tumor or stromal cells based on the cytokeratin signal. The tumor cells were then classified into γ- H2AX (or cleaved caspase 3 or cleaved PARP) positive or negative based on the intensity of the selected marker. Co-staining with the MM-302 standard allowed to interpolate the # liposomes/cell from the PEG intensity values on the individual tumor cells (Figure 14C).

For human tissue analysis, frozen patient biopsies from MM-302 phase I trial MM- 302-02-01 -01 were used. Patients enrolled in trial NCT01304797 were dosed with MM-302 (8, 16, 30, 40 or 50 mg/m²). 72 h following the first received dose of MM-302, a biopsy was collected from the primary or metastatic lesion of the patients. The biopsy was frozen in liquid nitrogen and stored at -80°C until further processing to cryosections (7-μιτι thick). Tissues were stained to determine HER2 and PEG expression (Figures 14D and E). Higher amounts of MM-302 were seen inside the HER-2+ tumors of mice that received MM-302, whereas the liposomal doxorubicin seemed to accumulate in the stroma of tumors following PLD injection (Figure 14E).

Figures 15A-15B show MM-302 activity in a HER2 1+ metastatic model. Figure 15A is a pictorial representation of frozen sections of lung tissue after staining. Figure 15B is a scatter plot comparing the anti-tumor activity in ovarian, lung, brain, adrenal gland, and liver metastases relative to a control, PLD, DOX, and T-DM1.

Figures 16A-16C show HER2 expression and MM-302 uptake into tumor cells from MM-302 patient biopsies from the Phase 1 trial NCT01304797. Figure 16A is a bar graph of the histopathology evaluation of the frozen biopsies of each patient in terms of the percentage of biopsied cells that were normal, necrotic, or tumor cells. Figure 16B is dual graph providing the distribution of HER2 expression within the biopsies. Figure 16C is pictorial representation of examples of staining on selected biopsies. DETAILED DESCRIPTION

MM-302 is a HER2 -targeted antibody-liposomal doxorubicin conjugate that offers targeted delivery of PEGylated Liposomal Doxorubicin (PLD) to a cancer cell

overexpressing the human epidermal growth factor receptor 2 (HER2; also known as ErbB2) proteinMM-302 is a HER2 -targeted PEGylated liposome that encapsulates doxorubicin to facilitate its delivery to HER2-overexpressing tumor cells while limiting exposure to non- target tissues, including the heart, while limiting exposure to non-target tissues, including the heart. MM-302 liposomes are prepared and loaded with doxorubicin using an ammonium sulfate gradient as previously described (Kirpotin et. al, Cancer Res. 2006; Park et al., Clin. Cancer Res. 2002, the disclosures of which are incorporated herein by reference in their entireties). The MM-302 HER2-targeted liposome encapsulates approximately 20,000 molecules of doxorubicin in its core and an average of 45 single chain anti-HER2 antibodies (scFv) conjugated to its surfaceMM-302 is a HER2 -targeted PLD and has been designed to provide improved efficacy while maintaining the safety profile of PLD. It is a sterile, injectable parenteral, liquid formulation of doxorubicin encapsulated into long-circulating

immunoliposomes conjugated to human recombinant scFv, specific to c-ErbB-2 oncoprotein. The anti-HER2 scFv present on the liposome targets the MM-302 liposome to HER2- overexpressing cells and promotes internalization. It binds to a different epitope than trastuzumab and does not bind to cardiomyocytes. The liposomal component stably encapsulates doxorubicin and extends the half-life relative to free doxorubicin.

Liposomal encapsulation dramatically alters the pharmacokinetics and biodistribution of doxorubicin. By virtue of their comparatively large size, MM-302 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. The normal vasculatures of healthy organs such as the heart typically prevent leakage and significant accumulation of liposomes. The amount of HER2 receptors needed per cell to optimize the binding of MM-302 is approximately 200,000 HER2 receptors per cell. Below this level, binding is comparable with that of untargeted PLD (UT-PLD). MM- 302 does not effectively bind to or enter human cardiomyocytes. The level of MM-302 uptake into human cardiomyocytes is on the same order as UT-PLD. In contrast, the uptake of free doxorubicin is relatively much higher compared to both MM-302 and UT-PLDMM- 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 tumor cells expressing HER2 above a critical threshold of - 200,000 HER2, with a resultant increase in anti-tumor activity relative to PLD in multiple preclinical models. 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. Importantly, cardiomyocytes do not possess the requisite number of HER2 receptors required to efficiently mediate the uptake of MM- 302, potentially mitigating safety concerns. The level of MM-302 uptake into human cardiomyocytes is on the same order as PLD. In contrast, uptake of free doxorubicin is significantly higher compared to both MM-302 and PLD. 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 U.S.

Each MM-302 liposome encapsulates approximately 20,000 molecules of doxorubicin in its core and an average of 45 single chain anti-HER2 antibodies (scFv) are conjugated to its surface. The doxorubicin in MM-302 is doxorubicin sulfate, as described in Kirpotin et al, Cancer Res. 2006 and Park et al., Clin. Cancer Res. 2002.

MM-302 is created by covalently conjugating single-chain antibody variable fragments scFv; denoted F5 having the following amino acid sequence:

VQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISGRGDN TYYADSVKGRFTISRDNSK TLYLQMNSLRAEDTAVYYCAKMTSNAFAFDYWGQG TLVTV S S GGGGS GGGGS GGGGS QSVLTQPPSVS GAPGQRVTI S CTGS S SNIGAGYGV HWYQQLPGTAPKLLIYGNTNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYYCQS YD S S L S GW VF GGGTKLT VLGGS GGC (SEQ ID NO: 1), an anti-HER2 scFv selected for the ability to promote internalization) to a long-circulating, doxorubicin loaded liposome via a polyethylene glycol (PEG) spacer.

The MM-302 drug product is a sterile, injectable parenteral, liquid formulation of doxorubicin encapsulated into long-circulating immunoliposomes conjugated to human recombinant scFv, specific to the C-ErbB-2 oncoprotein. The scFv F5, was selected from a fully human scFv phage display library for the efficient internalization into HER2- overexpressing cells. The liposome particles have the average size in the range of 75-110 nm and each consist of a single bilayer membrane composed of fully hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and small amount of PEG (molecular weight 2000)-derivatized distearoylphosphatidylethanolamine (PEG-DSPE). The liposome membrane encloses an interior space where doxorubicin molecules are contained and, due to their high concentration, form a fibrous crystalline or gel-like precipitate. The anti-HER2 scFv F5 is conjugated to the liposome surface through the poly (ethylene glycol) spacer using unique C-terminal conjugation site. A schematic representation of MM-302 liposome is shown in Figure 1A. The MM-302 drug product contains doxorubicin in the amount equivalent to 2 mg/mL of doxorubicin HC1. It also contains 10.2 mg/mL of HSPC, 3.39 mg/mL of cholesterol, 3.39 mg/mL of methoxy -terminated polyethylene glycol (MW 2000)- distearoylphosphatidylethanolamine (PEG-DSPE), PEG-DSPE-conjugated scFv F5 in the concentration of 0.2 mg/mL of antibody protein, 10 mM L-histidine-HCl buffer (pH 6.5), 100 mg/niL sucrose to maintain isotonicity, ammonium sulfate in the concentration of less than 0.8 mg/mL, and sodium citrate in the concentration of less than 0.5 mg/mL.

MM-302 liposome is a unilamellar lipid bilayer vesicle of approximately 75-1 10 nm in diameter that encapsulates an aqueous space, which contains doxorubicin sulfate in a gelated or precipitated state. The lipid membrane is composed of phosphatidylcholine, cholesterol, and a polyethylene glycol-derivatized distearoylphosphatidylethanolamine. There is approximately one PEG molecule for 200 phospholipid molecules, of which approximately one PEG chain for each 1780 phospholipid molecules bears at its end a scFv antibody fragment that binds to c-ErB-2 protein on the surface of a cancer cell and internalizes the liposome into the cell. MM-302 drug product is sterile dispensed into 10 mL clear glass vials closed with crimped rubber caps at about 10-mL (20 mg doxorubicin HC1) per vial, labeled, and stored refrigerated in a dark place.

The number of HER2 receptors on a cell is one of the critical parameters determining MM-302 uptake into target and non-target cells. Experiments were conducted to identify the level of HER2 receptor expression needed for the optimal binding of MM-302 to a cancer cell (see, e.g., U. S. Patent No. 9,226,966). MM-302 was taken up by cells as measured by cell-associated doxorubicin in a dose- and time-dependent manner. The results demonstrated that increasing levels of HER2 are associated with significant increases in uptake of MM-302 by cells in vitro. The uptake of PLD was low in all the cell lines tested and did not correlate with HER2 expression. Similar results were obtained for the same cell lines incubated for shorter periods of time and with lower concentrations of MM-302. These results

demonstrated that MM-302 is selectively taken up into cells that express HER2, specifically in cells in the 2+ range and above of HER2.

The HER2IErbB2 gene is amplified and/or overexpressed in many types of human malignancies, including but not limited to breast, ovarian, endometrial, pancreatic, colorectal, prostate, salivary gland, kidney, and lung. HER2-overexpressing cancers are designated as HER2 3+ or HER2 2+ depending on the level of ErbB2 receptor overexpression, with HER2 3+ indicating the highest levels of HER2 expression. HER2 3+ and HER2 2+ status are typically determined by an immunoassay such HercepTest®. HER2 gene amplification can be determined by FISH (fluorescence in situ hybridization, e.g., HER2 IQFISH pharmDx™ kit, Dako), with HER2-amplified cancer cells being those that have more than two HER2 gene copies being HER2-amplified, and cells and/or tumors comprising HER2-amplified cancer cells being referred to as "FISH positive." "HER2 -intermediate" tumors as used herein correspond to a HER2 score of 1+ or 2+ by immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USAand HER2 FISH negative as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA), or one that corresponds to a score of 1+ by immunohistochemistry using for example, a HercepTest^® kit in accordance with the manufacturer's instructions. The HER2 -intermediate tumor can be further characterized using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA) For example, a HER2 intermediate tumor can be HER2 FISH negative as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions.

Alternatively, a HER2 intermediate tumor can be HER2 FISH positive as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions.

In various embodiments, the present disclosure provides methods for treating breast cancer or gastric cancer in a human patient having HER2 -intermediate breast cancer or HER2 -intermediate gastric cancer. In some embodiments, the frequency and dosing of MM- 302 to a human patient having HER2 -intermediate breast cancer or HER2 -intermediate gastric cancer is provided in Table 1.

"mg/m²" indicates mg of doxorubicin (formulated as MM-302) per square meter of body surface area of the patient. For HER2 -intermediate breast cancer, dose 1, 2, 3, or 4 is preferred. For Kaposi's sarcoma dose 1, 2, or 3 is preferred, for ovarian cancer, dose 3, 4, or 5 is preferred and for multiple myeloma dose 2, 3, 4, or 5 is preferred. For HER2- intermediate gastric cancer, dose 1, 2, 3, or 4 is preferred. Dosing regimens may vary in patients with solid tumors that are "early" (pre-metastatic, e.g., adjuvant breast cancer) as compared to "advanced" (metastatic tumors). . In some embodiments, the patient thusly treated has HER2-intermediate metastatic breast cancer (MBC), and/or HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions. In various embodiments, the method comprises administering to the human patient, for example, once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER2-targeted doxorubicin immunoliposome in one or more treatment cycles to treat the HER-2 intermediate breast cancer, the HER2 -intermediate metastatic breast cancer, or the HER2-intermediate metastatic breast cancer harboring a number of metastatic lesions, in the patient.

In these related embodiments, the HER2-intermediate breast cancer, HER2- intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions, are characterized as preferably having a HER2 score of 1+ or 2+ by immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions(Dako, Agilent Technologies, Santa Clara, California USA) and are HER2 FISH negative as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA). In altemative embodiments, the HER2 -intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions all have a HER2 score of 1+ by

immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA) and are HER2 FISH negative as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA). In other altemative embodiments, the HER2-intermediate breast cancer, HER2-intermediate metastatic breast cancer, or the HER2-intermediate metastatic breast cancer harboring a number of metastatic lesions all have a HER2 score of 2+ by immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA) and are HER2 FISH negative as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA). In other altemative

embodiments, the HER2-intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2-intermediate metastatic breast cancer harboring a number of metastatic lesions all have a HER2 score of 1+ by immunohistochemistry using for example, a

HercepTest® kit in accordance with the manufacturer's instructions (Dako, Agilent

Technologies, Santa Clara, California USA) and are HER2 FISH positive as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA). In still other altemative embodiments, the HER2-intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions all have a HER2 score of 2+ by immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA) and are HER2 FISH positive as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA).

In other related embodiments, the HER2-intermediate gastric cancer, HER2- intermediate metastatic gastic cancer, the HER2 -intermediate metastatic gastric cancer harboring a number of metastatic lesions, are characterized as preferably having a HER2 score of 1+ or 2+ by immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions(Dako, Agilent Technologies, Santa Clara, California USA) and are HER2 FISH negative as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA). In altemative embodiments, the HER2 -intermediate gastric cancer, HER2 -intermediate metastatic gastric cancer, or the HER2 -intermediate metastatic gastric cancer harboring a number of metastatic lesions all have a HER2 score of 1+ by

immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA) and are HER2 FISH negative as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA). In other altemative embodiments, the HER2-intermediate gastric cancer, HER2-intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions all have a HER2 score of 2+ by immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions (Dako,

Agilent Technologies, Santa Clara, California USA) and are HER2 FISH negative as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA). In other altemative

embodiments, the HER2-intermediate gastric cancer, HER2-intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions all have a HER2 score of 1+ by immunohistochemistry using for example, a

HercepTest® kit in accordance with the manufacturer's instructions (Dako, Agilent

Technologies, Santa Clara, California USA) and are HER2 FISH positive as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA). In still other alternative embodiments, the HER2-intermediate gastric cancer, or HER2 -intermediate metastatic gastric cancer, or the HER2 -intermediate metastatic gastric cancer harboring a number of metastatic lesions all have a HER2 score of 2+ by immunohistochemistry using for example, a HercepTest® kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA) and are HER2 FISH positive as tested using a HER2 IQFISH pharmDx™ kit in accordance with the manufacturer's instructions (Dako, Agilent Technologies, Santa Clara, California USA).

In each of these HER2 -intermediate breast and gastric cancers, the present disclosure provides a method of treating breast or gastric cancer in a human patient having HER2- intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2- intermediate metastatic breast cancer harboring a number of metastatic lesions or HER2- intermediate gastric cancer, HER2 -intermediate metastatic gastric cancer, or the HER2- intermediate metastatic gastric cancer harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome for one or more treatment cycles.

In some embodiments, the present disclosure provides a method of treating breast cancer or gastric cancer in a human patient having HER2 -intermediate breast cancer, HER2- intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions or HER2-intermediate gastric cancer, HER2- intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM- 302 HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg

trastuzumab, to treat the HER-2 intermediate MBC or HER2 -intermediate gastric cancer in the patient.

In another aspect is provided a method of treating gastric cancer in a human patient having HER2-intermediate gastric cancer, HER2 -intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome for one or more treatment cycles. In another aspect is provided a method of treating gastric cancer in a human patient having HER2 -intermediate gastric cancer, HER2-intermediate metastatic gastric cancer, or the HER2 -intermediate metastatic gastric cancer harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate gastric cancer in the patient.

Thus, in another aspect is provided a method of treating breast cancer in a human patient having HER2-intermediate breast cancer, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome, to treat the HER-2 intermediate MBC in the patient using one or more treatment cycles.

In another aspect is provided a method of treating breast cancer in a human patient having HER2-intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome for one or more treatment cycles. In another aspect is provided a method of treating breast cancer in a human patient having HER2 -intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2- intermediate metastatic breast cancer harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate MBC in the patient.

EXAMPLES

Unless otherwise indicated, the following Materials and Methods were used in the

Examples.

Example 1: Tumor Growth Inhibition and Liposome Delivery Study in Mice

Cell culture

BT-474-M3 is a HER2-overexpressing cell line received as a gift from Hermes Biosciences (CA). NCI-N87 cells were purchased from ATCC (VA) 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

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. (Orlando, FL) and labeled per vendor's instructions with Alexa Fluor®-488 or AlexaFluor-555. (Life Technologies).

In vitro fluorescence microscopy: 10,000 cells were plated per chamber (Nunc Lab Tek II Slides) and simultaneously treated with Ι μΜ DiI5-MM-302 and Ι μΜ Alexa-488-trastuzumab for lh on ice at 4°C, fixed with 3.7% formaldehyde for 15min at RT and stained with Hoechst 33342. (Life Technologies®). 20X and 40X images were captured with a Nikon Eclipse TE2000U microscope (Tokyo, Japan).

Nuclear doxorubicin/liposome imaging by high content microscopy

BT-474-M3 and NCI-N87 cells were plated in 96-well TC 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. 24h later cells were fixed with 3.7% formaldehyde

(15min/RT), incubated with 1 : 1000 Hoechst 33342 (Life Technologies) and 1 : 10,000 Whole Cell Blue Dye. (8403501 ; Life Technologies). Plates were scanned using an Array Worx® High Content Scanner (Applied Precision, Issaquah, WA) with a 10X objective for Hoechst® 33342 (460nm), doxorubicin (595nm), and APC/DiI5 (657nm). Identification of nuclei, cell segmentation and signal quantitation were performed using ImageRail® software. Data are presented as the mean pixel intensity for all cells in a given well for the indicated channel (approx. 4000 cells evaluated per well).

In vitro viability

BT-474-M3 and NCI-N87 cells were plated in 96-well TC 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 72h. CellTiter-Glo® (CTG) assay was performed per vendor instructions (Promega, Madison, WI). Luminescence was measured using an EnVision® 2103 Multilabel plate reader (Perkin Elmer, Waltham, MA). 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® (Germantown, NY) and seven- week-old female nu/nu mice were purchased from Charles River

Laboratories. (Wilmington, MA). The care and treatment of experimental animals was in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines.

Establishment of xenografts: NCR/nu mice were implanted with a 17 -estradiol pellet (0.74mg; 60- day release; Innovative Research of America (Sarasota, FL)) 2-3 days before inoculation with ΙΟχΙΟ⁶ BT-474-M3 cells into the mammary fat pad (m.f.p.). Nu/nu mice were inoculated with 7.5x10⁶ NCI-N87 cells subcutaneously into the right flank of the mouse.

Tumor growth inhibition: Once tumors reached a volume of 200-300 mm³, mice were randomized into groups of 8-10 mice/group of equal average tumor volume and dosed intravenously (IV) with: PBS (control), MM-302 (3mg/kg (dox), IV q7d for 3 doses), trastuzumab (7mg/kg loading dose, then 3.5mg/kg q3d 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: width²xlengthx0.52. Bliss (synergy) analysis was performed as follows: Tumor growth inhibition (TGI) in each treatment group was calculated using the following formula:

where, V_treated and V_control 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: TGI_Bii_SS Additivity ^{~ TGI}MM-302 ^{+ TGI}Tiastuzumab ^{~ TGI} MM-

Values less than additive indicate antagonism, while values above additive indicate synergy.

Delivery studies: Once tumors reached a volume of 200-300 mm³, mice were dosed IV with PBS (control), trastuzumab (7mg/kg), DiI5-MM-302 (3mg/kg), or trastuzumab (7mg/kg) and DiI5-MM-302 (3mg/kg); or purified human IgG (7mg/kg); 1-001 -A, R&D Systems) or trastuzumab-DMl (7mg/kg; CuraScript). Alternatively, mice were dosed with Alexa-555- trastuzumab (7mg/kg) and DiI5-MM-302 (3mg/kg). Tumors were collected 4, 24 and 72h post injection of agents. Five minutes prior to sacrifice, mice were dosed IV with 200 μΐ of FITC-lectin (Vector Laboratories, Inc., Burlingame, CA) 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 HPLC was performed as previously described (Reynolds JG, et al. Toxicol. Appl. Pharmacol. 2012). HPLC quantification of doxorubicin. Tissues are weighed and disaggregated with lmL H2O using a TissueLyser® (Qiagen®) for 3min. Afterwards 100 μΐ of the homogenate were transferred into a new tube and 900 μΐ of 1% acetic acid/methanol was added. For the cultured cells, cells were treated with drug, as described, trypsinized and lysed using 1.0% acetic acid in methanol. Lysates were vortexed for 10 s and placed at -80 °C for 1 h. Samples were spun at RT for lOmin at 10,000 RPM. Supernatants and doxorubicin standards are analyzed by HPLC (Dionex) using a C18 reverse phase column (Synergi Polar-RP 80A 250x4.60mm 4 μπι column).

Doxorubicin is eluted running a gradient from 30% acetonitrile; 70% 0.1% trifluoroacetic acid (TFA)/H20 to 55% acetonitrile; 45% 0.1 % TFA/H2O during a 7 min span at a flow rate of l .Oml/min. The doxorubicin peak is detected using an in-line fluorescence detector excited at 485 nm, and emitting at 590 nm.

Histological analysis of tumor liposome deposition

Tumors were embedded in O.C.T. compound (Tissue-Tek®, Torrance, CA), frozen in liquid nitrogen immediately after collection and prepared for cryosection (10-μιτι thickness). Slides were air-dried for 30min at RT and counterstained with Hoechst 33342 diluted 1 :5000 in ProLong Gold mounting media (Molecular Probes, Eugene, OR). Slides were imaged on an Aperio ScanScope FL scanner (Leica Biosystems, Buffalo Grove, IL) at 20X magnification. Images were analyzed using custom rule sets written in Definiens® Developer XD. (Definiens, Munich, Germany). 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 sub-objects and each sub- object was assigned into different classes based on the liposome MFI: <1000, 1000-2000, 2000-3000, 3000-4000, 4000-6000 MFI and >6000. The relative % of area occupied by different MFI classes was then determined.

Confocal microscopy of BT-474-M3 xenografts

Slides with 10-μιτι thick frozen tumor sections were air-dried at RT for 20min, counterstained and mounted with Hoechst 33342 diluted 1 :5000 into ProLong Gold anti-fade reagent (Molecular Probes, Eugene, OR). Slides were imaged on a Leica SP8 X inverted confocal system at 405nm (solid-state diode laser), 487nm, 555nm and 647nm (WhiteLight lasers) with a 40X/1.3 oil objective (Harvard Medical School, Boston, MA). Images were visualized with open source Fiji software (http://fiji.se/Fiji).

Phosphoprotein analysis: p-p53 (Serl5) and p-Akt (Ser473)

p-p53 (K15113D-1) or p-Akt (K150MND-1) assays were performed per vendor instructions (Meso Scale Discovery® (MSD), Rockville, MD).

Example 1 describes a study of tumor growth inhibition and MM-302 liposome delivery study in mice.

First, the results described in this Example indicate that 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 (Figure 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 (Figure IB). First, fluorescent DiI5-MM-302 liposomes and AlexaFluor-488-trastuzumab were incubated simultaneously with HER2-overexpressing BT-474-M3 cells (breast) and membrane-bound signal was evaluated using fluorescence microscopy. Empty liposomes without doxorubicin were used to minimize autofluorescence and experiments were performed on ice to minimize internalization. BT-474-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 (Figure 1C, MM-302, top and middle panels). Trastuzumab is present in the same peripheral locations, as well as between individual cells (Figure 1C, trastuzumab, top and middle panels). These observations are consistent with the physical size differences between the antibody trastuzumab and MM-302. When individual BT-474-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 (Figure 1 C, bottom row). 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.

Next, the binding of MM-302 and delivery of doxorubicin to the cell nucleus in the presence of trastuzumab was quantitatively evaluated. BT-474-M3 and HER2-overexpressing NCI-N87 cells (gastric) were simultaneously incubated with DiI5 -MM-302 (0-2 μΜ) and trastuzumab (0, 1 or 10 μg/mL) for 24, 48 and 72h and liposome signal (DiI5) and nuclear doxorubicin were then evaluated by high throughput fluorescence microscopy. A dose- dependent increase in liposome signal (Figure 2A) and nuclear doxorubicin (Figure 2B) was observed with increasing MM-302 concentration in both cell lines at 24h (circles). The addition of trastuzumab at 1 μg/mL (light gray squares) or 10 μg/mL (black squares) did not alter the amount of MM-302 liposome bound or nuclear doxorubicin in either BT-474-M3 or NCIN87 cells (Figures 2A and 2B). Similar results were observed at 48 and 72h.

The results discussed above indicate that MM-302, but not trastuzumab, activates p53. p53 protein levels and phosphorylation/activation are tightly controlled by the cell under normal conditions. However, in response to DNA damage (induced by UV, IR or chemical agents including doxorubicin), p53 can become phosphorylated (p-p53) in the N-terminal domain leading to its activation and accumulation. To investigate this response, BT-474-M3 and NCI-N87 cells were treated with l uM MM-302 for 0-24h and p-p53 (Serl5) 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 (Figure 2C). The increase in p-p53 observed with MM-302 is not altered by co-treatment with trastuzumab at 1 or 10 μg/mL (Figure 2C). Trastuzumab alone has no effect on p-p53 under the conditions tested (Figure 2C).

Third, the results described in this Example indicate that 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. Treatment of BT-474-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 24h (Figure 2D). Co-treatment with MM-302 did not alter the decrease in p-Akt signal attributed to trastuzumab (Figure 2D), while MM-302 alone has no effect on p-Akt signal at the same time points (Figure 2D).

Fourth, the results described in this Example indicate that 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, BT-474- M3 and NCI-N87 cells were treated with trastuzumab alone (10 μg/mL), MM-302 alone (0.5μΜ) or MM-302 and trastuzumab.

Viability was evaluated 72h following treatment and calculated relative to untreated cells. Trastuzumab alone reduced viability in both cell lines by approximately 15% (Figure 3, black bar) while MM-302 alone reduced BT-474-M3 viability by 45% and NCI-N87 by 65% (Figure 3 light gray bars, center). In both BT-474-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; Figure 3, dark gray right hand bars), consistent with an additive effect based on percent viability.

Fifth, the results described in this Example indicate that MM-302 and trastuzumab can bind the same cells in human xenograft tumors. Next was investigated whether both MM-302 and trastuzumab could bind to the same cells in vivo as were observed in vitro. Mice bearing BT-474-M3 tumors were injected with fluorescent DiI5- MM-302 ("empty"; 3mg/kg) and Alexa-555-trastuzumab (7mg/kg) for 4 or 24h 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 24h (Figure 4, MM-302/ far left panels). Trastuzumab is widely distributed and observed on the surface of most tumor cells within the section (Figure 4, trastuzumab, second panels from left), but is not as evident within vessels as MM-302 (consistent with faster distribution of an antibody; center panels). Co-localization of MM-302 and trastuzumab is readily apparent on multiple cells within proximity to the blood vessels (Figure 4, merge/right hand panels). Similar co-localization is observed at 4h.

Sixth, the results described in this Example indicate that combining MM-302 and trastuzumab increases DNA damage signaling in vivo. Building on the in vitro findings, next was evaluated whether changes in p-p53 levels could be detected within tumor xenografts following treatment with MM-302. Mice bearing BT-474-M3 or NCI-N87 human xenograft tumors were treated with trastuzumab alone, MM-302 alone or MM-302 and trastuzumab for 4, 24 or 72h. Tumors were collected and levels of p-p53 (Serl5) evaluated. No changes in p- p53 were observed in the untreated or trastuzumab treated ly sates at any time point (Figure 5A, squares and diamonds, respectively). Significant p-p53 was observed 24h following MM- 302 treatment (p < 0.0001), with a comparable increase in signal from 4 to 24h with or without trastuzumab co-administration (Figure 5A, triangles and circles, respectively).

Interestingly, while both treatments increase p-p53 signal at 72h relative to the 24h time point, the signal is greater with trastuzumab co-administration (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 72h time point (p = 0.0112).

Seventh, the results described in this Example indicate that trastuzumab acutely increases deposition of MM-302 in human xenograft tumors. The in vitro combination experiments described herein 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 to the question of whether trastuzumab was influencing the amount of MM-302 reaching the tumor in vivo. Mice bearing human BT-474-M3 or NCI- N87 xenograft tumors were treated with MM-302 alone or simultaneously with trastuzumab; tumors were collected 4, 24 or 72h after drug administration and doxorubicin levels analyzed by HPLC. When MM-302 was administered as a monotherapy to BT-474-M3 tumors, approximately 5% of the injected doxorubicin/gram tissue (i.d./g) was detected in tumors at 4h (Figure 5B left, hatched bars). Consistent with the p-p53 results, when trastuzumab was injected simultaneously with MM-302 there was a greater than two-fold increase in the amount of doxorubicin detected in the BT-474-M3 tumor at 4h relative to MM-302 alone (p = 0.0076; Figure 5B left, striped bars). These results are consistent with a mechanism wherein trastuzumab increases MM-302 delivery, which in turn results in the increased p-p53 which was experimentally observed. Doxorubicin levels increase from 4 to 24h 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 BT-474-M3 cells at 24h with co-administration of trastuzumab relative to MM-302 alone, although the values were not statistically significant. By 72h, there is little difference between the amounts of doxorubicin in BT-474-M3 tumors treated with MM-302 or MM-302 and trastuzumab. In the gastric NCI-N87 xenograft model, a nearly two-fold increase in doxorubicin levels was observed at 4h when trastuzumab is co-administered with MM-302 (Figure 5B right; p = 0.0449), while a significant increase is also observed at 24h with the combination (p = 0.0024). Changes in MM-302 deposition were not observed with co-injection of a nonspecific human IgG, a monoclonal human antibody against EGFR, or PBS, but an increase was seen with trastuzumab-DMl (T-DM1). Additionally, 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.

As further confirmation, frozen sections from BT-474-M3 tumors treated with DiI5- MM-302, alone or in combination with trastuzumab, were collected 4, 24 or 72h post- injection, then imaged and analyzed to quantify the mean fluorescence intensity (MFI) per tumor. Consistent with the doxorubicin results, co-administration of trastuzumab increased DiI5-MM-302 signal in the tumor at both 4 and 24h relative to MM-302 alone (Figure 6A; p = 0.0159 and p = 0.0317, respectively). A trend towards increased MM-302 deposition was also observed at 72h with trastuzumab (p = 0.0517, not significant (n.s.)). Because of their large size, nanoparticles are known to have challenges effectively penetrating into tumors. Therefore we sought to investigate if 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 0-1000 up to >6000 MFI). This facilitated generation of an image 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 24h time point (Figure 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 (see Figure 6C). The % tumor area with the highest liposome intensity (>6000 MFI) increased from 0.2% to 0.7% at 4h (p=0.0159), from 0.2% to 2.2% at 24h (p=0.0317) and from 0.2% to 2.5% at 72h (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.

Eighth, the results disclosed in this Example indicate that combining MM-302 and trastuzumab improves anti-tumor activity. DNA damage from MM-302 and reduced p-Akt signaling from trastuzumab was observed when MM-302 and trastuzumab were combined in vitro, as well as increased liposome deposition when MM-302 and trastuzumab were coadministered in vivo. Next was investigated whether these observations would translate into increased anti-tumor activity. Mice bearing either BT-474-M3 or NCI-N87 xenograft tumors were treated with MM-302 alone, trastuzumab alone or co-administration of both drugs. Each monotherapy was effective at reducing tumor volume relative to control in both models (Figures 7A and 7B, circles (MM-302 alone) and triangles (trastuzumab alone)). In the BT-474-M3 model, the combination of MM-302 and trastuzumab had significantly greater anti-tumor activity than either drug alone (p < 0.0001), including 6 out of 10 tumors with greater than 50% reduction in tumor volume and 2 complete regressions (Figure 7A, diamonds). Similarly, in the NCI-N87 model the combination had greater anti-tumor activity than either trastuzumab (p < 0.0001) or MM- 302 alone (p=0.0002), including 2 out of 9 tumors with greater than 50% reduction in tumor volume (Figure 7B, diamonds). 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. In the case of the BT-474- 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 (Figures 7A and 7B).

Liposomes are significantly larger than both therapeutic antibodies such as trastuzumab as well as the target HER2 receptor. Based on fluorescence microscopy, both agents are present on the same BT-474-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, co-localization was readily apparent. The HER2-overexpressing cell line SUM- 190, which tends to grow as individual cells, was then evaluated. Co-localization of trastuzumab and MM-302 on single cells was readily observed by fluorescence microscopy. Images of binding of MM-302 and trastuzumab to the same tumor cells were captured in vivo by injecting fluorescent DH5-MM-302 and Alexa-555-trastuzumab into mice carrying BT- 474-M3 xenografts. By 4h, 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 co-localization is not absolute but is clearly evident in locations where both drugs are present.

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. Treatment of BT-474-M3 or NCI-N87 cells with trastuzumab reduced basal pAkt signaling in vitro and this inhibition was unchanged with co-administration of MM-302. The additive killing effect when both drugs are co-administered 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, it is possible that an even greater combinatorial benefit may be observed in the presence of a functional immune system.

Example 2: MM-302 Effectively Targets and Reduces Pulmonary Metastatic Burden in Breast Cancer Models Expressing Intermediate Levels of HER2

MM-302 is a HER2 -targeted antibody-liposomal doxorubicin conjugate designed to target doxorubicin to HER2- expressing cancer cells. The objective of this study was to compare the relative efficacy of MM-302 and its non-targeted counterpart, PEGylated liposomal doxorubicin (PLD), in treating HER2-intermediate MBC (corresponding to 1+/2+ by IHC) using models that closely mimic how HER2-overexpressing metastatic tumors are established in humans. MM-302 is currently being evaluated in HER2 -positive locally advanced breast cancer (LABC)/MBC patients in the registration-directed HERMIONE trial.

To establish metastatic disease, the murine 4T1-HER2 cell line engineered to express intermediate levels of HER2 (median of -lxl O⁵ HER2 receptors/cell), and the human MDA- MB-453 cells that endogenously express intermediate levels of HER2 (2+ by IHC and median of ~3xl 0⁵ HER2 receptors/cell), were inoculated orthotopically into the right and left mammary fat pads of immunocompromised mice. Study schema are found in Figure 8A (nude mice) and Figure 9A (BRG mice). Primary tumors then spontaneously seeded cancer cells in distant visceral organs such as the lung. When primary tumor volumes reached -150 mm³ (4T1 -HER2) or -270 mm³ (MDA-MB-453), mice were randomized and treated with vehicle control, PLD or MM-302 (Figures 8B and 9B) or free (unencapsulated) doxorubicin (Figure 8B). At the end of the study, primary tumors and lungs were harvested to assess liposome delivery and quantify pulmonary metastatic burden. In Figure 9C (top) is shown representative images of immunohistochemical human cytokeratin (CK) stain segmentation in fixed lung sections. The bottom panel is a graph showing quantification of human CK positive cells in lung sections indicating that the lung percent of area occupied by CK positive cells was approximately 10-fold lower in the MM-302-treated group than in the PLD group. P value corresponds to Student's t-test comparing metastatic burden in the PLD vs MM-302 -treated groups. Figure 9D is a graph with tumor growth curves indicating that treatment with either MM-302 or PLD significantly inhibited primary tumor growth. The data represent the mean tumor volume +/- SEM. Mice were treated on days indicated by triangles on the x-axis. Figure 9E shows the delivery and anti-tumor activity of MM-302 vs. PLD to HER-2+ tumors in vivo. Mice that received MM-302 had a 16x higher liposome delivery and a 12x higher anti -tumor activity as compared to mice that received PLD.

The study described below shows that MM-302 effectively targets and reduces pulmonary metastatic burden in breast cancer models expressing intermediate levels of HER2. MM-302 is more effective than PLD at reducing total pulmonary metastatic burden in both HER2 -intermediate models as evidenced by the lower number of surface metastases in the 4T1-HER2 model and the lower number of human cytokeratin positive cells per lung in the MDA-MB-453 model. MM-302 and PLD are equally effective at slowing (4T1-HER2) and inhibiting (MDA-MB-453) primary tumor growth.

4Tl-GFPluc2-HER2 cells were inoculated into the right and left #4 mammary fat pad

(mfp) of 6-week old nude mice that were then randomized and treated with vehicle control (PBS), 3 mg/kg non-targeted PEGylated liposomal doxorubicin (PLD) or 3 mg/kg MM-302 once weekly for 3 weeks starting at day 20. Thirty -nine days following inoculation, the mice were sacrificed and their lungs were harvested for enumeration of surface metastases. Figure 8B shows gross images of Bouin's solution fixed lungs illustrating surface metastases (white nodules). In Figure 8C is shown the total number of surface metastases per lung (left) and the lung weight (right) for each mouse in each group. The number of surface lung metastases was 2. lx lower in the MM-302 -treated mice than the PLD-treated mice while the lung weights were 1.3x lower. Tumor growth curves indicate that treatment with free doxorubicin had no effect on primary tumor growth while MM-302 and PLD both slowed tumor growth slightly and to the same degree (Figure 8D).

MDA-MB-453 cells were inoculated into the right and left #4 mammary fat pad (mfp) of 7 to 10-week CIEA BALB/c-Rag2null-IL2Rgnull (BRG) mice that were then randomized and treated with vehicle control (PBS), 6 mg/kg non-targeted PEGylated liposomal doxorubicin (PLD) or 6 mg/kg MM-302 once weekly for 3 weeks starting at day 79. One hundred days following inoculation, the mice were sacrificed and their lungs were harvested for quantification of lung metastases. B. (Left) Shown in Figure 9B (top) are gross images of Bouin's fixed lungs illustrating a blanket of surface micro metastases in control and PLD- treated mice but not in MM-302-treated mice; and on the bottom, a graph depicting the percentage of mice with surface micrometastases in each group. Representative images of immunohistochemical human cytokeratin (CK) stain segmentation in fixed lung sections are shown in Figure 9C. Quantification of human CK positive cells in lung sections indicating that the lung area (percent) occupied by CK positive cells was approximately 10-fold lower in the MM-302-treated group than in the PLD group. P value corresponds to Student's t-test comparing metastatic burden in the PLD vs MM-302 -treated groups. D. Tumor growth curves indicating that treatment with either MM-302 or PLD significantly inhibited primary tumor growth. The data represent the mean tumor volume +/- SEM. Mice were treated on days indicated by triangles on the x-axis.

Figure 10A shows the results of H&E stain of frozen lung, matching primary tumor and liver tissues harvested from a 4Tl-GFPluc2-HER2 tumor-bearing mouse 24h after injection of fluorescently-labeled MM-302 -DiI5. Figure 10B shows the results of fluorescent staining of serial sections, illustrating that in lungs harboring sheet-like metastases, liposome distribution is uniformly high throughout the entire lung, while in primary tumors, liposome distribution is low and limited to the periphery of the tumor. Figure IOC shows the results of H&E staining of lungs harboring nodular metastatic lesions (arrows). Figure 10D shows the results of fluorescent staining of serial sections illustrating that in lungs harboring large nodular metastatic lesions, liposome distribution is peripheral while in small lesions, it is uniformly high. Note that there is no measurable liposome accumulation in normal lung tissue. Liposomes are shown in cyan and nuclei in blue.

Figure 11 shows fluorescent images of primary tumors harvested from a 4T1- GFPluc2-HER2 tumor-bearing mouse 36h after co-injection of both PLD and MM-302, each fluorescently-labeled with a different fluorescent probe (DiR and DiI5, respectively). The images illustrate preferential accumulation of both PLD-DiR and MM-302-DiI5 on the periphery of the tumors, which is consistent with them being equally efficacious at slowing tumor growth.

Figure 12A shows H&E staining of frozen lungs harvested from 4Tl-GFPluc2-HER2 metastasis-bearing mice 24h after injection of fluorescently labeled PLD-DiI5 (left) or MM- 302-DiI5 (right). Figure 12B shows fluorescent staining of serial sections illustrating the distribution of fluorescently labeled liposomes (cyan) in lung metastases. Figure 12C shows quantification of mean liposome fluorescence intensity in lung metastases. The data represent the mean fluorescence intensity per pixel +/- SEM (p<0.05).

Figures 13A-13B depict the correlation between the receptor number of various cell lines and cell viability following treatment with MM-302. Figure 13A shows the results of an in vitro binding assay showing the binding of MM-302 and PLD to various cell lines. Cells were collected, pelleted at 2000 rpm and divided into four parts, each incubated (50 x 10⁶ cells/ml) for 1 h at 37°C under rotation with the either fluorescently labeled MM-302 (MM- 302-DiI5; 5μg/ml), PEGylated liposomal doxorubicin (PLD-DiI5; 5ug/ml), T-DM1 (T-DM1- DiI5; 27.8 μg/ml). At the end of the incubation, cells were pelleted at 2000 rpm, an aliquot of supernatant and cell pellet was collected to determine the unbound and bound drug fraction by means of DiI5 fluorescence measurement with an EnVision® Plate Reader. Figure 13B is an in vitro viability assay in the same cell lines. Here, cells were plated in 96-well plates at a density of 5,000 cells/well. The following day, media was replaced with fresh media alone (untreated), media containing MM-302 (0.08 to 10 ug/ml) or T-DMl (0.08 to 10 μg/ml). Each treatment was performed in duplicate. Plates were incubated at 37°C for 72 hours. Media was then removed, replaced with 200 μΐ/well of a 1 : 1 mixture of RPMI and Cell Titer- Glo reagent, followed by incubation at RT for 30 min. Luminescence was measured using an EnVision plate reader. Alternatively, cells were plated in 96-well plates at a density of 5,000 cells/well. The following day, media was replaced with fresh media alone (untreated), media containing MM-302 (5.8 x 10^Λ-5 to 5.8 ug/ml; dox equiv) or T-DMl (1 χ10^Λ-4 to 10 ug/ml). Plates were incubated at 37°C for 96 hours. Media was then removed, replaced with 200 μΐ/well of a 1 : 1 mixture of RPMI and Cell Titer-Glo reagent, followed by incubation at RT for 30 min. Luminescence was measured using an EnVision plate reader.

MM-302 induces tumor cell death across the entire range of HER2 expression T-DMl cell-killing activity is observed in 2+/3+, but not in 1+/2+ cell lines.

Example 3: MM-302 activity in HER2 1+ metastatic model

Mice were inoculated with 1 x 10⁶ T47D-luc cells by tail vein injection. At day 50 following injection, MM-302 or PLD were administered at a dosage of 5 mg/kg, or PBS control. 24 hours following the administration, organs were harvested and prepared into frozen sections for immunofluorescent staining with antibodies against HER2, PEG, and cytokeratin (CK). The organs collected and stained were lungs (shown stained in Figure 15 A), ovaries, kidneys/adrenal glands, liver, and brain. Image acquisition was then performed using Aperio FL at 20x. MM-302 demonstrated increased tumor cell uptake relative to PLD (Figure 15 A).

In a parallel experiment, the tail-vein injected mice were treated on day 50 following injection with 3 doses of MM-302, PLD, or free doxorubicin (5 mg/kg), 1 dose of T-DMl (10 mg/kg) or PBS control. On day 71, lungs, ovaries, kidney s/adrenal glands, liver, and brain were collected for quantification of metastases by bioluminescence (BLI, IVIS). MM-302 showed increased anti-tumor activity in ovarian, adrenal gland and liver metastases relative to both PLD and T-DMl (Figure 15B). Example 4: A PEG-HER2 assay that enables detection of HER2-mediated MM-302 internalization on clinical samples.

A PEG-HER2 assay was developed that enables detection of HER2-mediated MM- 302 internalization on clinical samples. (A pictographical representation of the assay is shown in Figure 14A. As shown in the Figure, the assay uses a HER2 standard cell pellet array (left side of figure), a PEG Standard Cell Array (right side of figure), and a

representative sample of the experimental tissue stained according to the methods of the assay (center). The arrays are used to determine a standard curve for HER2 or PEG staining in the tumor tissue from animals or patients. For the HER2 standard cell array, MDA-MB- 231, MCF7, T47D, DU145, IGROV1, MDA-MB-175-VII, MDA-MB-453, MDA-MB-361, MCF-7 clone 18 and SKOV3 were grown to -80% confluence (total of 2-4 x 10⁸ cells), rinsed with PBS and dissociated with Accutase™. Cells were then collected, pelleted at 2000 rpm and fixed with 10% neutral buffered formalin (NBF) for 4 h at 4°C. Following this, the cells were pelleted at 2000 rpm, washed with PBS, and packed by centrifugation at 2000 rpm. The cell pellets were resuspended with Histogel™ and transferred into a cloning ring until polymerization was accomplished. The resulting pellets were subsequently placed into cassettes, surrounded by OCT (Optimal Cutting Temperature) compound, and frozen in an isopentane-dry ice slurry. Frozen cell pellets were stored at -80°C until processing. For the MM-302 standard cell pellet array, BT474-M3 were grown to -80% confluence (total of 1.6 x 10⁹ cells), rinsed with PBS and dissociated with Accutase™. Cells were collected, pelleted at 2000 rpm and divided into 10 parts, each incubated (50 x 10⁶ cells/ml) for 1 h at 37°C under rotation with the following MM-302 concentrations: 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320 μg/ml. At the end of the incubation, cells were pelleted at 2000 rpm, an aliquot of supernatant and cell pellet was collected to determine the unbound and bound doxorubicin fraction of each reaction by HPLC. HPLC quantification of doxorubicin was performed as previously described (Reynolds JG, Toxicol Appl Pharmacol 2012, incorporated by reference herein in its entirety) and allowed to determine the bound ng dox/cell and the #

liposomes/cell for the individual reactions. To obtain the # liposomes/cell the following formula was used:

# liposomes/cell= (ng dox/10⁹)/580)/20000)*6.022* 10²³, where 580 is the molecular weight of doxorubicin, 20,000 is the number of doxorubicin molecules packed in each liposome, and 6.022* 10²³ is the Avogadro constant. The remaining cell pellet from each reaction mix was fixed with 10% NBF for 4 h at 4°C. Cells were pelleted at 2000 rpm, washed with PBS, and packed by centrifugation at 2000 rpm. The cell pellets were resuspended with Histogel and transferred into a cloning ring until polymerization was accomplished. The resulting pellets were subsequently placed into cassettes, surrounded by OCT compound and frozen in an isopentane-dry ice slurry. Frozen cell pellets were stored at -80°C until processing. For the generation of tumor xenografts, 7-week-old female NCR/nu, CIEA BRG nude mice, and CB 17.Cg-PrkdcscidLyst bg/Cr were used to establish tumor xenografts using BT474-M3, SUM190 and MDA-MB-361 cells as previously described (Geretti E Mol Cancer Ther. 2015, incorporated by reference herein in its entirety). MCF7 (7.5x 10⁶) tumor cells were inoculated into the right mammary fat pad #2 of NCR/nu mice. MDA-MB-231 (1 x 10⁶) into the right mammary fat pad #4 of CB 17.Cg-PrkdcscidLyst bg/Cr. IGROV1 (5xl0⁶) were inoculated subcutaneously into the right flank of CIEA BRG mice. When tumors reached a volume of about 300 mm³, mice were dosed with IV with fluorescently labeled MM-302 (DiI5-labeled) (3 mg/kg) or fluorescently labeled PLD (PEGylated liposomal doxorubicin) (3 mg/kg), or PBS as indicated. Mice were sacrificed 24 h or 72 h following liposome injection.

Immediately after sacrificing, mice were perfused with PBS to remove liposomes still in circulation. Tumors were collected immediately after sacrificing. One part of each tumor was fixed for 24 h at RT in 10% NBF, cryoprotected for 24 h at 4°C in 15% sucrose, and subsequently frozen after being embedded in OCT compound. Frozen blocks were stored at -80°C until further processing to cryosections (7-μιτι thick) (Figure 14B). Tumors were stained to detect HER2. PEG and cytokeratin and images acquired using an Aperio

ScanScope FL. To determine Γ-Η2ΑΧ (or cleaved caspase 3 or cleaved PARP)-PEG- cytokeratin stained images, tumors were stained with anti phospho-histone H2A.X, anti- human cleaved caspase 3, and mouse anti-human cytokeratin. Cell nuclei were detected using Hoescht® staining, followed by classification of the cells into tumor or stromal cells based on the cytokeratin signal. The tumor cells were then classified into γ-Η2ΑΧ (or cleaved caspase 3 or cleaved PARP) positive or negative based on the intensity of the selected marker. Co- staining with the MM-302 standard allowed to interpolate the # liposomes/cell from the PEG intensity values on the individual tumor cells (Figure 14C).

For human tissue analysis, frozen patient biopsies from MM-302 Phase I trial MM- 302-02-01 -01, Patients enrolled in trial NCT01304797 were dosed with MM-302 (8, 16, 30, 40 or 50 mg/m²). 72 h following the first received dose of MM-302, a biopsy was collected from the primary or metastatic lesion of the patients. The biopsy was frozen in liquid nitrogen and stored at -80°C until further processing to cryosections (7-μιτι thick). Tissues were stained to determine HER2 and PEG expression (Figure 14D, top) and one for a patient biopsy with HER2 IHC 1+ and no HER2 amplification (Figures 14 D and 14E). Higher amounts). Internalization of MM-302 was detected in tumor cells at various levels of HER2 expression (Figure 14E). Example 5: MM-302 patient biopsies from Phase 1 trial NCT01304797: HER2 expression and MM-302 uptake into tumor cells.

Frozen patient biopsies with tumor content > 5% were stained for HER2, PEG (surrogate for MM-302) and cytokeratin, followed by Image Analysis (Definiens Developer XD). The distribution of HER2 expression within the biopsies is shown in Figure 16B (color- coded by the HER2 IHC score determined on correspondent formalin-fixed, paraffin- embedded (FFPE) samples). Examples of staining on selected biopsies are shown in Figure 16C. Uptake of MM-302 was detected in tumor cells at various levels of HER2 expression, including in a HER2 1+, FISH NEG patient.

Two biopsies were taken from recurrent breast carcinoma or a metastatic site of each patient. Pass 1 : biopsies preserved as frozen; pass 2: biopsies preserved as formalin-fixed, paraffin-embedded (FFPE) samples. The table below provides the intended dose of MM-302 administered to each patient, as well as the dose of Trastuzumab and Cyclophosphamide administered to each patient, where applicable. Figure 16A provides the histopathology evaluation of the frozen biopsies of each patient in terms of the percentage of biopsied cells that were normal, necrotic, or tumor cells.

500-1031 50 mg/m2, q4w 1 5

100-1040 30 mg/m2, q4w 4 mg/kg, q2w 2 1

300-1037 30 mg/m2, q4w 4 mg/kg, q2w 2 1

500-1043 40 mg/ni2, q4w 4 mg/kg, q2w 2 2

500-1045 40 mg/ni2, q4w 4 mg/kg, q2w 2 2

200-1048 30 mg/m2, q3w 6 mg/kg, q3w 3 1

100-1063 30 mg/m2, q3w 6 mg/kg, q3w 3 2

100-1066 30 mg/m2, q3w 6 mg/kg, q3w 3 2

200-1053 30 mg/m2, q3w 6 mg/kg, q3w 3 2

200-1067 30 mg/m2, q3w 6 mg/kg, q3w 3 2

600-1050 30 mg/m2, q3w 6 mg/kg, q3w 3 2

600-1051 30 mg/m2, q3w 6 mg/kg, q3w 3 2

200-1062 30 mg/m2, q3w 6 mg/kg, q3w 450 mg/m2, q3w 4 1

200-1064 30 mg/m2, q3w 6 mg/kg, q3w 450 mg/m2, q3w 4 1

200-1073 30 mg/m2, q3w 6 mg/kg, q3w 450 mg/m2, q3w 4 1

300-1069 30 mg/m2, q3w 6 mg/kg, q3w 450 mg/m2, q3w 4 1

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features set forth herein. The disclosure of each and every U.S., international, or other patent or patent application or publication referred to herein is hereby incorporated herein by reference in its entirety.

Embodiments

1. A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising:

a. administering to the patient once on day 1 of a first 21-day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER2 -targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by b. administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER2 -targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate metastatic breast cancer in the patient.

2. The method of embodiment 1, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC.

3. The method of embodiment 2, wherein the HER2-intermediate metastatic breast cancer is FISH negative.

4. The method of any one of embodiments 1-3, wherein the HER2 -intermediate

metastatic breast cancer is characterized by a score of 2+ by IHC.

5. The method of any one of embodiments 1-4, wherein comprising treating the patient with subsequent treatment 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

6. A method of treating gastric cancer in a human patient having HER2-intermediate gastric cancer, the method comprising:

a. administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by

b. administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate gastric cancer in the patient.

7. The method of embodiment 1, wherein the HER2-intermediate gastric cancer is characterized by a score of 1+ or 2+ by IHC.

8. The method of embodiment 2, wherein the HER2-intermediate gastric cancer is FISH negative.

9. The method of any one of embodiments 1-3, wherein the HER2 -intermediate gastric cancer is characterized by a score of 2+ by IHC.

10. The method of any one of embodiments 1-4, wherein comprising treating the patient with subsequent treatment 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle. A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer and harboring a number of metastatic lesions, the method comprising:

a. administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by

b. administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate metastatic breast cancer in the patient.

The method of embodiment 11 , wherein the number of metastatic lesions is reduced following treatment.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising:

a. administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a HER2 -targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by

b. administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a HER2-targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER- 2 intermediate metastatic breast cancer in the patient.

The method of embodiment 13, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC.

The method of embodiment 14, wherein the HER2-intermediate metastatic breast cancer is FISH negative.

The method of any one of embodiments 13-15, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 13-16, wherein comprising treating the patient with subsequent treatment 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle. A method of treating gastric cancer in a human patient having HER2-intermediate gastric cancer, the method comprising:

a. administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by

b. administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER- 2 intermediate gastric cancer in the patient.

The method of embodiment 18, wherein the HER2-intermediate gastric cancer is characterized by a score of 1+ or 2+ by IHC.

The method of embodiment 19, wherein the HER2-intermediate gastric cancer is FISH negative.

The method of any one of embodiments 18-20, wherein the HER2 -intermediate gastric cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 18-21, wherein comprising treating the patient with subsequent treatment 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer and harboring a number of metastatic lesions, the method comprising:

a. administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by

b. administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER- 2 intermediate metastatic breast cancer in the patient.

The method of embodiment 23, wherein the number of metastatic lesions is reduced following treatment. The method of any one of embodiments 1-24, wherein the HER-2 targeted doxorubicin immunoliposome is a unilamellar lipid bilayer vesicle of approximately 75-110 nm in diameter that encapsulates an aqueous space which contains doxorubicin sulfate with an amount of doxorubicin equivalent to 2 mg/mL of doxorubicin HC1, the unilamellar lipid bilayer vesicle composed of

phosphatidylcholine, cholesterol, and a polyethyleneglycol-derivatized phosphatidyl- ethanolamine and encapsulates approximately 20,000 molecules of doxorubicin in its core and an average of 45 single chain anti-HER2 antibodies (scFv) of SEQ ID NO: 1 conjugated to its surface.

The method of embodiment 25, wherein the HER-2 targeted doxorubicin immunoliposome comprises one PEG molecule for 200 phospholipid molecules, of which approximately one PEG chain for each 1780 phospholipid molecules bears at its end a scFv antibody fragment of SEQ ID NO: 1 that binds to c-ErB-2 protein. The method of any one of embodiments 25-26, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10.2 mg/mL of HSPC, 3.39 mg/mL of cholesterol, 0.18 mg/mL of methoxy -terminated polyethylene glycol (MW2000)- (PEGDSPE), and PEG-DSPE-conjugated scFv F5 in the concentration of 0.2 mg/mL of antibody protein of SEQ ID NO: 1.

The method of any one of embodiments 25-27, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10 mM L-histidine-HCl buffer (pH 6.5), 100 mg/mL sucrose, ammonium sulfate in the concentration of less than 0.8 mg/mL, and sodium citrate in the concentration of less than 0.5 mg/mL.

The method of any one of embodiments 25-28, wherein the HER-2 targeted doxorubicin immunoliposome is administered from a 10 mL clear glass vial closed with crimped rubber caps at 10-mL (20 mg doxorubicin HC1) per vial.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a MM-302 HER2-targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer in the patient.

The method of embodiment 30, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC. The method of embodiment 31 , wherein the HER2-intermediate metastatic breast cancer is FISH negative.

The method of embodiment 31 , wherein the HER2-intermediate metastatic breast cancer is 1+ by IHC and is FISH positive.

The method of any one of embodiments 30-33, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 30-34, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

A method of treating gastric cancer in a human patient having HER2-intermediate gastric cancer, the method comprising: administering to the patient once on day 1 of a first 21-day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin

immunoliposome to treat the HER-2 intermediate gastric cancer in the patient.

The method of embodiment 36, wherein the HER2-intermediate gastric cancer is characterized by a score of 1+ or 2+ by IHC.

The method of embodiment 37, wherein the HER2-intermediate gastric cancer is FISH negative.

The method of embodiment 38, wherein the HER2-intermediate gastric cancer is 1+ by IHC and is FISH positive.

The method of any one of embodiments 36-39, wherein the HER2 -intermediate gastric cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 30-41, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer and harboring a number of metastatic lesions, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer harboring a number of metastatic lesions in the patient.

The method of embodiment 42, wherein the number of metastatic lesions is reduced following treatment. A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a HER2-targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer in the patient.

The method of embodiment 44, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC.

The method of embodiment 44, wherein the HER2-intermediate metastatic breast cancer is FISH negative.

The method of embodiment 44, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ by IHC and is FISH positive.

The method of any one of embodiments 44-47, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 44-48, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

A method of treating gastric cancer in a human patient having HER2-intermediate gastric cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a HER-2 targeted doxorubicin immunoliposome to treat the HER-2 intermediate gastric cancer in the patient.

The method of embodiment 50, wherein the HER2-intermediate gastric cancer is characterized by a score of 1+ or 2+ by IHC.

The method of embodiment 50, wherein the HER2-intermediate gastric cancer is FISH negative.

The method of embodiment 50, wherein the HER2-intermediate gastric cancer is characterized by a score of 1+ by IHC and is FISH positive.

The method of any one of embodiments 50-53, wherein the HER2 -intermediate gastric cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 50-54, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle. A method of treating gastric cancer in a human patient having HER2-intermediate gastric cancer and harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a HER-2 targeted doxorubicin immunoliposome to treat the HER-2 intermediate gastric cancer and metastatic lesions in the patient.

The method of embodiment 56, wherein the number of metastatic lesions is reduced following treatment.

The method of embodiment 56, wherein the HER2-intermediate gastric cancer is characterized by a score of 1+ or 2+ by IHC.

The method of any one of embodiments 56-58, wherein the HER2 -intermediate gastric cancer is FISH negative.

The method of embodiment 56, wherein the HER2-intermediate gastric cancer is characterized by a score of 1+ by IHC and is FISH positive.

The method of any one of embodiments 56-60, wherein the HER2 -intermediate gastric cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 56-61, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

The method of any one of embodiments 30-62, wherein the HER-2 targeted doxorubicin immunoliposome is a unilamellar lipid bilayer vesicle of approximately 75-110 nm in diameter that encapsulates an aqueous space which contains doxorubicin sulfate with an amount of doxorubicin equivalent to 2 mg/mL of doxorubicin HC1, the unilamellar lipid bilayer vesicle composed of

phosphatidylcholine, cholesterol, and a polyethyleneglycol-derivatized phosphatidyl- ethanolamine and encapsulates approximately 20,000 molecules of doxorubicin in its core and an average of 45 single chain anti-HER2 antibodies (scFv) of SEQ ID NO: 1 conjugated to its surface.

The method of embodiment 63, wherein the HER-2 targeted doxorubicin

immunoliposome comprises one PEG molecule for 200 phospholipid molecules, of which approximately one PEG chain for each 1780 phospholipid molecules bears at its end a scFv antibody fragment of SEQ ID NO: 1 that binds to c-ErB-2 protein. The method of any one of embodiments 63-64, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10.2 mg/mL of HSPC, 3.39 mg/mL of cholesterol, 3.39 mg/mL of methoxy-terminated polyethylene glycol (MW 2000)- distearoylphosphatidylethanolamine (PEG-DSPE), and PEG-DSPE-conjugated scFv F5 in the concentration of 0.2 mg/mL of antibody protein of SEQ ID NO: 1.

The method of any one of embodiments 63-65, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10 mM L-histidine-HCl buffer (pH 6.5), 100 mg/mL sucrose, ammonium sulfate in the concentration of less than 0.8 mg/mL, and sodium citrate in the concentration of less than 0.5 mg/mL.

The method of any one of embodiments 63-66, wherein the HER-2 targeted doxorubicin immunoliposome is administered from a 10 mL clear glass vial closed with crimped rubber caps at 10-mL (20 mg doxorubicin HC1) per vial.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a MM-302 HER2-targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer in the patient.

The method of embodiment 68, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC.

The method of embodiment 69, wherein the HER2-intermediate metastatic breast cancer is FISH negative.

The method of embodiment 69, wherein the HER2-intermediate metastatic breast cancer is 1+ by IHC and is FISH negative.

The method of any one of embodiments 68-71, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 68-72, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer and harboring a number of metastatic lesions, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer harboring a number of metastatic lesions in the patient. The method of embodiment 74, wherein the number of metastatic lesions is reduced following treatment.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a HER2-targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer in the patient.

The method of any one of embodiments 74-76, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC.

The method of any one of embodiments 74-77, wherein the HER2 -intermediate metastatic breast cancer is FISH negative.

The method of any one of embodiments 74-77, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 1+ by IHC and is FISH negative.

The method of any one of embodiments 74-77, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 74-80, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

The method of any one of embodiments 68-81, wherein the HER-2 targeted doxorubicin immunoliposome is a unilamellar lipid bilayer vesicle of approximately 75-110 nm in diameter that encapsulates an aqueous space which contains doxorubicin sulfate with an amount of doxorubicin equivalent to 2 mg/mL of doxorubicin HC1, the unilamellar lipid bilayer vesicle composed of

phosphatidylcholine, cholesterol, and a polyethyleneglycol-derivatized phosphatidyl- ethanolamine and encapsulates approximately 20,000 molecules of doxorubicin in its core and an average of 45 single chain anti-HER2 antibodies (scFv) of SEQ ID NO: 1 conjugated to its surface.

The method of embodiment 82, wherein the HER-2 targeted doxorubicin

immunoliposome comprises one PEG molecule for 200 phospholipid molecules, of which approximately one PEG chain for each 1780 phospholipid molecules bears at its end a scFv antibody fragment of SEQ ID NO: 1 that binds to c-ErB-2 protein. The method of any one of embodiments 82-83, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10.2 mg/mL of HSPC, 3.39 mg/mL of cholesterol, 3.39 mg/mL of methoxy-terminated polyethylene glycol (MW 2000)- distearoylphosphatidylethanolamine (PEG-DSPE), and PEG-DSPE-conjugated scFv F5 in the concentration of 0.2 mg/mL of antibody protein of SEQ ID NO: 1.

The method of any one of embodiments 82-84, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10 mM L-histidine-HCl buffer (pH 6.5), 100 mg/mL sucrose, ammonium sulfate in the concentration of less than 0.8 mg/mL, and sodium citrate in the concentration of less than 0.5 mg/mL.

The method of any one of embodiments 82-85, wherein the HER-2 targeted doxorubicin immunoliposome is administered from a 10 mL clear glass vial closed with crimped rubber caps at 10-mL (20 mg doxorubicin HC1) per vial.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a MM-302 HER2-targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer in the patient.

The method of embodiment 87, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC.

The method of embodiment 88, wherein the HER2-intermediate metastatic breast cancer is FISH negative.

The method of embodiment 88, wherein the HER2-intermediate metastatic breast cancer is 1+ by IHC and is FISH negative.

The method of any one of embodiments 87-90, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 2+ by IHC.

The method of any one of embodiments 87-91, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer and harboring a number of metastatic lesions, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer harboring a number of metastatic lesions in the patient.

The method of embodiment 93, wherein the number of metastatic lesions is reduced following treatment.

A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a HER2-targeted doxorubicin

immunoliposome to treat the HER-2 intermediate metastatic breast cancer in the patient.

The method of any one of embodiments 93-95-9, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC.

The method of any one of embodiments 93-96, wherein the HER2 -intermediate metastatic breast cancer is FISH negative.

The method of any one of embodiments 93-96, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 1+ by IHC and is FISH negative.

The method of any one of embodiments 93-96, wherein the HER2 -intermediate metastatic breast cancer is characterized by a score of 2+ by IHC.

. The method of any one of embodiments 93-99, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

. The method of any one of embodiments 87-100, wherein the HER-2 targeted doxorubicin immunoliposome is a unilamellar lipid bilayer vesicle of approximately 75-110 nm in diameter that encapsulates an aqueous space which contains doxorubicin sulfate with an amount of doxorubicin equivalent to 2 mg/mL of doxorubicin HC1, the unilamellar lipid bilayer vesicle composed of

phosphatidylcholine, cholesterol, and a polyethyleneglycol-derivatized phosphatidyl- ethanolamine and encapsulates approximately 20,000 molecules of doxorubicin in its core and an average of 45 single chain anti-HER2 antibodies (scFv) of SEQ ID NO: 1 conjugated to its surface.

. The method of embodiment 101 , wherein the HER-2 targeted doxorubicin immunoliposome comprises one PEG molecule for 200 phospholipid molecules, of which approximately one PEG chain for each 1780 phospholipid molecules bears at its end a scFv antibody fragment of SEQ ID NO: 1 that binds to c-ErB-2 protein.. The method of any one of embodiments 101-102, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10.2 mg/mL of HSPC, 3.39 mg/mL of cholesterol, 3.39 mg/mL of methoxy-terminated polyethylene glycol (MW 2000)- distearoylphosphatidylethanolamine (PEG-DSPE), and PEG-DSPE-conjugated scFv F5 in the concentration of 0.2 mg/mL of antibody protein of SEQ ID NO: 1.

. The method of any one of embodiments 101-102, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10 mM L-histidine-HCl buffer (pH 6.5), 100 mg/mL sucrose, ammonium sulfate in the concentration of less than 0.8 mg/mL, and sodium citrate in the concentration of less than 0.5 mg/mL.

. The method of any one of embodiments 101-104, wherein the HER-2 targeted doxorubicin immunoliposome is administered from a 10 mL clear glass vial closed with crimped rubber caps at 10-mL (20 mg doxorubicin HC1) per vial.

Claims

CLAIMS We claim:

1. A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER2 -targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer in the patient.

2. The method of claim 1, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 1+ or 2+ by IHC.

3. The method of claim 2, wherein the HER2-intermediate metastatic breast cancer is FISH negative.

4. The method of claim 2, wherein the HER2-intermediate metastatic breast cancer is 1+ by IHC and is FISH negative.

5. The method of any one of claims 1-3, wherein the HER2-intermediate metastatic breast cancer is characterized by a score of 2+ by IHC.

6. The method of any one of claims 1-5, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

7. A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer and harboring a number of metastatic lesions, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer harboring a number of metastatic lesions in the patient.

8. The method of claim 7, wherein the number of metastatic lesions is reduced following treatment.

9. A method of treating breast cancer in a human patient having HER2 -intermediate breast cancer, HER2-intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions, the method comprising:

a. administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m² of doxorubicin HC1 encapsulated in a MM-302 HER2 -targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by

b. administering to the patient on day 1 of a subsequent 21 -day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m² of doxorubicin HCl encapsulated in a MM-302 HER2 -targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate metastatic breast cancer in the patient.

10. The method of any one of claims 7-9, wherein the HER2-intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions is characterized by a score of 1+ or 2+ by IHC.

11. The method of any one of claims 7-10, wherein the HER2-intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions is FISH negative.

12. The method of any one of claims 7-10, wherein the HER2-intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions is characterized by a score of 1+ by IHC and is FISH negative.

13. The method of any one of claims 7-10, wherein the HER2-intermediate breast cancer, HER2 -intermediate metastatic breast cancer, or the HER2 -intermediate metastatic breast cancer harboring a number of metastatic lesions is characterized by a score of 2+ by IHC.

14. The method of any one of claims 7-13, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

15. The method of any one of claims 1-14, wherein the HER-2 targeted doxorubicin immunoliposome is a unilamellar lipid bilayer vesicle of approximately 75-110 nm in diameter that encapsulates an aqueous space which contains doxorubicin sulfate with an amount of doxorubicin equivalent to 2 mg/mL of doxorubicin HCl, the unilamellar lipid bilayer vesicle composed of phosphatidylcholine, cholesterol, and a

polyethyleneglycol-derivatized phosphatidyl-ethanolamine and encapsulates approximately 20,000 molecules of doxorubicin in its core and an average of 45 single chain anti-HER2 antibodies (scFv) of SEQ ID NO: l conjugated to its surface.

16. The method of claim 15, wherein the HER-2 targeted doxorubicin immunoliposome comprises one PEG molecule for 200 phospholipid molecules, of which

approximately one PEG chain for each 1780 phospholipid molecules bears at its end a scFv antibody fragment of SEQ ID NO: 1 that binds to c-ErB-2 protein.

17. The method of any one of claims 15-16, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10.2 mg/mL of HSPC, 3.39 mg/mL of cholesterol, 3.39 mg/mL of methoxy-terminated polyethylene glycol (MW 2000)- distearoylphosphatidylethanolamine (PEG-DSPE), and PEG-DSPE-conjugated scFv F5 in the concentration of 0.2 mg/mL of antibody protein of SEQ ID NO: 1.

18. The method of any one of claims 15-17, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10 mM L-histidine-HCl buffer (pH 6.5), 100 mg/mL sucrose, ammonium sulfate in the concentration of less than 0.8 mg/mL, and sodium citrate in the concentration of less than 0.5 mg/mL.

19. The method of any one of claims 15-18, wherein the HER-2 targeted doxorubicin immunoliposome is administered from a 10 mL clear glass vial closed with crimped rubber caps at 10-mL (20 mg doxorubicin HC1) per vial.

20. A method of treating gastric cancer in a human patient having HER2-intermediate gastric cancer, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HC1 encapsulated in a MM-302 HER-2 targeted doxorubicin

immunoliposome to treat the HER-2 intermediate gastric cancer in the patient.

21. The method of claim 20, wherein the HER2 -intermediate gastric cancer is

characterized by a score of 1+ or 2+ by IHC.

22. The method of claim 21 , wherein the HER2 -intermediate gastric cancer is FISH

negative.

23. The method of claim 21 , wherein the HER2 -intermediate gastric cancer is 1+ by IHC and is FISH positive.

24. The method of any one of claims 21 -23, wherein the HER2 -intermediate gastric cancer is characterized by a score of 2+ by IHC.

25. The method of any one of claims 20-24, wherein the method further comprises

treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

26. A method of treating breast cancer in a human patient having HER2 -intermediate metastatic breast cancer and harboring a number of metastatic lesions, the method comprising: administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome to treat the HER-2 intermediate metastatic breast cancer harboring a number of metastatic lesions in the patient.

27. The method of claim 26, wherein the number of metastatic lesions is reduced

following treatment.

28. A method for treating gastric cancer in a human patient having HER2-intermediate gastric cancer, HER2 -intermediate metastatic gastric cancer, or the HER2- intermediate metastatic gastric cancer harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome for one or more treatment cycles. In another aspect is provided a method of treating gastric cancer in a human patient having HER2 -intermediate gastric cancer, HER2- intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions, the method comprising

administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 8 mg/kg trastuzumab; followed by administering to the patient on day 1 of a subsequent 21- day treatment cycle, following the first treatment cycle, an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HCl encapsulated in a MM-302 HER-2 targeted doxorubicin immunoliposome in combination with 6 mg/kg trastuzumab, to treat the HER-2 intermediate gastric cancer in the patient.

29. The method of claim 28, wherein the HER2 -intermediate gastric cancer, HER2- intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions is characterized by a score of 1+ or 2+ by IHC.

30. The method of claim 28, wherein the HER2 -intermediate gastric cancer, HER2- intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions is FISH negative.

31. The method of claim 28, wherein the HER2 -intermediate gastric cancer, HER2- intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions is characterized by a score of 1+ by IHC and is FISH positive.

The method of any one of claims 28-31, wherein the HER2 -intermediate gastric cancer, HER2 -intermediate metastatic gastric cancer, or the HER2-intermediate metastatic gastric cancer harboring a number of metastatic lesions is characterized by a score of 2+ by IHC.

The method of any one of claims 28-32, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

A method of treating gastric cancer in a human patient having HER2-intermediate gastric cancer and harboring a number of metastatic lesions, the method comprising administering to the patient once on day 1 of a first 21 -day treatment cycle: an amount of doxorubicin contained in 30 mg/m2 of doxorubicin HCl encapsulated in a HER-2 targeted doxorubicin immunoliposome to treat the HER-2 intermediate gastric cancer and metastatic lesions in the patient.

The method of claim 34, wherein the number of metastatic lesions is reduced following treatment.

The method of claim 34, wherein the HER2 -intermediate gastric cancer is characterized by a score of 1+ or 2+ by IHC.

The method of any one of claims 34-36, wherein the HER2 -intermediate gastric cancer is FISH negative.

The method of claim 34, wherein the HER2 -intermediate gastric cancer is characterized by a score of 1+ by IHC and is FISH positive.

The method of any one of claims 34-38, wherein the HER2 -intermediate gastric cancer is characterized by a score of 2+ by IHC.

The method of any one of claims 34-39, wherein the method further comprises treating the patient with subsequent 21 -day treatment cycles for a total of up to about 6 months from day 1 of the first treatment cycle.

The method of any one of claims 20-40, wherein the HER-2 targeted doxorubicin immunoliposome is a unilamellar lipid bilayer vesicle of approximately 75-110 nm in diameter that encapsulates an aqueous space which contains doxorubicin sulfate with an amount of doxorubicin equivalent to 2 mg/mL of doxorubicin HCl, the unilamellar lipid bilayer vesicle composed of phosphatidylcholine, cholesterol, and a

polyethyleneglycol-derivatized phosphatidyl-ethanolamine and encapsulates approximately 20,000 molecules of doxorubicin in its core and an average of 45 single chain anti-HER2 antibodies (scFv) of SEQ ID NO: l conjugated to its surface.

42. The method of claim 41 , wherein the HER-2 targeted doxorubicin immunoliposome comprises one PEG molecule for 200 phospholipid molecules, of which

approximately one PEG chain for each 1780 phospholipid molecules bears at its end a scFv antibody fragment of SEQ ID NO: 1 that binds to c-ErB-2 protein.

43. The method of any one of claims 41 -42, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10.2 mg/mL of HSPC, 3.39 mg/mL of cholesterol, 3.39 mg/mL of methoxy-terminated polyethylene glycol (MW 2000)- distearoylphosphatidylethanolamine (PEG-DSPE), and PEG-DSPE-conjugated scFv F5 in the concentration of 0.2 mg/mL of antibody protein of SEQ ID NO: 1.

44. The method of any one of claims 41 -43, wherein the HER-2 targeted doxorubicin immunoliposome comprises 10 mM L-histidine-HCl buffer (pH 6.5), 100 mg/mL sucrose, ammonium sulfate in the concentration of less than 0.8 mg/mL, and sodium citrate in the concentration of less than 0.5 mg/mL.

45. The method of any one of claims 41 -44, wherein the HER-2 targeted doxorubicin immunoliposome is administered from a 10 mL clear glass vial closed with crimped rubber caps at 10-mL (20 mg doxorubicin HC1) per vial.