HER2-targeted Liposomal Doxorubicin Research Articles

Abstract OT3-01-01: HERMIONE: A phase 2, randomized, open label trial comparing MM-302 plus trastuzumab with chemotherapy of physician's choice plus trastuzumab, in anthracycline naive HER2-positive, locally advanced/metastatic breast cancer patients previously treated with pertuzumab and ado-trastuzumab emtansine (T-DM1)

Abstract Background: Although HER2-targeted therapies such as pertuzumab and T-DM1 have improved patient outcomes, treatment resistance typically occurs. MM-302 is a HER2-targeted liposomal doxorubicin in development by Merrimack Pharmaceuticals. In a Phase 1 study, patients with HER2-positive metastatic breast cancer (MBC) were treated with MM-302 alone and in combination with trastuzumab with or without cyclophosphamide. MM-302 had an acceptable safety profile and promising efficacy was observed in patients not previously exposed to an anthracycline. Trial design: HERMIONE (NCT02213744) is a randomized Phase 2, two-arm, open-label trial designed to evaluate if MM-302 can address an unmet medical need in patients with anthracycline naïve, trastuzumab-, pertuzumab- and T-DM1-pretreated HER2-positive locally advanced breast cancer (LABC)/MBC. Patients are randomized 1:1 to receive MM-302 (30mg/m2, Q3W) plus trastuzumab (6mg/kg, Q3W) or chemotherapy of physician's choice (vinorelbine, capecitabine, or gemcitabine) plus trastuzumab (6mg/kg, Q3W). Eligibility criteria: Centrally confirmed HER2-positive LABC/MBC, no prior anthracycline exposure, prior trastuzumab in any setting, prior pertuzumab and T-DM1 in the LABC/MBC setting, unlimited prior lines of therapy, ECOG 0-1 and LVEF ≥50%. CNS metastases are permitted if stable and without symptoms or steroids for 4 weeks. Specific aims: The primary endpoint is independently assessed progression free survival (PFS). Secondary endpoints include investigator assessed PFS, overall survival, response rate, safety and patient related outcomes. Statistics: 250 patients will be enrolled to observe 191 PFS events for 90% power to detect a Hazard Ratio of 0.625. The MM-302 arm will be compared to the control arm on the primary endpoint of PFS using a stratified log-rank test at one-sided 0.025 level. Accrual status: First patient in was December 2014 and enrollment is expected to be complete in late 2016. Sites are open in the US, Canada and Western Europe and are currently enrolling patients. Citation Format: Miller K, Cortes J, Hurvitz SA, Krop IE, Tripathy D, Verma S, Riahi K, Reynolds JG, Wickham T, Molnar I, Yardley DA. HERMIONE: A phase 2, randomized, open label trial comparing MM-302 plus trastuzumab with chemotherapy of physician's choice plus trastuzumab, in anthracycline naive HER2-positive, locally advanced/metastatic breast cancer patients previously treated with pertuzumab and ado-trastuzumab emtansine (T-DM1). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT3-01-01.

Cyclophosphamide-Mediated Tumor Priming for Enhanced Delivery and Antitumor Activity of HER2-Targeted Liposomal Doxorubicin (MM-302).

Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that effective tumor delivery and penetration are critical barriers to their clinical activity. HER2-targeted PEGylated liposomal doxorubicin (MM-302, HER2-tPLD) is an antibody-liposomal drug conjugate designed to deliver doxorubicin to HER2-overexpressing cancer cells while limiting uptake into nontarget cells. In this work, we demonstrate that the administration and appropriate dose sequencing of cyclophosphamide can improve subsequent MM-302 delivery and enhance antitumor activity in preclinical models without negatively affecting nontarget tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Furthermore, the effect was found to be unique to cyclophosphamide and related analogues, and not shared by other agents, such as taxanes or eribulin, under the conditions examined. Analysis of the cyclophosphamide-treated tumors suggests that the mechanism for improved MM-302 delivery involves the induction of tumor cell apoptosis, reduction of overall tumor cell density, substantial lowering of interstitial fluid pressure, and increasing vascular perfusion. The novel dosing strategy for cyclophosphamide described herein is readily translatable to standard clinical regimens, represents a potentially significant advance in addressing the drug delivery challenge, and may have broad applicability for nanomedicines. This work formed the basis for clinical evaluation of cyclophosphamide for improving liposome deposition as part of an ongoing phase I clinical trial of MM-302 in HER2-positive metastatic breast cancer.

Abstract CT234: A phase I study of MM-302, a HER2-targeted PEGylated liposomal doxorubicin, in patients with HER2+ metastatic breast cancer

Abstract Background: MM-302 is an antibody drug conjugated HER2-targeted liposomal doxorubicin in development by Merrimack Pharmaceuticals. MM-302 is designed to deliver doxorubicin to HER2-overexpressing cancer cells with minimal exposure to healthy cardiomyocytes. Results of the MM-302 phase I study as a monotherapy, in combination with trastuzumab and trastuzumab plus cyclophosphamide, are presented. Methods: 69 patients with HER2-positive metastatic breast cancer (mBC) were treated with either MM-302 alone (8, 16, 30, 40 and 50 mg/m2, Q4W) (Arm 1), MM-302 (30 and 40 mg/m2, Q4W) plus trastuzumab (4 mg/kg, Q2W) (Arm 2), MM-302 (30 mg/m2, Q3W) plus trastuzumab (6 mg/kg, Q3W) (Arm 3), MM-302 (30 mg/m2, Q3W) plus trastuzumab (6 mg/kg, Q3W) and cyclophosphamide (450 mg/m2, Q3W) (Arm 4). Patients on Arms 3 and 4 received a single 3-7 mg/m2 (approximately 10.8 mCi) dose of 64Cu-MM-302 followed by PET/CT to assess for MM-302 tumor deposition. Results: Patients received a median of 4 prior regimens for mBC. The most common Grade 3/4 side effect was neutropenia observed in 8 patients with 1 patient experiencing febrile neutropenia. Adverse events of any grade occurring in >20% of the population were constipation, cough, decreased appetite, diarrhea, dyspnea, fatigue, nausea, neutropenia, stomatitis and vomiting. Alopecia and hand foot syndrome were observed in 10% and 4% of patients respectively. 1 patient experienced a dose limiting toxicity DLT (febrile neutropenia) and a MTD was not reached at 50 mg/m2. 11 patients received cumulative anthracycline (previous exposure plus MM-302) exposure >550 mg/m2. LVEF reductions below 50% or a >10 percentage point drop in LVEF from baseline occurred in 6 patients. 1 patient experienced Grade 1 cardiac failure resulting in treatment discontinuation. In patients treated with ≥30 mg/m2 MM-302 (n = 49), alone or in combination with trastuzumab, the response rate (RR) was 12% and median progression free survival (mPFS) was 7.6 months (95% CI: 3.6-11.0). mPFS was 10.6 months (95% CI: 1.8-10.6) in the 13 patients receiving MM-302 plus trastuzumab and cyclophosphamide in Arm 4. Conclusions: MM-302 had a manageable safety profile in this study as a monotherapy, in combination with trastuzumab and with trastuzumab and cyclophosphamide. RR and mPFS in this heavily pretreated mBC population suggest further study is warranted. MM-302 at a dose of 30 mg/m2 Q3W in combination with trastuzumab is currently being evaluated in a randomized phase II trial (HERMIONE) in anthracycline naïve HER2-positive locally advanced/mBC patients previously treated with trastuzumab, pertuzumab and T-DM1. Citation Format: Patricia LoRusso, Ian Krop, Kathy Miller, Cynthia Ma, Barry A. Siegel, Anthony F. Shields, Istvan Molnar, Thomas Wickham, Joseph Reynolds, Karen Campbell, Bart Hendriks, Ty McClure, Victor Moyo, Pamela Munster. A phase I study of MM-302, a HER2-targeted PEGylated liposomal doxorubicin, in patients with HER2+ metastatic breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT234. doi:10.1158/1538-7445.AM2015-CT234

Abstract P4-15-04: Effect of pre-treatment with cyclophosphamide on MM-302 (HER2-targeted liposomal doxorubicin) deposition in HER2-positive metastatic breast cancer patients assessed by 64Cu-MM-302 PET/CT

Abstract Background: Liposomal encapsulation of doxorubicin has addressed the cardiotoxicity of free doxorubicin, but has not achieved an improvement in anti-tumor activity in metastatic breast cancer. MM-302 is a HER2-targeted liposomal doxorubicin, specifically designed to target tumor cells overexpressing HER2 and minimize uptake into normal cells such as cardiomyocytes, which express low levels of HER2. MM-302 and MM-302 plus trastuzumab are being studied in patients as part of an ongoing Phase 1 clinical trial. Published reports indicate that deposition into solid tumors is a rate-limiting step in liposome-mediated delivery of drug to tumor cells. In order to understand drug deposition into solid tumors, we have radiolabeled MM-302 with 64Cu for imaging by PET/CT. Further, preclinical work has demonstrated that pretreatment with cyclophosphamide has the ability to improve liposomal drug delivery by making the tumor microenvironment permissive for deposition and retention of liposomes. This Phase 1 study evaluates the delivery of 64Cu-MM-302 to solid tumors and the ability of cyclophosphamide pretreatment to increase delivery of 64Cu-MM-302 to tumors. Methods: Patients aged ≥ 18 years with histologically confirmed HER2-positive advanced breast cancer that has progressed or recurred on standard therapy or for which no standard therapy exists, who have adequate performance status, bone marrow reserve and organ function, were eligible for the study. Patients received 30 mg/m2 of MM-302 plus 6 mg/kg trastuzumab, q3w and 400 MBq (10.8 mCi; 3-5 mg/m2, doxorubicin basis) of 64Cu-MM-302 (cycle 1 only) with or without pretreatment of 450 mg/m2 cyclophosphamide. Patients underwent PET/CT on the day of administration and on day 2, day 3 or both. The primary goal of this analysis was to study delivery of 64Cu-MM-302 and the effect of cyclophosphamide pretreatment. Other endpoints being studied include safety, dosimetry, and treatment response. Results: Combination of MM-302 with cyclophosphamide was well tolerated and no issues were reported related to 64Cu-MM-302 administration or imaging. Uptake of 64Cu-MM-302 in tumor lesions increased over time with significant deposition at 24 and 48 h while activity in the blood decreased over time. Median lesion deposition of 64Cu-MM-302 (as % i.d./kg) in the patients with cyclophosphamide pretreatment was higher than in those without pretreatment: 6.0 (n=5 patients/20 lesions) vs. 4.4 (n=7 patients/36 lesions) at 24 h and 7.6 (n=4 patients/16 lesions) vs. 4.0 (n=4 patients/17 lesions) at 48 h. Conclusions: PET/CT imaged 64Cu-MM-302 deposition into diverse tumor lesions, including liver, brain and bone metastases. Our preliminary data thus far suggest that cyclophosphamide pretreatment increases delivery of 64Cu-MM-302 to patient tumors, as predicted by preclinical models. Correlation between 64Cu-MM-302 tumor deposition and lesion/patient response is currently being investigated. Citation Format: Kathy Miller, Patricia M LoRusso, Pamela Munster, Ian Krop, Cynthia Ma, Helen Lee, Joe Reynolds, Karen Campbell, Victor Moyo, Bart Hendriks, Thomas Wickham, Barry A Siegel, Anthony F Shields. Effect of pre-treatment with cyclophosphamide on MM-302 (HER2-targeted liposomal doxorubicin) deposition in HER2-positive metastatic breast cancer patients assessed by 64Cu-MM-302 PET/CT [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-04.

Abstract P6-11-05: Tumor priming with cyclophosphamide for enhanced tumor delivery, penetration and anti-tumor activity of MM-302, HER2-targeted liposomal doxorubicin

Abstract MM-302, HER2-targeted PEGylated liposomal doxorubicin, is a liposomal antibody drug conjugate designed to target doxorubicin to HER2-overexpressing cancer cells, while limiting uptake into non-target cells. Effective tumor delivery and penetration are critical barriers to the clinical activity of nanomedicines, including liposomes. Cyclophosphamide has been successfully combined with both doxorubicin and liposomal doxorubicin in breast cancer therapy, with the two drugs being administered on the same day to patients. We have evaluated a novel sequential combination regimen of cyclophosphamide with MM-302 with the goal of improving tumor delivery and penetration of MM-302. Methods: Biodistribution studies were carried out in multiple tumor xenograft models to assess the delivery of MM-302 and free doxorubicin, either as single agents or in combination with cyclophosphamide at different dosing schedules. The total doxorubicin within tumors was quantified by HPLC and microscopically by determining the number of doxorubicin-positive nuclei within frozen tumor sections. Induction of DNA damage/repair, tumor cell apoptosis, and changes in the tumor architecture in response to drug treatment (tumor cell density and vascular parameters) were quantified by automated image analysis. Interstitial fluid pressure measurements were carried out to evaluate changes in the tumor physiology upon cyclophosphamide treatment. Anti-tumor activity studies in BT474-M3 tumor-bearing mice were performed to evaluate the ability of the different dosing regimens to inhibit tumor growth. Results: Pre-dosing of tumors with cyclophosphamide enhanced subsequent MM-302 delivery to tumor xenografts (2-3-fold) without affecting delivery to non-target tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Analysis of cyclophosphamide-treated tumors suggests that the mechanism for improved MM-302 delivery involves the induction of tumor cell apoptosis, reduction of overall tumor cell density, substantial lowering of interstitial fluid pressure and increase in vascular perfusion. Finally, treatment of tumors xenografts with cyclophosphamide followed by MM-302 resulted in a significantly greater tumor growth inhibition compared to either single agent alone. Conclusions: Rational combination of MM-302 with cyclophosphamide results in an active and tolerable regimen in preclinical models that enhances the tumor delivery and activity of MM-302, without affecting doxorubicin exposure to non-target organs. This novel sequential dosing strategy represents an advance in addressing the critical challenge for tumor delivery of nanomedicines. This work provided data supporting the initiation of a clinical evaluation of the effect of cyclophosphamide on MM-302 delivery as part of an on-going Phase 1 clinical trial of MM-302 in HER2-positive metastatic breast cancer (http://clinicaltrials.gov/show/NCT01304797). Citation Format: Elena Geretti, Shannon C Leonard, Nancy Dumont, Christopher W Espelin, Daniel F Gaddy, Thomas J Wickham, Bart S Hendriks. Tumor priming with cyclophosphamide for enhanced tumor delivery, penetration and anti-tumor activity of MM-302, HER2-targeted liposomal doxorubicin [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-11-05.

Whole-body organ-level and kidney micro-dosimetric evaluations of (64)Cu-loaded HER2/ErbB2-targeted liposomal doxorubicin ((64)Cu-MM-302) in rodents and primates.

BackgroundFeatures of the tumor microenvironment influence the efficacy of cancer nanotherapeutics. The ability to directly radiolabel nanotherapeutics offers a valuable translational tool to obtain biodistribution and tumor deposition data, testing the hypothesis that the extent of delivery predicts therapeutic outcome. In support of a first in-human clinical trial with 64Cu-labeled HER2-targeted liposomal doxorubicin (64Cu-MM-302), a preclinical dosimetric analysis was performed.MethodsWhole-body biodistribution and pharmacokinetic data were obtained in mice that received 64Cu-MM-302 and used to estimate absorbed radiation doses in normal human organs. PET/CT imaging revealed non-uniform distribution of 64Cu signal in mouse kidneys. Kidney micro-dosimetry analysis was performed in mice and squirrel monkeys, using a physiologically based pharmacokinetic model to estimate the full dynamics of the 64Cu signal in monkeys.ResultsOrgan-level dosimetric analysis of mice receiving 64Cu-MM-302 indicated that the heart was the organ receiving the highest radiation absorbed dose, due to extended liposomal circulation. However, PET/CT imaging indicated that 64Cu-MM-302 administration resulted in heterogeneous exposure in the kidney, with a focus of 64Cu activity in the renal pelvis. This result was reproduced in primates. Kidney micro-dosimetry analysis illustrated that the renal pelvis was the maximum exposed tissue in mice and squirrel monkeys, due to the highly concentrated signal within the small renal pelvis surface area.ConclusionsThis study was used to select a starting clinical radiation dose of 64Cu-MM-302 for PET/CT in patients with advanced HER2-positive breast cancer. Organ-level dosimetry and kidney micro-dosimetry results predicted that a radiation dose of 400 MBq of 64Cu-MM-302 should be acceptable in patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-015-0096-0) contains supplementary material, which is available to authorized users.

Abstract P4-12-29: Assessment of safety and activity in an expanded phase 1 study of MM-302, a HER2-targeted liposomal doxorubicin, in patients with advanced HER2-positive (HER2+) breast cancer

Abstract Background: MM-302 is a novel antibody drug conjugate (ADC) combining PEGylated liposomal doxorubicin with anti-HER2 scFvs. MM-302 was designed to overcome the limitations associated with using anthracyclines in the treatment of HER2-positive breast cancer. Anthracyclines have been an effective backbone of breast cancer therapies for decades. However, cardiotoxicity issues associated with free anthracyclines have limited their effective use. While liposomal doxorubicin formulations have succeeded in reducing cardiotoxicity, they have failed to demonstrate clear-cut efficacy advantages. MM-302 specifically targets tumor cells overexpressing HER2 with minimal uptake into normal cells such as cardiomyocytes which express low levels of HER2. This Phase 1 study evaluates the safety of MM-302 in patients with HER2+ advanced breast cancer. Methods: Patients aged ≥ 18 years with histologically confirmed HER2+ advanced breast cancer that have progressed or recurred on standard therapy or for which no standard therapy exists having measurable disease, adequate performance status, bone marrow reserve and organ function, were eligible for the study. A post-treatment biopsy was required. A 3 + 3 dose escalation design (8, 16, 30, 40 and 50 mg/m2 administered i.v. q4w) is used to identify a Phase 2 dose followed by expansion arms at 40 and 50 mg/m2 for a preliminary indication of activity. Secondary endpoints included determination of dose-limiting toxicity, adverse event(s), and PK of MM-302, as well as overall response and clinical benefit rates of MM-302. Results: We report data from MM-302 dose levels (30, 40 and 50 mg/m2 as monotherapy) that are expected to be therapeutically relevant. Eight patients dosed at presumed sub-therapeutic dose levels of 8 and 16 mg/m2 were excluded from the efficacy analysis. Patients had received a median of 9.5 prior agents (range 3-19) in all settings. All patients had been treated with trastuzumab, 96% with prior taxanes, 67% lapatinib, 54% anthracyclines and 33% T-DM1. At the time of this analysis (n = 27), overall response rate was 17% (4 out of 24 evaluable patients) and the estimated median Progression Free Survival (PFS) was 5.7 months, with six patients still receiving treatment. Eight patients (30%) had a PFS of greater than 6.0 months. Neutropenia was the most common treatment-related grade 3/4 toxicity and occurred in 15% of patients. The most common adverse events include fatigue, nausea, vomiting, and decreased appetite. Constipation, stomatitis, headache and rash were also observed in greater than 20% of patients. No patients discontinued treatment for toxicity. There was no protocol defined cardiotoxicity observed across all dose cohorts and five patients have had cumulative anthracycline exposure exceeding 550 mg/m2 without a reduction in left ventricular ejection fraction. Conclusion: Overall MM-302 appears to be well tolerated with promising activity in this ongoing trial. Further development is continuing combining MM-302 with trastuzumab and/or cyclophosphamide. In addition, we are evaluating the use of PET imaging to correlate liposomal tumor deposition as an additional potential patient preselection strategy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-29.

Impact of Tumor HER2/ERBB2 Expression Level on HER2-Targeted Liposomal Doxorubicin-Mediated Drug Delivery: Multiple Low-Affinity Interactions Lead to a Threshold Effect

Numerous targeted nanotherapeutics have been described for potential treatment of solid tumors. Although attention has focused on antigen selection and molecular design of these systems, there has been comparatively little study of how cellular heterogeneity influences interaction of targeted nanoparticles with tumor cells. Antigens, such as HER2/ERBB2, are heterogeneously expressed across different indications, across patients, and within individual tumors. Furthermore, antigen expression in nontarget tissues necessitates optimization of the therapeutic window. Understanding the performance of a given nanoparticle under different regimens of antigen expression has the ability to inform patient selection and clinical development decisions. In this work, HER2-targeted liposomal doxorubicin was used as a model-targeted nanoparticle to quantitatively investigate the effect of HER2 expression levels on delivery of doxorubicin to the nucleus. We find quantitatively greater nuclear doxorubicin delivery with increasing HER2 expression, exhibiting a threshold effect at approximately 2 × 10(5) HER2 receptors/cell. Kinetic modeling indicated that the threshold effect arises from multiple low-affinity interactions between the targeted liposome and HER2. These results support previous data showing little or no uptake into human cardiomyocytes, which express levels of HER2 below the threshold. Finally, these results suggest that HER2-targeted liposomal doxorubicin may effectively target tumors that fall below traditional definitions of HER2-positive tumors, thereby expanding the potential population of patients that might benefit from this agent.

Abstract 3271: Tumor priming with cyclophosphamide increases delivery and activity of Her2-targeted liposomal doxorubicin (MM-302) in preclinical models of breast cancer.

Abstract MM-302 is a HER2-targeted liposomal doxorubicin formulation designed to target doxorubicin to HER2-overexpressing cancer cells while limiting uptake into non-target cells. The combination of doxorubicin with cyclophosphamide has been widely used in both the early- and late-stage breast cancer settings. Recent clinical trials have shown promising response rates to the combination of cyclophosphamide, pegylated liposomal doxorubicin (PLD) and trastuzumab in HER2-positive disease. The purpose of this study is to investigate the effects of cyclophosphamide on the tumor delivery of MM-302, and to evaluate the anti-tumor activity of this drug combination in preclinical models of breast cancer. Methods: Biodistribution studies were carried out in BT474-M3, SUM190, and MDA-MB-453 tumor-bearing mice to assess the delivery of MM-302, PLD and free doxorubicin as single agents or in combination with cyclophosphamide at different dosing schedules. The total doxorubicin within tumors was quantified by HPLC and, microscopically, by automated image analysis in order to determine the extent of doxorubicin-positive nuclei. Automated image analysis was also used to quantify tumor cell apoptosis, changes in tumor cell density, and induction of DNA-damage in response to drug treatment. Activity studies in BT474-M3 tumor-bearing mice were performed to evaluate the ability of the different dosing regimens to inhibit tumor growth. Results: Pre-dosing of tumors with cyclophosphamide significantly increased the delivery of MM-302 to tumors. The increase in delivery was tumor-specific, and the cyclophosphamide pre-dose had no effect on doxorubicin accumulation to non-target tissues such as heart or skin. Pre-treatment of tumors with cyclophosphamide also increased the delivery of PLD to tumors, but not of free doxorubicin. However, the increase in nuclear doxorubicin delivery via MM-302 compared to PLD was significantly higher in the HER2-positive breast cancer models analyzed. Histological analysis of the cyclophosphamide-treated tumors suggests that induction of tumor cell apoptosis and reduction of overall tumor cell density are among the mechanisms for improved MM-302 delivery and tumor cell response to doxorubicin in this preclinical model. Finally, treatment of BT474-M3 tumors with cyclophosphamide followed by MM-302 resulted in a significantly higher tumor growth inhibition compared to both single agents as well as compared to a MM-302 and cyclophosphamide co-injection regimen. Conclusions: Rational combination of MM-302 with cyclophosphamide is an active and tolerable combination in preclinical models that enhances the HER2-targeting properties of MM-302. This study suggests that cyclophosphamide may be a promising combination partner for MM-302 in clinical studies. Citation Format: Elena Geretti, Shannon C. Leonard, Daniel F. Gaddy, Nancy Dumont, Thomas J. Wickham, Bart S. Hendriks. Tumor priming with cyclophosphamide increases delivery and activity of Her2-targeted liposomal doxorubicin (MM-302) in preclinical models of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3271. doi:10.1158/1538-7445.AM2013-3271

Abstract P4-02-05: A novel 64Cu-liposomal PET agent (MM-DX-929) predicts response to liposomal chemotherapeutics in preclinical breast cancer models

Abstract Background: Liposomal anthracyclines (such as pegylated liposomal doxorubicin (PLD) or HER2-targeted liposomal doxorubicin (MM-302)) are being used and/or evaluated for the clinical management of breast cancer, but responses vary from patient to patient. It is hypothesized that variability in the deposition of liposomal therapeutics within tumors leads to differences in drug exposure, thereby directly influencing tumor response. We have developed MM-DX-929, a novel 64Cu-liposomal PET imaging agent, as a clinically-implementable tool to investigate whether image-based quantification of liposome deposition in tumors can predict treatment response to liposomal chemotherapeutics, including PLD, MM-302 and/or liposomal irinotecan (MM-398). Objectives: Our primary objective is to demonstrate that the extent of tumor uptake of MM-DX-929 is predictive of tumor response to MM-302 in preclinical breast cancer xenograft models. A secondary objective is to enable clinical translation of MM-DX-929 to enable incorporation into existing therapeutic trials. Methods: Mice bearing BT474-M3 mammary and subcutaneous tumors were injected intravenously with MM-DX-929 prior to dosing with MM-302. PET/CT imaging was performed at 16h post MM-DX-929 injection, and tumor uptake was determined. Response to treatment was quantified as tumor volume changes measured over a 2-month period by MRI. Results: Tumor deposition of MM-DX-929 administration correlated well with treatment response to MM-302 (Spearman correlation coefficient of −0.891 and a p-value of 0.0004). MM-DX-929 accumulation in tumors prior to the start of the MM-302 treatment successfully predicted improved tumor growth inhibition following MM-302 treatment. Conclusion: These findings support trials of MM-DX-929 as a predictive imaging agent to select patients who are most likely to respond to liposomal therapies. The clinical development of MM-DX-929 for identification of breast cancer patients likely to respond to liposomal therapeutics is currently being pursued. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-02-05.

HER2-targeted liposomal doxorubicin displays enhanced anti-tumorigenic effects without associated cardiotoxicity

Anthracycline-based regimens are a mainstay of early breast cancer therapy, however their use is limited by cardiac toxicity. The potential for cardiotoxicity is a major consideration in the design and development of combinatorial therapies incorporating anthracyclines and agents that target the HER2-mediated signaling pathway, such as trastuzumab. In this regard, HER2-targeted liposomal doxorubicin was developed to provide clinical benefit by both reducing the cardiotoxicity observed with anthracyclines and enhancing the therapeutic potential of HER2-based therapies that are currently available for HER2-overexpressing cancers. While documenting the enhanced therapeutic potential of HER2-targeted liposomal doxorubicin can be done with existing models, there has been no validated human cardiac cell-based assay system to rigorously assess the cardiotoxicity of anthracyclines. To understand if HER2-targeting of liposomal doxorubicin is possible with a favorable cardiac safety profile, we applied a human stem cell-derived cardiomyocyte platform to evaluate the doxorubicin exposure of human cardiac cells to HER2-targeted liposomal doxorubicin. To the best of our knowledge, this is the first known application of a stem cell-derived system for evaluating preclinical cardiotoxicity of an investigational agent. We demonstrate that HER2-targeted liposomal doxorubicin has little or no uptake into human cardiomyocytes, does not inhibit HER2-mediated signaling, results in little or no evidence of cardiomyocyte cell death or dysfunction, and retains the low penetration into heart tissue of liposomal doxorubicin. Taken together, this data ultimately led to the clinical decision to advance this drug to Phase I clinical testing, which is now ongoing as a single agent in HER2-expressing cancers.

Abstract 3637: MM-302, a HER2-targeted liposomal doxorubicin, shows binding/uptake and efficacy in HER2 2+ cells and xenograft models

Abstract Introduction: MM-302 is an ErbB2/HER2-targeted liposomal doxorubicin formulation designed to target doxorubicin to HER2-overexpressing cancer cells while limiting uptake into non-target cells. The characterization of the binding and uptake of MM-302 as a function of HER2 surface levels on various cell lines and in tumor models will help determine the minimum HER2 receptor levels required for MM-302 patient selection. Methods: Cellular models included 16+ established human tumor cell lines as well as 4T1 murine breast cancer and HeLa human cervical cancer cells engineered to overexpress HER2 from 1E05-1E06 receptors/cell. Cells were incubated with MM-302, untargeted liposomes or free doxorubicin and binding/uptake was measured by HPLC or flow cytometry. In vivo efficacy was determined in select mouse xenograft models. Tumors were mechanically and enzymatically dissociated, followed by flow cytometry to quantify the uptake of targeted and untargeted liposomes into HER2-expressing tumor cells, macrophages and other cell types. Results: Binding and uptake of MM-302 increased with rising HER2 surface levels in cells of common parental origin (4T1, HeLa), with significant uptake into cells with >2E05 HER2/cell. Further, total cell binding/uptake of MM-302 also increased with rising HER2 expression across a panel of 16+ cell lines. There was negligible uptake of untargeted liposomes under all conditions that showed no dependence on HER2 expression. Improved anti-tumor efficacy of MM-302 was observed in BT474-M3 and NCI-N87 mouse xenograft models relative to untargeted liposomal doxorubicin and free doxorubicin. Further, using disaggregated tumors, the relationship between HER2 expression and MM-302 uptake was confirmed in vivo in both models. Conclusions: Our studies identified a HER2 threshold for uptake of MM-302 of approximately 2E05 receptors/cell, and suggest a role for MM-302 not only in HER2 3+ cancers, but also in HER2 2+ cancers as well. The ability to treat HER2 2+ tumors offers an exciting opportunity to serve a major unmet clinical need. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3637. doi:10.1158/1538-7445.AM2011-3637

Abstract 3638: MM-302, HER2-targeted liposomal doxorubicin, does not impair cardiomyocyte function in vitro

Abstract In HER2+ (ErbB2+) breast cancer, the ability to combine anthracyclines, such as doxorubicin, with anti-HER2 targeted therapies is limited clinically due to potential synergistic cardiotoxicity: doxorubicin directly damages heart cells and HER2 is an important mediator of cardiac repair signaling. Herein we detail findings for MM-302, a novel HER2-targeted liposomal doxorubicin formulation. The liposomal encapsulation is designed to protect the heart from doxorubicin exposure and the HER2-targeting component mediates uptake in HER2-positive cells without blocking HER2 signaling. In order to study the potential effects of MM-302 on the heart, we performed studies using Embryonic Stem Cell (ESC)-derived cardiac cells. Total cell uptake of free doxorubicin and MM-302 into ESC-derived cardiac cells was measured using HPLC. We found that, despite expressing low levels of HER2, the ESC-derived cardiac cells do not take up MM-302, whereas doxorubicin freely enters the cells in a time and dose-dependent manner over a 3 hour time course. Subsequent to delivery, cell viability was assessed in response to free doxorubicin or MM-302 at 24 hours post 3-hour exposure. Doxorubicin resulted in cell death in a dose-dependent manner, whereas MM-302 had no effect on cardiac cell viability. To understand the mechanism underlying the cardiotoxicity associated with doxorubicin exposure, and to differentiate MM-302 from existing therapies, we measured cell functional responses in ESC-derived cardiac cells, including cell stress (phopsho-p53), DNA damage (gamma-H2AX) and apoptosis (cleaved-PARP) with high-content microscopy. We demonstrated that, while doxorubicin treatment results in DNA damage, cell stress, apoptosis, and ultimately cell death in a dose-dependent manner, MM-302 does not. Our findings demonstrate that MM-302 provides a means to deliver doxorubicin to high HER2-expressing cancer cells, while sparing exposure, accumulation, and toxicity in non-target (low HER2-expressing) cells, such as cardiomyocytes. This opens an exciting avenue for potentially introducing a novel anthracycline product for the treatment of HER2+ breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3638. doi:10.1158/1538-7445.AM2011-3638

Abstract P3-14-09: Preclinical Safety and Activity of MM-302, a HER2-Targeted Liposomal Doxorubicin Designed To Have an Improved Safety and Efficacy Profile over Approved Anthracyclines

Abstract Background: Anthracyclines have been an effective backbone of breast cancer therapies for decades. However, cardiotoxicity issues associated with free anthracyclines have limited their effective use in the clinic and ledto the exploration of anthracycline-free regimens, particularly with HER2- positive cancers that require treatment with another cardiotoxic agent, trastuzumab. While liposomal doxorubicin formulations have succeeded in reducing cardiotoxicity, they have failed to demonstrate clearcut efficacy advantages and can involve other toxicities. To address the safety and efficacy limitations of currently available anthracyclines, we have designed a new liposomal formulation, MM-302, that targets doxorubicin to HER2- overexpressing tumor cells. Antibody fragments that bind to HER2 without blocking HER2-mediated signaling are coupled to the outer surface of pegylated liposomal doxorubicin. MM-302 specifically binds and enters tumor cells overexpressing HER2 with minimal uptake into normal cells such as cardiomyocytes which express low levels of HER2. Materials and methods: MM-302, untargeted pegylated liposomal doxorubicin (PLD), and free doxorubicin were compared for their uptake into target and nontarget cells, pharmacokinetics, biodistribution, tumor microdistribution using FACS and confocal microscopy, and anti-tumor activity in BT-474 and NCI-N87 xenograft models. Results: Cellular uptake studies in tumor cell lines expressing various levels of HER2 demonstrate that MM-302 delivers significantly higher doxorubicin levels to HER2 over-expressing tumor cells compared to PLD as well as similar or higher levels than highly permeable free doxorubicin. However, in human cardiomyocytes, while free doxorubicin was again taken up at high levels, doxorubicin uptake was dramatically lower with both MM-302 and PLD. Pharmacokinetic studies in mice demonstrate that MM-302 has a similar half life, clearance, and organ distribution compared to PLD. In HER2-overexpressing BT-474 breast and NCI-N87 gastric tumor xenografts, MM-302 exhibits superior anti-tumor activity to both free anthracyclines and PLD. Tumor microdistribution studies further suggest that differences in the localization of doxorubicin in the tumor may be responsible for the enhanced activity of MM-302 compared to free doxorubicin and PLD. Discussion: These preclinical data suggest that MM-302 may provide in clinical practice a new opportunity to specifically deliver an anthracycline to HER2-overexpressing tumors with a potentially cleaner safety profile and improved efficacy over currently available free or liposomal doxorubicin. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-14-09.