Statistical analyses of tumor growth curves in mouse study 2 (Figure 5)

Heregulin is expressed in MCF-7Ca and LTLT-Ca cells

Quantified target bands from Figure 2

Brief descriptions of the supplementary figure data provided

Pharmacodynamic analyses of HRG and ERBB3 expression in tumors harvested post-treatment

Dosing everolimus in combination with letrozole and seribantumab causes weight loss in mice

ERBB3 levels are significantly higher following co-treatment with letrozole and everolimus

BACKGROUND:Seribantumab (MM-121) is a fully human IgG2 monoclonal antibody that binds to human epidermal growth factor receptor 3 (HER3/ErbB3) to block heregulin (HRG/NRG)-mediated ErbB3 signaling and induce receptor downregulation. This open-label, randomized phase 1/2 study evaluated safety and efficacy of seribantumab plus erlotinib in advanced non-small cell lung cancer (NSCLC). Here, we report the activity of seribantumab plus erlotinib, versus erlotinib alone, in patients with EGFR wild-type tumors and describe the potential predictive power of HRG.MATERIALS AND METHODS:Patients with EGFR wild-type NSCLC were assigned randomly to receive seribantumab + erlotinib or erlotinib alone. Patients underwent pretreatment core needle biopsy and archived tumor samples were collected to support prespecified biomarker analyses.RESULTS:One hundred twenty-nine patients received seribantumab + erlotinib (n = 85) or erlotinib alone (n = 44). Median estimated progression-free survival (PFS) in the unselected intent-to-treat (ITT) population was 8.1 and 7.7 weeks in the experimental and control arm, respectively (hazard ratio [HR], 0.822; 95% confidence interval [CI], 0.37-1.828; p = 0.63), and median estimated overall survival was 27.3 and 40.3 weeks in the experimental and control arm, respectively (HR, 1.395; 95% CI, 0.846 to 2.301; p = .1898) In patients whose tumors had detectable HRG mRNA expression, treatment benefit was observed in the seribantumab + erlotinib combination (HR, 0.35; 95% CI, 0.16-0.76; p = .008). In contrast, in patients whose tumors were HRG negative, the HR was 2.15 (95% CI, 0.97-4.76; p = .059, HRG-by-treatment interaction, p value = .0016).CONCLUSION:The addition of seribantumab to erlotinib did not result in improved PFS in unselected patients. However, predefined retrospective exploratory analyses suggest that detectable HRG mRNA levels identified patients who might benefit from seribantumab. An ongoing clinical trial of seribantumab, in combination with docetaxel, is underway in patients with advanced NSCLC and high HRG mRNA expression (NCT02387216).IMPLICATIONS FOR PRACTICE:The poor prognosis of patients with non-small cell lung cancer (NSCLC) underscores the need for more effective treatment options, highlighting the unmet medical need in this patient population. The results of this study show that a novel biomarker, heregulin, may help to identify patients with advanced NSCLC who could benefit from treatment with seribantumab. On the basis of the observed safety profile and promising clinical efficacy, a prospective, randomized, open-label, international, multicenter phase II trial (SHERLOC, NCT02387216) is under way to investigate the efficacy and safety of seribantumab in combination with docetaxel in patients with heregulin-positive advanced adenocarcinoma.

This phase 1 dose-escalation trial studied MM-302, a novel HER2-targeted PEGylated antibody–liposomal doxorubicin conjugate, in HER2-positive locally advanced/metastatic breast cancer. Patients were enrolled in four cohorts: MM-302 monotherapy (8, 16, 30, 40, and 50 mg/m2 every 4 weeks [q4w]); MM-302 (30 or 40 mg/m2 q4w) plus trastuzumab (4 mg/kg q2w); MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) q3w; MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) and cyclophosphamide (450 mg/m2) q3w. Sixty-nine patients were treated. The most common adverse events (AEs) were fatigue and nausea. Grade 3/4 AEs of special interest included neutropenia, fatigue, mucosal inflammation, anemia, thrombocytopenia, febrile neutropenia, and palmar-plantar erythrodysesthesia. The MTD was not reached. With MM-302 ≥ 30 mg/m2, overall response rate (ORR) was 13% and median progression-free survival (mPFS) 7.4 months (95% CI: 3·5–10·9) in all arms. In 25 anthracycline-naïve patients, ORR was 28·0% and mPFS 10·9 months (95% CI: 1·8–15·3). Imaging with 64Cu-labeled MM-302 visualized tumor-drug penetrance in tumors throughout the body, including the brain. MM-302 monotherapy, in combination with trastuzumab, or trastuzumab plus cyclophosphamide, was well tolerated and showed promising efficacy. The selected phase 2 MM-302 dose was 30 mg/m2 plus 6 mg/kg trastuzumab q3w.

2524 Background: Seribantumab (MM-121) is an anti-ErbB3 human monoclonal antibody being tested as an anti-cancer therapy for patients with high expression of heregulin mRNA in NSCLC (SHERLOC study, 3g every 3 weeks [Q3W]+docetaxel) and in and in HR+/ HER2- metastatic breast cancer (mBC) (SHERBOC study, 3g every 2 weeks [Q2W]+fulvestrant). Here we aimed to evaluate seribantumab dose regimens based on PK and safety. Methods: Pharmacokinetics (PK) and safety were evaluated for fixed and weight-based dose: in NSCLC, 3 g Q3W vs. 20 mg/kg Q2W; in mBC, 3 g Q2W vs. 40 mg/kg loading dose, followed by 20 mg/kg QW. PK was quantified using nonlinear mixed effect with the following covariates: sex, race, age, weight, dose, study, and hepatic functions. Adverse events (AEs) were evaluated for associations to PK and weight: Grade 1+ and 3+ diarrhea, fatigue, hypokalemia, hypomagnesemia, nausea, pulmonary embolism, rash, stomatitis, and vomiting. Results: Seribantumab PK from 499 patients in 7 previous trials identified associations with sex and weight. Fixed and weight-based doses showed similar variability. With higher weight, weight-based dose resulted in higher exposure (average concentration [Cavg] and maximum concentration [Cmax]), and fixed dose resulted in lower exposure. Steady-state Cmax were higher with 3 g than with 20 mg/kg; these values were comparable to Cmax with 40 mg/kg. Steady-state Cavg were equal to or higher for fixed dose than comparable weight-based dose. All doses tested had minimum concentrations (Cmin) higher than the nonclinical target concentration of 100 mg/L. Among AEs evaluated, significant associations with seribantumab exposure were observed for G1+ diarrhea, fatigue, nausea, rash, and stomatitis. Weight-based dose showed higher AE rates with increasing weight. Fixed dose is predicted to reduce AE rates in high-weight patients and to increase AE rates in low-weight patients, with the latter rates still lower than those in patients with high weight dosed by weight. Conclusions: The recommended dose of seribantumab is 3 g Q3W+docetaxel in NSCLC and 3 g Q2W+fulvestrant in mBC. The fixed dose of seribantumab allows acceptable concentrations and safety profiles and potentially reduces drug waste and dosing errors.

Nanoliposomal irinotecan (nal-IRI) is a liposomal formulation of irinotecan with a longer half-life (t(1/2)), higher plasma total irinotecan (tIRI), and lower SN-38 maximum concentration (C-max) compared with nonliposomal irinotecan. Population pharmacokinetic (PK) analysis of nal-IRI was performed for tIRI and total SN-38 (tSN38) using patient samples from six studies. PK-safety association was evaluated for neutropenia and diarrhea in 353 patients. PK-efficacy association was evaluated from a phase III study in pancreatic cancer NAPOLI1. Efficacy was associated with longer duration of unencapsulated SN-38 (uSN38) above a threshold and higher C-avg of tIRI, tSN38, and uSN38. Neutropenia was associated with uSN38 C-max and diarrhea with tIRI C-max. Baseline predictive factors were race, body surface area, and bilirubin. Analysis identified PK factors associated with efficacy, safety, and predictive baseline factors. The results support the benefit of nal-IRI dose of 70mg/m(2) (free-base; equivalent to 80mg/m(2) salt base) Q2W over 100mg/m(2) Q3W.

Abstract Heregulin (HRG) is the cognate ligand of the human epidermal growth factor receptor 3 (ErbB3) and has been identified as a potent driver of a distinct tumor cell phenotype characterized by enhanced survival that appears to be inherently more resistant to standard of care therapies. Preclinical studies indicate that not only can HRG induce proliferation of cellular cancer models but also that it can mediate insensitivity to numerous classes of anticancer agents including chemotherapies and targeted therapeutics. Furthermore, we and others have found that HRG-ErbB3 signaling is adept at mediating insensitivity to several classes of anti-hormonal agents that currently represent the mainstay of treatment options for hormone receptor (HR+), HER2 negative advanced breast cancer. This finding, coupled with the fact that HRG is expressed in almost half of all HR+, HER2 negative advanced breast cancers, indicates that HRG-ErbB3 signal transduction may be contributing to loss of sensitivity to endocrine based therapies in this disease. Mechanistically, anti-hormonal drugs including, fulvestrant have been implicated in increasing ErbB3 expression levels, resulting in a compensatory mechanism where cells become resistant to endocrine treatments due to estrogen independent proliferation. Seribantumab is a fully human monoclonal antibody designed to block HRG from binding to ErbB3 and prevent the establishment of HRG-driven cancer cell survival in response to standard of care therapies, including chemotherapies and anti-endocrine therapies. Clinical results from a randomized, Phase 2 study in women with metastatic breast cancer who received seribantumab plus exemestane or placebo plus exemestane highlighted the ability to sensitize HRG-positive tumors to exemestane by co-administration with seribantumab. Overall, data indicated that there was a positive trend in prolonging progression-free survival (PFS) and a statistically significant difference in overall survival (OS) in this randomized and unselected or “all-comers” patient population for those patients who had seribantumab added to their exemestane therapy rather than placebo. Safety analysis indicated that seribantumab plus exemestane was well tolerated, with manageable diarrhea being the most frequent adverse event observed. A preplanned biomarker analysis using archived tissue samples showed that women who have HRG-positive tumors were less sensitive to exemestane alone and derived substantial clinical benefit when receiving the combination of seribantumab plus exemestane, illustrated by a statistically significant improvement in PFS in this HRG-positive population. Further clinical subgroup analysis suggested that patients who received prior chemotherapy and patients whose disease had progressed in the metastatic setting may be more likely to benefit from the addition of seribantumab to their exemestane. To identify patients with HRG-driven cancers, an RNA in situ hybridization (ISH) diagnostic assay has been developed and is currently being utilized to select patients in a Phase 2 non-small cell lung cancer trial. We will present updates on clinical outcomes based on recent patient subgroup analyses along with a clinical development plan for seribantumab in HR+, HER2- advanced breast cancer. Citation Format: Greg Finn, Hong Zhang, Anna Blois, sara Mathews, Art Kudla, Jason Baum, Mike Cieslewicz, gavin macbeath, Bambang Adiwijaya, Akos Czibere. A randomized trial of exemestane +/- seribantumab (MM-121) in postmenopausal women with locally advanced or metastatic ER/PR+ HER2- breast cancer: Final analysis and extended subgroup analysis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A14.

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR/ErbB1), human EGFR 2 (HER2/ErbB2), ErbB3/HER3, and ErbB4/HER4. The first two members of this family, EGFR and HER2, have been implicated in tumorigenesis and cancer progression for several decades, and numerous drugs have now been approved that target these two proteins. Less attention, however, has been paid to the role of this family in mediating cancer cell survival and drug tolerance. To better understand the complex signal transduction network triggered by the ErbB receptor family, we built a computational model that quantitatively captures the dynamics of ErbB signaling. Sensitivity analysis identified ErbB3 as the most critical activator of phosphoinositide 3-kinase (PI3K) and Akt signaling, a key pro-survival pathway in cancer cells. Based on this insight, we designed a fully human monoclonal antibody, seribantumab (MM-121), that binds to ErbB3 and blocks signaling induced by the extracellular growth factors heregulin (HRG) and betacellulin (BTC). In this article, we present some of the key preclinical simulations and experimental data that formed the scientific foundation for three Phase 2 clinical trials in metastatic cancer. These trials were designed to determine if patients with advanced malignancies would derive benefit from the addition of seribantumab to standard-of-care drugs in platinum-resistant/refractory ovarian cancer, hormone receptor-positive HER2-negative breast cancer, and EGFR wild-type non-small cell lung cancer (NSCLC). From preclinical studies we learned that basal levels of ErbB3 phosphorylation correlate with response to seribantumab monotherapy in mouse xenograft models. As ErbB3 is rapidly dephosphorylated and hence difficult to measure clinically, we used the computational model to identify a set of five surrogate biomarkers that most directly affect the levels of p-ErbB3: HRG, BTC, EGFR, HER2, and ErbB3. Preclinically, the combined information from these five markers was sufficient to accurately predict which xenograft models would respond to seribantumab, and the single-most accurate predictor was HRG. When tested clinically in ovarian, breast and lung cancer, HRG mRNA expression was found to be both potentially prognostic of insensitivity to standard therapy and potentially predictive of benefit from the addition of seribantumab to standard of care therapy in all three indications. In addition, it was found that seribantumab was most active in cancers with low levels of HER2, consistent with preclinical predictions. Overall, our clinical studies and studies of others suggest that HRG expression defines a drug-tolerant cancer cell phenotype that persists in most solid tumor indications and may contribute to rapid clinical progression. To our knowledge, this is the first example of a drug designed and clinically tested using the principles of Systems Biology.

Abstract Background/Objectives: Children with relapsed or refractory solid tumors have a poor prognosis. Irinotecan is active in some pediatric solid tumors and synergizes with alkylating agents. nal-IRI encapsulates irinotecan into long-circulating, liposome-based nanoparticles. In adults, nal-IRI demonstrated extended plasma exposure compared with non-liposomal irinotecan. In pediatric solid tumor models, nal-IRI had robust preclinical activity and synergized with cyclophosphamide, and therefore merits testing in children with relapsed and refractory solid tumors. Herein we describe a phase 1 dose-escalation study of nal-IRI in combination with cyclophosphamide (NCT02013336) and preliminary pharmacokinetic and safety results. Methods: Cyclophosphamide was administered on days 1-5 of each cycle (250 mg/m2/d intravenously [IV]) with a single 90-min IV infusion of nal-IRI on day 3 of a Q3-week schedule, escalating from 60 mg/m2 to 210 mg/m2 (expressed as irinotecan HCL trihydrate salt), in a standard 3+3 dose-escalation design to determine the maximum tolerated dose. To date, the nal-IRI dose has been escalated from 60 mg/m2 to 150 mg/m2. Samples for pharmacokinetic analysis were collected during the first cycle of chemotherapy before infusion and at 4h, 24h, 48h, 120h, and 168h post-infusion. Plasma pharmacokinetics of total irinotecan and SN-38 were quantified using mixed effect modeling, and were compared with adult values from a population pharmacokinetic analysis of 6 clinical studies of nal-IRI.1 Results: To date, 10 males and 6 females with a median age of 12.8 years (range: 5-19) have been enrolled: 10 with Ewing sarcoma, 2 with neuroblastoma, 3 with osteosarcoma, and 1 with rhabdomyosarcoma. The estimated total irinotecan volume of distribution (Vd) was 1.9 L, clearance (CL) was 10.3 L/week, and half-life (t1/2) was 21.2 h, which were 42% (Vd and CL) of adult values and comparable to adult values (t1/2). The corresponding Cmax was 72% higher than that observed in adults. SN-38 clearance was 11.4 L/week (comparable to adults), t1/2 was 19.3 h (48% of adult values), and Cmax was 68% of adult values. Thrombocytopenia leading to treatment delay was a dose-limiting toxicity at 150 mg/m2 (n=1); other systemic toxicity attributed to chemotherapy within the 1st cycle was nausea/vomiting (n=1). Conclusions: Preliminary safety and pharmacokinetic data support continued investigation of nal-IRI in pediatric oncology. Clinical outcomes including safety of patients treated in this study will be reported once a maximum tolerated dose is achieved. 1. Adiwijaya B et al. Clin Pharmacol Ther. 2017. In press. Citation Format: Paul D. Harker-Murray, William H. Meyer, Patrick Leavey, Min H. Kang, Hwangeui Cho, Bambang S. Adiwijaya, Jonathan B. Fitzgerald, J Marc Pipas, Daryl C. Drummond, C. Patrick Reynolds. Plasma pharmacokinetics of liposomal irinotecan (nal-IRI) in pediatric oncology patients with recurrent or refractory solid tumors: South Plains Oncology Consortium Study 2012-001 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT146. doi:10.1158/1538-7445.AM2017-CT146

Abstract This abstract was withdrawn by the authors.

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR/ErbB1), human EGFR 2 (HER2/ErbB2), ErbB3/HER3, and ErbB4/HER4. The first two members of this family, EGFR and HER2, have been implicated in tumorigenesis and cancer progression for several decades, and numerous drugs have now been approved that target these two proteins. Less attention, however, has been paid to the role of this family in mediating cancer cell survival and drug tolerance. To better understand the complex signal transduction network triggered by the ErbB receptor family, we built a computational model that quantitatively captures the dynamics of ErbB signaling. Sensitivity analysis identified ErbB3 as the most critical activator of phosphoinositide 3-kinase (PI3K) and Akt signaling, a key pro-survival pathway in cancer cells. Based on this insight, we designed a fully human monoclonal antibody, seribantumab (MM-121), that binds to ErbB3 and blocks signaling induced by the extracellular growth factors heregulin (HRG) and betacellulin (BTC). In this article, we present some of the key preclinical simulations and experimental data that formed the scientific foundation for three Phase 2 clinical trials in metastatic cancer. These trials were designed to determine if patients with advanced malignancies would derive benefit from the addition of seribantumab to standard-of-care drugs in platinum-resistant/refractory ovarian cancer, hormone receptor-positive HER2-negative breast cancer, and EGFR wild-type non-small cell lung cancer (NSCLC). From preclinical studies we learned that basal levels of ErbB3 phosphorylation correlate with response to seribantumab monotherapy in mouse xenograft models. As ErbB3 is rapidly dephosphorylated and hence difficult to measure clinically, we used the computational model to identify a set of five surrogate biomarkers that most directly affect the levels of p-ErbB3: HRG, BTC, EGFR, HER2, and ErbB3. Preclinically, the combined information from these five markers was sufficient to accurately predict which xenograft models would respond to seribantumab, and the single-most accurate predictor was HRG. When tested clinically in ovarian, breast and lung cancer, HRG mRNA expression was found to be both potentially prognostic of insensitivity to standard therapy and potentially predictive of benefit from the addition of seribantumab to standard of care therapy in all three indications. In addition, it was found that seribantumab was most active in cancers with low levels of HER2, consistent with preclinical predictions. Overall, our clinical studies and studies of others suggest that HRG expression defines a drug-tolerant cancer cell phenotype that persists in most solid tumor indications and may contribute to rapid clinical progression. To our knowledge, this is the first example of a drug designed and clinically tested using the principles of Systems Biology.

Purpose Seribantumab is a fully human immunoglobulin G2 monoclonal antibody that binds to human epidermal growth factor receptor (HER) 3 (ErbB3), blocking heregulin (HRG) –mediated ErbB3 signaling and inducing ErbB3 receptor downregulation. This open-label randomized phase II study evaluated progression-free survival (PFS) with seribantumab in combination with once-per-week paclitaxel compared with paclitaxel alone in patients with platinum-resistant or -refractory ovarian cancer. A key secondary objective was to determine if any of five prespecified biomarkers predicted benefit from seribantumab. Patients and Methods Patients with platinum-resistant or -refractory epithelial ovarian, fallopian tube, or primary peritoneal cancer were randomly assigned at a ratio of two to one to receive seribantumab plus paclitaxel or paclitaxel alone. Patients underwent pretreatment core needle biopsy; archival tumor samples were also obtained to support biomarker analyses. Results A total of 223 patients were randomly assigned (seribantumab plus paclitaxel, n = 140; paclitaxel alone, n = 83). Median PFS in the unselected intent-to-treat population was 3.75 months with seribantumab plus paclitaxel compared with 3.68 months with paclitaxel alone (hazard ratio [HR], 1.027; 95% CI, 0.741 to 1.425; P = .864). Among patients whose tumors had detectable HRG mRNA and low HER2 (n = 57 [38%] of 151 with available biomarker data), increased treatment benefit was observed in those receiving seribantumab plus paclitaxel compared with paclitaxel alone (PFS HR, 0.37; 95% CI, 0.18 to 0.76; P = .007). The HR in patients not meeting these criteria was 1.80 (95% CI, 1.08 to 2.98; P = .023). Conclusion The addition of seribantumab to paclitaxel did not result in improved PFS in unselected patients. Exploratory analyses suggest that detectable HRG and low HER2, biomarkers that link directly to the mechanism of action of seribantumab, identified patients who might benefit from this combination. Future clinical trials are needed to validate this finding and should preselect for HRG expression and focus on cancers with low HER2 levels.

MM-302 is a HER2-targeted antibody–liposomal doxorubicin conjugate designed to target doxorubicin to HER2-expressing tumor cells. MM-302 showed an acceptable safety profile and promising activity in a Phase I study in HER2-positive metastatic breast cancer (NCT01304797), and it is now being evaluated in a Phase II trial in the same setting (NCT02213744). The goal of this work is to determine the correlation between single-cell HER2 expression and liposome uptake on MM-302 patient biopsies as well as on preclinical tumor models. Frozen biopsies were collected 3 days post infusion of MM-302 (8-50 mg/m2; alone or in combination with trastuzumab, with or without cyclophosphamide), and stained for PEG (surrogate for MM-302), HER2 and cytokeratin, side-by-side with two cell standard arrays: A PEG array, obtained by cell incubation with increasing amounts of MM-302, and a HER2 array, containing a panel of cell lines at various HER2 levels. The HER2 expression and liposome uptake in individual tumor cells of the human samples was quantified based on the PEG and HER2 fluorescent intensities of the standards. MM-302 cellular delivery was investigated in vivo in IHC 0, 1 + , 2+ and 3+ tumor models. Uptake of MM-302 into HER2-expressing cells was detected in ∼80% of patient biopsies. Interestingly, HER2 expression within individual samples was found to be heterogeneous, ranging from ∼1 X 105 to over 1 X 106 HER2 receptors per cell. Evaluation of cellular HER2 expression and MM-302 uptake within the same sample revealed that the magnitude of MM-302 tumor cell uptake was comparable across the range of HER2 expression from ∼1 X 105 to over 1 X 106 HER2 receptors per cell. These findings were in line with preclinical in vivo studies showing HER2-mediated delivery of MM-302 to IHC 1 + , 2 + , and 3+ tumors but not to IHC 0 tumors. Our data suggest that MM-302 effectively targets tumor cells expressing various levels of HER2 in patients' tumors. Hence, in addition to treating HER2-positive patients, MM-302 may be a promising agent for treating patients with intermediate HER2 expression (HER2 IHC 1 + /2 + , FISH-negative).

Introduction: MM-302 is an antibody-liposomal drug conjugate designed specifically to target doxorubicin to HER2-overexpressing tumor cells. MM-302 is currently being evaluated in a Phase II trial in HER2 positive metastatic breast cancer (NCT02213744). HER2-positive breast cancer accounts for about 15-20% of breast cancer cases and is defined as IHC 3+ or 2+ and HER2 FISH amplified. A substantial percentage (∼30%) of breast cancer patients show positive HER2 IHC (1+/2+) without HER2 gene amplification (“HER2 intermediate”). This population is not eligible for treatment with currently approved HER2-targeted therapies. The purpose of this study is to investigate the in vitro and in vivo delivery/activity of MM-302 in the HER2 intermediate population. Methods: In vitro binding and viability studies were performed with MM-302, PEGylated liposomal doxorubicin (PLD) and T-DM1 with a panel of cell lines representing a range of HER2 expression. HER2-mediated cellular delivery of MM-302 was investigated in vivo in different HER2 expressing tumor models using a novel PEG immunofluorescent assay herein described. Frozen tumor tissues were stained for PEG, HER2 and cytokeratin, side-by-side with two cell standard arrays: A PEG array, obtained by cell incubation with increasing concentrations of MM-302, and a HER2 array, built with a panel of cell lines at different HER2 expression (from ∼50,000 to over 1,000,000 HER2). Image analysis and subsequent regression of the PEG and HER2 fluorescent intensities from the respective standards allowed for the quantification of the number of liposomes in individual tumor cells at distinct HER2 receptor numbers. Tumor cell apoptosis following MM-302 cellular delivery was measured by immunofluorescence followed by image analysis. Results: MM-302 efficiently bound to, and induced, tumor cell death across a panel of cell lines, with no significant distinction between cell lines expressing 300-400,000 HER2 or above 1,000,000 HER2 (IHC 3+). Conversely, T-DM1, used as control, significantly bound to and induced cell death only to cells above 1,000,000 HER2 (IHC 3+). In vivo evaluation of HER2-mediated cellular delivery via PEG immunofluorescent staining showed that MM-302 can be equally efficiently internalized in tumor cells above 1,000,000 HER2 (IHC 3+) and in the HER2 intermediate expression range. Preliminary analysis on post-treatment patient biopsies collected during a MM-302 Phase I study (NCT01304797) support clinical translation of these preclinical observations. Conclusions: Treatment with MM-302 results in efficient HER2 binding and liposome cellular delivery across a panel of HER2 models that extend beyond the traditional HER2 positive definition. This study suggests that MM-302 may be a promising candidate for the treatment of patients with intermediate HER2 expression who represent a significant unmet medical need. Citation Format: Elena Geretti, Christopher Espelin, Bambang Adiwijaya, Nancy Dumont, Silvia Coma, Zachary Koncki, Minh Pham, Gabriela Garcia, Troy Bloom, Victoria Rimkunas, Joe Reynolds, Karen Campbell, Victor Moyo, Istvan Molnar, Patricia LoRusso, Ian Krop, Kathy Miller, Cynthia Ma, Pamela Munster, Thomas Wickham. HER2-targeted PEGylated liposomal doxorubicin (MM-302) efficiently targets the HER2 intermediate cell population in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-061.