Victor Moyo

Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene

The product of the SLC40A1 gene, ferroportin 1, is a main iron export protein. Pathogenic mutations in ferroportin 1 lead to an autosomal dominant hereditary iron overload syndrome characterized by high serum ferritin concentration, normal transferrin saturation, iron accumulation predominantly in macrophages, and marginal anemia. Iron overload occurs in both the African and the African-American populations, but a possible genetic basis has not been established. We analyzed the ferroportin 1 gene in 19 unrelated patients from southern Africa (N = 15) and the United States (N = 4) presenting with primary iron overload. We found a new c. 744 C-->T (Q248H) mutation in the SLC40A1 gene in 4 of these patients (3 Africans and 1 African-American). Among 22 first degree family members, 10 of whom were Q248H heterozygotes, the mutation was associated with a trend to higher serum ferritin to amino aspartate transferase ratios (means of 14.8 versus 4.3 microg/U; P = 0.1) and lower hemoglobin concentrations (means of 11.8 versus 13.2 g/dL; P = 0.1). The ratio corrects serum ferritin concentration for alcohol-induced hepatocellular damage. We also found heterozygosity for the Q248H mutation in 7 of 51 (14%) southern African community control participants selected because they had a serum ferritin concentration below 400 microg/L and in 5 of 100 (5%) anonymous African-Americans, but we did not find the change in 300 Caucasians with normal iron status and 25 Caucasians with non-HFE iron overload. The hemoglobin concentration was significantly lower in the African community controls with the Q248H mutation than in those without it. We conclude that the Q248H mutation is a common polymorphism in the ferroportin 1 gene in African populations that may be associated with mild anemia and a tendency to iron loading.

Combination of Imatinib Mesylate with Autologous Leukocyte-Derived Heat Shock Protein and Chronic Myelogenous Leukemia

Purpose: To test the feasibility, safety, immunogenicity, and clinical efficacy of an autologous vaccine of leukocyte-derived heat shock protein 70-peptide complexes (Hsp70PC), in conjunction with imatinib mesylate, in patients with chronic myeloid leukemia (CML) in chronic phase. Experimental Design: Patients had cytogenetic or molecular evidence of disease, despite treatment with imatinib mesylate for all except one patient, at the beginning of study. Hsp70PCs were purified from the leukopheresed peripheral blood mononuclear cells and were administered in eight weekly intradermal injections at 50 μg/dose without adjuvant. Clinical responses were assessed by bone marrow analysis before and after vaccinations. An IFN-γ enzyme-linked immunospot assay was used to estimate the effect of treatment on natural killer cells and T cells against CML. Results: Twenty patients were treated. The manufacturing of Hsp70PCs was successful and the administration was safe for all patients. Minimal or no side effects were reported. Clinical responses were seen in 13 of 20 patients as measured by cytogenetic analysis of bone marrow Philadelphia chromosome–positive cells in metaphases and/or, when possible, the level of Bcr/Abl transcript by PCR. Immunologic responses were observed in 9 of 16 patients analyzed, characterized by an increase in the frequency of CML-specific IFN-γ-producing cells and IFN-γ-secreting natural killer cells in the blood. A significant correlation between clinical responses and immunologic responses was observed. Conclusions: Autologous Hsp70PC vaccination is feasible and safe. When combined with imatinib mesylate, it is associated with immunologic and possible clinical responses against CML in chronic phase.

64Cu-MM-302 positron emission tomography quantifies variability of enhanced permeability and retention of nanoparticles in relation to treatment response in patients with metastatic breast cancer

Purpose: Therapeutic nanoparticles are designed to deliver their drug payloads through enhanced permeability and retention (EPR) in solid tumors. The extent of EPR and its variability in human tumors is highly debated and has been proposed as an explanation for variable responses to therapeutic nanoparticles in clinical studies. Experimental Design: We assessed the EPR effect in patients using a 64Cu-labeled nanoparticle, 64Cu-MM-302 (64Cu-labeled HER2-targeted PEGylated liposomal doxorubicin), and imaging by PET/CT. Nineteen patients with HER2-positive metastatic breast cancer underwent 2 to 3 PET/CT scans postadministration of 64Cu-MM-302 as part of a clinical trial of MM-302 plus trastuzumab with and without cyclophosphamide (NCT01304797). Results: Significant background uptake of 64Cu-MM-302 was observed in liver and spleen. Tumor accumulation of 64Cu-MM-302 at 24 to 48 hours varied 35-fold (0.52–18.5 %ID/kg), including deposition in bone and brain lesions, and was independent of systemic plasma exposure. Computational analysis quantified rates of deposition and washout, indicating peak liposome deposition at 24 to 48 hours. Patients were classified on the basis of 64Cu-MM-302 lesion deposition using a cut-off point that is comparable with a response threshold in preclinical studies. In a retrospective exploratory analysis of patient outcomes relating to drug levels in tumor lesions, high 64Cu-MM-302 deposition was associated with more favorable treatment outcomes (HR = 0.42). Conclusions: These findings provide important evidence and quantification of the EPR effect in human metastatic tumors and support imaging nanoparticle deposition in tumors as a potential means to identify patients well suited for treatment with therapeutic nanoparticles. Clin Cancer Res; 23(15); 4190–202. ©2017 AACR.

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

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 naive 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

Systems biology driving drug development: from design to the clinical testing of the anti-ErbB3 antibody seribantumab (MM-121)

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.

Seribantumab, an anti-ERBB3 antibody, delays the onset of resistance and restores sensitivity to letrozole in an estrogen receptor-positive breast cancer model

Heregulin-driven ERBB3 signaling has been implicated as a mechanism of resistance to cytotoxic and antiendocrine therapies in preclinical breast cancer models. In this study, we evaluated the effects of seribantumab (MM-121), a heregulin-blocking anti-ERBB3 monoclonal antibody, alone and in combination with the aromatase inhibitor letrozole, on cell signaling and tumor growth in a preclinical model of postmenopausal estrogen receptor–positive (ER+) breast cancer. In vitro, heregulin treatment induced estrogen receptor phosphorylation in MCF-7Ca cells, and long-term letrozole-treated (LTLT-Ca) cells had increased expression and activation levels of EGFR, HER2, and ERBB3. Treatment with seribantumab, but not letrozole, inhibited basal and heregulin-mediated ERBB receptor phosphorylation and downstream effector activation in letrozole-sensitive (MCF-7Ca) and -refractory (LTLT-Ca) cells. Notably, in MCF-7Ca–derived xenograft tumors, cotreatment with seribantumab and letrozole had increased antitumor activity compared with letrozole alone, which was accompanied by downregulated PI3K/MTOR signaling both prior to and after the development of resistance to letrozole. Moreover, the addition of an MTOR inhibitor to this treatment regimen did not improve antitumor activity and was not well tolerated. Our results demonstrate that heregulin-driven ERBB3 signaling mediates resistance to letrozole in a preclinical model of ER+ breast cancer, suggesting that heregulin-expressing ER+ breast cancer patients may benefit from the addition of seribantumab to antiendocrine therapy. Mol Cancer Ther; 14(11); 2642–52. ©2015 AACR.

246PA META-ANALYSIS OF BIOMARKERS IN THREE RANDOMIZED, PHASE 2 STUDIES OF MM-121, A LIGAND-BLOCKING ANTI-ERBB3 ANTIBODY, IN PATIENTS WITH OVARIAN, LUNG, AND BREAST CANCERS

ABSTRACT Aim: Heregulin (HRG)-driven ErbB3 signaling mediates resistance to standard-of-care (SOC) cancer therapy in a variety of preclinical models. MM-121, a HRG-blocking anti-ErbB3 antibody, underwent clinical evaluation to determine if patients with advanced malignancies would derive benefit from the addition of MM-121 to their standard therapy. Potential predictive biomarkers were identified from a pre-specified set of mechanistic markers. Study BM+ n/N* BM+ Prevalence PFS HR in study PFS HR in measured BM PFS HR in BM+ PFS HR in BM- Ovarian 69/150 46% 1.03 [0.74-1.42] 1.10 [0.74-1.63] 0.40 [0.21-0.76] 2.02 [1.17-3.50] Lung 36/67 54% 0.82 [0.55-1.21] 0.91 [0.51-1.61] 0.39 [0.18-0.82] 2.43 [1.07-5.55] Breast 21/57 37% 0.75 [0.48-1.15] 0.68 [0.38-1.23] 0.35 [0.13-0.94] 0.99 [0.47-2.08] *n = BM + , N = patients with measured biomarkers Methods: Randomized Phase 2 studies were conducted in: i) platinum-resistant ovarian cancer, ii) EGFR wt NSCLC, and iii) ER/PR + , HER2- mBC. Patients were randomized to SOC therapy, with or without MM-121. Safety and clinical activity data from these studies have previously been presented. Tumor tissue was acquired from each patient and five pre-specified biomarkers were measured and correlated with clinical benefit: HRG, betacellulin, EGFR, HER2, and ErbB3. Protein levels were determined by quantitative IHC (qIHC) and mRNA levels by RT-PCR and RNA in-situ hybridization (RNA-ISH). Results: Among 464 patients (220 ovarian, 115 breast, 129 lung), RNA-ISH data were available from 224 patients (157 ovarian, 67 lung), qIHC from 252 (174 ovarian, 78 lung), and RT-PCR from 175 (105 ovarian, 57 breast, 13 lung). Of the five biomarkers, the most predictive of response was HRG mRNA: patients with detectable HRG in pre-treatment biopsies or high HRG in archived tissue blocks responded poorly to SOC therapy and benefited most from MM-121. In addition, benefit was largely restricted to patients with low ErbB2. Hazard ratios for PFS were calculated, defining biomarker positive (BM+) patients: Ovarian cancer, detectable HRG and low ErbB2; Lung cancer, detectable HRG; and, Breast cancer, high HRG. PFS hazard ratios with 95% CI and prevalence of the BM+ and BM- subpopulations are provided below. Conclusions: Heregulin is a potential biomarker for poor response to SOC therapy and a potential predictor of clinical benefit from MM-121 in late-stage ovarian, lung, and breast cancers. Disclosure: G. MacBeath is an employee of Merrimack Pharmaceuticals and holds stock in the company; B. Adiwijaya is an employee of Merrimack Pharmaceuticals, the sponsor of this clinical research, and holds stock in the company; J. Liu is an unpaid advisor for Merrimack Pharmaceuticals and was a Principal Investigator for one of the clinical studies included in this analysis; L.V. Sequist is an upaid advisor for Merrimack Pharmaceuticals and was a Principal Investigator of one of the clinical studies included in this analysis; E. Pujade-Lauraine serves as an upaid advisor to Merrimack Pharmaceuticals and Sanofi. He was also a lead investigator for one of the clinical studies included in this analysis; M. Higgins is an unpaid advisor to Merrimack Pharmaceuticals and Sanofi. She was a Principal Investigator of one of the clinical studies included in this analysis; I. Tabah-Fisch is an employee of Sanofi, a sponsor of the clinical studies summarized in this abstract, and holds stock in the company; J. Pearlberg is an employee of Sanofi, a sponsor of this clinical research, and holds stock in the company; V. Moyo, W. Kubasek, R. Nering and A. Czibere: Is an employee of Merrimack Pharmaceuticals, a sponsor of the clinical research, and holds stock in the company.

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

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.

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

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 A6: Sustained intratumoral activation of MM-398 results in superior activity over irinotecan demonstrated by using a systems pharmacology approach

MM-398 is a stable nanotherapeutic encapsulation of the prodrug irinotecan with an extended plasma half-life and higher intratumoral deposition compared with free-irinotecan. MM-398 is currently in multiple clinical trials, including a phase 3 trial for patients with advanced gemcitabine-resistant pancreatic cancer (NAPOLI-1). Pancreatic cancer has been described as being notoriously difficult to treat, potentially due to inadequate drug penetration through the dense stroma, or because the hypoxic tumor microenvironment suppresses cytotoxic activity. We sought to better understand how MM-398, a relatively large (100nm) liposomal nanotherapeutic, could potentially treat pancreatic cancer by determining the relative roles of systemic vs. local tumor activation of irinotecan in contributing to the activity of MM-398. Using a systems pharmacology approach, we developed a mechanistic pharmacokinetic (PK) model of MM-398 and free-irinotecan to predict both plasma and intratumoral levels of irinotecan and SN-38. The model was trained with PK and biodistribution data from mice bearing HT-29 xenografts, which were administered intravenously with varying doses of MM-398 or free-irinotecan. Model simulations predicted that MM-398 resulted in equivalent SN-38 exposure (area under curve, AUC) in tumor at a fivefold lower dose than free-irinotecan. However, an in vivo animal activity study showed that 15-fold lower dose of MM-398 was sufficient to yield equal growth inhibition of HT-29 xenografts, which reveals the limit of relating simple AUC-based exposure to in vivo tumor response. While intratumoral SN-38 exposure from free-irinotecan was limited to the first 48 hours after dosing, MM-398 maintained high levels of SN-38 throughout the week-long time window. Further analysis of the exposure-response identified that the duration of intratumoral SN-38 levels above the threshold was a valid predictive marker for xenograft tumor response. Identifying the source of intratumoral SN38 is confounded by the fact that the mouse species has an additional carboxylesterase (CES) that can convert irinotecan to SN-38 in serum. The serum SN-38/irinotecan ratio in mice is tenfold higher than that observed in humans. In order to translate this preclinical observation into the clinic, it is critical to identify the role of mouse-specific serum CES on intratumoral SN-38 exposure. Thus, we performed a PK study with knockout mice lacking the Ces1c gene, which encodes serum CES, and then retrained our mechanistic PK model. Serum SN-38 levels in the Ces1c knockout mice were measurably decreased by ˜85% in the central compartment. In contrast, simulating the effect of knock-out of either serum CES or tumor CES, predicts that the duration of intratumoral residence of SN-38 is significantly affected by tumor CES, rather than serum CES. This suggests that local activation to SN-38 by tumor CES as the main driver for SN-38 tumor residence, which in turn drives response. In summary, we applied a systems pharmacology approach to identify the importance of tumor CES (local SN-38 generation) as one of the determinants of MM-398 response. Liposomal encapsulation of irinotecan dramatically alters the pharmacokinetic profile of SN-38 in the tumor, as well as tumor response, by maintaining SN-38 levels above the response threshold. Local, sustained activity of this active irinotecan metabolite could result in prolonged cytotoxic and tumor microenvironment modifications with beneficial effects on treatment of pancreatic cancer and other solid tumors. Citation Format: Jaeyeon Kim, Eliel Bayever, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald, Ashish Kalra, Milind Chalishazar, Stephan Klinz, Nancy Paz, Bart Hendriks, Daryl Drummond, Dmitri Kirpotin, Victor Moyo. Sustained intratumoral activation of MM-398 results in superior activity over irinotecan demonstrated by using a systems pharmacology approach [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A6.