Ashish Kalra

Therapeutically Targeting ErbB3: A Key Node in Ligand-Induced Activation of the ErbB Receptor–PI3K Axis

Computational modeling of the ErbB signaling network affirms ErbB3 as a therapeutic target. Zooming In on ErbB3 Aberrant signaling involving the ErbB family of receptors, which can signal as homo- or heterodimers to activate the phosphatidylinositol 3-kinase (PI3K) signaling pathway, has been implicated as contributing to various cancers. Using a systems approach, Schoeberl et al. implicated ErbB3—a member of the ErbB family that is catalytically inactive—as critical to signaling stimulated by ligands that bind either ErbB1 or ErbB3. Computational analysis suggested that inhibiting ligand binding to ErbB3 might represent a more successful approach to treating cancers associated with ligand-induced stimulation of ErbB-PI3K signaling mediated by combinatorial receptor activation than do current therapies that target overexpressed or mutationally activated ErbB-family receptors. Moreover, experimental analysis revealed that a monoclonal antibody developed on the basis of this strategy could stop the growth of tumors grafted into immunodeficient mice. The signaling network downstream of the ErbB family of receptors has been extensively targeted by cancer therapeutics; however, understanding the relative importance of the different components of the ErbB network is nontrivial. To explore the optimal way to therapeutically inhibit combinatorial, ligand-induced activation of the ErbB–phosphatidylinositol 3-kinase (PI3K) axis, we built a computational model of the ErbB signaling network that describes the most effective ErbB ligands, as well as known and previously unidentified ErbB inhibitors. Sensitivity analysis identified ErbB3 as the key node in response to ligands that can bind either ErbB3 or EGFR (epidermal growth factor receptor). We describe MM-121, a human monoclonal antibody that halts the growth of tumor xenografts in mice and, consistent with model-simulated inhibitor data, potently inhibits ErbB3 phosphorylation in a manner distinct from that of other ErbB-targeted therapies. MM-121, a previously unidentified anticancer therapeutic designed using a systems approach, promises to benefit patients with combinatorial, ligand-induced activation of the ErbB signaling network that are not effectively treated by current therapies targeting overexpressed or mutated oncogenes.

Preclinical Activity of Nanoliposomal Irinotecan Is Governed by Tumor Deposition and Intratumor Prodrug Conversion

A major challenge in the clinical use of cytotoxic chemotherapeutics is maximizing efficacy in tumors while sparing normal tissue. Irinotecan is used for colorectal cancer treatment but the extent of its use is limited by toxic side effects. Liposomal delivery systems offer tools to modify pharmacokinetic and safety profiles of cytotoxic drugs. In this study, we defined parameters that maximize the antitumor activity of a nanoliposomal formulation of irinotecan (nal-IRI). In a mouse xenograft model of human colon carcinoma, nal-IRI dosing could achieve higher intratumoral levels of the prodrug irinotecan and its active metabolite SN-38 compared with free irinotecan. For example, nal-IRI administered at doses 5-fold lower than free irinotecan achieved similar intratumoral exposure of SN-38 but with superior antitumor activity. Tumor response and pharmacokinetic modeling identified the duration for which concentrations of SN-38 persisted above a critical intratumoral threshold of 120 nmol/L as determinant for antitumor activity. We identified tumor permeability and carboxylesterase activity needed for prodrug activation as critical factors in achieving longer duration of SN-38 in tumors. Simulations varying tumor permeability and carboxylesterase activity predicted a concave increase in tumor SN-38 duration, which was confirmed experimentally in 13 tumor xenograft models. Tumors in which higher SN-38 duration was achieved displayed more robust growth inhibition compared with tumors with lower SN-38 duration, confirming the importance of this factor in drug response. Overall, our work shows how liposomal encapsulation of irinotecan can safely improve its antitumor activity in preclinical models by enhancing accumulation of its active metabolite within the tumor microenvironment.

Comparing routes of delivery for nanoliposomal irinotecan shows superior anti-tumor activity of local administration in treating intracranial glioblastoma xenografts

BACKGROUND Liposomal drug packaging is well established as an effective means for increasing drug half-life, sustaining drug activity, and increasing drug efficacy, whether administered locally or distally to the site of disease. However, information regarding the relative effectiveness of peripheral (distal) versus local administration of liposomal therapeutics is limited. This issue is of importance with respect to the treatment of central nervous system cancer, for which the blood-brain barrier presents a significant challenge in achieving sufficient drug concentration in tumors to provide treatment benefit for patients. METHODS We compared the anti-tumor activity and efficacy of a nanoliposomal formulation of irinotecan when delivered peripherally by vascular route with intratumoral administration by convection-enhanced delivery (CED) for treating intracranial glioblastoma xenografts in athymic mice. RESULTS Our results show significantly greater anti-tumor activity and survival benefit from CED of nanoliposomal irinotecan. In 2 of 3 efficacy experiments, there were animal subjects that experienced apparent cure of tumor from local administration of therapy, as indicated by a lack of detectable intracranial tumor through bioluminescence imaging and histopathologic analysis. Results from investigating the effectiveness of combination therapy with nanoliposomal irinotecan plus radiation revealed that CED administration of irinotecan plus radiation conferred greater survival benefit than did irinotecan or radiation monotherapy and also when compared with radiation plus vascularly administered irinotecan. CONCLUSIONS Our results indicate that liposomal formulation plus direct intratumoral administration of therapeutic are important for maximizing the anti-tumor effects of irinotecan and support clinical trial evaluation of this therapeutic plus route of administration combination.

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.

Abstract 2065: Magnetic resonance imaging with an iron oxide nanoparticle demonstrates the preclinical feasibility of predicting intratumoral uptake and activity of MM-398, a nanoliposomal irinotecan (nal-IRI)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Sustained intratumoral delivery of cytotoxic agents is a major challenge for effective cancer treatment, and motivated the development of MM-398, a stable nanoliposomal irinotecan (nal-IRI) with an extended plasma half-life and greater tumor deposition than free irinotecan. By using a systems pharmacology approach, we have previously shown that tumor deposition of nal-IRI and the subsequent conversion of irinotecan to the active metabolite, SN-38, by carboxylesterases are important determinants for nal-IRI activity in vivo. Ferumoxytol (FMX) is a 30nm iron-oxide, super-paramagnetic nanoparticle, known to be taken up by macrophages (as is nal-IRI), and for exhibiting magnetic resonance imaging properties. Since the size of a nanoparticle affects the rate of transcapillary transport significantly, we hypothesized that nal-IRI tumor biodistribution may be predicted by FMX-based MRI (Fe-MRI). Biodistribution and imaging studies were performed in mice bearing cell-line derived (A2780, HT29, A549) and patient-derived (pancreatic adenocarcinoma) tumor xenografts. The protocol consisted of a baseline MRI scan, i.v. injection of FMX (20mg/kg), and then i.v. injection of fluorescently labeled nal-IRI (10mg/kg) 24hr later. Mice were sacrificed 24hr and 72hr after nal-IRI injection, and irinotecan and SN-38 concentrations were determined in plasma, tumor, and tissues by HPLC analysis. The presence of FMX did not interfere with nal-IRI PK or biodistribution. Cellular distribution of liposomes within tumors was also not affected by FMX at up to 50mg/kg as measured by flow cytometry. Furthermore, immunohistochemistry showed that both liposomes and FMX were co-localized with tumor-associated macrophages. The drug metabolite measurements from tissue samples showed that the xenograft tumor models display wide ranges of nal-IRI deposition capacity (irinotecan concentrations at 24hr: ∼2,104 to 20,096ng/g). A2780 tumors displayed highest concentration of both iron (3.92 μg/ml) and irinotecan (9,466 ng/g) at 72hr after nal-IRI injection, whereas A549 tumors displayed lowest levels of both iron (0.23 μg/ml ) and irinotecan (436 ng/g). We observed a correlation between the tumor Fe-MRI signal and intratumoral levels of irinotecan 72hr after nal-IRI injection (R2=0.9, p<0.001). Furthermore, in vivo activity studies confirmed that xenograft models having higher intratumoral levels of irinotecan and SN-38 at 72hr showed greater tumor growth inhibition. In summary, preclinical studies demonstrate the potential of utilizing Fe-MRI as a potential diagnostic tool to identify patients with higher tumor permeability. Based on encouraging preclinical data, a pilot study in patients with advanced solid tumors with extensive Fe-MRI scanning and paired tumor biopsies (NCT # 01770353) is being conducted. Citation Format: Ashish V. Kalra, Joseph Spernyak, Jaeyeon Kim, Arnold Sengooba, Stephan Klinz, Nancy Paz, Jason Cain, Walid Kamoun, Ninfa Straubinger, Yang Qu, Sheryl Trueman, Eliel Bayever, Ulrik Nielsen, Daryl Drummond, Jonathan Fitzgerald, Robert Straubinger. Magnetic resonance imaging with an iron oxide nanoparticle demonstrates the preclinical feasibility of predicting intratumoral uptake and activity of MM-398, a nanoliposomal irinotecan (nal-IRI). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2065. doi:10.1158/1538-7445.AM2014-2065

Photodynamic priming mitigates chemotherapeutic selection pressures and improves drug delivery

Physiologic barriers to drug delivery and selection for drug resistance limit survival outcomes in cancer patients. In this study, we present preclinical evidence that a subtumoricidal photodynamic priming (PDP) strategy can relieve drug delivery barriers in the tumor microenvironment to safely widen the therapeutic window of a nanoformulated cytotoxic drug. In orthotopic xenograft models of pancreatic cancer, combining PDP with nanoliposomal irinotecan (nal-IRI) prevented tumor relapse, reduced metastasis, and increased both progression-free survival and 1-year disease-free survival. PDP enabled these durable improvements by targeting multiple tumor compartments to (i) increase intratumoral drug accumulation by >10-fold, (ii) increase the duration of drug exposure above a critical therapeutic threshold, and (iii) attenuate surges in CD44 and CXCR4 expression, which mediate chemoresistance often observed after multicycle chemotherapy. Overall, our results offer preclinical proof of concept for the effectiveness of PDP to minimize risks of tumor relapse, progression, and drug resistance and to extend patient survival.Significance: A biophysical priming approach overcomes key treatment barriers, significantly reduces metastases, and prolongs survival in orthotopic models of human pancreatic cancer. Cancer Res; 78(2); 558-71. ©2017 AACR.

Abstract C293: Irinotecan sucrosofate liposome injection, MM-398, demonstrates superior activity and control of hypoxia as measured through longitudinal imaging using [18F]FAZA PET compared to free irinotecan in a colon adenocarcinoma xenograft model.

Tumor hypoxia is strongly linked to aggressive disease progression and resistance to therapy. Positron emission tomography (PET) imaging with hypoxia tracers such as [18F]fluoroazomycin arabinoside (FAZA) allows for non-invasive quantification of tumor hypoxia during treatment. We and others have previously shown by immunohistochemical methods that treatments with longer lasting camptothecin formulations reduce tumor hypoxia after either single or multiple treatment cycles. Here we evaluated the kinetics and magnitude of hypoxia changes in tumors after treatment with irinotecan sucrosofate liposome injection (MM-398) which has shown an extended plasma half-life and higher intratumoral deposition in animal models relative to free pro-drug and compare it to the effects of free irinotecan at equivalent exposure levels. FAZA-PET/CT was used for longitudinal monitoring of tumor hypoxia changes in the HT29 mouse colon cancer xenograft model over a 21-day period following weekly chemotherapy administrations of either MM-398 (5 & 10mg/kg) or free irinotecan (50mg/kg). These dosages were predicted to result in comparable SN-38 exposure in either plasma or tumor based on a mechanistic pharmacokinetic model of MM-398 and free irinotecan that was developed using a systems pharmacology approach. Baseline levels of FAZA uptake in tumors were similar across treatment groups. Significant differences in tumor FAZA uptake were observed between these groups as early as Day 7 post treatment initiation, with increased FAZA uptake seen in tumors treated with free irinotecan. In contrast, differences in tumor volume only became statistically significant on Day 16. MM-398 at 10mg/kg was the most effective treatment for control of tumor volume and also minimized changes in FAZA uptake at all time-points. Background FAZA levels in the muscle were consistent over time across all treatment groups (0.78±0.18 %ID/g, 0.81±0.11 %ID/g and 0.71±0.20 %ID/g). However, normalization with muscle signal did not improve quantification of FAZA uptake differences in tumors. Tumor-specific hypoxia status at the study end point was confirmed by co-staining for CA9 and EF5 levels, which were, as expected, highly correlated. Average EF5 intensity/tumor area was lowest in the MM-398 (10mg/kg) treatment group, while being highest in the irinotecan (50mg/kg) treatment group. This study demonstrated the feasibility of performing longitudinal and repeated tumor hypoxia assessment using FAZA-PET imaging. Treatment with MM-398, but not free irinotecan, led to significant changes in the tumor microenvironment as measured by reduced hypoxia levels that occurred far earlier than anatomical changes assessed by tumor volume. Imaging of hypoxia levels after anti-cancer therapy with MM-398 has the potential to allow early assessment of treatment activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C293. Citation Format: Stephan G. Klinz, Jinzi Zheng, Raquel De Souza, Michael Dunne, Jason Cain, Jaeyeon Kim, Nancy Paz, Ashish Kalra, David Jaffray, Jonathan Fitzgerald. Irinotecan sucrosofate liposome injection, MM-398, demonstrates superior activity and control of hypoxia as measured through longitudinal imaging using [18F]FAZA PET compared to free irinotecan in a colon adenocarcinoma xenograft model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C293.

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.

Abstract A63: MM-398/PEP02, a novel liposomal formulation of irinotecan, demonstrates stromal-modifying anticancer properties.

MM-398 is a stable nanotherapeutic encapsulation of the prodrug irinotecan (CPT-11) with longer plasma half-life and higher tumor deposition due to an enhanced permeability and retention effect. Pancreatic cancer has responded poorly to many therapeutics, largely because of inadequate drug penetration due to poor vascularization and the highly aggressive, hypoxic nature of the disease. We sought to better understand how MM-398, a relatively large (100nm) liposomal nanotherapeutic, could be used treat pancreatic cancer. We have tested MM-398 in several pancreatic xenograft models: BxPC3 (KRAS wild type), AsPC-1(KRAS G12D) , Panc-1 (KRAS G12D) and MiaPaCa (KRAS G12C). All models demonstrated complete tumor regression at 20 mg/kg or a human equivalent dose of 60-120 mg/m2. At this same dose, MM-398 suppresses tumor growth in a gemcitabine insensitive AsPC-1 xenograft. MM-398 functionally blocked AsPC-1 tumor cell proliferation as measured by ki-67 staining; however, gemcitabine administered at its maximum tolerated dose did not impact proliferation. MM-398 is currently in multiple clinical trials, including a phase 3 trial for patients with advanced gemcitabine-resistant pancreatic cancer (NAPOLI-1). In order to further understand mechanisms driving response to MM-398, we screened and ranked several cell lines for their ability to convert irinotecan into the active metabolite, SN38. BxPC3 and HT-29 tumors ranked highest in ability to convert irinotecan to SN-38, as measured by HPLC. In a BxPC3 pancreatic orthotopic model which spontaneously metastasizes, 10 mg/kg MM-398 significantly reduced both primary and metastatic tumor load as measured by ex vivo biophotonic imaging of BxPC3luc cells to spleen, lung, liver, diaphragm and GI associated lymph nodes (p In summary, MM-398 induces tumor regression in multiple mouse models of pancreatic cancer, including an orthotopic metastatic model. MM-398 activity may be driven in part by the ability to modify tumor microenvironment parameters, such as hypoxia and vascularization, both of which limit efficacy of chemotherapeutic agents in the treatment of pancreatic cancer. These data support the continued investigation of MM-398 in pancreatic cancer. Citation Format: Nancy Paz, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald, Ashish Kalra, Milind Chalishazar, Stephan Klinz, Jaeyeon Kim, Daryl Drummond, Dmitri Kirpotin, Victor Moyo, Eliel Bayever. MM-398/PEP02, a novel liposomal formulation of irinotecan, demonstrates stromal-modifying anticancer properties. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A63.

Abstract C207: Identifying differential mechanisms of action for MM-398/PEP02, a novel nanotherapeutic encapsulation of irinotecan.

MM-398 (aka PEP02) is a stable, nanotherapeutic encapsulation of the pro-drug CPT-11 (irinotecan) that is currently in clinical development. In preclinical experiments, treatment with MM-398 resulted in significantly higher intratumoral concentrations of both irinotecan (142-fold) and its major metabolite, SN-38 (9-fold) and exhibited superior anti-tumor activity compared to free irinotecan in multiple tumor xenografts. Subsequently, multiple phase 1 and 2 studies have established a pharmacokinetic and safety profile that supports continued clinical development, including in pancreatic, gastric, colorectal and potentially other cancers. Because current evidence suggests that resistance to pancreatic cancer is driven largely by inadequate drug penetration into these often poorly vascularized, stromally dense and hypoxic tumors, we sought to better understand how this relatively large (100nm) liposomal nanotherapeutic could potentially result in increased efficacy in very advanced gemcitabine-resistant pancreatic cancer and other cancer types. We developed a mechanism-based PK model of MM-398 and free irinotecan designed to predict intratumor SN-38 levels. Sensitivity analysis revealed that for MM-398 local activation of irinotecan to SN-38 was a far more important parameter than systemic activation. Through cellular uptake studies, we demonstrated in vitro that MM-398 was preferentially internalized by phagocytic macrophage/monocyte cell lines and, to a far lesser extent, by tumor cell lines. Furthermore, tumor microdistribution studies by flow cytometry and IHC showed uptake of MM-398 liposomes in both tumor cells and tumor-associated macrophages with more liposomal material being present in the macrophages. This distribution also suggests that macrophages may contribute to the postulated rate limiting process of irinotecan activation. The sensitivity analysis also suggested that tumor permeability and vascularization are important determinants of tumor-associated SN-38 levels for both free irinotecan and MM-398. To determine the effect of MM-398 on these parameters we treated mice bearing HT29 (colorectal cancer) xenografts with a single dose of MM-398 and measured hypoxic markers (CAIX) and microvessel density (CD31) by IHC. Tumors treated with MM-398 showed a greater degree of CD31 staining and lower CAIX staining, indicating that MM-398 may be able to affect tumor characteristics that traditionally have contributed to therapy resistance and limited the delivery of cancer therapeutics and resistance. In summary, encapsulation of irinotecan alters rate-limiting processes that determine tumoral SN-38 levels. Delivery of MM-398 is believed to alter tumor microvessel density and decrease hypoxia. These intriguing mechanisms of action findings support the continued clinical development of MM-398 as a differentiated therapeutic for several cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C207.