Matthew Onsum

An ErbB3 Antibody, MM-121, Is Active in Cancers with Ligand-Dependent Activation

ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in epidermal growth factor receptor (EGFR; ErbB1), ErbB2 [human epidermal growth factor receptor 2 (HER2)], and [hepatocyte growth factor receptor (MET)] addicted cancers, and reactivation of ErbB3 is a prominent method for cancers to become resistant to ErbB inhibitors. In this study, we evaluated the in vivo efficacy of a therapeutic anti-ErbB3 antibody, MM-121. We found that MM-121 effectively blocked ligand-dependent activation of ErbB3 induced by either EGFR, HER2, or MET. Assessment of several cancer cell lines revealed that MM-121 reduced basal ErbB3 phosphorylation most effectively in cancers possessing ligand-dependent activation of ErbB3. In those cancers, MM-121 treatment led to decreased ErbB3 phosphorylation and, in some instances, decreased ErbB3 expression. The efficacy of single-agent MM-121 was also examined in xenograft models. A machine learning algorithm found that MM-121 was most effective against xenografts with evidence of ligand-dependent activation of ErbB3. We subsequently investigated whether MM-121 treatment could abrogate resistance to anti-EGFR therapies by preventing reactivation of ErbB3. We observed that an EGFR mutant lung cancer cell line (HCC827), made resistant to gefitinib by exogenous heregulin, was resensitized by MM-121. In addition, we found that a de novo lung cancer mouse model induced by EGFR T790M-L858R rapidly became resistant to cetuximab. Resistance was associated with an increase in heregulin expression and ErbB3 activation. However, concomitant cetuximab treatment with MM-121 blocked reactivation of ErbB3 and resulted in a sustained and durable response. Thus, these results suggest that targeting ErbB3 with MM-121 can be an effective therapeutic strategy for cancers with ligand-dependent activation of ErbB3.

Computational Modeling of ERBB2-Amplified Breast Cancer Identifies Combined ErbB2/3 Blockade as Superior to the Combination of MEK and AKT Inhibitors

Computational modeling of signaling feedback in ErbB2-positive breast cancer predicts improved combination therapies. Modeling Optimal Therapeutic Strategies Drug resistance is a common cause of therapy failure in cancer, and identifying optimal therapeutic strategies is difficult because of complex feedback, crosstalk, and redundancy in cellular signaling networks. Using cellular data, Kirouac et al. constructed an in silico model of signaling circuits activated by the ErbB family of receptors in cells with a genomic amplification of ERBB2. Predicted in silico and validated in cultured ERBB2-amplified cells, ErbB3 was activated in response to kinase-targeted therapeutics, such as the ErbB2 inhibitor lapatinib, and ErbB3 activity promoted drug resistance in breast cancer cells. Adding an ErbB3 inhibitor (MM-111) either to lapatinib and trastuzumab treatment or to inhibitors of the kinases AKT and MEK effectively reduced tumor growth in mice bearing ErbB2-overexpressing xenografts. The findings indicate that combination therapies inhibiting ErbB3 are an improved therapeutic option for HER2-positive breast cancer patients. Crosstalk and compensatory circuits within cancer signaling networks limit the activity of most targeted therapies. For example, altered signaling in the networks activated by the ErbB family of receptors, particularly in ERBB2-amplified cancers, contributes to drug resistance. We developed a multiscale systems model of signaling networks in ERBB2-amplified breast cancer to quantitatively investigate relationships between biomarkers (markers of network activity) and combination drug efficacy. This model linked ErbB receptor family signaling to breast tumor growth through two kinase cascades: the PI3K/AKT survival pathway and the Ras/MEK/ERK growth and proliferation pathway. The model predicted molecular mechanisms of resistance to individual therapeutics. In particular, ERBB2-amplified breast cancer cells stimulated with the ErbB3 ligand heregulin were resistant to growth arrest induced by inhibitors of AKT and MEK or coapplication of two inhibitors of the receptor ErbB2 [Herceptin (trastuzumab) and Tykerb (lapatinib)]. We used model simulations to predict the response of ErbB2-positive breast cancer xenografts to combination therapies and verified these predictions in mice. Treatment with trastuzumab, lapatinib, and the ErbB3 inhibitor MM-111 was more effective in inhibiting tumor growth than the combination of AKT and MEK inhibitors and even induced tumor regression, indicating that targeting both ErbB3 and ErbB2 may be an improved therapeutic approach for ErbB2-positive breast cancer patients.

Single-Cell Quantitative HER2 Measurement Identifies Heterogeneity and Distinct Subgroups within Traditionally Defined HER2-Positive Patients

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues.

Abstract 4633: MM-111, a bispecific HER2 and HER3 antibody, synergistically combines with trastuzumab and paclitaxel in preclinical models of gastric cancer.

Increasing evidence suggests amplification or overexpression of human epidermal growth factor receptor-2 (HER2), and HER3 levels are correlated to decreased survival in gastric cancers. Our previous studies established that MM-111, a novel bispecific antibody that specifically targets the HER2/HER3 heterodimer, blocks heregulin (HRG) binding to HER3 and corresponding downstream signaling pathways. In this study we used computational and experimental biology to assess the activity of MM-111 combination therapies in treating gastric cancer. First, we utilized a multi-scale computational network model of HER2-positive tumors that relates HER family signal transduction with cell growth to predict that MM-111 plus trastuzumab would synergistically inhibit tumor growth. The mechanism underlying this prediction is that the combinatorial blockade of HER2 and HER3 suppresses signal propagation through both the AKT and ERK cascades and this leads to synergistic cell growth arrest. Cell viability and signaling experiments in a gastric cancer model (NCI-N87) confirmed these predictions. In particular, MM-111 plus trastuzumab caused 25% greater cell growth inhibition than the additive effects of the individual treatments (compared to Bliss independence; p −8 ). We then assessed the effect of adding paclitaxel to the trastuzumab plus MM-111 combination. Our key finding was that activation of HER3 by HRG greatly reduced paclitaxel9s activity. MM-111, but not trastuzumab, restored cell sensitivity to paclitaxel in the presence of HRG. A synergistic effect (35% greater than Bliss independence; p −8 ) was observed for the combination of MM-111, trastuzumab and paclitaxel. Similar combination effects were also achieved using a three-dimensional NCI-N87 spheroid assay: Inhibition of HER2, HER3, and the AKT and ERK pathways correlated with spheroid growth inhibition. Furthermore, HRG-overexpressing NCI-N87 (NCI-N87-HRG) xenografts were less sensitive to paclitaxel than the wild type xenografts. MM-111 restored the sensitivity to paclitaxel in these xenografts, even when given as a second line treatment regimen after tumor progression on the front-line regimen (trastuzumab plus 5-fluorouracil). Trastuzumab alone did not overcome the HRG-induced paclitaxel resistance, but its combination with MM-111 showed significantly greater activity than either drug alone. Taken together, our data suggest that combination of MM-111, trastuzumab and paclitaxel is worthy of investigation as a potential therapeutic strategy for the treatment of HER2-positive gastric cancer. Citation Format: Bo Zhang, Johanna Lahdenranta, Jinyan Du, Daniel Kirouac, Stephanie Nguyen, Ryan Overland, Violette Paragas, Arthur Kudla, Ulrik Nielsen, Charlotte McDonagh, Matthew Onsum. MM-111, a bispecific HER2 and HER3 antibody, synergistically combines with trastuzumab and paclitaxel in preclinical models of gastric 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 4633. doi:10.1158/1538-7445.AM2013-4633

Abstract LB-410: Phase I dose escalation study of MM-121, a fully human monoclonal antibody to ErbB3, in patients with advanced solid tumors

Introduction: ErbB3 has been identified as a key driver of the PI3K/AKT signaling pathway, resulting in cell growth and survival. MM-121 is a fully human monoclonal antibody targeting ErbB3. By binding to ErbB3, MM-121 blocks heregulin, thereby inhibiting ligand-induced ErbB3 heterodimerization and activation of receptors. Binding of MM-121 also induces downregulation of ErbB3 from the cell surface. Methods: Patients ≥ 18 years with refractory advanced solid tumors were given MM-121 weekly in 4-week cycles at 6 dose levels. Dose escalation was based on dose limiting toxicities (DLTs) during cycle 1. Primary objectives were to determine the maximum tolerated dose (MTD) of MM-121 and describe any objective response and progression free survival. Secondary objectives were to describe the safety, pharmacokinetic (PK), and pharmacodynamic profile of MM-121. A dose expansion cohort at the highest dose cohort was also included for selected tumor types, with required pre- and post-treatment biopsies to assess MM-121 target effects, ErbB3 pathway markers, and guide dose schedule decisions. Results: Between July 2008 to January 2011, 38 patients have been enrolled in the study. Twenty-five patients were enrolled in the dose escalation part of the study and treated until unacceptable toxicity or progression. Six cohorts were completed and an MTD was not reached. Grade 1/2 nausea, diarrhea, fatigue and rash were the most commonly observed adverse events. Grade 1 hypomagnesemia was observed in 4 patients at different doses. One patient experienced a DLT of confusion at the lowest dose, considered possibly related to MM-121 treatment but confounded by other comorbidities. No other treatment related serious AEs were reported. An additional 13 patients have been enrolled to date in an expansion cohort. The overall safety profile for patients in the expansion cohort is similar to that observed for the dose escalation cohorts. Conclusion: In this single agent, first in human, dose escalation phase I study MM-121 was well tolerated with a favorable safety profile. Enrollment in the dose expansion cohort is ongoing. 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 LB-410. doi:10.1158/1538-7445.AM2011-LB-410

Abstract P1-07-03: Quantification of HER2 expression at the single cell level and HER2 intratumoral heterogeneity of breast cancer tissue samples using automated image analysis

HER2 is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or FISH testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to accurate HER2 assessment and patient stratification may include intratumoral heterogeneity, lack of assays that are quantitative and/or objective, and differences between measuring HER2 at the protein vs. DNA level. We have developed a novel immunofluorescence method for absolute quantitation of HER2 protein expression at the single cell level on formalin-fixed, paraffin-embedded (FFPE) patient samples. Our assay utilizes automated image analysis software to identify cells, classify them as tumor or non-tumor cells, and quantitate the level of HER2 staining for each tumor cell. The HER2 staining level is then converted to absolute HER2 receptor numbers per cell using an array composed of cell lines that span a range of HER2 levels. This cell pellet array standard is stained in parallel with each tissue sample. With this approach we quantitate HER2 expression at the single cell level and describe the heterogeneity of HER2 expression within breast cancer tissue samples. Our assay provides additional insight into the absolute level of HER2 expression and heterogeneity of HER2 expression within tumors, and allows for direct comparison with the currently existing HER2 assessments by standard IHC and/or FISH. This new assay could be used to increase understanding of the relationship between HER2 expression and patient response to HER2-targeted therapies. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-03.

Abstract 1888: MM-111, a bispecific HER2 and HER3 antibody, inhibits trastuzumab-resistant tumor cell growth

Amplification of human epidermal growth factor receptor 2 (HER2) occurs in approximately 25% of breast cancers and is associated with increased disease recurrence and poor prognosis. Trastuzumab, a monoclonal antibody targeting HER2, has demonstrated clinical benefit in HER2 over-expressing tumors. However, acquired resistance and disease progression is widely observed in patients treated with trastuzumab. In this experiment our objectives were to dissect the dynamic, molecular mechanisms involved in acquired resistance to trastuzumab and to determine whether MM-111, a novel bispecific antibody fusion protein that specifically targets the HER2/HER3 heterodimer and blocks heregulin binding to HER3, has activity in trastuzumab-resistant tumor cells. BT474 cells were cultured in the presence of trastuzumab and cells were periodically tested for response to trastuzumab and MM-111. Samples were also collected for protein and RNA analyses. Resistance to trastuzumab gradually increased in BT474 cells after four months of exposure to trastuzumab, as measured by a cell proliferation assay. Quantitative flow cytometry analysis showed EGFR, HER2, and HER3 levels on the cell surface of resistant cells were similar to the parental cells. Phospho-protein kinase antibody arrays revealed that signaling pathways associated with the ERK cascade were activated during the development of drug resistance. Western blotting further confirmed that phosphorylation of EGFR, ERK, CREB, c-Jun, and AFT-1 was increased in the resistant cells. Real-time polymerase chain reaction also showed transcript levels of HER ligands, including HRG1α, HRG1α, betacellulin, amphiregulin, epigen, TGFβ, and HB-EGF, dramatically increased in tumor cells that acquired resistance to trastuzumab. Compared to the BT474 parental cells, MM-111 showed a greater inhibition in trastuzumab-resistant cells in a spheroid growth assay. Furthermore, trastuzumab-resistant cells became more sensitive to gefitinib and erlotinib, both EGFR inhibitors. The combination of gefitinib or erlotinib with MM-111 showed greater inhibition than either drug alone. In conclusion, our data suggest that one mechanism by which HER2 overexpressing breast cancer cells develop resistance to trastuzumab is to up-regulate ligand-dependent EGFR and HER3 signaling pathways. The use of MM-111 and EGFR inhibitors may provide an effective therapeutic strategy for the treatment of trastuzumab-resistant cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1888. doi:1538-7445.AM2012-1888

Abstract A10: In silico design of biomarker-optimized drug combinations in ERBB2+ cancers

Crosstalk and compensatory circuits within cancer signaling networks fundamentally limit the activity of targeted therapies. Combination drug regimens are thus required to fully inhibit an oncogenic network. However, rationally designing optimal combinations, given the large number of targeted agents available remains a challenge. Previously, we developed a multi-scale systems model of ERBB2-high breast cancer to quantitatively interrogate relationships between biomarkers (proxies for the mode of network activation) and combination drug efficacy. We found that targeting the ERBB2-ERBB3 heterodimer with a combination of ERBB2 inhibitors (trastuzumab and lapatinib) and an ERBB3 inhibitor (MM-111) was more effective at inducing tumor regression than the combination of an AKT and MEK inhibitor (MK2206 and GSK1120212), and was significantly better tolerated. However, the model was parameterized based on data collected from one cell line. Here we have performed profiling of an 18-cell line panel to assess whether our model and results are extendable to other ERBB2-high cancers. We monitored cell viability and signaling events upon AKT and MEK inhibitor treatments in the presence or absence of the ERBB3 ligand heregulin in these cell lines. While all cells were ERBB2+, we observed widely variable phenotypic and signaling dependencies on the PI3K/AKT and MAPK/ERK pathway activities across the panel. At the phenotypic level, cells primarily depend on either AKT or ERK signaling in basal conditions. Interestingly, upon heregulin stimulation some cells lines switch pathway dependency from AKT to ERK. Adaptive feedback circuits downstream of ERK and AKT were identified in all cell lines, though the identity and strength vary extensively. ERBB3 signaling and total AKT were consistently up-regulated to various degrees upon AKT inhibitor treatment. In contrast, multiple ERBB receptors as well as other RTKS, AKT signaling, and total ERK were upregulated in the response to MEK inhibition. While it is plausible that signaling pathways beyond AKT and ERK are modulating cell viability, we are able to quantitatively describe cell growth regulation based on AKT and ERK pathway activity using quantitative logic-based modeling framework. Our results will help us better understand how signaling events are decoded by cancer cells into phenotypic responses, and enable in silico drug combination screening across molecularly and functionally heterogeneous cancers. Citation Format: Jinyan Du, Daniel Kirouac, Johanna Lahdenranta, Ryan Overland, Matthew Onsum, Charlotte McDonagh. In silico design of biomarker-optimized drug combinations in ERBB2+ cancers. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A10.

Abstract 4945: Targeting ErbB3-addicted cancers across the HER2 spectrum

ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in EGFR-, HER2-, and MET-addicted cancers, and ligand-driven activation of ErbB3 is a prominent method by which cancers become resistant to targeted inhibitors of ErbB signaling, as well as anti-hormonal and chemotherapeutic agents. With the goals of inhibiting survival signaling in cancer cells, delaying the onset of resistance to standard-of-care therapies, and re-sensitizing cancer cells to prior therapies, we developed an anti-ErbB3 monoclonal antibody, MM-121, that effectively blocks ligand-dependent activation of ErbB3 and induces internalization and degradation of the receptor. The activity of single-agent MM-121 was examined in xenograft models and a machine-learning algorithm was developed that accurately predicts which preclinical models of cancer will respond to MM-121 based on five protein-based biomarkers. By simulating ErbB3 inhibition using a computational model of ErbB signaling, we predicted that a bispecific molecule that docks onto HER2 and subsequently binds to ErbB3 would effectively block ErbB3-driven signaling in HER2-amplified tumors. We therefore developed a bispecific antibody comprising two human scFv antibodies linked by a modified form of human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with HER2 and ErbB3, effectively inhibiting ErbB3 signaling and demonstrating antitumor activity in preclinical models that are dependent on HER2 overexpression. Both MM-121 and MM-111 are now in early stages of clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4945. doi:1538-7445.AM2012-4945

Abstract C27: Targeting ErbB3 and EGFR in lung cancer patients: A phase I trial of MM-121 in combination with erlotinib in patients with non-small cell lung cancer (NSCLC).

Background: The benefit of EGFR tyrosine kinase inhibitors (TKIs) is largely restricted to EGFR mutation-positive cancers and resistance invariably develops. A central theme of acquired resistance is persistent ErbB3 signaling, resulting in activation of the PI3K/AKT survival pathway. MM-121 is a fully human IgG1 monoclonal antibody (mAb) to ErbB3 with pre-clinical activity as a single agent and in combination with erlotinib in NSCLC, particularly in cancers with ligand-dependent activation of EGFR. This phase 1 study evaluated the safety and tolerability of MM-121 and erlotinib in NSCLC, as well as PK, immunogenicity, efficacy endpoints and exploratory biomarker evaluation. Methods: Patients with advanced NSCLC, good performance status and adequate organ function were enrolled. Patients were EGFR TKI-naive, unless they were EGFR mutant, in which case acquired resistance was allowed. MM-121 was administered weekly and erlotinib was administered daily. Seven cohorts were enrolled, evaluating varying dose levels of the combination, as well as alternate MM-121 infusion schedules. Dose levels were determined by safety and pharmacokinetic (PK) data. Results: Between February 2010 and July 2011, 33 patients were enrolled. Median age was 64 years and there were 19 (57.5%) women. Twenty-four patients were erlotinib-naive and 1 patient was an EGFR mutant. The most frequent adverse events were rash, diarrhea, nausea and fatigue. As of 31 July 2001, 16 patients remain on study. Full results will be presented at the meeting. Conclusions: In this phase 1 dose escalation study, MM-121 plus erlotinib was well tolerated by the majority of patients. A phase 2 study is planned. Reference:NCT00994123 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 C27.