Arthur J. Kudla

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.

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 3485: MM-111, an ErbB2/ErbB3 bispecific antibody with potent activity in ErbB2-overexpressing cells, positively combines with trastuzumab to inhibit growth of breast cancer cells driven by the ErbB2/ErbB3 oncogenic unit

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC MM-111 is a novel bispecific antibody fusion protein which targets the ErbB2/ErbB3 oncogenic unit, blocking activation of the phosphatidylinositol 3-kinase (PI3K) pro-survival pathway. The anti-ErbB2 arm of MM-111 binds with high affinity to the ErbB2 receptor, which localizes the bispecific molecule to ErbB2 over-expressing tumor cells and promotes binding of the anti-ErbB3 arm to the ErbB3 receptor. MM-111 binding to ErbB3 results in inhibition of ErbB3 signaling by blocking the binding of the ErbB3 physiological ligand heregulin. MM-111 treatment of ErbB2 overexpressing cancer cells inhibits activation of the PI3K pathway with sub-nanomolar potency, blocks cell cycle progression and attenuates tumor cell growth in multiple xenograft models. ErbB2 over-expressing tumor cells are addicted to growth signals provided by the ligand-activated ErbB2/ErbB3 heterodimer. Activation of downstream PI3K pathway signaling also occurs through ligand-independent ErbB2/ErbB2 homodimers and ErbB2/ErbB3 heterodimers. Recently the ErbB2-targeted therapeutic antibody trastuzumab was shown to inhibit basal ErbB3 signaling in the absence of ligand stimulation, purportedly by interrupting ligand-independent ErbB2/ErbB3 heterodimers formed through overexpression of ErbB2. However, in these studies trastuzumab did not effectively block ligand-induced activation of the ErbB2/ErbB3 oncogenic unit. Indeed, there is emerging evidence that ligand-induced ErbB3 activation may have an important role in resistance to trastuzumab. As MM-111 and trastuzumab have distinct and potentially complimentary effects on signaling in cells overexpressing ErbB2, we hypothesized that their combination may synergistically effect inhibition of tumor cell growth driven by the ErbB2/3 signaling network. Our data demonstrate that MM-111 and trastuzumab positively combine to inhibit breast cancer growth in multiple in vitro breast cancer models expressing ErbB2 and ErbB3. Further, in the BT-474 breast cancer xenograft model the combination of MM-111 and trastuzumab results in greater inhibition of tumor growth and an increased number of completely regressed tumors compared to the monotherapy treatment groups. Pharmacodynamic analysis of samples from these studies show that repeated administration of the combined therapeutic agents results in strong inhibition of the ErbB3/PI3K pathway. In conclusion, we show that MM-111 and trastuzumab inhibit the growth of ErbB2 over-expressing breast tumors with distinct mechanisms that act synergistically in combination. Concurrent treatment with MM-111 and trastuzumab may provide a potent therapeutic regimen for ErbB2-overexpressing breast cancer patients and potentially deter acquired resistance to trastuzumab through ErbB3 activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3485.

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 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 655: Combination of MM-111, an ErbB2/ErbB3 bispecific antibody, with endocrine therapies as an effective strategy for treatment of ER+/HER2+ breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Approximately 75% of breast cancers are estrogen receptor (ER) positive. Although endocrine therapies such as tamoxifen, fulvestrant, and letrozole have demonstrated significant efficacy in treating ER+ breast cancer patients, intrinsic or acquired resistance has limited their success. Recent studies suggest that crosstalk between ErbB receptor signaling and ER signaling may contribute to resistance to endocrine therapy. Overexpression of human epidermal growth factor receptor 2 (HER2, synonymous with ErbB2) and upregulation of the ErbB3 ligand heregulin are associated with poor prognosis and reduced overall survival. MM-111 is a novel bispecific antibody fusion protein that specifically targets the ErbB2/ErbB3 heterodimer and blocks heregulin binding to ErbB3. MM-111 inhibits ligand-induced ErbB3 phosphorylation, tumor cell cycle progression, and tumor growth when ErbB2 is overexpressed. We hypothesized that combination of endocrine therapies with MM-111 may improve anti-tumor efficacy. In an estrogen-stimulated BT474-M3 ER-positive breast cancer cell three-dimensional spheroid assay, MM-111, when used as a single agent, showed growth inhibitory effects similar to the anti-estrogen drugs tamoxifen and fulvestrant. Combination of MM-111 with anti-estrogen therapy showed superior activity to either drug alone. In the presence of heregulin, MM-111 maintained its growth inhibitory activity, whereas the inhibitory effect of tamoxifen and fulvestrant was diminished. This suggests that activation of ErbB3 confers tumor cell resistance to anti-estrogen therapies. When both estrogen and heregulin were present, the combination of MM-111 and the anti-estrogen drugs demonstrated a significantly greater inhibitory effect than either drug alone. Western blot analysis showed that treatment of BT-474-M3 cells with the combination of MM-111 and the anti-estrogen drugs significantly increased apoptosis markers such as cytochrome C and BAX. Furthermore, an in vivo BT474-M3 xenograft model showed resistance to tamoxifen treatment (5 mg/pellet, 60-day release). In this xenograft model MM-111 sensitized tumor response to tamoxifen and the combination treatment dramatically inhibited tumor growth. In conclusion, the combination of MM-111 and endocrine therapies may provide a potent regimen that overcomes acquired resistance to endocrine therapies in ER+, ErbB2-overexpressing breast cancer patients. 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 655. doi:10.1158/1538-7445.AM2011-655

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 654: MM-111, an ErbB2/ErbB3 bispecific antibody, effectively combines with lapatinib to inhibit growth of ErbB2-overexpressing tumor cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The oncogenic receptor ErbB2 (HER2) is frequently overexpressed in a variety of cancer indications, including breast, gastric, bladder, and lung. ErbB3 (HER3), the preferred dimerization partner of ErbB2, is critical for the survival and growth of ErbB2-overexpressing tumors, but is poorly inhibited by current ErbB2-targeted therapies. MM-111 is a novel bispecific antibody designed to specifically inhibit function of the ErbB2/ErbB3 heterodimer in ErbB2-overexpressing tumors. MM-111 binds simultaneously to ErbB2 and ErbB3 and blocks the interaction of ErbB3 with its ligand, heregulin. Inhibiting the ErbB2/ErbB3 heterodimer prevents ligand-induced activation of the phosphotidylinositol 3-kinase (PI3K) pro-survival pathway, altering cell cycle progression and inhibiting tumor growth. Upregulation of both heregulin and ErbB3 has been observed in tumors that have acquired resistance to the ErbB2-targeted therapies trastuzumab and lapatinib. In addition, ErbB2 and ErbB3 are frequently upregulated in estrogen receptor positive breast cancers that have developed resistance to anti-estrogen therapies. These data indicate that the ErbB2/ErbB3 receptor heterodimer potently activates growth of tumors characterized by ErbB2 overexpression and implicate ErbB3 as a mechanism of resistance to current therapies. Because of its ability to inhibit ErbB3 activity, MM-111 could be effective in combination with ErbB2-targeted therapies by overcoming ErbB3-mediated resistance. Previous data demonstrating that MM-111 acts in concert with trastuzumab to inhibit growth of ErbB2-overexpressing human tumor models support this hypothesis. We now show that MM-111 effectively combines with lapatinib to inhibit growth of tumors driven by the ErbB2/ErbB3 heterodimer. MM-111, as a single agent, is more effective than lapatinib at inhibiting heregulin-driven activation of ErbB3 and AKT in ErbB2-overexpressing cancer cell lines. In these same cell lines, the combination of MM-111 and lapatinib results in greater AKT inhibition than either treatment alone. In addition, we have demonstrated in ErbB2-overexpressing 3D spheroid cancer models that treatment with the MM-111/ lapatinib combination increases the number of cells in early phase apoptosis as compared to single-drug treatments. Finally, in an ErbB2-overexpressing cancer xenograft model, the combination of MM-111 and lapatinib results in greater inhibition of tumor growth than either therapy alone. In conclusion, we show that MM-111 and lapatinib positively combine to inhibit the growth of ErbB2-overexpressing tumors. Our results suggest that concurrent treatment with MM-111 and lapatinib may provide a potent therapeutic regimen for cancer patients whose tumors overexpress ErbB2 by deterring ErbB3-mediated resistance to lapatinib. 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 654. doi:10.1158/1538-7445.AM2011-654

Abstract A19: Identification of Heregulin (HRG) expression as a driver of a difficult-to-treat cancer phenotype and development of a companion diagnostic for the HRG-ErbB3 targeting drug seribantumab

Heregulin (HRG) is the cognate ligand of the ErbB3 receptor 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. Seribantumab (MM-121) is a fully human monoclonal antibody designed to block HRG from binding to ErbB3 and to prevent the establishment of HRG-driven cancer cell survival in response to cytotoxic therapies. Clinical evidence from randomized studies suggests that patients who receive standard therapy have shorter progression-free survival (PFS) when their tumors are positive for HRG RNA expression compared to patients receiving standard therapy with HRG-negative tumors. The addition of seribantumab was able to enhance the anti-tumor effect of these respective standard therapies and prolong PFS in patients with HRG-positive tumors. Profiling data indicates that positive HRG expression can be found in more than a third of most solid tumors identifying a high unmet medical need. In order to identify HRG-driven tumors with a more difficult-to-treat phenotype, an RNA in situ hybridization (ISH) diagnostic assay has been developed. Details will be presented illustrating the benefits of RNA ISH over alternate detection methods for HRG. This includes the visualization of tumor cell HRG expression with high sensitivity and specificity in small tissue specimens such as fine needle aspirates and core needle biopsies. This assay is currently being used to select patients for inclusion into diagnostic-driven clinical studies. Citation Format: Sara Mathews, Gregory Finn, Arthur J. Kudla, Victoria Rimkunas, Peter Laivins, Gavin MacBeath, Akos Czibere, Jason Baum. Identification of Heregulin (HRG) expression as a driver of a difficult-to-treat cancer phenotype and development of a companion diagnostic for the HRG-ErbB3 targeting drug seribantumab. [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 A19.