Disentangling ERBB Signaling in Breast Cancer Subtypes-A Model-Based Analysis.

Targeted therapies have shown striking success in the treatment of cancer over the last years. However, their specific effects on an individual tumor appear to be varying and difficult to predict. Using an integrative modeling approach that combines mechanistic and regression modeling, we gained insights into the response mechanisms of breast cancer cells due to different ligand-drug combinations. The multi-pathway model, capturing ERBB receptor signaling as well as downstream MAPK and PI3K pathways was calibrated on time-resolved data of the luminal breast cancer cell lines MCF7 and T47D across an array of four ligands and five drugs. The same model was then successfully applied to triple negative and HER2-positive breast cancer cell lines, requiring adjustments mostly for the respective receptor compositions within these cell lines. The additional relevance of cell-line-specific mutations in the MAPK and PI3K pathway components was identified via L1 regularization, where the impact of these mutations on pathway activation was uncovered. Finally, we predicted and experimentally validated the proliferation response of cells to drug co-treatments. We developed a unified mathematical model that can describe the ERBB receptor and downstream signaling in response to therapeutic drugs targeting this clinically relevant signaling network in cell line that represent three major subtypes of breast cancer. Our data and model suggest that alterations in this network could render anti-HER therapies relevant beyond the HER2-positive subtype.

Stromal NRG1 in luminal breast cancer defines pro-fibrotic and migratory cancer-associated fibroblasts.

HER3 is highly expressed in luminal breast cancer subtypes. Its activation by NRG1 promotes activation of AKT and ERK1/2, contributing to tumour progression and therapy resistance. HER3-targeting agents that block this activation, are currently under phase 1/2 clinical studies, and although they have shown favorable tolerability, their activity as a single agent has proven to be limited. Here we show that phosphorylation and activation of HER3 in luminal breast cancer cells occurs in a paracrine manner and is mediated by NRG1 expressed by cancer-associated fibroblasts (CAFs). Moreover, we uncover a HER3-independent NRG1 signaling in CAFs that results in the induction of a strong migratory and pro-fibrotic phenotype, describing a subtype of CAFs with elevated expression of NRG1 and an associated transcriptomic profile that determines their functional properties. Finally, we identified Hyaluronan Synthase 2 (HAS2), a targetable molecule strongly correlated with NRG1, as an attractive player supporting NRG1 signaling in CAFs.

Effects of HER Family-targeting Tyrosine Kinase Inhibitors on Antibody-dependent Cell-mediated Cytotoxicity in HER2-expressing Breast Cancer.

PURPOSE: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. EXPERIMENTAL DESIGN: Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array-determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry-based protocols. RESULTS: Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell-mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. CONCLUSIONS: TKIs differentially alter tumor cell phenotype which can impact NK cell-mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

A phase Ib/II study of HER3-targeting lumretuzumab in combination with carboplatin and paclitaxel as first-line treatment in patients with advanced or metastatic squamous non-small cell lung cancer.

PURPOSE: This study investigated the safety and clinical activity of lumretuzumab, a humanised antihuman epidermal growth factor receptor 3 (HER3) monoclonal antibody, in combination with carboplatin and paclitaxel in first-line treatment of patients with squamous non-small cell lung cancer (sqNSCLC). HER3 ligand heregulin and HER3 protein expression were evaluated as potential biomarkers of clinical activity. PATIENTS AND METHODS: This open-label, phase Ib/II study enrolled patients receiving lumretuzumab at 800 mg (flat) in combination with carboplatin (area under the curve (AUC) 6 mg/mL×min) and paclitaxel (200 mg/m2) administered intravenously on a every 3-week schedule. Adverse event (AE) rates and tumour responses were determined. Heregulin messenger RNA (mRNA) and HER3 protein expression were investigated in archival tumour biopsies. RESULTS: Altogether, 12 patients received lumretuzumab in combination with carboplatin and paclitaxel. The most frequent AEs were gastrointestinal, haematological and nervous system toxicities, which were generally mild and manageable. Partial responses were observed in 3 of 12 patients lasting 81, 177 and 207 days. All responses were achieved in tumours expressing higher heregulin mRNA levels. CONCLUSION: Lumretuzumab in combination with carboplatin and paclitaxel was well tolerated. Objective responses were enriched in tumours expressing higher heregulin mRNA levels.

Clinical development of HER3-targeting monoclonal antibodies: Perils and progress.

The human epidermal growth factor receptor (HER) family consists of four transmembrane receptor tyrosine kinases: epidermal growth factor receptor (EGFR), HER2, HER3, and HER4. They are part of a complex signalling network and stimulate intracellular pathways regulating cell growth and differentiation. So far, monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors targeting EGFR and HER2 have been developed and approved. Recently, focus has turned to HER3 as it may play an important role in resistance to EGFR- and HER2-targeting therapies. HER3-targeting agents have been undergoing clinical evaluation for the last 10 years and currently thirteen mAbs are in phase 1 or 2 clinical studies. Single agent activity has proven to be limited, however, the tolerability was favourable. Thus, combinations of HER3-binding mAbs with other HER-targeting therapies or chemotherapies have been pursued in various solid tumor entities. Data indicate that the HER3-binding ligand heregulin may serve as a response prediction marker for HER3-targeting therapy. Within this review the current status of clinical development of HER3-targeting compounds is described.

Mechanistic Investigations of Diarrhea Toxicity Induced by Anti-HER2/3 Combination Therapy.

Combination of targeted therapies is expected to provide superior efficacy in the treatment of cancer either by enhanced antitumor activity or by preventing or delaying the development of resistance. Common challenges in developing combination therapies include the potential of additive and aggravated toxicities associated with pharmacologically related adverse effects. We have recently reported that combination of anti-HER2 and anti-HER3 antibodies, pertuzumab and lumretuzumab, along with paclitaxel chemotherapy in metastatic breast cancer, resulted in a high incidence of diarrhea that ultimately limited further clinical development of this combination. Here, we further dissected the diarrhea profile of the various patient dose cohorts and carried out in vitro investigations in human colon cell lines and explants to decipher the contribution and the mechanism of anti-HER2/3 therapeutic antibodies to intestinal epithelium malfunction. Our clinical investigations in patients revealed that while dose reduction of lumretuzumab, omission of pertuzumab loading dose, and introduction of a prophylactic antidiarrheal treatment reduced most severe adverse events, patients still suffered from persistent diarrhea during the treatment. Our in vitro investigations showed that pertuzumab and lumretuzumab combination treatment resulted in upregulation of chloride channel activity without indication of intestinal barrier disruption. Overall, our findings provide a mechanistic rationale to explore alternative of conventional antigut motility using medication targeting chloride channel activity to mitigate diarrhea of HER combination therapies. Mol Cancer Ther; 17(7); 1464-74. ©2018 AACR.

Phase Ib study evaluating safety and clinical activity of the anti-HER3 antibody lumretuzumab combined with the anti-HER2 antibody pertuzumab and paclitaxel in HER3-positive, HER2-low metastatic breast cancer.

Purpose To investigate the safety and clinical activity of comprehensive human epidermal growth factor receptor (HER) family receptor inhibition using lumretuzumab (anti-HER3) and pertuzumab (anti-HER2) in combination with paclitaxel in patients with metastatic breast cancer (MBC). Methods This phase Ib study enrolled 35 MBC patients (first line or higher) with HER3-positive and HER2-low (immunohistochemistry 1+ to 2+ and in-situ hybridization negative) tumors. Patients received lumretuzumab (1000 mg in Cohort 1; 500 mg in Cohorts 2 and 3) plus pertuzumab (840 mg loading dose [LD] followed by 420 mg in Cohorts 1 and 2; 420 mg without LD in Cohort 3) every 3 weeks, plus paclitaxel (80 mg/m2 weekly in all cohorts). Patients in Cohort 3 received prophylactic loperamide treatment. Results Diarrhea grade 3 was a dose-limiting toxicity of Cohort 1 defining the maximum tolerated dose of lumretuzumab when given in combination with pertuzumab and paclitaxel at 500 mg every three weeks. Grade 3 diarrhea decreased from 50% (Cohort 2) to 30.8% (Cohort 3) with prophylactic loperamide administration and omission of the pertuzumab LD, nonetheless, all patients still experienced diarrhea. In first-line MBC patients, the objective response rate in Cohorts 2 and 3 was 55% and 38.5%, respectively. No relationship between HER2 and HER3 expression or somatic mutations and clinical response was observed. Conclusions Combination treatment with lumretuzumab, pertuzumab and paclitaxel was associated with a high incidence of diarrhea. Despite the efforts to alter dosing, the therapeutic window remained too narrow to warrant further clinical development. TRIAL REGISTRATION: on ClinicalTrials.gov with the identifier NCT01918254 first registered on 3rd July 2013.

Direct estrogen receptor (ER) / HER family crosstalk mediating sensitivity to lumretuzumab and pertuzumab in ER+ breast cancer.

Bidirectional cross talk between members of the human epidermal growth factor family of receptors (HER) and the estrogen receptor (ER) is believed to underlie resistance mechanisms that develop in response to treatment with anti-HER agents and endocrine therapy. We investigated the interaction between HER2, HER3 and the ER in vitro using human embryonic kidney cells transfected with human HER2, HER3, and ERα. We also investigated the additive efficacy of combination regimens consisting of anti-HER3 (lumretuzumab), anti-HER2 (pertuzumab), and endocrine (fulvestrant) therapy in vivo. Our data show that both HER2 and HER3 can directly complex with the ER and can mediate phosphorylation of the ER. Phosphorylation of the ER was only observed in cells that expressed both HER2 and ERα or in heregulin-stimulated cells that expressed both HER3 and ERα. Using a mouse xenograft model of ER+/HER2-low (HER2 immunohistochemistry 1+ or 2+ without gene amplification) human breast cancer we show that the combination of lumretuzumab and pertuzumab is highly efficacious and induces long-lasting tumor regression in vivo and adding endocrine therapy (fulvestrant) to this combination further improved efficacy. In addition, a prolonged clinical response was observed with the combination of lumretuzumab and pertuzumab in a patient with ER+/HER2-low breast cancer who had failed endocrine therapy. These preclinical data confirm that direct cross talk exists between HER2/HER3 and ER which may explain the resistance mechanisms to endocrine therapy and monoclonal antibodies that target HER2 and HER3. Our data also indicate that the triplet of anti-HER2, anti-HER3, and endocrine therapy might be an efficacious combination for treating patients with ER+/HER2-low breast cancer, which is an area of significant unmet medical need.

Detection of truncated HER2 forms in formalin-fixed, paraffin-embedded breast cancer tissue captures heterogeneity and is not affected by HER2-targeted therapy.

Truncated forms of HER2, previously identified in subsets of HER2-positive breast cancer, originate from proteolytic extracellular domain (ECD) cleavage or alternative translation initiation. They lack ECD but may retain intracellular domain functionality, potentially associated with unfavorable prognosis, metastasis, and decreased sensitivity to antibody-based HER2-targeted therapy. To study the distribution of truncated HER2 in breast cancer, we detected loss of membrane-bound ECD independently of its molecular origin in paraffin sections, combining multispectral unmixing of chromogenic duplex IHC for HER2 ECD and intracellular domain with advanced image analysis. HER2 C-terminal fragment 611-transfected MCF7 and 4-aminophenylmercuric acetate-treated SKBR3 cell lines were used as controls. Applying a prototype work flow to whole sections, paired surgical resection/core needle biopsy samples, and paired samples from 69 patients of a phase 2 neoadjuvant clinical trial, we observed unexpected heterogeneity of ECD loss at the single-cell level, and in different areas of individual tumors, indicating that extent and localization of HER2 ECD loss add relevant information to averaging truncated HER2 across whole sections. We show acceptable run-to-run variation (coefficient of variation, <0.15), image analysis results in moderate agreement with conventional slide assessment (Cohen's κ = 0.59), and no obvious interference with previous HER2-ECD-targeted therapy. We conclude that duplex IHC and digital image processing extend current approaches of truncated HER2 detection.

Pertuzumab counteracts the inhibitory effect of ErbB2 on degradation of ErbB3.

Overexpression of ErbB2 and ErbB3 is found in several human cancers, and ErbB2-ErbB3 heterodimers are known as the most potent signaling units among ErbB dimers. While ErbB2 probably undergoes weak endocytosis, ErbB3 is readily internalized even in the absence of added ligand and without requirement for kinase activity. Overexpression of ErbB2 has been demonstrated to inhibit epidermal growth factor-induced internalization and degradation of epidermal growth factor receptor. This happens due to epidermal growth factor receptor-ErbB2 dimerization and can be counteracted by the anti-ErbB2 antibody pertuzumab, which binds the dimerization arm of ErbB2. Pertuzumab does also inhibit ErbB2-ErbB3 dimerization, but to what extent this has effect on constitutive and/or ligand-induced downregulation of ErbB3 is not known. In this study, we demonstrate that expression of ErbB2 as such did not block constitutive internalization of ErbB3, but that heregulin-induced degradation of ErbB3 was significantly slowed in cells expressing high levels of ErbB2. Incubation with pertuzumab did, however, counteract this effect. This indicates that the formation of ErbB2-ErbB3 heterodimers inhibits downregulation of ErbB3 and supports the notion that pertuzumab inhibits ErbB2 dimerization. The inhibitory effect of pertuzumab on ligand-induced ErbB2-ErbB3 heterodimerization was confirmed by the observation that pertuzumab inhibited heregulin-induced phosphorylation of ErbB3 in cells expressing ErbB2 and efficiently reduced heregulin-induced downstream signaling in cells expressing low levels of ErbB2. Altogether the results indicate that pertuzumab can be a valuable therapeutic agent not only in cancers overexpressing ErbB2 but also in cancers co-expressing ErbB2 and ErbB3.

Molecular determinants of trastuzumab efficacy: What is their clinical relevance?

Trastuzumab-containing therapy is a standard of care for human epidermal growth factor receptor-2 (HER2)-positive breast cancer. In pre-clinical models, a wide range of molecular mechanisms have been associated with reduced sensitivity to trastuzumab in vitro. These include expression of the truncated HER2 receptor fragment p95HER2, activating mutation of the gene encoding the class 1A catalytic subunit of phosphatidylinositol 3-kinase (PIK3CA), loss of phosphatase and tensin homolog (PTEN), activation of other downstream signal transducers, prevention of cell cycle arrest, increased signaling through alternative (HER or non-HER) tyrosine kinase receptors, and resistance to antibody-dependent cellular cytotoxicity. However, the clinical significance of these mechanisms as determinants of trastuzumab efficacy in vivo has been unclear. Here, we review clinical studies of potential predictive biomarkers of trastuzumab efficacy in HER2-positive breast cancer and consider whether evaluation of such markers might inform patient selection for therapy. We find that clinical evidence relating to potential predictive biomarkers is mostly limited to small, retrospective studies, many of which have yielded conflicting findings. Some trends are evident in the retrospective data and in biomarker analyses from randomized clinical trials, particularly relating to activation of the phosphatidylinositol 3-kinase pathway, but none is sufficiently strong to form a basis for patient selection. This may be explained by the fact that multiple mechanisms of action determine the clinical efficacy of trastuzumab. In the absence of novel, validated biomarkers of efficacy, trastuzumab eligibility should continue to be based on evaluation of HER2 status according to standard methods.

Lipoplex mediated silencing of membrane regulators (CD46, CD55 and CD59) enhances complement-dependent anti-tumor activity of trastuzumab and pertuzumab.

The therapeutic potential of anticancer antibodies is limited by the resistance of tumor cells to complement-mediated attack, primarily through the over-expression of membrane complement regulatory proteins (mCRPs: CD46, CD55 and CD59). Trastuzumab, an anti- HER2 monoclonal antibody, approved for the treatment of HER2-positive breast and gastric cancers, exerts only minor complement-mediated cytotoxicity (CDC). Pertuzumab is a novel anti-HER2 monoclonal antibody, which blocks HER2 dimerization with other ligand-activated HER family members. Here, we explored the complement-mediated anti-tumor effects of trastuzumab and pertuzumab on HER2-positive tumor cells of various histological origins. Delivery of chemically stabilized anti-mCRP siRNAs using cationic lipoplexes, AtuPLEXes, to HER2-over-expressing BT474, SK-BR-3 (breast), SKOV3 (ovarian) and Calu-3 (lung) cancer cells reduced mCRPs expression by 85-95%. Knockdown of individual complement regulators variably led to increased CDC only upon combined treatment with trastuzumab and pertuzumab. The combined down-regulation of all the three regulators augmented CDC by 48% in BT474, 46% in SK-BR-3 cells, 78% in SKOV3 cells and by 30% in Calu-3 cells and also increased complement-induced apoptosis and caspase activity on mCRP neutralized tumor cells. In addition, antibody-induced C3 opsonization of tumor cells was significantly enhanced after mCRP silencing and further augmented tumor cell killing by macrophages. Our findings suggest that siRNA-induced inhibition of complement regulator expression clearly enhances complement- and macrophage-mediated anti-tumor activity of trastuzumab and pertuzumab on HER2-positive tumor cells. Thus - if selectively targeted to the tumor - siRNA-induced inhibition of complement regulation may serve as an innovative strategy to potentiate the efficacy of antibody-based immunotherapy.

Pertuzumab Increases 17-AAG-Induced Degradation of ErbB2, and This Effect Is Further Increased by Combining Pertuzumab with Trastuzumab.

ErbB2 is an important oncogenic protein involved in carcinogenesis of, among others, breast, gastric, and ovarian carcinoma. Over-expression of ErbB2 is found in almost 20% of breast cancers, and this results in proliferative and anti-apoptotic signalling. ErbB2 is therefore an important treatment target. Antibodies recognizing full-length ErbB2 are clinically established, and drugs targeting the ErbB2 stabilizing heat shock protein 90 (Hsp90) are under clinical evaluation. We have investigated effects of the ErbB2-binding antibodies trastuzumab and pertuzumab alone and in combination, as well as the effect of the antibodies in combination with the Hsp90 inhibitor 17-AAG. Our results confirm the notion that combination of different ErbB2-binding antibodies more efficiently down-regulates ErbB2 than does one antibody in isolation. Additionally, our data demonstrate that ErbB2 is most efficiently down-regulated upon incubation with anti-ErbB2 antibodies in combination with Hsp90 inhibitors. The combination of anti-ErbB2 antibodies, and especially the combination of antibodies with 17-AAG, did also increase the inhibition of Akt activation of either agent, which could suggest an anti-proliferative effect. In such case, combining these agents could be beneficial in treatment of tumors not responding to trastuzumab only.

Trastuzumab and pertuzumab produce changes in morphology and estrogen receptor signaling in ovarian cancer xenografts revealing new treatment strategies.

PURPOSE: The aim of this study was to investigate the antitumor effects of HER2-directed combination therapy in ovarian cancer xenograft models to evaluate their potential. The combinations of trastuzumab and pertuzumab, and trastuzumab and aromatase inhibitor therapy were investigated. EXPERIMENTAL DESIGN: The effects of trastuzumab, pertuzumab, and letrozole on growth response, apoptosis, morphology, and gene and protein expression were evaluated in the SKOV3 ovarian cancer cell line xenograft and a panel of five human ovarian xenografts derived directly from clinical specimens. RESULTS: The combination of HER2-directed antibodies showed enhanced antitumor activity compared with single antibody therapy in the SKOV3 xenograft model. Apoptosis, morphology, and estrogen-regulated gene expression were modulated by these antibodies in both spatial and temporal manners. A panel of ovarian cancer xenografts showed differential growth responses to the combination of trastuzumab and pertuzumab. High HER2 expression and increasing HER3 protein expression on treatment were associated with growth response. In trastuzumab-treated SKOV3 tumors, there was a change in tumor morphology, with a reduction in frequency of estrogen receptor alpha (ERα)-negative clear cell areas. Trastuzumab, but not pertuzumab, increased expression of ERα in SKOV3 xenografts when analyzed by quantitative immunofluorescence. ERα and downstream signaling targets were modulated by trastuzumab alone and in combination. Trastuzumab enhanced the responsiveness of SKOV3 xenografts to letrozole when given in combination. CONCLUSIONS: These data suggest that trastuzumab in combination with pertuzumab could be an effective approach in high HER2-expressing ovarian cancers and could also enhance sensitivity to endocrine therapy in ERα-positive ovarian cancer.

Strongly enhanced antitumor activity of trastuzumab and pertuzumab combination treatment on HER2-positive human xenograft tumor models.

The human epidermal growth factor receptor (HER) family plays an important role in cell survival and proliferation, and is implicated in oncogenesis. Overexpression of HER2 is associated with aggressive disease and poor prognosis. Trastuzumab is a humanized monoclonal antibody targeting HER2 and has proven survival benefit for women with HER2-positive early and metastatic breast cancer. Pertuzumab, another monoclonal antibody, is a HER2 dimerization inhibitor that binds to a different epitope on HER2 than trastuzumab and inhibits HER2 dimer formation with other HER family members such as HER3 and HER1. We investigated the antitumor activity of these agents alone and in combination in HER2-positive breast and non-small cell lung cancer xenografts. Our data show that the combination of trastuzumab and pertuzumab has a strongly enhanced antitumor effect and induces tumor regression in both xenograft models, something that cannot be achieved by either monotherapy. The enhanced efficacy of the combination was also observed after tumor progression during trastuzumab monotherapy. Near-IR fluorescence imaging experiments confirm that pertuzumab binding to tumors is not impaired by trastuzumab pretreatment. Furthermore, we show by in vitro assay that both trastuzumab and pertuzumab potently activate antibody-dependent cellular cytotoxicity. However, our data suggest that the strongly enhanced antitumor activity is mainly due to the differing but complementary mechanisms of action of trastuzumab and pertuzumab, namely inhibition of HER2 dimerization and prevention of p95HER2 formation.

Modulation of HER3 is a marker of dynamic cell signaling in ovarian cancer: implications for pertuzumab sensitivity.

This study was designed to evaluate the expression of HER receptors as a marker of sensitivity to the humanized anti-HER2 monoclonal antibody pertuzumab in ovarian cancer cells. In a recent clinical trial, low levels of HER3 mRNA have been shown to associate with pertuzumab response when combined with gemcitabine. We sought to define how pertuzumab modulated HER expression levels in ovarian cancer using cell line models to better understand differential and dynamic receptor expression in therapeutic response. Changes in HER3 mRNA expression were also assessed in pertuzumab-treated xenografts. HER3 mRNA and, to a lesser extent, HER2, were down-regulated after stimulation both with heregulin-beta1 and epidermal growth factor in a range of ovarian cancer cell lines either growth sensitive or growth resistant to pertuzumab. Pertuzumab reversed this down-regulation and the magnitude of the reversal correlated with pertuzumab sensitivity. The change in HER3 mRNA expression correlated inversely to how much the extracellular signal-regulated kinase and phosphoinositide 3-kinase pathways were dynamically activated with stimulation. Finally, up-regulation of HER3 mRNA was found in cancer xenografts treated with pertuzumab. We conclude that HER3 mRNA is down-regulated by both heregulin-beta1 and epidermal growth factor activation. This suggests that in some tumors, low HER3 mRNA expression is driven by, or dependent on, growth factor. HER3 mRNA expression is effectively reversed in pertuzumab-sensitive tumors. These data are consistent with low HER3 mRNA identifying a pertuzumab-sensitive phenotype.

Pertuzumab increases epidermal growth factor receptor down-regulation by counteracting epidermal growth factor receptor-ErbB2 heterodimerization.

Epidermal growth factor receptor (EGFR) and ErbB2 readily form heterodimers when both are expressed in the same cell and the EGFR is activated by one of its ligands. Our data show that such heterodimers are constitutively formed also in a ligand-independent manner on overexpression of EGFR and ErbB2 in porcine aortic endothelial cells. Interestingly, cross-linking experiments showed that incubation with the antibody pertuzumab, which has been shown to bind the dimerization arm of ErbB2, resulted in dissolution of EGFR-ErbB2 heterodimers. Incubation with pertuzumab also increased the amount of EGF-induced EGFR homodimers, and under these conditions, endocytosis of radiolabeled EGF was increased. This increase was significant, although slightly more EGF was internalized in cells expressing EGFR only compared with pertuzumab-treated cells expressing both EGFR and ErbB2. By confocal microscopy analysis, more EGF was observed in endosomes on incubation with pertuzumab, and under similar conditions, immunoblotting experiments showed increased EGFR degradation on incubation with both EGF and pertuzumab. These results show that pertuzumab enhanced the endocytic down-regulation of EGFR by counteracting EGFR-ErbB2 heterodimerization. Our previous results showing that ErbB2 counteracts EGFR endocytosis can therefore be explained by tethering of EGFR to ErbB2 at the plasma membrane.

Flow cytometric FRET analysis of erbB receptor interaction on a cell-by-cell basis.

Lateral interaction of c-erbB family receptors resulting in dimer formation is the key event initiating signal transduction. Consequently cross-activation and intracellular signaling is triggered with immediate impact on cell proliferation, migration, cell survival, and differentiation. In order to elucidate the connection of signal input (receptor activation) and signal output (altered cellular behavior) we dynamically assessed cell proliferation of BT474 and SK-BR-3 breast cancer cell lines. We quantitated c-erbB2 receptor homodimerization upon treatment with the therapeutic monoclonal anti-c-erbB2 antibodies trastuzumab (Herceptin) and pertuzumab by flow cytometric FRET (FCET) measurements on a cell-by-cell basis and calculated the extent of antibody-induced cell cycle exit. The results confirm that trastuzumab does not decrease c-erbB2 homodimers despite its strong potency to drive c-erbB2-overexpressing cells into quiescence. Pertuzumab, however, is able to prevent c-erbB2 homodimerization and thereby enhance the antiproliferative effect of trastuzumab when administered in combination.

Sensitivity to pertuzumab (2C4) in ovarian cancer models: cross-talk with estrogen receptor signaling.

Pertuzumab (Omnitarg, rhuMab 2C4) is a humanized monoclonal antibody, which inhibits HER2 dimerization. Because it has shown some clinical activity in ovarian cancer, this study sought to identify predictors of response to this agent in a model of ovarian cancer. A panel of 13 ovarian cancer cell lines was treated with heregulin beta1 (HRGbeta1) or transforming growth factor-alpha, and cell proliferation was assessed. Both agents increased cell number in the majority of cell lines studied, the response to both being similar (r = 0.83; P = 0.0004, Pearson test). HRGbeta1 stimulation could be partially reversed by pertuzumab in 6 of 13 cell lines, with complete reversal in PE04 and PE06 cells. Addition of pertuzumab to transforming growth factor-alpha-stimulated cells produced growth inhibition in 3 of 13 cell lines (PE01, PE04, and PE06). The magnitude of HRGbeta1-driven growth stimulation correlated significantly with an increase in extracellular signal-regulated kinase 2 (P = 0.037) but not Akt (P = 0.99) phosphorylation. Such HRGbeta1-driven phosphorylation of extracellular signal-regulated kinase 1/2 and Akt could be reduced with pertuzumab, accompanied by changes in cell cycle distribution. In cell lines responsive to pertuzumab, HRGbeta1-enhanced phosphorylation of HER2 (Tyr(877)) was reduced. Estrogen-stimulated changes in growth, cell cycle distribution, and signaling were reversed by pertuzumab, indicating cross-talk between HER2 and estrogen signaling. These data indicate that there is a subset of ovarian cancer cell lines sensitive to pertuzumab and suggest possible predictors of response to identify patients who could benefit from this therapy. Furthermore, we have identified an interaction between HER2 and estrogen signaling in this disease.

Combination treatment with erlotinib and pertuzumab against human tumor xenografts is superior to monotherapy.

In many solid tumors, overexpression of human epidermal growth factor receptors (e.g., HER1/EGFR and HER2) correlates with poor prognosis. Erlotinib (Tarceva) is a potent HER1/EGFR tyrosine kinase inhibitor. Pertuzumab (Omnitarg), a novel HER2-specific, recombinant, humanized monoclonal antibody, prevents heterodimerization of HER2 with other HERs. Both mechanisms disrupt signaling pathways, resulting in tumor growth inhibition. We evaluated whether inhibition of both mechanisms is superior to monotherapy in tumor cell lines expressing different HER levels. Human non-small cell lung cancer (NSCLC) cells (Calu-3: HER1/EGFR 0+, HER2 3+; QG56: HER1/EGFR 2-3+, HER2 0+) and breast cancer cells (KPL-4: HER1/EGFR 2-3+, HER2 3+) were implanted into BALB/c nu/nu mice and severe combined immunodeficient beige mice, respectively. Tumor-bearing mice (n = 12 or 15 per group) were treated with vehicle (Captisol or buffer), erlotinib (orally, 50 mg/kg/d), pertuzumab (i.p. 6 mg/kg/wk with a 2-fold loading dose), or erlotinib and pertuzumab for 20 (QG56), 27 (KPL-4), or 49 (Calu-3) days. Drug monotherapy had antitumor activity in all models. Tumor volume treatment-to-control ratios (TCR) with erlotinib were 0.36 (Calu-3), 0.79 (QG56), and 0.51 (KPL-4). Pertuzumab TCR values were 0.42, 0.51, and 0.64 in Calu-3, QG56, and KPL-4 models, respectively. Combination treatment resulted in additive (QG56: TCR 0.39; KPL-4: TCR 0.38) or greater than additive (Calu-3: TCR 0.12) antitumor activity. Serum tumor markers for NSCLC (Cyfra 21.1) and breast cancer (soluble HER2) were markedly inhibited by combination treatment (80-97% in Calu-3 and QG56; 92% in KPL-4), correlating with decreased tumor volume. Overall, erlotinib and pertuzumab are active against various human xenograft models, independently of HER1/EGFR or HER2 expression. A combination of these HER-targeted agents resulted in additive or greater than additive antitumor activity.