HER2 Antibody Research Articles

Dynamic disordering of liposomal cocktails and the spatio-temporal favorable release of cargoes to circumvent drug resistance

Multidrug resistance (MDR) has been a major impediment to the success of cancer chemotherapy. Extensive efforts have been devoted to the development of drug delivery systems using nanotechnology to reverse MDR in cancer. However, the spontaneous release of drug payloads was always a slow process, which leads to the low intracellular drug concentration resulting in consequent drug insensitivity. To circumvent this limitation, we described a liposomal cocktail (LMDHV) constructed by a pH-responsive molecule (i.e., malachite green carbinol base (MG)) and liposome conjugated with Her-2 antibody for codelivery of doxorubicin (DOX) and verapamil (VER) to suppress drug resistance in Her-2 positive breast cancer. MG inserted in the bilayer as pH responders greatly contributed to the destabilization of the vesicle membrane in low pH, followed by the rapid release of the payloads. LMDHV showed 6-fold reversal efficiency in DOX resistant breast cancer owing to the efficient tumor targeting delivery and rapid burst release of drug intracellularly. Compared to tumor inhibition ratio of treated groups by free DOX (32.4 ± 7.4%), our designed kinetically favorable drug release system exhibited significantly (P < 0.01) enhanced tumor inhibition ratio up to 83.9 ± 12.5%, which is attributed to the remarkably increased drug concentration in cells. The spatio-temporal favorable release of drugs resulted in synergistic inhibition of tumor growth in xenografts. We envision that this new type of liposomal cocktail might be potentially utilized to circumvent drug resistance in the future.

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (KadcylaTM). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA’) fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA’, was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA’-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

Immuno nanoparticles integrated electrical control of targeted cancer cell development using whole cell bioelectronic device.

Electrical properties of cells determine most of the cellular functions, particularly ones which occur in the cell's membrane. Manipulation of these electrical properties may provide a powerful electrotherapy option for the treatment of cancer as cancerous cells have been shown to be more electronegative than normal proliferating cells. Previously, we used an electrical impedance sensing system (EIS) to explore the responses of cancerous SKOV3 cells and normal HUVEC cells to low intensity (<2 V/cm) AC electric fields, determining that the optimal frequency for SKOV3 proliferation arrest was 200 kHz, without harming the non-cancerous HUVECs. In this study, to determine if these effects are cell type dependant, human breast adenocarcinoma cells (MCF7) were subjected to a range of frequencies (50 kHz-2 MHz) similar to the previously tested SKOV3. For the MCF7, an optimal frequency of 100 kHz was determined using the EIS, indicating a higher sensitivity towards the applied field. Further experiments specifically targeting the two types of cancer cells using HER2 antibody functionalized gold nanoparticles (HER2-AuNPs) were performed to determine if enhanced electric field strength can be induced via the application of nanoparticles, consequently leading to the killing of the cancerous cells without affecting non cancerous HUVECs and MCF10a providing a platform for the development of a non-invasive cancer treatment without any harmful side effects. The EIS was used to monitor the real-time consequences on cellular viability and a noticeable decrease in the growth profile of the MCF7 was observed with the application of the HER2-AuNPs and the electric fields indicating specific inhibitory effects on dividing cells in culture. To further understand the effects of the externally applied field to the cells, an Annexin V/EthD-III assay was performed to determine the cell death mechanism indicating apoptosis. The zeta potential of the SKOV3 and the MCF7 before and after incorporation of the HER2-AuNPs was also obtained indicating a decrease in zeta potential with the incorporation of the nanoparticles. The outcome of this research will improve our fundamental understanding of the behavior of cancer cells and define optimal parameters of electrotherapy for clinical and drug delivery applications.

Engineering multivalent antibodies to target heregulin-induced HER3 signaling in breast cancer cells

The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of “second generation” antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert.

Role of HER3 expression and PTEN loss in patients with HER2-overexpressing metastatic breast cancer (MBC) who received taxane plus trastuzumab treatment

Background:The aim of this study was to investigate the role of human epidermal growth factor receptor (HER3) and PTEN expression in patients with HER2-overexpressing metastatic breast cancer (MBC).Methods:One hundred twenty-five MBC patients who were treated with taxane plus trastuzumab chemotherapy as first-line therapy were included in this analysis. Immunohistochemical (IHC) staining with HER3 and PTEN antibodies were conducted retrospectively.Results:Patients who had negative HER3 staining (62.4%) had a better progression-free survival (PFS) than did those who had positive HER3 staining (P=0.001; median PFS, 21 vs 11 months). Patients who had a PTEN score >20 (78.1%) showed longer PFS than did those with a PTEN score ⩽20 (P=0.006; median PFS, 13 vs 9 months). Patients who had a PTEN score >20 exhibited a longer overall survival (OS) than did those with a PTEN score ⩽20 (P=0.005; median OS, 48 vs 25 months). HER3 negativity and PTEN loss were identified as independent risk factors for PFS. PTEN loss was identified as an independent risk factor for OS.Conclusion:HER3 and PTEN expressions may be predictive markers, and PTEN expression may be a predictive and prognostic biomarker for trastuzumab treatment in HER2-positive MBCs.

Combination of Antibody That Inhibits Ligand-Independent HER3 Dimerization and a p110α Inhibitor Potently Blocks PI3K Signaling and Growth of HER2+ Breast Cancers

We examined the effects of LJM716, an HER3 (ERBB3) neutralizing antibody that inhibits ligand-induced and ligand-independent HER3 dimerization, as a single agent and in combination with BYL719, an ATP competitive p110α-specific inhibitor, against HER2-overexpressing breast and gastric cancers. Treatment with LJM716 reduced HER2-HER3 and HER3-p85 dimers, P-HER3 and P-AKT, both in vitro and in vivo. Treatment with LJM716 alone markedly reduced growth of BT474 xenografts. The combination of LJM716/lapatinib/trastuzumab significantly improved survival of mice with BT474 xenografts compared with lapatinib/trastuzumab (P = 0.0012). LJM716 and BYL719 synergistically inhibited growth in a panel of HER2+ and PIK3CA mutant cell lines. The combination also inhibited P-AKT in HER2-overexpressing breast cancer cells and growth of HER2+ NCI-N87 gastric cancer xenografts more potently than LJM716 or BYL719 alone. Trastuzumab-resistant HER2+/PIK3CA mutant MDA453 xenografts regressed completely after 3 weeks of therapy with LJM716 and BYL719, whereas either single agent inhibited growth only partially. Finally, mice with BT474 xenografts treated with trastuzumab/LJM716, trastuzumab/BYL719, LJM716/BYL719, or trastuzumab/LJM716/BYL719 exhibited similar rates of tumor regression after 3 weeks of treatment. Thirty weeks after treatment discontinuation, 14% of mice were treated with trastuzumab/LJM716/BYL719, whereas >80% in all other treatment groups were sacrificed due to a recurrent large tumor burden (P = 0.0066). These data suggest that dual blockade of the HER2 signaling network with an HER3 antibody that inhibits HER2-HER3 dimers in combination with a p110α-specific inhibitor in the absence of a direct HER2 antagonist is an effective treatment approach against HER2-overexpressing cancers.

Immunohistochemical assessment of HER2 expression in gastric cancer in a cohort of 118 Brazilian patients

INTRODUCTION: Recent studies indicate a role of human epidermal growth factor receptor type 2 (HER2) in the development of numerous types of human cancer, including gastric cancer, and its overexpression correlates with poor prognosis and increased aggressiveness of this neoplasm. OBJECTIVES: Identify and evaluate the immunohistochemical expression of HER2 in gastric cancer from biopsies, surgical resection specimens, lymph node metastasis and distant metastasis, and evaluate their correlation with currently known clinical and histopathological prognostic factors. METHODS: Samples from 118 patients of both sexes and all age groups diagnosed with gastric cancer were analyzed. Immunohistochemistry (IHQ) was performed using the HER2 antibody, and its evaluation was made according to the modified gastric cancer testing protocol, taking into account incomplete basolateral staining or only lateral staining. RESULTS: HER2 expression did not correlate with any histological prognostic factors or clinical outcome, except for the N stage. By comparing the HER2 expression in biopsies, surgical specimen, lymph node and metastasis, 88.1% were in agreement. CONCLUSION: As anti-HER2 therapies are becoming the standard of care in gastric cancer, currently available data indicate that IHQ should be used as the screening test, and the pathologist has an important role to ensure an accurate testing of HER2 status in these tumors. In addition, the HER2 status can be tested using the available samples from biopsies, surgical specimens, nodal and extranodal metastatic disease.

An Antibody That Locks HER3 in the Inactive Conformation Inhibits Tumor Growth Driven by HER2 or Neuregulin

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

Human Breast Cancer Cells Harboring a Gatekeeper T798M Mutation in HER2 Overexpress EGFR Ligands and Are Sensitive to Dual Inhibition of EGFR and HER2

Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance. HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR/HER2/HER3/PI3K axis. A low 3% allelic frequency of the T798M mutant shifted 10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT, and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin, and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting that increased EGFR ligand production was causally associated with drug resistance. Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles.

HER2 in gastric cancer: a digital image analysis in pre-neoplastic, primary and metastatic lesions

The assessment of human epidermal growth factor receptor 2 (HER2) status in gastric cancer is crucial in selecting patients who may benefit from targeted therapy, yet heterogeneous expression could represent an important drawback for HER2 testing. We aimed to analyze (i) HER2 heterogeneity in primary gastric cancers, pre-neoplastic and metastatic lesions and (ii) HER2 prognostic role. We studied 292 surgically resected primary gastric carcinomas and constructed 21 tissue microarrays including tumor tissue cores, invasive front, paired lymph node metastasis, low- and high-grade dysplasia. Microarrays were immunohistochemically stained with HER2 antibody and digitally scanned. Novel digital analysis algorithms were developed to score HER2 expression. Fluorescence in situ hybridization was performed on equivocal cases. HER2-positive cases were 13% and heterogeneous HER2 expression was observed in 71% of positive samples. Analysis of HER2 status in tumor and tumor invasive front demonstrate concordance in 177 cases (88%). Comparison of HER2 expression in primary cancer and synchronous lymph node metastasis exhibited discordant status in 14% of cases. Dysplastic epithelium surrounding the tumor showed immunohistochemical score 2 or 3 in 19% of high-grade and in 9% of low-grade dysplastic samples. HER2 status was significantly associated with intestinal-type carcinomas (P=0.018) and prognosis since patients with primary HER2-positive tumor showed decreased overall survival (P=0.006). Intratumoral HER2 expression heterogeneity and variable lymph node metastases status strongly suggest evaluating more than one sample and, if available, metastatic foci for routinely HER2 testing.

A pilot study comparing circulation tumor cells (CTC) detection by AdnaTest prostate assay to CellSearch in patients (pts) with castration resistant prostate cancer (CRPC).

e16005 Background: The detection of CTC in blood is prognostic for survival in CRPC. CellSearch is an FDA cleared CTC enumeration assays that reports the number of cells meeting strict morphologic and immunohistochemical criteria. An unmet need is to increase detection rates in pts with favorable &lt; 4 cells/7.5 ml of blood. Here we studied CTC detection using AdnaTest PCR based assay in relation to CellSearch. Methods: Whole blood was collected under an IRB approved protocol from 40 pts with CRPC for the AdnaTest and CellSearch assays. The standard CellSearch platform was used for CTC detection. The AdnaTest includes immunomagnetic cell enrichment using EpCAM and Her2 antibodies followed by mRNA isolation and multiplex PCR amplification of 3 prostate associated transcripts (PSMA, PSA and EGFR). The measurement of one or more transcripts above 0.1ng/µl is considered a positive test. Results: CellSearch CTC counts and AdnaTest detection rates are presented in the Table. Overall AdnaTest detected CTC in 26 pts (65%, 95% CI 50-80%), including 6 of the 18 (33%, 95% CI 11-55%) pts with no CTC using CellSearch. Of 23 pts with ≤ 4 CTC by CellSearch, 10 pts (45%, 95% 30-61%) were positive by AdnaTest. In contrast, in 17 pts with ≥ 5 CTC by CellSearch, 16 pts (94%. 95% 87-100%) were positive by AdnaTest. Overall, 70% (28) of the 40 pts had detectable CTC by either AdnaTest or CellSearch, with positive agreement in 71%. AdnaTest positivity was based on detection of PSA, alone or in combination with PSMA or EGFR. Only 1 patient (4%) was positive for EGFR alone, and none by PSMA alone. Conclusions: AdnaTest demonstrated incremental detection of CTC in pts with no CTC identified by CellSearch. This is consistent with the difference between PCR detection vs the strict definition of CTC using CellSearch, which is known to represent only a small proportion of the EpCAM+ events in the chamber. Further studies to assess the utility of this assay as a biomarker for clinical outcome in pts with CPRP are warranted. [Table: see text]

C-MYC, HER2, and HER3 expression in localized prostate cancer (PC) treated with radical radiotherapy: Modulation by statins use and correlation with time to progression.

e16045 Background: There has been growing interest in the activity of statins (St) in PC patients receiving definitive radiation therapy. The oncogene Ras can be inhibited by HMG-CoA reductase inhibitors that in turn are also able to downregulate Myc. The aim of this study was to correlate St use and progression with the c-Myc, HER2 and HER3 expression in PC treated with radical radiotherapy (RT) with concurrent androgen deprivation. Methods: A total of 85 patients diagnosed with localised PC between 2000 and 2005 were selected for the study. Inmunohistochemical assay on core biopsy of each patient was performed using monoclonal antibodies for c-Myc, HER2 and HER3. The expression was evaluated using a semiquantitative method (Hscore scale: 0 to 300). Clinical and pathological variables were compared between St users and non-users and correlated with expression of c-Myc, HER2 and HER3. St users were defined as patients who had been taking St for at least 2 years before the diagnosis of PC and during follow up. Time to progression (TTP) was analyzed. Results: Mean age was 71 (56-82) years. Median follow-up was 75 months after RT. Twenty-five patients (29.4%) were using St. No statistical differences were found between treatment groups regarding median age, risk category, clinical T stage, Gleason score or median radiation dose. Median Hscore value of c-Myc was 40 (5-210) for St users vs. 72.5 (5-280) for non-users (p = 0.01). Only 8.6% of the cases showed HER2 expression and in those staining was mild intense and focal. Moderate expression of HER3 was observed in 36 cases (41.8%) and no correlation with St users nor with TTP was demonstrated (p=0.8). At the time of analysis, only 13 pts (15.3%) had biochemical relapse (1 low risk, 3 intermediate, and 9 high risk). St use (46.2%) was significantly associated with improved TTP (55 vs. 36.2 months, p = 0.022). Conclusions: Our work implicates a correlation of St use with a significant lower c-Myc expression and improvement in TTP in all groups, regardless the risk. No correlation was reported between HER2 or HER3, TTP and St use.

Role of HER3 expression and PTEN loss in patients with HER2-overexpressing metastatic breast cancer (MBC) patients who received taxane plus trastuzumab treatment.

637 Background: The HER3 receptor is a key member of ErbB family and preferentially signals through the PI3K pathway. Formation of dimers between HER3 and HER2 seems to be crucial for HER2-driven signals in HER2 overexpressing tumors. Given the fact that HER2-HER3 is considered the most active signalling dimer of the ErbB system, HER3 activity may contribute to the resistance of trastuzumab. PTEN plays a well-established role in the negative regulation of the PI3K pathway. Our aim of this study was to investigate the role of HER3 and PTEN expression in patients with HER2 overexpressed MBC. Methods: One-hundred twenty-five MBC patients who were treated with taxane plus trastuzumab chemotherapy as the first line therapy were included in this analysis. Immunohistochemical stainings (IHC) with HER3 and PTEN antibody were conducted retrospectively. Results: Median age was 48 years. HER3 IHC was graded from 0 to 3. PTEN IHC was scored as multiplication of intensity and proportion from 0 to 300. The patients who had negative HER3 stain showed better progression free survival (PFS) to taxane plus trastuzumab chemotherapy than those positive HER3 stain (p=0.001, median PFS 21 vs. 11 mo.). The patients who had PTEN score of more than 20 showed longer PFS than those PTEN score of 20 or less than 20 (p=0.006, median PFS 13 vs. 9 mo.). The patients who had PTEN score of more than 20 showed longer overall survival (OS) than those PTEN score of 20 or less than 20 (p=0.005, median OS 48 vs. 25 mo.). HER3 negativity and PTEN loss were identified as independent risk factors for PFS [Hazard Ratio (HR) 0.4 (95% CI 0.3-0.8 for HER3 negativity; HR 2.1 (95% CI 1.2-3.7) for PTEN loss]. However, PTEN loss (HR 3.1 [95% CI 1.6-6.3]) was identified as an independent risk factor for OS. Conclusions: HER3 and PTEN expression may be predictive markers for trastuzumab treatment in HER2-positive MBCs. PTEN expression may have a potential predictive and prognostic biomarker for trastuzumab treatment.

Aptamer to ErbB-2/HER2 enhances degradation of the target and inhibits tumorigenic growth

Aptamers, oligonucleotides able to avidly bind cellular targets, are emerging as promising therapeutic agents, analogous to monoclonal antibodies. We selected from a DNA library an aptamer specifically recognizing human epidermal growth factor receptor 2 (ErbB-2/HER2), a receptor tyrosine kinase, which is overexpressed in a variety of human cancers, including breast and gastric tumors. Treatment of human gastric cancer cells with a trimeric version (42 nucleotides) of the selected aptamer (14 nucleotides) resulted in reduced cell growth in vitro, but a monomeric version was ineffective. Likewise, when treated with the trimeric aptamer, animals bearing tumor xenografts of human gastric origin reflected reduced rates of tumor growth. The antitumor effect of the aptamer was nearly twofold stronger than that of a monoclonal anti-ErbB-2/HER2 antibody. Consistent with aptamer-induced intracellular degradation of ErbB-2/HER2, incubation of gastric cancer cells with the trimeric aptamer promoted translocation of ErbB-2/HER2 from the cell surface to cytoplasmic puncta. This translocation was associated with a lysosomal hydrolase-dependent clearance of the ErbB-2/HER2 protein from cell extracts. We conclude that targeting ErbB-2/HER2 with DNA aptamers might retard the tumorigenic growth of gastric cancer by means of accelerating lysosomal degradation of the oncoprotein. This work exemplifies the potential pharmacological utility of aptamers directed at cell surface proteins, and it highlights an endocytosis-mediated mechanism of tumor inhibition.

Abstract 5461: A HER3 antibody that blocks ligand-independent HER2-HER3 dimerization sensitizes to HER2 and PI3K inhibitors .

Abstract We hypothesized that complete inhibition of HER3 is required for the optimal action of HER2 or PI3K/Akt inhibitors against HER2+ tumors. The HER3 monoclonal antibody LJM716 (LJM) inhibits ligand-induced and ligand-independent dimerization of HER3. Treatment with LJM inhibited P-HER3 and P-Akt in a panel of HER2+ breast cancer cell lines. In BT474 and SKBR3 cells, LJM inhibited HER2-HER3 dimers as assessed by coprecipitation of HER2 with HER3 antibodies. Using a DUAL antibody proximity base assay, formalin-fixed sections of HER2+ xenografts treated with LJM showed a reduction of HER2-HER3 dimers and P-HER3 compared to untreated controls. Treatment with LJM alone also induced regression of established BT474 xenografts. Mice with BT474 xenografts were treated with lapatinib and trastuzumab (Lap/T), LJM and trastuzumab (LJM/T) or lapatinib, trastuzumab and LJM716 (Lap/T/LJM). After 3 weeks of treatment, mice from the 3 groups exhibited nearly complete tumor regression (volume &amp;lt;25 mm3). At 28 weeks of follow-up with no treatment, 8/12 mice in the Lap/T group and 5/7 mice in the LJM/T exhibited tumor regrowth (≥200 mm3), whereas only 2 of 15 mice in the Lap/T/LJM group did so (p=0.0046, Log-rank test). We expanded our studies to examine the combination of LJM716 with the p110α (PI3Kα) specific inhibitor BYL719 (BYL). LJM in combination with BYL inhibited growth in monolayer and 3-D Matrigel and S473 P-Akt in HER2+ breast cancer cells with mutations in the PI3K pathway better than either single agent. MDA-MB-453 xenografts regressed completely after 3 weeks of therapy with BYL/LJM whereas either single agent inhibited xenograft growth only partially. All mice bearing BT474 xenografts treated with T/LJM, T/BYL, LJM/BYL or T/LJM/BYL exhibited tumor regression after 24 days of treatment. Twenty weeks after treatment discontinuation, only 1/8 mice in the T/LJM/BYL exhibited tumor re-growth whereas &amp;gt;80% of mice in all other groups did so (p=0.0009, Log-rank test). Finally, we hypothesized that LJM would be at least equivalent to the HER2 antibody pertuzumab (P) in combination with T against HER2+ tumors. Inhibition of growth in vitro and P-Akt were superior with LJM/T compared to P/T in vitro. Mice bearing BT474 xenografts treated with either LJM/T or P/T had a similar nearly complete tumor regression after 4 weeks of treatment. We are currently monitoring potential tumor regrowth in these mice. In summary, treatment with LJM inhibited HER2-HER3 dimers, P-HER3 and P-Akt in HER2+ breast cancer cells with PI3K pathway mutations. As a single agent, LJM markedly inhibited HER2+ xenograft growth. Treatment with LJM in combination with Lap/T improved survival of mice with HER2+ xenografts compared to Lap/T. Further, LJM sensitized breast cancer cells and xenografts to a p110α-specific inhibitor. Finally, the HER3 antibody in combination with T inhibited PI3K/Akt and xenograft growth as well as the combination of P/T. Citation Format: Joan T. Garrett, Cammie R. Sutton, Carl Uli Bialucha, Seth A. Ettenberg, Jerry Wallweber, Lisa DeFazio-Eli, Carlos L. Arteaga. A HER3 antibody that blocks ligand-independent HER2-HER3 dimerization sensitizes to HER2 and PI3K inhibitors . [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 5461. doi:10.1158/1538-7445.AM2013-5461

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

Abstract 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&amp;lt; 0.05). Next, we assessed the effect of exogenous HRG on this combination therapy. HRG reduced the activity of trastuzumab but did not affect MM-111’s activity, and it enhanced the synergy of this combination (50% greater than Bliss independence; p &amp;lt; 10−8). 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 paclitaxel's 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 &amp;lt; 10−8) 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-79: Combined treatment with HER3 antibody MM-121/SAR 256212 and EGFR antibody cetuximab in pre-clinical models of head and neck cancer.

Abstract Background: The EGFR monoclonal antibody cetuximab is approved for treating head and neck squamous cell cancer (SCCHN) in combination with radiation therapy for locally advanced disease and chemotherapy for recurrent and metastatic disease. Intrinsic or compensatory HER3 signaling and sustained PI3K/Akt activation may play a role in resistance to EGFR-targeted therapy. However, the role of HER3 in resistance to EGFR-targeted therapy in SCCHN has not been elucidated. Therefore, we first tested whether the combination of MM-121/SAR 256212 (M), a HER3 antibody being co-developed by Merrimack Pharmaceuticals and Sanofi, with cetuximab (C) may be more effective than M or C alone in treatment of SCCHN. Methods: We screened for the expression of EGFR, HER3 and their activated forms in 12 SCCHN cell lines. We then evaluated the activity of C, M, and their combination (CM) both in vitro by colony formation assay and in vivo by using SCCHN xenograft models (Tu212 and SCC47). In the in vivo experiment, mice were treated by intraperitoneal injection (i.p.) of C, M or CM twice per week for 4 weeks. The experimental groups include a PBS control, C (6.25ug/dose), M at low dose (300ug/dose, LD), M at high dose (600ug/dose, HD), the combination with LD (CMLD), and the combination with HD (CMHD). In addition, we examined the effect of M and C on HER3 and its downstream pathways in SCCHN cell lines by western blot assay. Results: pHER3 was detected in 9/12 while pEGFR, EGFR and HER3 were expressed in all of 12 SCCHN cell lines. The colony formation assay showed that Tu212 cells were significantly inhibited by CM compared to the control (p &amp;lt;0.001), M (p&amp;lt;0.001) and C (p=0.009) alone. Similar results were also observed using the SCC47 cell line. Western blot analysis confirmed the down-regulation of activated HER3 and its downstream phospho-Akt and phospho-S6 ribosomal protein by CM. Our in vivo study showed significant tumor growth inhibition in both MM-121 LD (p&amp;lt;0.001) and MM-121HD (p&amp;lt;0.001) compared to the control. Moreover, the treatment with both CMLD and CMHD significantly suppressed Tu212 xenograft tumor growth compared to the PBS control (both p&amp;lt;0.0001), C alone (both p&amp;lt;0.0001), and MLD (p=0.0046 and 0.0008, respectively), but only CMHD showed significant inhibition compared to MHD (p = 0.02). There was no significant difference between CMLD and CMHD (p=0.5994). Studies using another SCCHN xenograft model (SCC47) showed a similar inhibitory effect from the treatments by C, M and CM. Conclusion: This study shows that the combination of cetuximab and MM-121 is significantly more active than MM-121 or cetuximab alone in models of head and neck cancer in which both EGFR and HER3 are activated. Further studies will be expanded to understand the underlying mechanisms of this combination. (This study was supported by Merrimack Pharmaceuticals Inc.) Citation Format: Ning Jiang, Dongsheng Wang, Zhongliang Hu, Aminur M. Rahman, Hongzheng Zhang, Ruhul A. Amin, Xiaojing Wang, Zhengjia Chen, Shin M. Dong, Gabriela Garcia, Gavin MacBeath, Jun Ma, Fadlo R. Khuri, Nabil F. Saba, Georgia Z. Chen. Combined treatment with HER3 antibody MM-121/SAR 256212 and EGFR antibody cetuximab in pre-clinical models of head and neck 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 LB-79. doi:10.1158/1538-7445.AM2013-LB-79

Abstract 2781: Champions TumorGraft™ models possess PIK3CA mutations - A great tool to explore the PI3K/AKT pathway.

Abstract Phosphatidylinositol 3-kinase (PI3K) is an important intracellular signal transducer that is involved in many cellular functions such as cell growth, proliferation, and differentiation. PI3K is a heterodimeric molecule composed of a regulatory and a catalytic subunit. PIK3CA, which encodes the p110α catalytic subunit, is often mutated in a range of human cancers, such as colorectal and non-small-cell lung cancers. Increasing our understanding of the biological implications of these mutations and the role of PI3K pathway in cancer proliferation or survival may lead to potential new therapeutic opportunities targeting PI3K signaling. Champions Oncology focuses on the development of TumorGraft models derived from the direct implantation of patient tumors into immunocompromised mice. Compared to traditional cell line-based xenograft models, the patient derived Champions TumorGraft™ models maintain stable gene-expression patterns and mutational status and correlate to clinical predictability. Through extensively characterization, Champions has identified 19 TumorGraft models with PIK3CA mutations. Out of these 19 models, there are 11 colorectal cancers, 3 non-small cell lung cancers, 3 pancreatic cancers, 1 gastric cancer and 1 melanoma. Genomics analyses using the Ampliseq platform identified the majority of PIK3CA mutations in exon 9 and 20. Mutations in these regions of PIK3CA are known to confer resistance to some anti-cancer agents, such as HER2 antibodies. Additionally, it has been reported that PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers. Upon further analysis, we identified the same pattern in Champions TumorGraft models where∼70% (13 out of 19) of the PIK3CA mutant models possess either concurrent RAS or BRAF mutations. Notably, PI3K activates the downstream kinase AKT which ultimately triggers the mTOR pathway. Mutations in cancer often result in constitutive activation of this pathway. Given the role of the PI3K/AKT signaling in cancer and the presence of these specific mutations in the Champions TumorGraft bank, utilization of Champions’ TumorGraft models in oncology drug development allows for greater insight into new therapeutic opportunities targeting the inhibition of PI3K/AKT signaling and offers new promise for patients with these activated pathways. Citation Format: Tin Oo Khor, Dhanrajan Tiruchinapalli, Jay Friedman, Nathan Anderson, David Sidransky, Elizabeth M. Bruckheimer. Champions TumorGraft™ models possess PIK3CA mutations - A great tool to explore the PI3K/AKT pathway. [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 2781. doi:10.1158/1538-7445.AM2013-2781

Abstract 3512: Dielectrophoretic capture and genetic analysis of individual disseminated solid tumor cells.

Abstract Background: The purpose of this study was to develop a novel approach for isolation and genetic analysis of individual circulating and disseminated tumor cells (CTC, DTC) from patient samples. This work is motivated by our desire to develop non-invasive molecular diagnostics and understand single tumor cell heterogeneity. Methods: Here we used the DEPArray microelectronics and microfluidics platform for the manipulation and capture of individual tumor cells. A suspension of live or fixed cells, prelabeled with fluorochrome-conjugated antibodies, was loaded into the DEPArray cartridge, and application of non-uniform electrical fields (dielectrophoresis) was employed to trap individual cells into movable micro-cages. Cell images were acquired via the incorporated fluorescence microscope and CCD camera, and cells were selected based on morphology and expression of tumor antigens. To determine sensitivity and specificity of the capture process, cell lines were spiked into normal donor white blood cells (WBC) over a 5-log range. Pre-enrichment by FACSAria was employed when the starting tumor cell concentration was below 1 in 1,000 WBC. The whole genome amplification (WGA) product of single cells was then subjected to targeted sequencing. To apply this approach to patient samples, we obtained blood and bone marrow samples from patients with refractory high-risk neuroblastoma, a cancer with a high rate and quantity of distant metastatic disease. Results: Single tumor cells were recovered from all spiking experiments, including spiked samples with a tumor cell concentration as low as 1 in 106 WBC. Recovery of EpCAM-negative single cells was accomplished with the neuroblastoma cell lines SY5Y, NB1643, and Kelly labeled with an antibody for the disialoganglioside GD2, the breast cancer cell line MCF-7 with a HER2 antibody, and for the lung cancer cell line H292 with EGFR labeling. Recovery of EpCAM-positive cells was achieved with the colon carcinoma cell line SW480. Captured single cells were WGA'd and specificity was verified using Sanger sequencing. For the 2 neuroblastoma spiking experiments for which sequencing has been completed, the known ALK mutation (F1174L) was detected in 100% of cells. The known KRAS mutation (G12V) was detected in 100% of single SW480 cells sequenced to date. No mutations were detected in isolated WBC. We next analyzed the post-Ficoll fraction of 12 patient bone marrow and 1 patient blood sample, with a median tumor cell concentration of 0.119% (range 0.003 - 80.0%). A minimum of 10 single tumor cells were captured per patient. Genome-wide analysis of DNA and RNA copy number and mutation status from these samples is ongoing. Conclusion: These data demonstrate the development of a novel workflow for the reliable capture and genetic analysis of rare CTC. Efforts are ongoing to join this approach with a more comprehensive genome-wide analysis of single cells from neuroblastoma and other cancers. Citation Format: Erica L. Carpenter, JulieAnn Rader, Kateryna Krytska, Jacob Ruden, Eric Rappaport, Yael Mosse, Francesca Fontana, Manuela Banzi, Gianni Medoro, Nicolo Manaresi, Peter O'Dwyer, John Maris. Dielectrophoretic capture and genetic analysis of individual disseminated solid tumor cells. [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 3512. doi:10.1158/1538-7445.AM2013-3512

Abstract 5646: Overcoming acquired resistance to cetuximab by dual targeting of HER family members using antibody based therapy.

Abstract The EGFR is recognized as a key mediator of proliferation and progression in many human tumors and strategies to inhibit EGFR signaling have emerged as highly promising cancer therapy approaches. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to treatment. In an effort to examine mechanisms of acquired resistance to anti-EGFR monoclonal antibody we developed a series of cetuximab-resistant clones using the NSCLC line NCI-H226. During efforts to elucidate molecular pathways that are involved in acquired resistance to cetuximab we found that 1) EGFR ubiquitination and degradation is altered in cetuximab-resistant cells resulting in increased steady-state expression of the EGFR and 2) enhanced signaling from the EGFR to HER3 and the PI(3)K/Akt pathway indicating that activation of HER3 may lead to escape from cetuximab therapy. To investigate the role of HER3 in acquired resistance to cetuximab we used both RNAi and targeted approaches using the anit-HER3 antibody U3-1287/AMG888. siRNA directed against HER3 indicated that resistant cells had a strong dependency on HER3. Further, targeting HER3 using U3-1287/AMG888 showed a dose-dependent anti-proliferative response in resistant cells. In studies using siRNA against the EGFR and HER3 resulted in robust inhibition of cell proliferation than did silencing of either receptor alone in resistant cells. Comparable effects on cell proliferation were observed after combinatorial treatment of cells by the EGFR antibody cetuximab and the HER3 antibody U3-1287/AMG888. In addition, combined EGFR and HER3 inhibition by either siRNAs or antibodies decreased the phosphorylation of downstream signaling pathways AKT and MAPK. Annexin-V binding assays showed a significant increase in apoptosis in U3-1287/AMG888 plus cetuximab treated cells as compared to either agent alone. To determine if U3-1287/AMG888 could have therapeutic benefit as a combination treatment with cetuximab in vivo, mice harboring NCI-H226 xenografts will be treated with cetuximab for approximately 70-100 days until cetuximab-resistant tumors develop. At this time mice with resistant tumors will be divided into two groups that will be treated with both cetuximab and U3-1287/AMG888 or cetuximab alone. The work presented herein suggests that cells with acquired resistance to cetuximab maintain their dependence on EGFR and HER3 signaling via the AKT and MAPK pathways. Citation Format: Mari Iida, Toni M. Brand, Megan M. Starr, Neha Luthar, Matthew J. Wleklinski, Deric L. Wheeler. Overcoming acquired resistance to cetuximab by dual targeting of HER family members using antibody based therapy. [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 5646. doi:10.1158/1538-7445.AM2013-5646