Different Levels Of HER2 Expression Research Articles

Block Copolymer Micelles Target Auger Electron Radiotherapy to the Nucleus of HER2-Positive Breast Cancer Cells

Intracellular trafficking of Auger electron emitting radionuclides to perinuclear and nuclear regions of cells is critical to realizing their full therapeutic potential. In the present study, block copolymer micelles (BCMs) were labeled with the Auger electron emitter indium-111 ((111)In) and loaded with the radiosensitizer methotrexate. HER2 specific antibodies (trastuzumab fab) and nuclear localization signal (NLS; CGYGPKKKRKVGG) peptides were conjugated to the surface of the BCMs to direct uptake in HER2 expressing cells and subsequent localization in the cell nucleus. Cell uptake and intracellular distribution of the multifunctional BCMs were evaluated in a panel of breast cancer cell lines with different levels of HER2 expression. Indeed cell uptake was found to be HER2 density dependent, confirming receptor-mediated internalization of the BCMs. Importantly, conjugation of NLS peptides to the surface of BCMs was found to result in a significant increase in nuclear uptake of the radionuclide (111)In. Successful nuclear targeting was shown to improve the antipoliferative effect of the Auger electrons as measured by clonogenic assays. In addition, a significant radiation enhancement effect was observed by concurrent delivery of low-dose MTX and (111)In in all breast cancer cell lines evaluated.

Abstract 5284: 68Ga-radiolabeled DOTA-affibody molecule for in vivo assessment of HER2 expression with PET

Abstract Overexpression of HER2, a transmembrane tyrosine kinase receptor, is correlated with poor prognosis in patients with breast cancer. HER2 expression may vary between primary tumors and metastatic lesions and also change during the treatment. Therefore, there is a need for methods that can assess global HER2 expression non-invasively and reproducibly. PET imaging is well suited to this task based on its high sensitivity. Fluorine-18 (18F) is the most commonly used PET emitter for in vivo imaging since it is relatively short lived radionuclide (109 min). However, its production requires a cyclotron. Gallium-68 (68Ga) is an attractive alternative to 18F. It has a similarly short half-life (68 min) but can be obtained from a generator. In this work, we sought to monitor HER2 expression in vivo with 68Ga-labeled DOTA-ZHER2:2891 Affibody in a panel of breast cancer xenografts with different levels of HER2 expression. ZHER2:2891 Affibody molecules were conjugated with the chelator, DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid), and labeled with 68Ga. The in vitro binding characteristic of the tracer was evaluated using saturation cell-binding assay. In vivo studies were conducted in athymic nude mice bearing subcutaneous human breast cancer tumors with four different levels of HER2 expression. PET/CT scans were performed 1, 2, 3 hour post tracer injection. After imaging, the animals were sacrificed and selected organs were dissected to characterize the relationship between the signal detected by PET, ex vivo assessment of the tracer uptake, and the HER2 expression (in tumors). Nonspecific uptake was analyzed 1 hour post His6-ZTaq-GS-Cys-DOTA-68Ga (non-HER2-specific Affibody molecules) injection. In vitro assays showed high binding affinity of 68Ga-DOTA-ZHER2:2891 Affibody probe to MDA-MB-361 cells (Kd = 1.4±0.19 nM). In vivo biodistribution and PET imaging studies demonstrated high radioactivity uptake in the HER2 overexpressing tumors. Tracer was eliminated quickly from the blood and normal tissues, providing high tumor-to-blood ratios. The highest normal tissue concentration of radioactivity was seen in the kidneys. High-contrast PET images were recorded in BT474 and MDA-MB361 tumors 1 h post tracer injection. Low, but still detectable, uptake was observed for MCF7 tumors. Very good correlation was observed between PET and ex vivo estimates of tracer concentration and the receptor expression in the tumor tissue, as assessed by, respectively, radioactivity accumulation and enzyme-linked immunosorbent assay (ELISA). Stability tests of 68Ga-DOTA- ZHER2:2891 Affibody in vivo revealed that the tracer is stable in blood. These results suggest that PET imaging using 68Ga-DOTA-ZHER2:2891 Affibody is sensitive enough to detect different levels of HER2 expression in vivo. 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 5284. doi:10.1158/1538-7445.AM2011-5284

Abstract LB-323: Subclassification of squamous cell carcinomas of the head and neck based on HER/ErbB and c-MET receptor protein expression and activation profiles

Abstract The response rates of HER1 antibody monotherapy (∼13%) are comparable to the added response rates of HER1 antibody therapy when used in combination with chemotherapy (∼16%) in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN). Measures of HER1/ErbB1/EGFR protein levels by immunohistochemistry (IHC) or gene copy number by fluorescent in-situ hybridization (FISH) do not correlate with response, therefore, alternative gene or protein expression signatures are needed to identify patients most likely to respond to HER1 targeted therapies. Previously, quantitative VeraTag proximity immunoassays performed on formalin-fixed, paraffin imbedded SCCHN tumor tissue identified an activated HER1 signature (HER1-HER1 homodimers; HER1pY1173) that was significantly more pronounced in SCCHN compared to other carcinoma of the head and neck. In this study, we analyzed the expression of HER1, 2, 3 and c-MET along with the activation of HER1 and HER3 in 56 SCCHN tumors. Our results identified two distinct protein expression profiles: an activated HER1 profile, and a second profile characterized by the different expression levels of HER2, HER3, phosphorylated HER3, and c-MET relative to activated HER1. Our working hypothesis is that the first profile correlates with sensitivity to HER1 targeted therapies while the second profile correlates with either sensitivity or resistance to HER1 targeted therapies. HER1 protein levels measured by VeraTag assay varied over a ∼20-fold dynamic range, and significantly correlated with HER1 IHC staining and mRNA levels determined by qRT-PCR. HER2 and HER3 protein expression spanned a 15- to 20-fold range, and correlated with mRNA levels. Several of the highest HER1 expressing tumors have increased gene copy number; however, similar high values were measured in tumors without amplification, indicating that additional mechanisms of high HER1 expression exist in SCCHN. Although high HER1 activation measured by HER1-HER1 dimer and HER1pY1173 correlates with HER1 levels (r=∼0.6, p<0.05), and are associated with the highest levels of HER1 expression measured by multiple methods, not all high HER1 expressing tumors displayed highly activated HER1. These observations may explain why total HER1 expression in SCCHN tumors, determined by IHC or FISH, does not correlate with response to targeted therapies. On the other hand, approximately 16% of the SCCHN tumors studied here (9/56) exhibit a highly activated HER1 profile; similar in magnitude to the response rate seen with HER1 targeted antibody therapy. Future studies are designed to further validate the HER1 activation signature as a predictor of response to HER1 targeted therapies and/or the attenuation of response due to HER2, HER3 or c-MET expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2011-LB-323

HER2 Targeted Molecular MR Imaging Using a De Novo Designed Protein Contrast Agent

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice models. In addition to its 100-fold higher dose efficiency compared to clinically approved non-targeting contrast agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention over reported contrast agents as demonstrated by even distribution of the imaging probe across the entire tumor mass. This contrast agent will provide a powerful tool for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.

Fc-DIRECTED ANTIBODY CONJUGATION OF MAGNETIC NANOPARTICLES FOR ENHANCED MOLECULAR TARGETING

In this study, we report the fabrication of engineered iron oxide magnetic nanoparticles (MNPs) functionalized with anti-human epidermal growth factor receptor type 2 (HER2) antibody to target the tumor antigen HER2. The Fc-directed conjugation of antibodies to the MNPs aids their efficient immunospecific targeting through free Fab portions. The directional specificity of conjugation was verified on a macrophage cell line. Immunofluorescence studies on macrophages treated with functionalized MNPs and free anti-HER2 antibody revealed that the antibody molecules bind to the MNPs predominantly through their Fc portion. Different cell lines with different HER2 expression levels were used to test the specificity of our functionalized nanoprobe for molecular targeting applications. The results of cell line targeting demonstrate that these engineered MNPs are able to differentiate between cell lines with different levels of HER2 expression.

Changes in HER2 expression in breast cancer xenografts after therapy can be quantified using PET and (18)F-labeled affibody molecules.

In vivo imaging of human epidermal growth factor receptor type 2 (HER2) expression may allow direct assessment of HER2 status in tumor tissue and provide a means to quantify changes in receptor expression after HER2-targeted therapies. This work describes the in vivo characterization of the HER2-specific N-2-(4-(18)F-fluorobenzamido)ethyl]maleimide ((18)F-FBEM)-Z(HER2:342) Affibody molecule and its application to study the effect of 17 (dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) on HER2 expression by PET. To assess the correlation of signal observed by PET with receptor expression, we administered the tracer to athymic nude mice bearing subcutaneous human breast cancer xenografts with different levels of HER2 expression. To study the downregulation of HER2, we treated the mice with 4 doses (40 mg/kg) of 17-DMAG, an inhibitor of heat-shock protein 90, known to decrease HER2 expression. The animals were scanned before and after treatment. After the last scan, the mice were euthanized and tumors were frozen for receptor analysis. The tracer was eliminated quickly from the blood and normal tissues, providing high tumor-to-blood and tumor-to-muscle ratios as early as 20 min after injection. The high-contrast images between normal and tumor tissue were recorded for BT474 and MCF7/clone18 tumors. Low but still detectable uptake was observed for MCF7 tumors, and none for MDA-MB-468. The signal correlated with the receptor expression as assessed by immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay. The levels of HER2 expression estimated by post-treatment PET decreased 71% (P < 4 x 10(-6)) and 33% (P < 0.002), respectively, for mice bearing BT474 and MCF7/clone18 tumors. These changes were confirmed by the biodistribution studies, enzyme-linked immunosorbent assay, and Western blot. Our results suggest that the described (18)F-FBEM-Z(HER2:342) Affibody molecule can be used to assess HER2 expression in vivo by PET and monitor possible changes of receptor expression in response to therapeutic interventions.

Effect of lapatinib on accumulation of inactive HER2 at the cell membrane and on antibody-dependent cellular cytotoxicity (ADCC) mediated by trastuzumab: A novel mechanism for the enhanced effects of combined anti-HER2 therapy

3594 Background: Lapatinib is a HER2 tyrosine kinase inhibitor that has clinical activity in HER2 overexpressing (HER2+) breast cancer. In vitro and clinical studies have shown that lapatinib enhances the effects of the monoclonal antibody trastuzumab suggesting partially non-overlapping mechanisms of action. In order to dissect the differential mechanims of these agents, we have studied the effects of lapatinib and trastuzumab on receptor expression and signaling and have explored a new potential mechanism underlying the profound antitumor activity of the combination. Methods: HER2+ breast cancer cells SKBR3 and MCF7-HER2 were treated with lapatinib, trastuzumab or both. Assays of receptor expression, phosphorylation, signalling and tumor growth were performed. Trastuzumab-dependent cellular cytotoxicity was measured with different levels of HER2 expression and doses of lapatinib. Results: Lapatinib treatment of HER2+ breast cancer cells inhibited HER2 phosphorylation, prevented ubiquitination and resulted in a marked accumulation of inactive receptors at the cell surface. By contrast, trastuzumab caused enhanced HER2 phosphorylation, ubiquitination and degradation of the receptor. By immunoprecipitation and computational protein modelling techniques we further demonstrated that the lapatinib-induced accumulation of HER2 lead to stabilization of inactive HER2 homo- and hetero-dimers. Accumulation of HER2 induced by lapatinib and downregulation of HER2 mediated by trastuzumab were also observed in vivo, where the combination of the two agents triggered complete tumor remission in all cases after 10 days of treatment. Lapatinib-induced accumulation of HER2 at the cell surface markedly enhanced trastuzumab-mediated ADCC. Conclusions: Lapatinib results in a marked accumulation of inactive HER2 receptors at the cell surface both in vitro and in vivo. This increase in receptors number at the cell surface enhances ADCC by trastuzumab. We propose that this is a novel mechanism that may be clinically relevant and exploitable in the therapy of patients with HER2+ tumors. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Genentech