Background and Significance Expression of HER2 receptors in breast cancers is correlated with poor prognosis and their expression may be different in distant metastases as compared to the primary tumor. This project will provide means to assess global expression of HER2 in breast cancers (including metastases) and to deliver therapeutic agents specifically to HER2-positve cells. As the targeting agent we propose to use Affibody molecules obtained from our CRADA partner in Sweden (www.affibody.com). These very stable and highly soluble α-helical proteins are relatively small (8.3 kDa) and can be readily expressed in bacterial systems or produced by peptide synthesis. The His6-Zher2:324 binds to HER2 receptors with high affinity (22 pM) and is available with cystein at the carboxy-terminal to facilitate conjugation. For imaging purposes, these molecules with be labeled with radionuclides. For therapy, the His6-Zher2:324 will be conjugated with thermo-sensitive liposomes that, labeled with beacons for in vivo imaging and loaded with therapeutic agents (e.g. toxins, radiosensitizers or kinase inhibitors), will allow local drug release defined by real-time monitoring of their distribution. In spite of repeated attempts to use Lipososmes for site-directed delivery of hydrophilic and hydrophobic drugs for cancer therapy, their application is limited due to poor understanding of lipososmes interactions with the cells and the sub-optimal biodistribution profile of various formulations. We will circumvent these problems using multifunctional liposomes with targeting, imaging and optimal drug release capabilities. Our strategy, involving assessment of target presence and distribution in an individual patient followed by optimized, target-specific drug delivery, may significantly improve efficacy of breast cancer treatment while reducing side effects. Experimental procedures Labeling with imaging agents The Affibody molecules are conjugated using maleimide chemistry with either AlexaFluor or 18F for, respectively, optical and PET imaging. Thermosensitive liposomes Liposomes with optimal lipid composition, sensitive to temperatures just above 37oC. The thermal destabilization of liposomes in a temperature range of 37oC-45oC will be determined by monitoring calcein leakage by spectrofluorometry methods. The next step will include optimization of techniques to conjugate HER2-specific Affibody molecules to the liposome surface via the maleimide group and their labeling with optical, PET, and MRI imaging agents. As the liposome size is an important determinant of their biodistribution, we will characterize the size of the resulting conjugates using the resources at Nanotechnology Characterization Laboratory. The liposomes will be loaded with therapeutic agents according to published methods. We will consult clinical oncologists at CCR regarding the best drugs available for treatment of breast cancers. In vitro and in vivo characterization Using binding, proliferation and clonogenic survival assays, as well as molecular biology methods we will thoroughly characterize in vitro the binding properties of the conjugates and their effects on the target cells. We will also investigate the impact of their binding on receptor expression and binding of therapeutic antibodies (Herceptin). Biodistribution of the conjugates will be studied using nude mice bearing xenografts of HER2-positive tumors. To test the in vivo imaging capacity of radioconjugates, we will monitor, by optical or PET imaging, the expected downregulation of HER2 in tumor xenografts following treatment with Herceptin or DMAG. The estimation of expression level obtained from imaging data will be verified by ex-vivo analysis of tumor tissue by immunohistochemistry and Western blots. Initial in vivo studies of our drug delivery system will be carried out using liposomes loaded with a combination of hydrophilic and hydrophobic fluorescent markers allowing to map and track in vivo tissue/organ compartmentalization of liposomes and to monitor drug leakage and/or release at 37oC-45oC. Noninvasive methods based on focused ultrasound techniques will be optimized to disintegrate liposomes. MRI, and micro-PET may be used to optimize the imaging capacity of the conjugates by those modalities. Experimental therapy of HER2-positive tumor-bearing animals will be carried out to assess the improvement of the efficacy of therapeutic agents delivered by tumor targeting, heat sensitive liposomes as compared with current application methods. For Exploratory IND Studies a single mammalian species can be used to establish a margin of safety. Project Status Probes for Optical Imaging Affibody-AlexaFluor conjugates have been developed and compared to Herceptin- AlexaFluor conjugates in vitro and in vivo. We showed in vitro that Affibody molecules bind to different epitomes of HER2 than does Herceptin and Affibody has only limited effect on HER2 signaling pathways as compared with Herceptin. Comparative in vivo imaging studies of AlexaFluor labeled ZHER2-Cys Affibody and Herceptin have been carried out using nude mice with human breast and ovarian cancer xenografts. While high tumor-specific accumulation of Heceptin was observed, only weak signal could be detected at the tumor, most likely, due to the fast clearance from the blood and high accumulation of AlexaFluor-labeled ZHER2-Cys Affibody in the kidneys. To increase the time of the Affibody molecules in the blood, molecules containing an albumin binding domain have been labeled with AlexaFluor. Biodistribution studies of these conjugates are carried out. Probes for PET Imaging Methods for 18F labeling of Affibody molecules are being developed. The results of in vitro studies showed that pre-incubation with non-labeled Affibody molecules inhibited the binding of the radioconjugates to the cells in a dose-dependent manner. Saturation analysis of His6-ZHer2:342-Cys-18F binding to the cell membranes indicated a single class of high-affinity binding sites. In vivo, high accumulation of the radioactivity in the tumor was observed as early as 20 min after injection as showed by PET imaging and did not reach the plateau after 60 min. That was confirmed by the results of the biodistribution studies indicating that, already 1 hr post-injection, the concentration of radioactivity (%ID/g) in tumor was approximately 10 times higher than that in the blood. Three hours later, that ratio increased to almost 17. On contrary, the initially high concentration in the kidneys (comparable to that in the tumor) decreased three fold at the same time. HER2-Specific Delivery System Conjugates of thermo-sensitive liposomes with HER2-specific, ZHER2-Cys Affibody molecules were characterized in vitro. We investigated the time-dependent uptake of Affibody-modified liposomes versus non modified liposomes in HER2-positive SKBR-3 cells and HER2-negative U251 cells. There was no significant difference liposome binding at two studied temperatures: 22oC and 37oC. After 30 min incubation, we were able to show 8-fold enrichment of Affibody-modified liposomes in HER-2-positive cells as comported to non-modified liposomes. With the decreasing concentration of Affibody we observed much lower non-specific banding. Cell fluorescence after 2h incubation with non-modified liposomes was comparable to untreated control [summary truncated at 7800 characters]
| Hassan, Moinuddin; Chernomordik, Victor; Zielinski, Rafal et al. (2012) In vivo method to monitor changes in HER2 expression using near-infrared fluorescence imaging. Mol Imaging 11:177-86 |
| Kramer-Marek, Gabriela; Kiesewetter, Dale O; Martiniova, Lucia et al. (2008) [18F]FBEM-Z(HER2:342)-Affibody molecule-a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography. Eur J Nucl Med Mol Imaging 35:1008-18 |
