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. Two different approaches will be used for therapy. 1. To target visible, well defined tumors, 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. 2. To eradicate widespread disease in the metastatic setting (in which even the disease with known locations may be too widespread to use local treatment modalities) and for treatment in the adjuvant setting to treat non-detectable micrometastases we will develop Affitoxins affibody-based recombinant proteins combining HER2-targeting capacity of Affibody with proteins having therapeutic potential such as, for example, bacterial toxins or pro-apoptotic cell signaling molecules. Initially well focus on development of proteins with optimal of subcellular localization capacity using Affiprobes- molecules contain fluorescent proteins. Then we will test appropriate effectors, for instance, PE38 and p53 for molecules localizing in cytosol and nucleus, respectively. Investigation of the later therapeutic modality will be facilitated by my collaboration with Dr. Peter Chumakov of the Learner Research Institute, who will share with me reagents and experimental tools developed in his laboratory including the hybrid p53 cDNA in various expression constructs and a high throughput test system for functional activity of p53 (lentivirus-based reporter systems that conveniently measure transcriptional activity of p53 in virtually any cell type, including various in vivo models). This approach will facilitate early identification of the most promising protein allowing focusing the effort on the most promising compounds. 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. Research Design 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 This task will be carried oput in collaboration with Dr. Robert Blumenthal and his team at CCR Nanobiology Program. 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. Affitoxins A recombinant DNA construct combining HER2-specific Affibody molecules with Pseudomonas toxin will be developed and cloned by PCR amplification of the PE38 part of HA22 immunotoxin following by ligation of the PCR product into vector containing the HER2-affibody under control of IPTG-inducible T7 promoter. Similar procedures will be used to produce recombinant proteins containing different effectors and sub-cellular localization domains. 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. Biodistribution of the novel tumor-targeted molecules 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. 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. The subcellular distribution of Affitoxin will be tested using confocal microscopy and their therapeutic efficacy will be assessed in vitro and in vivo. For Exploratory IND Studies a single mammalian species can be used to establish a margin of safety. Accomplishments: 1. We have proved that F-18-labeled Affibody molecules can be used for in vivo quantification of changes in HER2 expression following therapeutic intervention. The results of this work have been published in the Journal of Nuclear Medicine and their significance highlighted by an editorial in the same journal as well as press releases from NIH and American Nuclear Society. 2. We have genetically fused HER2-specific Affibody molecule with a truncated and optimized version of Pseudomonas Exotoxin A (PE38KDEL. The resulting recombinant protein called HER2-Affitoxin combines high HER2 specificity and affinity of Affibody molecules with the tumoricidal potential of PE38KDEL. Manuscript reporting cloning, overexpression, purification, and in vitro characterization of this novel recombinant anti-cancer agent has been accepted for publication in the Journal of Immunotherapy. 3. We have created photo-stable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called Affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of Affiprobes. Affiprobes are able to stain both live cells and frozen tumor xerograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/receptors. A manuscript describing this work has been submitted to a European Journal of Chemical Biology: ChemBioChem. 4. Several Affibody-based recombinant proteins containing fluorescent p [summary truncated at 7800 characters]
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