This proposal will test the hypothesis that the cell entry and cell penetration features of a novel recombinant protein derived from the adenovirus (Ad) capsid mediates non-viral delivery of a novel toxic corrole compound specifically to breast cancer cells for targeted breast cancer therapy. We will test this new therapeutic for targeting HER2+ breast cancer, which is characterized by aggressive tumor formation, metastasis, chemoresistance, a poor prognosis, high mortality, and amplification of the human epidermal growth factor receptor HER2 subunit. About 30% of breast cancers are HER2+, comprising a significant subset of breast cancers requiring novel alternatives to standard treatment. The targeting aspect of this conjugate will direct therapy specifically to HER2+ cells, thus avoiding normal cells and other dividing cells such as blood cells, hair cells, and gastrointestinal epithelium.
The Specific Aims of this project are to test the hypotheses that: ? 1) HerPBK10 targets corroles to HER2+ cells in vitro. Standard metabolic assays for measuring cell survival and proliferation will be used to determine the cytotoxicity of conjugates on a panel of HER2+ and HER2- cells in vitro, and to assess the effect of serum on conjugate activity, and determine optimal dosage. Spectrophotometric absorbance assays will be used to assess the assembly parameters of the conjugates under different storage times and temperatures. Flow cytometry, fluorescence microscopy, and cytotoxicity assays will be used to assess targeting specificity by competitive inhibition with a free ligand, delivery to HER2+ cells in a mixed cell culture, and delivery to isogenic cell lines expressing different levels of receptor subunits. Finally, apoptosis as a mechanism of cell death will be examined. 2) HerPBK10 targets corroles to HER2+ cells in vivo. A mouse xenograft tumor model of HER2+ breast cancer will be used to assess the therapeutic efficacy of conjugate delivery by both intratumoral injection and systemic delivery, and the presence of circulating heregulin will be assessed. The biodistribution and pharmacokinetics of conjugates will also be examined by harvesting and immunohistological processing of tissues, and collection of serum, after in vivo circulation. We will use ELISA-based assays to examine cytokine induction in both tumor model and immune competent mice and to examine induction of neutralizing antibodies in immune competent mice. Apoptosis as a mechanism of cell death in vivo will also be examined. This research is relevant to public health because the proposed project will develop a novel therapeutic that can specifically target HER2+ breast cancer. This targeted therapy should be improved over conventional treatment methods because normal cells should not be affected. As HER2+ breast cancer does not respond well to conventional ? therapies, this alternative therapy could provide a significant contribution to breast cancer treatment. ? ? ?
