Modified viral capsid protein mediates non-viral targeting of unique non-covalent drug conjugates to HER2+ breast cancer cells.
Authors: Hasmik Agadjanian, Altan Rentsendorj, Vinod Valluripalli, Jun Ma, Sam Bass, Beth Karlan, Lali Medina-Kauwe
Journal: Cancer Research (2007)

1505  The purpose of this study is to test whether a unique recombinant cell-penetrating viral protein can transport DNA intercalating drugs by non-covalent interaction via a tiny nucleic acid carrier, and target such drugs to HER2+ breast cancer cells.  Targeted conjugates were assembled by mixing the recombinant protein, HerPBK10, with a double-stranded oligonucleotide, and the chemotherapeutic agent, doxorubicin (Dox) at room temperature, then purified by size exclusion chromatography. Stability over time during different storage temperatures was tested by ultrafiltration through molecular weight limiting spin columns. Targeted toxicity was tested on separate and mixed cultures of HER2+ and HER2- human breast cancer cell lines by 4 hour exposure to conjugates (0.5 uM, with respect to Dox concentration) in cell media, then exchanging for fresh media, and measuring cytotoxicity by metabolic assay up to 3 days post-treatment. Receptor specificity was tested by competitive inhibition with excess free ligand. In vivo targeting was tested by IV injection in a mouse xenograft model of HER2+ human breast cancer and monitoring Dox fluorescence in live mice.  The conjugate, called HerDox, exhibited undetectable Dox release under high speed centrifugation and high pressure chromatography conditions, as well as under storage at 4°C, 25°C, and 37°C for up to 12 days. In separate wells of HER2+ and HER2- human breast cancer cells, HerDox reduced HER2+ cell survival by over 75% whereas HER2- cells were unaffected. Dox alone reduced both cell types by the same order of magnitude. In mixed cultures of HER2- and HER2+ cells, HerDox nearly completely inhibited HER2+ cell proliferation whereas HER2- cells were little to modestly affected. Free heregulin ligand at 10x molar excess (compared to HerDox) completely inhibited HER2+ cell killing by HerDox. IV injection of HerDox in tumor-bearing nude mice exhibited widespread Dox fluorescence by 10 min, followed by fluorescence accumulation in the tumors by 20 min, which persisted up to 100 min after injection. Tissues harvested at 4h after injection showed that tumors exhibited the highest intensity of Dox fluorescence while residual fluorescence was observed in the liver and kidneys. Fluorescence was undetectable in the heart, lung, spleen and skeletal muscle. Targeting ligand alone showed similar biodistribution.  These studies indicate that a stable non-covalent conjugate can assemble and deliver a well-established chemotherapy drug to target cells in serum-containing media. Toxicity can be targeted to HER2+ cells in a mixed cell culture, and is mediated via the heregulin receptor. HerDox exhibits preferential accumulation in HER2+ human breast cancer tumors in vivo, and appears to avoid other tissues including the heart, lung, spleen, and skeletal muscle, while exhibiting relatively low to negligible levels of delivery to the liver and kidney.