Targeting granzyme B to tumor cells using a yoked human chorionic gonadotropin

• Published in: Cancer chemotherapy and pharmacology 2011-10, Vol.68 (4), p.979-990
• Main Authors: Kanatani, Isao, Lin, Xinjian, Yuan, Xiaoqin, Manorek, Gerald, Shang, Xiying, Cheung, Lawrence H., Rosenblum, Michael G., Howell, Stephen B.
• Format: Article
• Language: English
• Subjects: Animals; Antineoplastic agents; Apoptosis - drug effects; Biological and medical sciences; Blotting, Western; Cancer Research; Caspase 3 - metabolism; Cell Line; Cell Line, Tumor; Cells, Cultured; Chorionic Gonadotropin - chemistry; Chorionic Gonadotropin - genetics; Chorionic Gonadotropin - metabolism; Drug Delivery Systems; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Granzymes - administration & dosage; Granzymes - metabolism; Granzymes - pharmacology; Humans; Medical sciences; Medicine; Medicine & Public Health; Mice; Microscopy, Confocal; Oncology; Original; Original Article; Ovarian Neoplasms - drug therapy; Ovarian Neoplasms - pathology; Pharmacology. Drug treatments; Pharmacology/Toxicology; Protein Binding; Receptors, LH - genetics; Receptors, LH - metabolism; Spodoptera
• ISSN: 0344-5704; 1432-0843
• DOI: 10.1007/s00280-011-1573-4

Purpose Luteinizing hormone receptor (LHR) is found in abundance on human ovarian, breast, endometrial and prostate carcinomas but at only low levels on non-gonadal tissues. To selectively kill LHR-expressing tumors, granzyme B (GrB) was linked to a protein in which both chains of human chorionic gonadotropin were yoked together (YCG). Methods GrB-YCG was expressed and secreted from insect Sf9 cells. Its GrB enzymatic activity and binding affinity for hLHR were then characterized. The differential cytotoxicity of GrB-YCG versus GrB alone was tested in a panel of LHR-expressing tumor cells by SRB assay, and the mechanisms involved in the cell death were investigated by confocal fluorescence microscopy, flow cytometry, and western blot analysis. Results GrB-YCG was successfully expressed and secreted from Sf9 insect cells and purified from cell culture supernatants. The serine protease activity of GrB-YCG was equivalent to that of human recombinant GrB. An in vitro hormone binding assay revealed that the GrB-YCG molecule also retained the ability to bind to the LHR receptor with an affinity similar to that of native hCG. Upon cell binding, GrB-YCG was rapidly internalized into LHR-expressing human ovarian cancer cells and produced selective and potent tumor cell killing by inducing apoptosis through activation of caspase-3. Conclusions These results validate LHR as a therapeutic target and indicate that delivery of the human pro-apoptotic enzyme GrB to tumor cells by yoked hCG has substantial selectivity and therapeutic potential for human tumors that express high levels of LHR such as ovarian carcinomas.