title: Targeting granzyme B to tumor cells using a yoked human chorionic gonadotropin.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Medicine and the Rebecca and John Moores UCSD Cancer Center, University of California, San Diego, La Jolla, CA 92093-0819, USA.
authors.forename: Isao
authors.initials: I
authors.lastname: Kanatani
authors.forename: Xinjian
authors.initials: X
authors.lastname: Lin
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Yuan
authors.forename: Gerald
authors.initials: G
authors.lastname: Manorek
authors.forename: Xiying
authors.initials: X
authors.lastname: Shang
authors.forename: Lawrence H
authors.initials: LH
authors.lastname: Cheung
authors.forename: Michael G
authors.initials: MG
authors.lastname: Rosenblum
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Howell
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016503:Drug Delivery Systems; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D053804:Granzymes / Q000008:administration & dosage* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000473:pathology; D011485:Protein Binding; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
chemical_list: D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D053804:Granzymes; D053148:Caspase 3