title: Effective targeted cytotoxicity of neuroblastoma cells.
abstract: OBJECTIVE
  Despite aggressive treatment with surgery, chemotherapy, and radiotherapy, the prognosis for many children with neuroblastoma remains poor. Targeted toxins represent novel cancer therapeutics designed to selectively target and kill cancer cells. The authors have developed a novel fusion toxin, DT5F11, consisting of truncated diphtheria toxin (DT(A)) linked to a single chain antibody (sc5F11) targeting the GD(2) antigen found on most neuroblastoma cells. This report describes the construction, expression, and in vitro function of DT5F11.
  
  
  METHODS
  Utilizing restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis, the prkDTL5F11 plasmid was created by the fusion of distinct coding sequences for a single-chain GD(2) targeting antibody (sc5F11) and truncated diphtheria toxin (DT(A)). DH5alpha Escherichi coli-competent cells were transformed with prkDTL5F11; DNA was amplified, isolated, and sequenced. The fusion protein was expressed and assayed by Western blot. Targeted cytotoxicity was analyzed on GD(2)-positive (SK-N-AS, IMR-32, SK-N-MC, LAN-1) and GD(2)-negative (HeLa) cells.
  
  
  RESULTS
  Fluorescent dye-labeled cycle sequencing identified the constructed fusion toxin gene. Western blot analysis using a mouse antihuman DT(A) antibody showed a 69-kD band identifying the fusion toxin, DT5F11. Targeted cell killing with DT5F11 was seen only in GD(2) positive cells.
  
  
  CONCLUSIONS
  This study demonstrates creation of a novel fusion toxin with effective GD(2)-targeted cellular toxicity. Further investigation of this fusion toxin as a therapeutic agent in the management of neuroblastoma is warranted.
authors.affiliation: Division of Pediatric Surgery, Department of Surgery, Medical University of South Carolina, Charleston, SC, USA.
authors.forename: Patrick B
authors.initials: PB
authors.lastname: Thomas
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Delatte
authors.forename: Aimee
authors.initials: A
authors.lastname: Sutphin
authors.forename: Arthur E
authors.initials: AE
authors.lastname: Frankel
authors.forename: Edward P
authors.initials: EP
authors.lastname: Tagge
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use; D000912:Antibodies, Neoplasm / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000276:immunology; D016923:Cell Death / Q000187:drug effects / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use; D005732:Gangliosides / Q000096:biosynthesis / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use*; D009447:Neuroblastoma / Q000188:drug therapy / Q000276:immunology* / Q000473:pathology*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use; D010957:Plasmids / Q000235:genetics / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D004167:Diphtheria Toxin; D005732:Gangliosides; D007140:Immunoglobulin Fab Fragments; D000922:Immunotoxins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C029795:diphtheria toxin fragment A; C019403:ganglioside, GD2