title: Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin.
abstract: BACKGROUND
  The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein.
  
  
  OBJECTIVE
  We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin.
  
  
  METHODS
  The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human kappa myeloma and lymphoma cells, and on human free kappa Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2).
  
  
  RESULTS
  Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone.
  
  
  CONCLUSIONS
  The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.
authors.affiliation: Department of Cell and Molecular Biology, University of Technology Sydney, NSW, Australia.
authors.forename: R D
authors.initials: RD
authors.lastname: Dunn
authors.forename: K M
authors.initials: KM
authors.lastname: Weston
authors.forename: T J
authors.initials: TJ
authors.lastname: Longhurst
authors.forename: G G
authors.initials: GG
authors.lastname: Lilley
authors.forename: D E
authors.initials: DE
authors.lastname: Rivett
authors.forename: P J
authors.initials: PJ
authors.lastname: Hudson
authors.forename: R L
authors.initials: RL
authors.lastname: Raison
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000378:metabolism*; D019677:Artificial Gene Fusion; D001483:Base Sequence; D003602:Cytotoxicity, Immunologic; D005803:Genes, Immunoglobulin; D006801:Humans; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D008555:Melitten / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000941:Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011994:Recombinant Proteins; D008555:Melitten