Chironex fleckeri toxin as immunotoxin by mikesanye

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									Chironex fleckeri toxin as
     immunotoxin
          IMMUNOTOXIN
• Cell-specific cytotoxic agents
• Cancer, viral disease
• Cell-specific-ligand + cytotoxic compound
  -> selective cell destruction.
 General principle of an
     immunotoxin

TARGET
          LIGAND   TOXIN
 CELL
                   AIM



• Chironex fleckeri toxin + anti-human
  myeloma & lymphoma cells
Chironex fleckeri
         • Australian sea wasp, Box-
           jellyfish
         • Lethal, kills in 3-8 min
         • Several toxins
         • Haematolytic,
           dermatonecrotic, lethal
           components
         • Membrane permeability
           changes
         • Respirotory failure &/or
           cardiac dysfunction
        Current immunotoxins

• Toxins from plants, bacteria and bees
  - plant derived ricin chain A
• Resting and dividing cells
  -> advantage over chemotherapeutic agents
• Most Intracellular acting inhibiting protein
  synthesis
• Bee venom & Bacillus thuringiensis
  lytic -> no internallisation
                Applications
• Saporin- molecular neurosurgery-> highly
  selective neuro lesions, pain perception
• Most RTA- phase I and II clinical trials
  - B-cell lymphoma, Hodgikin’s disease, T-cell
  lymphoma, B-cell leukemia and steroid-resistant-
  graft-vs-host disease.
• Bone marrow purging for autologous
  transplantation.
• Cytomegalo virus and HIV infected cells.
                Side-effects
•   Vascular leak syndrome (VLS)
•   Muscle pain
•   Decrease in white blood cells
•   Occasional neuronal degeneration
            Experimental plan
• Capture of jellyfish in Darwin, Australia
• Collection and rupturing of nematocysts
  -microscopic evaluation
• Pelleting of capsule material and determination of
  protein concentration by absorbance
  measurements.
• Fractionation of crude venom by CE and analysis
  by SDS-CGE
   Bloom et.al (2001)
Capillary electrophoresis
       Experimental plan cont.
• Gel filtration
• Cytotoxicity analysis on human lymphoblastoid
  cells
• Production of monoclonal anti-KMA antibodies
  (KMA = specific epitope expressed on human
  myeloma and lymphoma cells)
• Produce an anti-KMA antibody gene construct by
  reverse transcription.
      Dunn et.al (1996)
      Experimental plan cont.
• Protein sequencing
• MALDI-TOF
• Cloning of toxin gene
       Ye et.al (2000)
      Experimental plan cont.
• Construction of a antibody-toxin-FLAG fusion
  protein and expression in E. coli
• Anti-FLAG affinity chromatography, SDS-PAGE
  and Wester blot
• Cell binding analysis by flow cytometry
• Specificity and toxicity analysis on human
  lynphoblastoid cells
         Dunn et.al (1996)
         Additional methods
• Expression in Pichia pastoris- Gurkan and
  Ellar (2003)
• Genetically modified anti-KMA antibody
  fragments for chemical conjugation to the
  toxin- Better et.al (1993).
    Chironex fleckeri immunotoxin
•   Unspecific lytic properties
•   Very potent
•   Membrane active
•   Effects not dependent on cell division

								
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