Chironex fleckeri toxin as immunotoxin

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Chironex fleckeri toxin as immunotoxin
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IMMUNOTOXIN

• Cell-specific cytotoxic agents
• Cancer, viral disease
• Cell-specific-ligand cytotoxic compound-gt
  selective cell destruction.

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General principle of an immunotoxin
TARGET CELL
TOXIN
LIGAND
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AIM

• Chironex fleckeri toxin anti-human myeloma
  lymphoma cells

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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

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Current immunotoxins

• Toxins from plants, bacteria and bees- plant
  derived ricin chain A
• Resting and dividing cells -gt advantage over
  chemotherapeutic agents
• Most Intracellular acting inhibiting protein
  synthesis
• Bee venom Bacillus thuringiensislytic -gt no
  internallisation

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Applications

• Saporin- molecular neurosurgery-gt highly
  selective neuro lesions, pain perception
• Most RTA- phase I and II clinical trials- B-cell
  lymphoma, Hodgikins disease, T-cell lymphoma,
  B-cell leukemia and steroid-resistant-graft-vs-hos
  t disease.
• Bone marrow purging for autologous
  transplantation.
• Cytomegalo virus and HIV infected cells.

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Side-effects

• Vascular leak syndrome (VLS)
• Muscle pain
• Decrease in white blood cells
• Occasional neuronal degeneration

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Experimental plan

• Capture of jellyfish in Darwin, Australia
• Collection and rupturing of nematocysts-microscop
  ic 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)

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Capillary electrophoresis
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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)

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Experimental plan cont.

• Protein sequencing
• MALDI-TOF
• Cloning of toxin gene Ye et.al (2000)

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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)

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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).

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Chironex fleckeri immunotoxin

• Unspecific lytic properties
• Very potent
• Membrane active
• Effects not dependent on cell division

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