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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
"Chironex fleckeri toxin as immunotoxin"