Developing apoferritin into an imaging and drug delivery platform; Optimisation of production and efficiency of cell uptake

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Biopharmaceutics (aka biologics) are increasingly being used in the clinic. The majority of these compounds are currently antibodies or antibody derived structures which have the disadvantage of needing to be produced in eukaryotic expression systems and because they are made of multiple chains held together by disulphide bonds are difficult to obtain and hence very expensive as therapeutics. We have been investigating an alternative protein scaffold, that of human apoferritin, which can be produced in high yield in E. coli as an imaging and drug delivery vehicle. Apoferritin self assembles into a protein capsule from 24 subunits when above pH 5.0 and its natural cargo of iron(III)oxide can be replaced by imaging molecules including fluorescent quantum dots, MRI and PET imaging agents or drug molecules.Apoferritin is thought to be preferentially taken up by cells that over express the transferrin or TIM2 receptors which includes a number of cancer cell types. By fusing specific peptides to the N-terminal of apoferritin so that they appear on the surface of the protein capsule, it should be possible to redirect the apoferritin to different cell types. Apoferritins are normally endocytosed and it is thought that the protein capsule disassembles in the late endosome or lysosome realising its contents with the apoferritin proteins either being degraded by the proteasome or being recycled and released from the cell. In the first rotation (Thomas lab), new apoferritin-peptide fusions will be produced and their production optimised. The cellular targeting and uptake abilities of these new fusion proteins will be investigated with different cell types using confocal microscopy.. In the second linked rotation (Bradshaw lab) the effect of the modified apoferritins on cell viability and progression through the cell cycle will be determined and the location and disassembly of the apoferritin in the endosome-lysosome will be studied by fluorescence microscopy and HR-TEM. In the third rotation (Grabowska lab) the localisation of the apoferritin peptide fusion in vivo in mice with and without orthotopic tumours will be investigated using fluorescence/luminescence techniques.