The development and testing of a radio labelled anti-transferrin scFv for radiotherapy

• Main Author: Godfrey, Samuel Nathan
• Published: University of Kent 2012
• Subjects: 615.842
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594200

The human transferrin receptor (CD71) is a ubiquitously expressed internalising glycoprotein involved in iron homeostasis. Expression of the transferrin receptor is found to be greatly upregulated in tumour cells and, as such, it offers a useful target in the development of immunotherapeutic agents for tumour therapies. This laboratory has developed a Histagged anti-CD71 single-chain variable fragment (scFv) antibody, expressed in Pichia postoris, to be used in the investigation oftargeted radio-immunoconjugates against cancer. The production of an anti-CD71-scFv was confirmed using N-terminal sequencing, and the scFv was purified via a double His-tag using affinity chromatography to produce yields of 13.3mgfl of anti-C071-scFv. Specific binding of the scFv to the human transferrin receptor was evaluated by flow cytometry using C071+ HL-60 leukaemia cells and CHO cells that are CD7r. The anti-CD71-scFv was demonstrated to bind specifically to the CD71 TfR receptor with comparable binding to the monoclonal anti-C071 antibody using flow cytometry. No binding of either antibody to the COJ1- CHO cell line was observed. Saporin, a ribosome inactivating protein, was used to· demonstrate the internalising ability ofthe anti-C071-scFv. Saporin is highly cytotoxic but only if it can reach the cytosol of the target cell. The antiCD71- scFv was able to deliver a non-internalising monoclonal antibody-saporin conjugate intracellularly. This resulted in significant toxicity to CD71+ MCF-7 cells and confirmed that the anti-CD71-scFv could internalise.The anti-CD71-scFv was successfully conjugated to the y and Auger emitting isotope, 1:2.51. 12S1 was selected because the v-rays allow simple monitoring of the binding of the anti-CD71-scFvNal2S1 whilst the Auger electrons provide a potential cytotoxic effect if the radionuclide can get close to the nucleus. The anti-C071- scFv_Nal25 , was shown to retain specific binding ability to its target CD71 receptor, however Clonogenic and Comet assay systems did not show signs of DNA damage and cell cytotoxicity as a result of targeted radio-immunotherapy with the scFv-Naus, conjugate. The anti-CD71-scFv was shown to provide a useful delivery mechanism that is cheap to produce and purify and able to specifically bind and internalise into CD71+ tumour cell s. The isotope 12S1 was unable to demonstrate a cytotoxic effect on targeted cells, most likely due to the formation of monoiodotyrosine residues caused by the degradation of the anti-CD71-scFv intracellularly rapidly diffusing from the lysosome. Further investigation using alternative isotopes and the addition of nuclear localisation and endosomal escape sequences is warranted.