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Mutagenesis of the therapeutic antibody aTSI scFv
2003 (English)Independent thesis Basic level (professional degree)Student thesis
Abstract [en]

The monoclonal antibody _TS1 has been used successfully in vivo in human-tumour bearing mice, to clear the circulation from non-tumour targeted radio-labelled antibody TS1. The interaction between the anti-cytokeratin antibody TS1 and its antiidiotypic antibody _TS1 have previously been studied by chemical modification and 3D modelling, and interesting targets for mutagenesis have been identified. In this study site-directed mutagenesis on interesting target amino acids were performed on the single-chain of _TS1 variable domains (scFv), and 5 mutants were made; 2 lysine residues and 3 tyrosine residues were substituted by alanine. The wild-type _TS1 scFv and the mutants were expressed in E. coli and studied with ELISA, SDS-PAGE, Western Blot and BIAcoreĀ®. Despite that the expression was low and that it was difficult to quantitate the scFv, the ELISA and BIAcoreĀ® results indicate that the tyrosine residues are more important for the association than the dissociation, since the mutants after substituting to alanine showed slower on-rates and slower off-rates to its antigen. Two mutants showed similar affinity constants as the wild-type scFv despite the large changes in on and off rates. After substituting one of the lysine residues to alanine a slightly higher on rate and affinity was observed, whereas mutation of the other lysine residue resulted in a slower on rate and lower affinity. This also confirms that equivalent affinities can be maintained by compensatory differences in on and off rates, and a changed affinity can reflect changes in both the on and off rate. The results from this study contribute to the functional mapping of the _TS1 epitope and the aim in the future is to create antiidiotypic antibodies with different clearing qualities.

Place, publisher, year, edition, pages
2003. , 30 p.
Identifiers
URN: urn:nbn:se:kau:diva-52525Local ID: KEM-20OAI: oai:DiVA.org:kau-52525DiVA: diva2:1101052
Subject / course
Chemistry
Available from: 2017-05-29 Created: 2017-05-29

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