Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486114
Title: An investigation into making a bispecific scFv antibody that recognises both pIgR. and IgG Fc by phage display technology in order to transport IgG to mucosal surfaces
Author: Burden, Matthew
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2006
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Abstract:
The polymeric immunoglobulin receptor (pIgR) transports polymeric immunoglobulins across the epithelial cell layer into mucosal secretions. It was hoped that pIgR could be exploited to actively transport IgG by constructing a bispecific· molecule capable of simultaneously binding IgG and pIgR. Increasing the IgG' delivered to mucosal secretions may benefit patients undergoing intravenous immunoglobulin therapy by increasing the mucosal defence against invading pathogens. Phage display, using synthetic phagemid libraries (I and J), was used to generate scFv to secretory component (SC), the extracellular portion of pIgR. Phage raised against peptides of pIgR yielded no SC binding clones. Subsequent selections against SC were successful. An in vitro transport assay was used to measure transport of anti-SC clones. No transport could be detected in the initial assay, so it was optimised to increase sensitivity. Subsequent selections using this assay suggested that the dominant epitope recognised by the J library in SC was distinct from those that bound on pIgR that led to the highest level of transport. The dominant epitope recognised by the I library was present on both pIgR and SC. The clones were of weak binding possibly because of the restrictive design ofthe library and apparent bias seen in the sequences ofthe unselected clones. Affinity maturation was applied to the VL CDR3 in an attempt to improve the level of transcytosis of an anti-SC clone in the transport assay. A small improvement of transport was observed. Recombinant techniques used to create a bispecific scFv tandem were unsuocessful, in that its construction led to loss of binding specificities of both scFvs. Investigations in vivo were carried out as the first step of establishing an assay to test the final bispecific reagent. Study of the transport of an anti-pIgR phage suggested it was enriched compared to control phage in the faeces of mice injected intravenously.
Supervisor: Not available Sponsor: Not available
Qualification Name: University of Bristol, 2006 Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.486114  DOI: Not available
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