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Title: A study of camelid antibodies to reveal structural features of a cytokine
Author: Hancock, Laura Marie
Awarding Body: Birkbeck (University of London)
Current Institution: Birkbeck (University of London)
Date of Award: 2010
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Camel heavy chain only antibodies have evolved into a strikingly different structural arrangement compared to conventional antibodies. Heavy chain-only antibodies do not interact with a light chain, they lack a CHI domain and are regularly observed to have an extended CDR-H3 region. Due to the presence of both antibody types in the camel species, a unique opportunity is available to compare their structure/activity relationship in a single immune response. This thesis aimed to isolate VHH (antigen binding domain of heavy chain-only antibodies) and conventional camel antibody Fab fragments, to assess whether they bind to different structural features of a cytokine. Further work aimed to assess whether VHH fragments were capable of stabilising a putative cleft of the cytokine and neutralise signalling. A fusion protein of the cytokine and its alpha receptor was cloned, purified, and used in camel immunisations to generate an immune response. A method was developed that enabled isolation of antibody cDNA from individual antigen positive camel B-cells, which in turn yielded a panel of 38 specific camel heavy chain-only and two conventional antibodies. Following expression and purification, the antibodies’ activities were profiled using Biacore, ELISA and functional cell-based assays to map the expected sites of interaction on the cytokine. These sites of interaction were structurally confirmed using a rapid NMR epitope mapping technique. Crystal structures were achieved for five VHH fragments and one conventional Fab fragment in complex with the cytokine. The interaction of conventional antibodies with proteins is associated with large planar surfaces. Limited studies undertaken to examine the binding interactions of camelid VHH fragments have shown them to be able to bind to clefts on a protein surface. Data generated in this thesis show that both camel VHH and Fab fragments display a cleft-binding nature, with the former also able to interact with planar surfaces. Both Fab and VHH fragments displayed neutralising activity, with the cleft-binding VHH fragments stabilising the structure of the cytokine.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available