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Title: The effect of engineered inter-domain CL-CHI disulphide bonds on IgG4 stability, functionality and fab arm exchange
Author: Peters, Shirley Jane
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2012
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The work presented here describes novel IgG4 formats that may be preferred format of choice when IgG4 is employed as a therapeutic. These formats have also improved our knowledge of IgG4 biology and may be used as tools to further investigate IgG4. IgG4 is one of four human IgG isotypes and has a number of unique characteristics when compared to the other IgG isotypes. A particular distinctive feature is that the inter-heavy chain disulphide bonds are liable to form intra-heavy chain disulphide bonds which is partially responsible for driving IgG4 Fab arm exchange whereby two IgG4 antibodies of different specificity exchange to form monovalent bispecific molecules. Comparative thermal stability analysis between isotypes has also shown that IgG 1 is more stable than tgG4. Of particular interest is that fact that the interdomain CL-CH 1 disulphide bond arrangement also differs between the isotypes. This thesis reports on investigations into improving the Fab domain thermal stability of an JgG4 by altering the inter-domain disulphide bond to mimic that of an IgG I inter-domain disulphide bond. Upon the generat ion of such molecules, whether such an altered disu lphide bond arrangement and any related improved stability influenced Fab arm exchange was also investigated. The novel Ig04 molecules generated that contained an IgO I like inter-domain disulphide CL -CH I arrangement exhibited increased Fab domain thermal stability. The extent of the increase in Fab domain thermal stability was dependent on the associated variable region. The Fab domain thermal stability could be further increased by appropriate placement of the novel disulph ide bond and mutations to the residues surrounding the disulphide. Fab arm exchange data showed that the placement of the novel inter-domain disulphide can reduce Fab arm exchange activity, but that an increase in Fab domain thermal stabil ity does not necessari ly reduce Fab arm exchange activity. The novel Ig04 mutants with reduced disu lphide bond heterogeneity, increased Fab domain thermal stability and reduced Fab Arm Exchange activity generated here offer potential advantages over Ig04 wild type molecules as biotherapeutic agents.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available