Sainz Pastor, N.;
Generation and characterisation of designed ankyrin repeat proteins for targeting carcinoembryonic antigen.
Doctoral thesis, UCL (University College London).
Designed Ankyrin Repeat Proteins (DARPins) are synthetic ligand-binding proteins with potential to be the next generation antibodies. DARPins have the advantages of being thermodynamically stable and are able to be manipulated in size, valency, pharmacokinetic performance and effector function. The potential of DARPins for targeting clinically relevant tumour antigens is explored in this thesis. The tumour marker used is carcinoembryonic antigen (CEA), a human glycoprotein overexpressed in a number of adenocarcinomas and proven as a target in antibody-directed therapies. It was hypothesised that in vitro selection of binders to recombinant protein domains that are specific only to CEA would yield molecules with tumour targeting potential. Recombinant and protein engineering approaches were used to test this hypothesis. First, the feasibility of producing pure recombinant domains of CEA was examined. The two N-terminal domains of CEA (N-A1), which contain a clinically relevant epitope recognised by the scFv MFE-23, were expressed in yeast. A combination of Endo H carbohydrate digestion with lectin and affinity chromatographies was used to obtain pure, homogeneous, soluble N-A1 and show that glycosylation is needed to maintain conformational stability. Kinetic analysis of the interaction with MFE-23 by surface plasmon resonance confirmed that the conformation of N-A1 resembled that of human CEA. A site-specifically biotinylated form of N-A1 was then exploited for selection of binders from a DARPin library, using ribosome display. Six rounds of selection were sufficient to achieve enrichment of specific binders. A carboxyterminal tag was engineered on the lead DARPin to allow site-specific dimerisation and radioiodination. The addition of the peptide did not affect the stability of the DARPin and the resulting covalent dimer showed improved dissociation rates and superior permanence time on N-A1. It could also be rapid and efficiently 125I-labelled whilst preserving immunoreactivity. In addition, the anti-N-A1 DARPin lead specifically bound to CEA in human adenocarcinoma sections. These results support the hypothesis and suggest a clear targeting potential for anti-CEA DARPins.
|Title:||Generation and characterisation of designed ankyrin repeat proteins for targeting carcinoembryonic antigen|
|Additional information:||Authorisation for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Wolfson Institute and Cancer Institute Administration > Cancer Institute|
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