Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.537213
Title: Generation and characterization of recombinantly polysialylated antibody
Author: Chen, Chen
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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Abstract:
With high affinity and specificity, antibodies are now proven biotherapuetics for a wide range of diseases, such as cancer and immunological conditions. However, antibody Fc-domain mediated cross reactivity with associated side effects has hindered the development of antibody therapy in a number of applications. The development of engineered recombinant antibody fragments to address the problems seen with whole monoclonal antibodies (mAbs). Their smaller size enables rapid antigen localisation, tissue penetration and higher specificity ratios. Whereas, poor pharmacokinetics due to fast blood clearance from the circulation is an important obstacle to these small molecules, and modification of antibody fragments is required for improved tissue exposure and uptake. In order to improve protein pharmacokinetics whilst maintaining good tissue/tumour to blood ratios and low cross-reactivity, conjugation of antibody fragments with a natural polymer polysialic acid (PSA) composed by N-acetylneuraminic acid (Neu5Ac or NANA), has been investigated. Compared to polyethylene glycol (PEG) conjugation, which is the predominant approach, PSA appears to be a better alternative because it is biodegradable, non-toxic and highly hydrophilic. In this project, an anti-carcinoembryonic antigen scFv (MFE-23), and an anti-HER2 scFv (C6.5) were used as model antibody fragments to conjugate with PSA. To overcome the limitations of chemical conjugations, this project was focused on the investigation of recombinant polysialylation of single chain antibodies. ScFv genes were firstly incorporated with a naturally polysialylatable (Ig5 and FN1) domains from human neuronal cell adhesion molecule. After transfections into polysialyltransferases-expressing HEK-293 cells, the biosynthesized sialic acid was enzymatically added to the N-glycosylated fusion protein Ig5 domain for elongating the attached PSA chain. Expressed by monoclonal cell secretion, polysialylated scFvs were successfully generated and purified. Based on the negative charge of PSA, an ion exchange method was developed for separating different polysialylated scFv isoforms. The degree of polymerization (DP) was detected by mass spectrometry, which showed an average of recombinant DP of up to 20 NANA residues attached to a complex Nglycan core. Affinity binding tests by ELISA confirmed the antibody KD was significantly retained at approximately 5nM after polysialylation, however BIAcore kinetic analysis demonstrated influencing change to the KD due to the strong negative charge of PSA. In vitro experiments using both FACS and confocal microscopy suggested that recombinant polysialylation has no affect of targeting antigens expressed on the live cell surface. Molecular hydrodynamic radius increases of polysialylated scFvs were detected by size exclusion chromatography, which led to corresponding pharmacokinetic improvement from in vivo mice studies with enhanced serum half-lives.
Supervisor: Deonarain, Mahendra ; Saffell, Jane Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.537213  DOI: Not available
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