Generation and Characterization of Recombinantly Polysialylated Antibody
Author(s)
Chen, Chen
Type
Thesis
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.
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.
Date Issued
2011
Date Awarded
2011-07
Advisor
Deonarain, Mahendra
Saffell, Jane
Creator
Chen, Chen
Publisher Department
Cell and Molecular Biology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)