Khalili, Hanieh; (2012) Disulfide-bridging PEGylation of antibody fragments. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Monoclonal antibodies are routinely used in the clinic. There are also a small number of antibody fragments (e.g. Fabs) that are clinically used. In many applications where antibody binding is required to antagonise a receptor or simply to bind a ligand, there is no need for the Fc properties that are associated with effector functions. Unfortunately, without the Fc region, antibody fragments rapidly clear from the blood circulation. PEGylation is the most successful and clinically used approach to date for increasing the circulation half-life of therapeutic proteins. Disulfide bridging PEGylation is an appropriate method to conjugate site-specifically a molecule of PEG at the interchain disulfide of a Fab. Since a PEGylated Fab will be monovalent, it is not possible to exploit the cooperative binding or avidity that is associated with a full IgG which is bivalent. It was hypothesised that site- specific conjugation of a molecule of PEG between the cysteines of the interchain disulfide would allow the monovalent PEGylated Fab to bind effectively to its target. Disulfide bridging PEGylation is accomplished by PEG reagents that undergo bis-alkylation. It was further hypothesised that a PEG reagent with a bis-alkylation functional group at each terminus of the PEG could then be used to conjugate two Fabs one at each end of the PEG molecule, to generate either a homo Fab-PEG-Fab or a hetero Fab-PEG-Fab* conjugate. An important objective of this PhD was to determine the structure-property correlations of a small family of PEGylated-Fabs because it was hypothesised that since the PEGylation is site-specific, the PEG-Fab binding properties would remain constant with increasing PEG molecular weight. It was finally hypothesised that Fab-PEG-Fab conjugates would display comparable binding properties to the full parent IgG. To test these hypotheses, three Fabs were PEGylated by bis-alkylation at the free thiols that were generated by disulfide reduction. Two clinically used monoclonal antibodies, bevacizumab and trastuzumab were proteolytically digested to provide their respective Fabs, Fabbeva and Fabtrast for PEGylation. Sourcing the Fabs from an IgG allowed for comparative studies with the parent IgG. The third Fab was also obtained from a clinically used medicine. Ranibizumab is a Fab, so it was PEGylated directly to give PEG-Fabrani. Both Fabbeva and Fabrani bind to VEGF and Fabtrast binds to HER-2. After treament with DTT to open the interchain disulfide, each Fab Thesis abstract underwent reaction with a PEG mono-sulfone reagent capable of thiol specific, bis- alkylation. PEGylation was accomplished reproducibly at near quantitative conversion with 1-2 equivalents of reagent. A single step ion-exchange purification process was used to obtain purified mono PEG-Fabs. The PEG-Fab conjugates were stable during a 3 month stability study at 4°C with no observed de-PEGylation. A PEG2x20-Fab'beva construct was also generated by the conjugation of two molecules of PEG to intrechain disulfide bonds of the Fab'beva. BIAcore and ELISA studies confirmed that compared with the unPEGylated Fabs, the PEGylated Fabbeva, Fabrani and Fabtrast displayed a 2 fold decrease in binding affinity for their respective ligands. This decrease in binding affinity was much less than had been reported in the literature and was due presumably to the conjugation of PEG far away from the binding reagion of the Fab. PEG-Fabbeva conjugates comprising 20, 30 and 40 kDa PEG all displayed similar binding affinities. The binding affinity of the PEG2x20-Fab'beva was decreased compared with mono PEG20-Fabbeva as a result of a change in the dissociation rate constant. The homodimer Fab-PEG-Fab constructs were derived from the 6, 10 and 20 kDa PEG reagents and Fabbeva, Fabrani and Fabtrast. Both of the homodimers derived from Fabbeva and Fabtrast maintained binding affinities comparable to their parent IgGs. BIAcore kinetic studies showed there was greater binding affinity and slower dissociation rate for the Fabbeva-PEG-Fabbeva than the native Fabbeva- While a similar binding affinity to bevacizumab was observed for the Fabbeva-PEG-Fabbeva, the dissociation rates of the Fabbeva-PEG-Fabbeva were slower than for bevacizumab. It was also found that using a longer PEG (i.e. Fabbeva-PEG20-Fabbeva) resulted in slower dissociation. The synthesis of the heterodimer Fabbeva-PEG20-Fab*trast could only be achieved one time, but once it was purified it was found to maintain binding to both VEGF and HER-2. An in-vitro angiogenesis assay suggested that the Fabbeva-PEG20- Fabbeva and Fabrani-PEG20-Fabrani inhibit angiogenesis more effectively than bevacizumab. Using PEG as a linking molecule to conjugate two Fabs would appear to be a way to chemically fuse two Fabs to achieve bivalency that is comparable to a full IgG. These results for Fab-PEG-Fab homodimer are encouraging and together with the results for the Fab-PEG-Fab* suggest a potential hybrid strategy of recombinant protein synthesis and chemical conjugation to develop bivalent and bispecific protein-based medicines.

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