| Summary: | Monoclonal antibodies are routinely used in the clinic. There is 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 Fe properties that are associated with effector functions. Unfortunately, without the Fe region, monovalent 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 site-specifically conjugate 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 the bivalency of a full IgG. An objective of this PhD project was firstly to understand if a monovalent PEGylated Fab can bind effectively to its antigen. A bis-alkylation PEG reagent with a 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. It was hypothesised that Fab-PEG- Fab conjugates would display comparable binding properties to the full parent IgG. Three Fabs were PEGylated in this project. Fabbeva and Fabtrast were obtained by papain digestion of bevacizumab and trastuzumab respectively. Ranibizumab is a clinically used Fab and therefore did not require digestion of a full antibody. Both Fabbeva and Fabrani bind to VEGF and Fabtrast binds to HER-2. After treament with DTT to open the interchain disulfide, each Fab underwent reaction with a Fab reagent capable of thiol specific, bis-alkylation. PEGylation was accomplished at near quantitative conversion with 1-2 equivalents with reproducible result. A single step ion-exchange purification process was used to obtain purified mono PEG-Fabs. The PEG-Fab conjugates were stable during a 3 months stability study at 4 °c with no de- PEGylation. A PEG2x2o-Fab' beva construct was also generated by conjugation of two molecules of the PEG reagent to intrachain 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 I for their respective ligands. This decrease in binding affinity was much less than had been reported in the literature. 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 PEG2o-Fabbeva as a result of a change in the dissociation rate constant. The homodimer Fab-PEG-Fab constructs comprising 6, 10 and 20 kDa PEG and Fabbeva, Fabrani and Fabtrast maintained their binding affinities compared with the 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 similar binding affinity to bevacizumab was observed for the Fabbeva-PEG-Fabbeva, the dissociation rates of the the Fabbeva-PEG-Fabbeva were slower than for bevacizumab. It was also found that using a longer PEG in the Fabbeva-PEG-Fabbeva resulted in slower dissociation. The heterodimer Fabbeva-PEG2o-Fabtrast* that was produced maintained binding to 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 valid way to provide bivalency to the molecule, resulting in at least the functional activity expected of a full IgG. Since the PEG is a flexible coil, it may be the case that the two conjugated Fabs in the Fab-PEG-Fab homodimer are brought towards the VEGF in a manner that is more efficient than for a native IgG, resulting in a stronger binding interaction and hence enhanced functional activity. These results for Fab- PEG-Fab homodimer are encouraging and together with the results for the Fab-PEG- Fab* bring the potential to aid development of bivalent and bispecific protein-based medicines.
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