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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.

Peptide aptamers are alternative binding proteins, whose development is motivated by the desire to overcome the drawbacks of antibodies. Defined as variable peptides constrained by a constant protein scaffold, peptide aptamers ., have been developed by several groups over the past two decades and this thesis will detail my and my colleagues' contribution to the field. Chapter 1 will provide the reader with a brief account of the history and applications of peptide aptamers, as well as give a rational for the technology. In the second chapter the development of SQM, a novel peptide aptamer scaffold derived from the protease inhibitor Stefin A, is described. SQM is based on the previously engineered STM scaffold, but, different to its predecessor, is capable of presenting randomised peptides from three different insertion sites. The stability and functionality of the new scaffold is explored, a process which highlights some structural problems in SQM. In a successful attempt to overcome these issues SQM is re engineered to yield SQT and several applications of the new scaffold, such as the generation of large combinatorial peptide aptamer libraries, are described (chapter 3). In order to further the peptide aptamer field the advancement of auxiliary technologies, such as affinity peptide tags for protein detection in western blots, immunocytochemistry etc., is crucial. Thus, in chapter 4, the development of an improved streptavidin-binding tag - generated by simply exploiting the multimeric nature of streptavidin - is outlined. In the final chapter I make use of the biologically neutral SQT scaffold to present and constrain four different apoptotic BH3 domains, with the aim of generating new tools for the study of the intrinsic apoptotic pathway. In combination the work presented here establishes Stefin A derived peptide aptamers as viable binding proteins with potential applications in a range of laboratory and clinical settings.

Interest in using plants for the production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, which leads to a reduction in protein yield, a loss of functionality and complications in downstream processing, is a significant problem. Typically, fully assembled antibody expressed in the plant is accompanied by additional immunoglobulin species of lower molecular weight, which are predominantly generated by the enzymatic action of plant proteases. As plant proteases are pivotal to maintaining metabolic functions, expression of fully assembled antibodies, alien to the plant proteomic system, remains a challenging task. The present study examined two monoclonal antibodies expressed in transgenic Nicotiana tabacum; the murine IgG 1 K, Guy's 13, which recognises an epitope on the surface protein SAIIII of Streptococcus mutans; and the human IgG 1 K 2G 12, which targets high mannose structures within the HIV-l gp120 envelope glycoprotein. The stability of plant-expressed antibodies during downstream processing was investigated. These studies demonstrated that proteolytic degradation is predominantly an in planta event, taking place prior to extraction. Intracellular antibody trafficking was investigated in plant protoplasts to gain a further understanding of the site and mechanism of degradation. The results obtained indicated that the initial proteolytic cleavage occurred intracellularly. Antibody integrity was affected at an early stage in the secretory pathway (ER/Golgi) and also by transport to the vacuole. The apoplastic space is not a major site of antibody degradation. The composition of each lower molecular weight band from protein A/G affinity purified antibody was determined using western blotting, N-terminal sequencing and mass spectrometry. The latter two techniques revealed that, although no conserved target sequences were evident, the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to interdomain or solvent exposed regions in the antibody. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy chain and light chain mutants were expressed transiently in combination in Nicotiana tabacum and demonstrated alterations in fragmentation pattern. Some mutant combinations, (particularly Serll3Thr/Ala1l4Gly) in the heavy • 11 3 chain together with changes in the light chain (Leu104Ile/Glu105 Asp, Lysl03Ser or Ile106Leu) resulted in a significant increase in the ratio of assembled antibody to antibody fragments, compared to the non-mutated Guy's 13. Overall this thesis strengthens understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to reduce degradation.

A naïve human antibody fragment phage display library was screened against conjugated microcystin-LR (MCLR).  Phage antibodies were eluted with free MCLR to promote the isolation of free antigen binders.  Isolated antibody fragments were cloned into a soluble expression vector and single-chain antibody (scAb) expressed in a bacterial host.  All scAbs bound free antigen in an indirect competition ELISA and levels of sensitivity were determined.  The most responsive clone 3A8 had an 800-fold increase in sensitivity to MCLR than previously documented scAbs and was able to detect levels below guidelines for MCLR in drinking water set by the World Health Organisation (WHO) at 1 μg/L.  Despite its sensitivity to MCLR, full characterisation revealed low levels of cross-reactivity with other microcystin variants. <i>In vitro</i> affinity maturation was employed to increase cross-reactivity to other microcystin variants in scab 3A8.  A chain-shuffled library was constructed where a naïve V_L repertoire was shuffled with clone 3A8 V_H chain.  The library was screened and isolated phage antibodies cloned and expressed as scAbs.  Chain-shuffled clone CSB-D9 displayed a 50-fold increase in cross-reactivity for a single microcystin variant and was capable of detecting all variants available below the guideline levels of 1 μg/L. The highly cross-reactive scab was used for the determination of total microcystin content in cyanobacterial extracts and results showed good correlation with HPLC quantification.  Immunoaffinity chromatography utilising scab CSB-D9 was used for the concentration of trace amount of microcystin from large volumes of water, out-performing columns generated with anti-microcystin whole antibody molecules. The ability of scab CSB-D9 to protect cultured hepatocytes against microcystin-induced apoptosis was also determined.  Results indicated a possible therapeutic application for human antibody fragments isolated from phage display libraries.

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