The antibody response has evolved under constant pressure to recognize common pathogens and also remain adaptable to novel threats. Given the limited size of the germline antibody repertoire, adaptability requires that some antibodies must be polyspecific for multiple distinct antigens. Despite the profound importance of polyspecificity in the antibody response, the structural features that allow it are not well understood. Antibodies raised against glycoconjugates of Chlamydiaceae LPS oligosaccharides of the inner-core sugar Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) have been shown to cross-react with several inner-core oligosaccharides through conserved recognition of single Kdo residues in a germline-encoded pocket, with additional sugars accommodated by flexible side-chains. Two of these antibodies, S25-2 and S25-39, were observed to bind several Kdo oligosaccharides with an identical binding site conformation, but adopted unique conformations of the heavy chain complementarity determining region loop 3 (CDR H3) in the absence of ligand. Conformational flexibility of germline antibodies is believed to facilitate polyspecificity by generating multiple unique binding sites in a single antibody. This thesis research further explores the conformational flexibility of the antibodies S25-2 and S25-39 to gain insight into mechanisms of antigen recognition and how this feature may allow polyspecificity. This was achieved first by solving structures of S25-39 from crystals grown in unique conditions to observe alternate CDR H3 conformations, and second by designing synthetic Kdo-based antigens so as both to inhibit interaction with the previously observed liganded conformation of S25-2 and S25-39 and to be accommodated by their observed unliganded conformations. These structures reveal an unprecedented level of structural diversity of CDR H3, notably including the exact ‘liganded’ conformation in the absence of ligand. This is the first direct structural evidence that CDR H3 can exist in a conformational equilibrium with antigen binding through a selection mechanism, as opposed to induced fit where antigen causes the observed conformational change. Definitive evidence for binding the synthetic antigens was not obtained, however the resulting structures revealed several additional unique conformations of CDR H3 suggesting that ligands can alter conformational equilibria during crystallization. A unique conformation was also observed with CDR H3 coordinating multiple iodide ions, revealing another potential source of polyspecificity with unique binding paratopes generated by ion coordination. Finally, the unparalleled level of conformational diversity observed for these antibodies highlights the challenges of antibody structure classification and prediction, and stresses the need for additional in-depth studies of conformational diversity and binding mechanisms to advance these fields for therapeutic application. This is the first targeted structural study of flexibility in antibodies and provides insight into their conformational dynamics and antigen-binding mechanisms. These are of fundamental importance in understanding antibody structure and function, a critical consideration in practical applications such as modelling and design of therapeutic or diagnostic antibodies. / Graduate / 2019-11-27

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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The initial objective of this thesis was to generate Peyer's patch T cell hybridomas producing lymphokines that regulate IgA-secreting B lymphocytes. Unprimed Peyer's patch cells were fused with BW5147. Karyotype analysis and fluorescent staining of the thy-1.2 marker confirmed the generation of hybridomas. It was envisaged that these hybridomas would be tested for their effects on IgA production by LPS-stimulated B cells. However, when the panel of hybridomas was available for testing there were technical difficulties with this assay. Sendai virus-primed Peyer's patch T cells were used in a subsequent fusion, which was screened using an antigen-specific <i>in vitro</i> helper assay. A number of hybridomas stimulated the production of anti-Sendai antibodies by primed B cells. The same hybridomas secreted IL-2 on stimulation by syngeneic spleen cells in the absence of added virus. Recombinant IL-2 replaced the hybridomas in stimulating primed B cells in the helper assay. These studies showed that several of the hybridomas had apparent auto-reactivity. This was not due to viral contamination of the animal stocks since spleen cells from isolator-reared syngeneic mice gave similar results. The genes responsible for stimulating the hybridomas were mapped to the I-region of the MHC. It was important to elucidate whether these T cells were truly auto-reactive or whether they were in fact <i>in vitro</i> artefacts. Hybridomas adapted to grow in serum free medium and subsequently tested for their response to syngeneic cells in the abscence of serum, did not produce IL-2. The addition of foetal calf serum restored the response. The component of foetal calf serum which is necessary for the stimulation of the hybridomas has been partially purified. It can be separated from the main serum protein components by HPLC on a DEAE ion exchange column. It is eluted by high salt which suggests that it is highly acidic or is bound strongly by hydrophobic interactions. The material is trypsin sensitive. It is labile at 4<SUP>o</SUP> C and is unstable to freezing and thawing and this has hampered its further purification. The mode of action of the component has been studied using pulsing experiments and it has been shown to act on the stimulator cells and not on the hybridomas. The data suggests that the hybridomas do not recognise a self-antigen alone, but rather that they recognise a component of the xenogeneic serum as antigen with self-MHC restriction. However it has not been formally excluded that the foetal calf serum component stimulates the expression or processing of an auto-antigen.

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