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The aims of this thesis are to establish the therapeutic value of two anti-glycan mAbs produced in-house, to develop an immunisation protocol with the aim of improving the immunogenicity tumour-associated glycolipids with the intention of producing therapeutically valuable mAbs and to determine the implication of a mAb with the ability to induce apoptosis in colorectal cancer. The anti-glycan mAbs 692/29 and 505/4 have previously been produced in-house and this study aimed to determine their fine specificity using a glycan array. 692/29 displayed binding predominantly to Lewis b as well as Lewis y-containing glycans. 505/4 was discovered to bind to sialyl Lewis a as well as sialyl di-Lewis a, with no cross-reactivity with other blood group antigens. This was compared to other anti-Lewis mAbs, with differences in specificity being observed. Characterisation of 505/4 mAb distribution showed binding to 80% of colorectal tumours and low levels of binding to normal tissues by IHC, suggesting it may be therapeutically useful. This thesis aimed to assess the ability of 505/4 and 692/29 to meditate immune mediated and non-immune mediated cell death as well as to determine whether non-immune-mediated cell death would be a desirable therapeutic property. Resistance to apoptosis is one of the hallmarks of cancer cells and mAbs stimulating apoptosis may not be very effective. Alternatively, cancer cells are driven to initiate apoptosis by genomic and other aberrations thus if pro-apoptotic pathways are stimulated these cells may be more susceptible to death than normal cells. To investigate the significance of apoptosis in cancer a large tissue microarray of colorectal tumours was assessed for apoptosis and its relationship to patient prognosis. Cleaved caspase-3 is a good marker of apoptosis as it is the executioner caspase for both the extrinsic and intrinsic pathways. Immunohistochemical analysis of colorectal tumour samples revealed that a high expression of cleaved caspase-3 in tumour was associated with good prognosis in colorectal cancer. This suggested that some tumours were still susceptible to apoptotic death but some are resistant and an alternative mechanism of cell death may be an advantage in these tumours. High expression of cleaved caspase-3 in the tumour-associated stroma was also an independent marker of good prognosis in colorectal cancer. This may be because apoptosis of the tumour-associated stroma reduces the level of pro-tumour signals originating from tumour-associated immune cells and stromal cells. As the tumour microenvironment can act in an immunosuppressive and pro-tumour manner, the ability of a mAb to induce direct cell death without the need for effector cells or complement would be an advantage. Lewis y and Lewis b are blood group antigens commonly overexpressed on the surface of a range of cancers. Characterisation of effector functions of 505/4 and 692/29 demonstrated that both mAbs have the ability to mediate apoptosis by antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and cause direct cell death in an oncosis-like manner. Comparison with other anti-Lewis mAbs demonstrated that a number of anti-Lewis mAbs can induce direct cell death independently of apoptosis. Thus, they could effectively target apoptotic sensitive and resistant colorectal cancers. Tumours aberrantly express glycolipids and these molecules may be involved in a number of cellular pathways. In addition a large proportion of anti-glycan mAbs, including 505/4 and 692/29 in this thesis, have displayed the ability to induce direct cell death. Therefore this thesis aimed to develop an immunisation protocol capable of increasing the immunogenicity of tumour-associated glycolipid for the production of anti-tumour glycolipid mAbs directed against ovarian cancer. This study suggests that the incorporation of tumour glycolipid into liposomes and their immunisation along with the iNKT cell adjuvant α-galactosylceramide, elicits an anti-tumour glycolipid immune response, which can yield IgG mAbs capable of binding a high proportion of ovarian cancers. In summary, this thesis confirmed specificity of 692/29 to Lewis y and Lewis b and 505/4 to sialyl Lewis a and sialyl di-Lewis a. Furthermore, this thesis demonstrated a promising tissue distribution of 505/4 in vitro. Characterisation of mAb effector functions suggest that both Lewis y and sialyl Lewis a directed mAbs have the ability to cause direct cell death, independently of apoptosis in antigen positive cells, as well as the ability to cause immune-mediated cell death. This may be an important factor in the immune-suppressive tumour microenvironment. Furthermore, this thesis provides the basis for the production of new anti-glycolipid antibodies that may also be able to induce direct cell death.

The Wnt signalling pathway is involved in stem cell maintenance, differentiation and tissue development, and in so doing plays a key role in controlling the homeostasis of colorectal crypts. In response to an external Wnt stimulus, the intracellular levels of the protein beta-catenin are regulated by the proteins which make up the Wnt signalling pathway. Abnormalities in the Wnt signalling pathway have been implicated in the initiation of colorectal and other cancers. In this thesis we analyse and simplify existing models of the Wnt signalling pathway, formulate models for Wnt's control of the cell cycle in a single cell, and incorporate these into a multiscale model to describe how Wnt may control the patterns of proliferation in a colorectal crypt. A systematic asymptotic analysis of an existing ODE-based model of the Wnt signalling pathway is undertaken, highlighting the operation of different pathway components over three different timescales. Guided by this analysis we derive a simplified model which is shown to retain the essential behaviour of the Wnt pathway, recreating the accumulation and degradation of beta-catenin. We utilise our simple model by coupling it to a model of the cell cycle. Our findings agree well with the observed patterns of proliferation in healthy colon crypts. Furthermore, the model clarifies a mechanism by which common colorectal cancer mutations may cause elevated beta-catenin and Cyclin~D levels, leading to uncontrolled cell proliferation and thereby initiating colorectal cancer. A second model for the influence of the Wnt pathway on the cell cycle is constructed to incorporate the results of a recent set of knockout experiments. This model reproduces the healthy proliferation observed in crypts and additionally recreates the results of knockout experiments by additionally including the influence of Myc and CDK4 on the cell cycle. Analysis of this model leads us to suggest novel drug targets that may reverse the effects of an early mutation in the Wnt pathway. We have helped to build a flexible software environment for cell-based simulations of healthy and cancerous tissues. We discuss the software engineering approach that we have used to develop this environment, and its suitability for scientific computing. We then use this software to perform multiscale simulations with subcellular Wnt signalling models inside individual cells, the cells forming an epithelial crypt tissue. We have used the multiscale model to compare the effect of different subcellular models on crypt dynamics and predicting the distribution of beta-catenin throughout the crypt. We assess the extent to which a common experiment reveals the actual dynamics of a crypt and finally explain some recent mitochondrial-DNA experiments in terms of cell dynamics.

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