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Title: Structure function relationship of anti-CD40 monoclonal antibodies
Author: Orr, Christian Mark
ISNI:       0000 0004 8501 9092
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2018
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The use of monoclonal antibodies (mAbs) in cancer therapy is now well-established and is revolutionising cancer treatment. However, their precise mechanisms of action are incompletely understood, making agent optimisation difficult. In addition to this, not all patients and tumours respond to mAb treatment and some of these treatments carry a high risk of detrimental side affects. Therefore, further study is required. This study focuses on gaining a greater understanding of the structure : function relationship of anti-CD40 mAbs. Unlike checkpoint blocking mAbs such as Ipilimumab and Nivolumab which de-repress an existing anti-tumour response, anti-CD40 mAbs act agonistically in an immunostimulatory fashion to stimulate new responses by stimulating CD40 on antigen presenting cells. Anti-CD40 mAb elicit a diverse spectrum of immunostimulatory activity from low agonism to super-agonism. The exact mechanism dictating the level of activity elicited by a given anti-CD40 mAb has yet to be fully understood. Two areas of interest in relav tion to exploring these mechanisms are the isotype of anti-CD40 mAb and the epitope location on CD40. Determination of these structural components promises to reveal functional information on the mAb mechanisms. To address these structure function relationships, a number of anti-CD40 mAbs were analysed functionally utilising a number of cell based assays. Of particular interest was the IgG2 isotype, which is present as two subtypes; A and B, with the B form displaying much greater levels of immunostimulatory activity than the A form. The differences between these subtypes is understood to be in the hinge region disulphide bond structure, however this has not been structurally proven. To address this, the crystal structures of a number of A and B-like form anti-CD40 mAb F(ab')2 fragments were crystallised and the structures were solved. This provided atomic resolution data of the hinge region disulphide bonding patterns, yielding an unexpected geometry for the B form, with implications for function. Some of these hinge region disulphide bonds which were previously thought to form in an intraF(ab') manner (within the same F(ab')) were shown to be inter-F(ab') (between two F(ab')). This allows the model of this pattern to be updated. The effect the alternate disulphide bonding pattern had on F(ab') arm angle constraints was key to understanding the resulting function. To follow up on the structural analysis of the A and B form anti-CD40 mAb F(ab')2, the solution structures were obtained using small angle X-ray scattering (SAXS). Differences in flexibility between the A and B form were observed, suggesting the disulphide bonding pattern has an effect on F(ab')2 conformation, with the A form less strictly confined in angle accessibility than the B form. The epitope binding of anti-CD40 mAbs can also provide insight into activity as immunostimulatory mAbs have been shown to bind to CRD1. To further explore this relationship of epitope location and activity level, anti-CD40 mAb F(ab') fragments were crystallised in complex with CD40. The structure of one such mAb, ChiLob 7/4 was solved, revealing precise binding location and interacting residues. This provided a platform on which to compare the sequences of other anti-CD40 mAbs in an attempt to reveal a link between sequence, variable region loop length and immunostimulatory activity levels. This study increases the understanding of how the function of anti-CD40 mAb is effected by structural differences. Here, a new model of the disulphide bonding pattern within the hinge region of IgG2 A and B forms is proposed as elucidated by X-ray crystallography, and the effect this has on the conformation, as shown by SAXS. Additionally, the effect of epitope on anti-CD40 mAb agonistic activity was revealed, and a model of action was proposed. Overall, this study allows for the design of new mAb for which target stoichiometry and clustering is known to be an important factor. It also furthers our understanding of how disulphide bonding patterns effect flexibility and limit the availability of certain conformations.
Supervisor: Cragg, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available