Abstract
The focus of this thesis has been to elucidate the molecular basis for the specificity of an antibody termed 14F7 that exhibits potential for immunotherapy in cancer. When a healthy cell transforms into a cancerous cell, it exhibits altered gene expression and proliferation. Some of these changes can be utilised to distinguish a cancerous from a normal cell, and thus be exploited in cancer immunotherapy for diagnostic and therapeutic purposes. One such change, is the expression of the NeuGc GM3 ganglioside that is composed of a trisaccharide with a hydrophobic tail. Gangliosides are important components in a cell membrane, helping the cells communicate with each other. NeuGc GM3 is highly similar to the most common ganglioside in humans, namely the NeuAc GM3, differing only by a terminal hydroxyl-group at NeuGc instead of a hydrogen in NeuAc (Figure 1A). NeuGc GM3 is located at the cell surface of several different tumours, but does not exist in healthy human cells. The ganglioside is recognised by a monoclonal IgG antibody named 14F7. To be able to exploit the favourable properties of 14F7, it would be beneficial to know precisely how the NeuGc GM3 ganglioside and the 14F7 antibody interact at both the molecular and atomic level.
To investigate how they interact at the atomic level it is desirable to obtain a protein structure. To this end, we generated four recombinant 14F7 variants in single chain variable fragment (scFv) format, expressed the proteins using an established periplasmic expression system and crystallised one of the scFvs. Recently, we soaked the trisaccharide part of NeuGc GM3 into the scFv crystals, revealing the binding mode of the ganglioside, where 14F7 utilises water molecules to create an extensive hydrogen-network to recognise the special hydroxyl-group of carbohydrate (Figure 1BC).
In follow-up studies, we investigated the interaction between 14F7 and NeuGc GM3 when the ganglioside was embedded in a lipid bilayer. Here, we observed that there was a concentration threshold for 14F7 binding. However, this threshold could be reduced if the similar ganglioside NeuAc GM3 was present together with NeuGc in the membrane. Binding was also abrogated if the membrane curvature was high. Together, this indicates that the 14F7 binding is likely dependent on the conformation of the ganglioside that is affected by its lateral interactions with other molecules.
In this work we have designed, produced, characterised and crystallised 14F7 in scFv format. Then, we have used liposomes to investigate 14F7-NeuGc GM3 binding dynamics. Together, this provides new insight into the functionality of 14F7 and highlights its potential within immunotherapy.