The Development of Nanobody Hydras for Targeted Cancer Treatment

Alongside surgery and radiotherapy, chemotherapy is one of the main forms of treating cancer but due to the properties of cancer cells, they are quickly becoming resistant to this form of treatment. Traditional chemotherapy has many associated side effects, some being tiredness, nausea and vomiting, anaemia etc. Due to these factors it is of high importance to find new therapeutics that have an increased effectiveness with fewer side effects. Due to resistance and side effects of traditional chemotherapy, new targeted therapeutics such as antibody-drug conjugates (ADCs) are being developed and have been shown to have great success due to their ability to target cancer cells. This allows lower doses of chemotherapy agent to be used and reduces side effects, that were previously debilitating during treatment. This project has developed an ADC prototype, which includes a polyfunctional linker, a protein hydra, that can bind to multiple drug payloads and therefore have a more targeted cytotoxicity with lower resistance. The work in this thesis shows the development of both chemical and biochemical techniques to produce a trifunctional linker modified antibody fragment. A camelid nanobody (VHH) produced through recombinant protein production and purification techniques, which targets HER2; a receptor that is overexpressed in a number of breast cancers, is used as a model system to develop this chemistry. This trifunctional linker modified antibody fragment was assessed for further conjugation reactivity using PEG-trapping experiments. In parallel to this work the development of chemical methodologies for the production of a Valine-Citrulline based immolative linker were explored. The preliminary progress made in this thesis enables any future projects to focus on the production of ADC prototypes with different drug combinations to assess synergistic responses.