Developing Affimers for the detection of biomarkers in cancer

Biomarkers are molecules present in the patients’ samples. They are used to detect the presence of a disease or an infection. An array of laboratory tools is used to monitor changes in the biomarker levels for diagnostic and prognostic purposes. Affimers are relatively new tools, which can be used in similar ways to commonly used antibodies with many advantages over the later. The work described in this PhD thesis has focused on a number of methodologies for the generation of Affimers against purified proteins and cell-surface molecules, with the intention of using these to detect biomarkers in cancer. To ensure the development of functional Affimer reagents against different membrane target molecules, EGFR, HER2, and HER3 were used as models to optimise the process of Affimers isolation and selection using phage display technology. In vitro production of Affimers using bacterial cells was assessed and optimised. Following optimisation, a range of Affimers that bound to EGFR, HER2, and HER3 were generated and partially characterised by different molecular applications, including immunofluorescence, immunohistochemistry, and pull-down assay. Upon characterisation, the developed reagents were not only able to identify their targets on cells and to precipitate them down from cell lysates, but also exhibited a complete inhibition of the downstream signalling activation of both EGFR and HER3. In addition, this thesis provides clear evidence of the potential of Affimers in biomarker discovery studies. After multiple rounds on non-tumourigenic (HB2) and cancerous (MDA-MB-453) breast-cell lines, eight novel anti-breast-cancer Affimers were successfully isolated and characterised. Following pull-down assay and mass spectrometry analysis, the target proteins to which these Affimers were found to bind was identified as CK18 and 19. Upon identification, the specificity of Affimers was further validated on a panel of breast cells, tissues, and on multiple breast-tissue microarrays (TMAs). There is a need to develop the knowledge to utilise this new Affimers-based technology to encourage adoption of this useful tool. With the aid of such technology, several novel detection reagents were generated, partially validated and proved to be promising tools for biomarker detection in different conventional assays