Nucleosomal arrays : a novel method to detect PD-L1 on the cell surface : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
Cancer is a complex group of diseases that can be difficult to therapeutically target as cancer cells develop a myriad of mechanisms to spread and survive. Immune evasion is one such mechanism cancer cells use to bypass the immune system and continue to invade other tissues. Evading the immune system is achieved through over-expression of the immune checkpoint protein programmed death ligand-1 (PD-L1). This protein is used as a predictive bio-marker to determine whether cancer patients are viable for PD-L1 immunotherapy, and over-expression of PD-L1 is determined used immunohistochemistry assays. Given that these assays use different PD-L1 antibodies that recognise different epitopes, it introduces variability to staining patterns and scoring matrices. This study has taken advantage of the endogenous high affinity interaction between PD-L1 and its receptor, programmed death receptor-1 (PD-1), to create a novel technique to detect PD-L1 on the surface of cancer cells. Nucleosomal arrays were formed with PD-1 peptide tagged to histone H2B containing octamers and fluorescently labelled DNA. These nucleosomal assemblies had correct nucleosome formation when using biotin-labelled positioning DNA and were able to be detected by binding Avidin-Alexa 488. These nucleosomal arrays bound specifically to PD-L1 on the surface of cancer cells shown by fluorescent confocal microscopy and demonstrated greater specificity than Wild Type H2B containing nucleosomal arrays. The use of nucleosomal arrays in this manner has an advantage over an antibody as there are a greater number of PD-L1 binding sites, and the fluorescent signal is able to be amplified due to positioning DNA being labelled at multiple sites. Though this study used the PD-L1/PD-1 interaction as a proof of principle, other short specific binding domains could be attached to the N-terminal tail of histones that could detect other extracellular proteins. This novel detection method is efficient and specific, and further optimisation in other fluorescent detection platforms may yield a more consistent and high-throughput method to detect extracellular proteins to determine patient response to immunotherapy treatments.