Yon, Alexander Robert; (2019) Single-molecule Study of Antibody Drug Binding to Cell Surface Receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Ion channels are critical modulators of cellular function and are implicated in a range of medical conditions. The ion channel studied in this thesis is the human P2X4 receptor, which is involved in signalling pathways associated with pain perception. The P2X4 receptor is a target for antibody-based therapeutics against neuropathic pain, a chronic pain condition. The development of a therapeutic can be facilitated by an understanding of its interaction with the target. In addition, such knowledge is important for the progression of candidate therapeutics through trial and approval processes. We use the atomic force microscope (AFM) to study P2X4 receptors reconstituted into model membranes of well controlled composition. AFM is a powerful technique for imaging single biomolecules at sub-nanometre resolution in near-native conditions. We visualise P2X4 receptors at high resolution in the presence and absence of the ligand ATP. In addition, the thesis reports our efforts to directly visualise the binding of candidate antibodies to P2X4 receptors using AFM. Furthermore, we apply super-resolution optical microscopy (principally total internal reflection fluorescence microscopy, TIRFM) to visualise the binding of labelled antibodies to P2X4 receptors in our model membranes. TIRFM, combined with single-particle tracking algorithms, allows us to determine valuable kinetic parameters (dissociation constant, on-rate, off-rate and Hill coefficient). Finally, we apply the acoustic technique known as quartz crystal microbalance with dissipation monitoring (QCM-D) to measure the binding of unlabeled antibodies to P2X4 receptors, validating our TIRFM data. In summary, the thesis reports the use of three biophysical methods to characterise the binding of an antibody therapeutic to a human protein implicated in a chronic pain condition. Our data yields information that has proven non-trivial to acquire using typical work-horse techniques; the results presented have contributed directly to a drug discovery project with our industrial partner MedImmune.

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