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Soave, Mark (2017) Characterisation of a monoclonal antibody raised against a thermostabilised β1-adrenoceptor. PhD thesis, University of Nottingham.

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Abstract

The β1-adrenoceptor (β1-AR) is an important regulator of cardiac function. Chronic stimulation and subsequent downregulation of cardiac β1-ARs have been implicated in heart failure. Recently, it has been demonstrated that antibodies which target the second extracellular loop (ECL2) of the human β1-AR play a role in the pathogenesis of some forms of cardiomyopathy. Heptares Therapeutics Ltd. developed a murine monoclonal antibody (mAb3) raised against the thermostabilised turkey β1-adrenoceptor which had ECL2 as the epitope. MAb3 was previously characterised in CHO cells transiently expressing the turkey β1-AR. This thesis characterises the pharmacology of mAb3 at the human and turkey β1-AR in stable CHO cell lines.

Initial studies confirmed mAb3 was able to bind to the ECL2 of the turkey β1-AR and allow for the direct visualisation of these receptors expressed in whole CHO cells. MAb3 was specific for the turkey β1-AR with an affinity circa 20-30nM. The epitope of mAb3 represented an allosteric binding site on the turkey β1-AR, which was confirmed with whole cell radioligand binding. Applying a model of functional allosterism, mAb3 behaved as a negative allosteric modulator of an orthosteric ligand in functional assays in CHO cells stably expressing the turkey β1-AR, whilst having no effect on the human β1-AR. MAb3 did not affect the secondary binding site agonistic responses mediated by the partial agonist CGP 12177.

The recently developed NanoBRET technique was applied here to monitor ligand binding at the human β1-AR. Specific binding of three fluorescent ligands could be measured with this technique. These ligands were then used as probes to accurately calculate the affinities of a panel of unlabelled β-adrenoceptor ligands. Affinity values obtained appeared to show probe dependence, however when the incubation time was increased the differences in affinities disappeared. This highlighted the importance of ensuring equilibrium was achieved before analyses of probe dependence could be made.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Hill, Stephen J. Woolard, Jeanette
Subjects:	Q Science > QP Physiology > QP351 Neurophysiology and neuropsychology Q Science > QR Microbiology > QR180 Immunology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	39890
Depositing User:	Soave, Mark
Date Deposited:	17 Jul 2017 04:40
Last Modified:	12 Oct 2017 22:06
URI:	https://eprints.nottingham.ac.uk/id/eprint/39890

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