Florey Department of Neuroscience and Mental Health - Theses
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ItemTowards biologics targeting vasopressin family receptors( 2022)G protein-coupled receptors (GPCRs) are integral membrane proteins that play a critical role in transducing information into a cell, and as such are important drug targets. Biophysical characterisation and drug discovery against GPCRs is challenging as they express at low levels in recombinant systems and demonstrate low stability when removed from the native lipid bilayer environment, which together, can mean that the routine generation of purified receptor protein is difficult. The inability to routinely generate purified GPCR protein is a bottleneck to the application of many biophysical techniques. In particular, this thesis focused on the application of anti-GPCR biologics discovery. The oxytocin receptor (OTR) and vasopressin 1A receptor (V1AR) are emerging therapeutic targets for social disorders such as schizophrenia and autism. The native ligands of these GPCRs are closely related nonapeptides oxytocin and arginine vasopressin. Despite therapeutic interest, there is a lack of selective compounds targeting OTR and V1AR, limitted understanding about how these peptides bind and activate their receptors, and a lack of delineation regarding their expression patterns in the brain. There is therefore a need for selective compounds for OTR and V1AR. I propose that OTR and V1AR binding antibodies, and nanobodies are an interesting modality to explore to these ends. Antibodies are an emerging method of targeting GPCRs, both therapeutically, and as tools for biomedical research. Theoretically, antibodies should bind to GPCR subtypes with improved selectivity compared to small molecules, while also providing benefits such as restricted biodistribution, an extended half-life, and additional functionality via the design of antibody-drug conjugates. However, the identification of antibodies against GPCRs has been hindered by the challenges associated with generating GPCR antigens that represent the 3D conformation of the receptor. The ability to generate a sufficient yield of stable, functional, purified GPCR protein for immunisation or panning is central to this challenge. Thus, the development of antibodies targeting GPCRs has lagged behind other protein families. Nanobodies are single VHH domain camelid antibodies, which are encoded by single gene. Domain antibodies such as nanobodies have emerged as a novel alternative for targeting GPCRs, as the smaller size, and extended complement determining region 3 (CDR3) can enable binding to smaller sites and can be advantageous over small molecule, or traditional antibody approaches. In this thesis I developed methodologies that aim to circumvent the main bottlenecks in receptor antibody and nanobody discovery, working towards the discovery of biologics targeting vasopressin family receptors. Firstly, I enhanced receptor protein expression of the OTR, using a novel method of lentivirus assisted mammalian cell directed evolution. Using this method, I discovered the variant OTR(3A) which has 8-fold enhanced expression compared to OTR(WT) by the introduction of only four amino acid point mutations. Then, I optimised the expression and purification of high expressing variants of V1AR and OTR from mammalian cells. Strategies for generating fluorescent receptor protein preparations were also explored. Subsequently, purified V1AR and OTR were used to generate an immune repertoire of anti V1AR and OTR nanobodies following alpaca immunisation. I developed a custom mammalian cell display panning methodology and demonstrated the feasibility of using a mammalian cell display approach for the identification of nanobodies, based on binding to a target antigen. Finally, in a pilot study, FPR1 and cognate antagonistic antibody Fpro0165 were used to demonstrate that a Fab expressed on the cell surface of a mammalian cell can bind to its cognate receptor expressed on the same cell and Fab-GPCR binding was detected using fluorescent ligand competition, and blockage of peptide induced signalling, laying the foundations for further development of this mammalian display method for anti-GPCR antibody discovery. Together, the work in this thesis has made significant contributions towards developing tools and methodologies that will aid in the discovery of selective binders to OTR and V1AR, and furthermore, will contribute to the generation of anti-GPCR biologics more generally.
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ItemProtein engineering of arginine vasopressin receptor V1A for structural biology( 2021)Peptide hormone arginine vasopressin (AVP) and its cognate G protein-coupled receptor (GPCR), V1A, belong to the vasopressin-oxytocin signalling system – a highly complex and widespread endocrine system in the human body. AVP activation of V1A causes a stimulatory contractile effect on vascular and uterine smooth muscle, while, in the brain, AVP and V1A play an important role in anxiety, aggression and social behaviours. Due to these physiological mechanisms, AVP and V1A have been of therapeutic interest for decades; initially for the treatment of peripheral conditions such as heart failure and menstrual cramps, while recently, V1A antagonists have shown promise for treating aspects of autism spectrum disorder (ASD). Despite this longstanding interest, there are currently no approved drugs selectively targeting V1A. Subtype-selectivity is important among the vasopressin-oxytocin receptor family, as these receptors share a high degree of sequence similarity and structural homology, yet have differing, and sometimes opposing, effects. Furthermore, as there are currently no experimental 3D structures of V1A, there is a lack of understanding on exactly how AVP binds to V1A at the molecular level, which limits the design and optimisation of new, V1A-selective drugs. GPCR structural biology is a rapidly developing field; however, the low-expression level and instability of many GPCRs, including V1A, can be problematic for purification and subsequent structural study. This thesis aimed to apply various protein engineering techniques to V1A, in order to facilitate the purification and subsequent biochemical study of this important, but long unfulfilled, potential therapeutic target. Chapter 2 covers the first GPCR-engineering venture, which was to introduce expression and stability augmenting missense mutations to V1A via a well-characterised method – directed evolution. This method, which uses E. coli display of receptor mutants, has successfully produced high-expressing, stabilised receptor mutants for the structural study of other neuropeptide receptors, including NTS1 and NK1. However, upon application of this method to V1A, stabilised V1A mutants were not produced. Instead, a V1A truncate was selected, which contained a small section of the V1A N terminus. The reason this protein was selected was unclear – but can primarily be attributed to the issues that arise when using E. coli for GPCR expression. It was evident that further engineering of V1A would require the use a different cell type, such as insect or mammalian cells. Chapter 3 explores the development of a mammalian cell-based directed evolution method. This new method was developed to overcome the expression issues encountered using E. coli, and required optimisation and innovation to accommodate the new cell type, including the use of lentivirus as the gene-delivery method. The method was developed, optimised and applied to V1A – producing V1A mutants that exhibited improved expression levels compared to wild-type V1A. Furthermore, several mutations were identified that appeared in multiple V1A mutants, indicating that they were contributing to this expression-boosting effect. Chapter 4 covers the purification of V1A using a combination of protein engineering approaches – including the introduction of the expression-boosting point mutations discovered in Chapter 3. Ultimately, functional V1A was able to be purified with the aid of these mutations, which provided a substantial increase in functional yield of purified receptor. The two main outcomes of this thesis are: the development of a mammalian cell-based directed evolution method that improves the functional yield of purified receptor – and the successful purification of functional V1A. Firstly, the new, mammalian cell-based selection method presents as a generic, rapid and viable means of improving the heterologous expression of low-expressing GPCRs – and potentially other complex membrane proteins with expression issues. Secondly, the substantial increase the yield of purified, functional V1A is a significant breakthrough in the biochemical study of V1A, as it enables the application of structural biology techniques, such as cryo-electron microscopy (cryo-EM), as well as ligand screening methods for new, V1A-selective ligands.