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dc.contributor.authorNess, Samuel
dc.date.accessioned2022-01-14T13:59:41Z
dc.date.available2022-01-14T13:59:41Z
dc.date.submitted2019-09-30
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/332720
dc.description.abstractA wide range of human disorders, including Alzheimer’s and Parkinson’s diseases, are associated with the process of misfolding and aggregation of proteins. Therefore, modulating the aggregation pathway of disease-related proteins, in particular the Amyloid-β peptide (A) in Alzheimer’s disease and α-Synuclein (αSyn) in Parkinson’s disease, is key to establishing therapeutics for these disorders. One promising method is the blockade of aggregation-prone regions of proteins using antibodies, but a major challenge in establishing antibodies against pre-selected epitopes is the effectiveness of the target regions to behave as antigens. To address this problem, a method of rational design was recently established in our group to construct complementary peptides in the complementarity determining regions (CDRs) of single-domain antibodies (sdAbs) towards the target regions. This was shown to bind disordered epitopes specifically and with good affinity. The binding was also shown to inhibit the aggregation of αSyn. This approach was also utilized to create multiple sdAbs binding different epitopes across the length of Aβ. It was found that systematically ‘scanning’ the sequence of Aβ in this way led to markedly different effects upon the Aβ aggregation pathway and the specificity of the sdAbs towards different aggregated species. To build on these advances and improve the designed sdAbs, in my work I combined this rational design method with established methods of protein evolution to improve binding affinities and specificity. The expectation was that this strategy would enable the establishment of a repertoire of high-affinity antibodies towards epitopes, which until recently, were deemed to be ‘un-targetable’. Using a rationally designed αSyn antibody as a template for evolution, it was found that protein engineering via directed evolution and phage display is indeed capable of producing stable variants of the designed antibodies with improved affinity and crucially, retention of the pre-selected and poorlyimmunogenic epitope that it was originally designed to bind. Next, we aimed to alter the specificity of two of the rationally designed Aβ antibodies towards maximising the inhibition of secondary protein nucleation, in order to tune the specificity of the antibodies away from binding the monomer, and towards the toxic oligomeric species. Indeed, using directed evolution and phage display with competitive selection, it was found that it is possible to further alter the inhibition of the microscopic nucleation events that govern protein aggregation, albeit with compromised sdAb thermal stability. This was likely due to both the library design strategy and the heterogeneous selection employed. Nevertheless, these results show that combining rational design and directed evolution can offer novel opportunities in antibody discovery against targets not readily accessible through existing methods.
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectRational Design
dc.subjectAntibody
dc.subjectDisordered epitopes
dc.subjectDirected evolution
dc.subjectPhage display
dc.subjectAffinity maturation
dc.subjectProtein aggregation
dc.subjectDrug development
dc.titleSystematic development of rationally designed antibodies targeting predetermined epitopes of interest and protein aggregation
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.date.updated2022-01-12T13:35:10Z
dc.identifier.doi10.17863/CAM.80163
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
rioxxterms.typeThesis
dc.type.qualificationtitlePh.D in Biophysics
cam.supervisorVendruscolo, Michele
cam.supervisor.orcidVendruscolo, Michele [0000-0002-3616-1610]
cam.depositDate2022-01-12
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement


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