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dc.contributor.authorKulenkampff, Klara
dc.date.accessioned2022-03-02T16:39:51Z
dc.date.available2022-03-02T16:39:51Z
dc.date.submitted2021-07-01
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/334595
dc.description.abstractAlzheimer's and Parkinson's diseases (AD and PD) are the most common neurodegenerative disorders, affecting millions of patients worldwide with a lack of disease-modifying treatments currently available. These diseases are characterised by the deposition of protein aggregates, which originate from misfolded proteins such as amyloid beta (Abeta), tau, and alpha-synuclein (aSyn). Although these protein deposits correlate to some extend with disease progression and cognitive decline, increasing evidence suggests that small intermediate aggregate species called oligomers are the primary driver of cytotoxicity and neuronal death. This evidence makes oligomers an attractive therapeutic target. Moreover, since oligomers can be found in human biofluids such as cerebrospinal fluid and serum, they offer potential as biomarkers for diagnostics and a measure of drug engagement in clinical trials. Yet, it remains challenging to capture, quantify, and characterise oligomers because of their heterogeneity, low abundance, and transient nature. Antibodies offer powerful tools in biological research and in diagnostic and therapeutic applications due to their ability to bind targets with high affinity and specificity. In earlier studies, an oligomer-specific antibody against Abeta was created to detect and quantify oligomers by applying a rational antibody design approach known as the cascade method. Here, we used this method to design a library of antibodies (DesAbs) systematically covering the length of wild-type tau (2N4R) and aSyn. The designed antibody sequences were grafted onto the CDR3 loop of a single domain antibody scaffold and are characterised in various biophysical assays, including kinetic aggregation assays, binding assays, and using fluorescence microscopy. Scanning the protein sequence in this way uncovered that different antibodies selectively bind to specific conformational states. The antibodies designed against tau were found to bind to late-stage aggregates. In contrast, the ones designed against aSyn showed to inhibit secondary nucleation, the dominant mechanism involved in oligomer production. Binding assays demonstrated that antibodies designed against aSyn bind with low affinity to aSyn monomers and aSyn fibrils, confirming that these DesAbs most likely interact with intermediate aggregate species. Finally, these antibodies were used to image aggregates present in PD patients' serum using fluorescence microscopy and one antibody showed similar detection levels of aggregates compared to a commercially available antibody. Identifying a DesAb as an oligomer-specific antibody against aSyn presents a valuable tool to study and further characterise the cytotoxic aggregate species in PD. In the future, the antibodies described here can be employed in different immunoassays as a diagnostic tool and, due to their high potential to inhibit the aggregation of aSyn, offer a promising prospect for therapeutic applications.
dc.description.sponsorshipMedical Research Council
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectAlzheimer's
dc.subjectParkinson's
dc.subjectAntibody
dc.subjectFluorescence Microscopy
dc.titleRational Design of Antibodies against Tau and Alpha-Synuclein
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.publisher.institutionUniversity of Cambridge
dc.date.updated2022-03-01T15:23:20Z
dc.identifier.doi10.17863/CAM.82014
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.contributor.orcidKulenkampff, Klara [0000-0001-7121-2957]
rioxxterms.typeThesis
dc.publisher.collegeEmmanuel
pubs.funder-project-idMedical Research Council (1945654)
cam.supervisorKlenerman, David
cam.supervisorVendruscolo, Michele
cam.supervisor.orcidKlenerman, David [0000-0001-7116-6954]
cam.supervisor.orcidVendruscolo, Michele [0000-0002-3616-1610]
cam.depositDate2022-03-01
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement
rioxxterms.freetoread.startdate2023-03-02


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