Abstract
dc:description.abstractThe transforming growth factor β (TGF-β) superfamily comprises the most structurally diverse family of growth factors and plays critical roles in embryogenesis and tissue homeostasis. A key feature of TGF-β growth factors is the regulatory pro-domain, which modulates the signalling activity of the mature growth factor domain. Through interactions with extracellular matrix components, pro-domains facilitate growth factor storage, localisation, and activation through mechanisms driven by distinct structural properties. Activin A is a TGF-β growth factor essential to development and healthy tissue function. Activin A signalling also contributes to the progression of many diseases, generating interest in therapeutic inhibition. Toward the goal of creating more effective activin A inhibitors, guided by an improved understanding of in vivo localisation and activation, I characterised novel α-activin A antibodies with biophysical and structural techniques. These binders included the dual-specific B52 antibody, which targets the growth factor domain of activin A and closely-related isoform activin B, in addition to eight non-neutralising antibodies against the activin A growth factor in complex with its pro-domain (pro-activin A). A protein refolding strategy was utilised to produce the antibodies as recombinant fragment antibodies (Fabs) in E. coli with high purity and homogeneity for downstream characterisation. B52 was discovered to bind activin A at its type II receptor binding site, which is well-conserved between activin A and activin B. Among the α-pro-activin A antibodies, α-pA5 emerged as a high-affinity binder of the activin A pro-domain at the shoulder. A sandwich ELISA was developed using the α-pA5 Fab to detect pro-activin A in solution, and full-length α-pA5 was validated for the specific detection of pro-activin A and the pro-domain in western blot. Together, the insights and tools established in this thesis further the development of more potent, context-dependent activin A inhibitors to treat disease and aid future studies on the extracellular activity of the activin A pro-form and pro-domains.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2025
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Pyeatt, Gwendolyn
- Advisor dc:contributor.advisor
-
- Hyvonen, Marko
Subjects
dc:subject × 4Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.123528
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/393025