Engineering the antibody-TRIM21 interface for the improved intracellular clearance of pathogenic targets

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting >55 million people worldwide, a number projected to rise sharply with an ageing population. A central hallmark of AD is the accumulation of misfolded tau protein deposits inside neurons and glia. Current therapies provide only modest clinical benefit and do not directly target tau, which is a key pathogenic driver in AD and other primary tauopathies, for which no disease-modifying treatments exist. Consequently, there is an urgent need for effective tau-targeting treatments for patients and their caregivers. Tau immunotherapy is among the most promising strategies and is under evaluation in several clinical trials. However, limited mechanistic understanding of antibody action has constrained clinical success. Recent work shows that extracellular tau-antibody complexes can be internalised by neurons and degraded via the intracellular Fc receptor and E3 ubiquitin ligase TRIM21, and that passive tau immunotherapy requires this pathway for in vivo efficacy. These findings indicate that enhancing antibody engagement with TRIM21 could substantially improve efficacy yet engineering strategies to optimise this interaction remain poorly defined. This thesis aimed to identify antibody-engineering approaches that enhance TRIM21 affinity and promote intracellular degradation of pathogenic targets. A single Fc mutation in human IgG1, T256P, increases affinity for human TRIM21. X-ray co-crystal structures of the TRIM21 PRYSPRY domain bound to Fc-T256P reveal a conformational change in IgG1 Fc that stabilises interactions between the antibody CH2 domain and TRIM21 PRY pocket. Despite residing within the binding site of FcRn, T256P does not affect FcRn binding or in vivo half-life. Functionally, enhanced TRIM21 affinity improves intracellular neutralisation of diverse pathogenic targets. In cellular models, hIgG1-T256P increases clearance of adenovirus infection and seeded tau aggregation via the TRIM21 pathway. Combining T256P with Fc-silencing mutations or the less inflammatory IgG4 isotype reduces FcγR and C1q engagement while preserving enhanced TRIM21-mediated activity, enabling potent antiviral protection in vivo that relies exclusively on TRIM21. Application of this dual-engineering strategy to tau antibodies dampens inflammatory responses in human iPSC-derived microglia and promotes robust intracellular clearance of seeded tau aggregation in primary neuronal cultures. Together, these findings establish IgG1-T256P as a generalisable strategy for enhancing TRIM21-mediated intracellular targeting across neurodegenerative disease and virology. This approach expands the intracellular immunotherapeutic toolkit without compromising FcRn binding or in vivo stability and can be combined with Fc-silencing strategies, offering a clear path toward safer, more potent next-generation tau immunotherapies.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Higher Doctorate

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2026

Author dc:creator

• Keeling, Sophie

Advisor dc:contributor.advisor

• McEwan, William

• Antibody Engineering
• Immunotherapy
• Neurodegeneration
• TRIM21

Licence

• https://www.repository.cam.ac.uk/bitstreams/93e08979-1826-4384-b470-de01de8e7f8f/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.131655

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/405433