Structural investigation of teleost immunoglobulin M tetramer assembly and engineering of chimeric antibodies consisting of teleost motifs

This thesis investigates novel structural and functional aspects of teleost and chimeric immunoglobulin M (IgM), providing critical insights into antibody assembly mechanisms and antibody therapeutic engineering applications. Through single-particle cryo-electron microscopy (cryo-EM) and functional analyses, we have characterized previously unknown structural features of teleosts polymeric immunoglobulins and applied structural insights into engineering chimeric antibodies. The research presents the first high-resolution cryo-EM structure of teleost IgM-Fc, revealing a joining chain (JC)-independent assembly mechanism distinct from human counterparts. The teleost IgM tetramer comprises eight heavy chains with identical amino acid sequences that adopt two distinct CHμ4 conformational folds, facilitating a unique assembly where four IgM-Fcs are linked through C-terminal tailpieces (Tps) forming a β-sandwich-like domain. This arrangement differs significantly from the human pentameric IgM-J complex, highlighting an alternative polymeric (p) Ig assembly mechanism. Further structural investigations of chimeric IgM-Fc scaffolds comprising human and teleost sequences (cFcμs) and hexameric human IgM revealed additional representatives of JC-independent pIgM assemblies. The cFcμs form pentamers with distinctive configurations where C-terminal tailpieces protrude from the plane shared by cFcμ monomers—a structural characteristic unique to these chimeric constructs. Comparative analyses across teleost and human IgM assemblies provided insights into the evolutionary diversification of pIg assembly mechanisms and supported the role of JC as a template for Tps association into β-sheets. To explore the potential of cFcμs in antibody engineering, we engineered cFcμ scaffolds fused with single-domain antibodies (sdAbs) targeting clinically relevant pathogens: Clostridioides difficile and influenza A virus. These sdAb-cFcμ fusion constructs formed polymers that exhibited promising functions, including bacterial crosslinking and viral neutralization with high potency. These findings demonstrated the practical applications of cFcμ scaffolds and highlight their potential as antimicrobial therapeutics. This research contributes significantly to structural immunology by revealing the remarkable structural plasticity of polymeric immunoglobulin throughout the evolution of jawed vertebrates and highlighting the potential of IgM-based engineered polymeric antibodies for therapeutic applications. The cFcμ scaffolds developed in this study offer a promising platform for antibody engineering, particularly for applications requiring high avidity and multivalent target binding.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Lyu, Mengfan

Contributors dc:contributor

• Stadtmueller, Beth M
• Wu, Nicholas C
• Huang, Raven H
• Tajkhorshid, Emad

• structural biology
• polymeric immunoglobulins
• immunoglobulin evolution
• cryo-EM
• antibody engineering
• chimeric antibodies

Statement dc:rights

• Copyright 2025 Mengfan Lyu

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129731

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129731