Exploring the interactions of engineered antibodies with FcRn and TRIM21 for new avenues in antibody design, analysis, and gene therapy applications

Reusch, Johannes (2024): Exploring the interactions of engineered antibodies with FcRn and TRIM21 for new avenues in antibody design, analysis, and gene therapy applications. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Monoclonal antibodies (mAbs) have emerged as pivotal therapeutic agents, with their effectiveness hinging on complex pharmacokinetic properties and interactions with immune receptors. This thesis investigates the nuanced interplay between mAbs and two key receptors: the neonatal Fc receptor (FcRn) and the tripartite motif-containing protein 21 (TRIM21). FcRn is known to affect the serum half-life of mAbs, while TRIM21 is involved in antibody-dependent intracellular neutralization (ADIN). Research has often focused on the IgG-FcRn affinity, neglecting the combined impact of both affinity and avidity, as well as the potential role of TRIM21 in antiviral therapy through Fc engineering. The research presented in this thesis aims to deepen our understanding of mAb interactions with FcRn and TRIM21, focusing on the mechanisms that govern these interactions. It employs advanced methodologies to elucidate the relationship between affinity and avidity. Specifically, it investigates the impact of Fc modifications that alter interactions with FcRn, affecting serum half-life, and with TRIM21, which is involved in viral neutralization within cells. The findings aim to guide the development of more effective therapeutic antibodies, with broad implications for the treatment of diseases. In the publication titled 'Insight into the Avidity-Affinity Relationship of the Bivalent, pH-Dependent Interaction Between IgG and FcRn,' we explore the intricate binding dynamics between IgG and FcRn, advancing beyond traditional analyses that consider only single-affinity interactions. Utilizing switchSENSE technology, which closely mimics the membrane orientation of FcRn, we conducted a comprehensive examination of both affinity and avidity across the broad endosomal pH spectrum (pH 5.8–7.4). Our findings reveal that the engineered IgG1-YTE (M252Y/S254T/T256E) variant demonstrates a critical affinity shift at pH 7.2, indicative of its enhanced design for FcRn interaction. It also exhibits a marked avidity switch at pH 6.2, which is absent at pH 7.4. This dual engagement capability distinguishes IgG1-YTE from the wild-type, demonstrating the impact of Fc engineering on binding properties. Our research emphasizes the importance of avidity in IgG recycling, which is dictated by the variable expression of FcRn and its higher density in endosomes, necessitating a 2:1 stoichiometry for an extended serum half-life. The switchSENSE platform emerges as a powerful analytical tool, superior to traditional surface plasmon resonance (SPR), capturing a full range of kinetic parameters and accurately differentiating between monovalent and bivalent binding modes. This methodological advancement is crucial for understanding the dynamics of IgG binding in physiological contexts. The superior binding characteristics of the YTE variant suggest improved pharmacokinetics, potentially leading to increased therapeutic efficacy through an optimized recycling mechanism. The variant's higher affinity and significant contribution to avidity, especially during endosomal acidification, result in more stable FcRn complex formation, a desirable feature for antibodies engineered for extended serum half-life. The findings confirm the importance of pH-dependent binding in antibody design and have significant implications for the development of antibodies with improved recycling and extended half-life. Future research will utilize switchSENSE to further explore molecular interactions in various antibody mutants and formats, aiming to refine FcRn-mediated recycling for next-generation antibody therapies. In our publication titled 'TRIM21 and Fc-Engineered Antibodies: Decoding its Complex Antibody Binding Mode with Implications for Viral Neutralization, we explore the complex role of TRIM21 within the immune system, focusing on its interaction with Fc-engineered antibodies and the subsequent impact on viral neutralization. Utilizing a combination of biosensor assays, mass photometry, electron microscopy, and structural predictions, our study dissects the intricate binding dynamics between TRIM21 and various antibody Fc variants, revealing a novel binding mechanism that is pivotal for developing effective viral neutralization strategies. Our investigation employs optimized SPR assays to establish precise affinities and avidities, underscoring the importance of assay conditions in accurately analyzing interactions. We demonstrate that TRIM21 PRYSPRY domains (monomers) bind symmetrically to a single IgG Fc homodimer in a non-cooperative manner, adhering to a 2:1 stoichiometry. This symmetry is consistent with crystallographic evidence of TRIM21 PRYSPRY-IgG interactions. Significantly, we identify that Fc mutations, such as YTE and HH (T307H, N434H), reduce TRIM21 binding while enhancing interaction with FcRn in a pH-dependent manner. This dual effect underlines the complexity of antibody engineering, where mutations can differentially influence receptor interactions, which is crucial for optimizing antibody recycling and immune defense. The mutation Y436A within the Fc CH2-CH3 domain notably decreases the affinity to both FcRn and TRIM21, with the latter by 180-fold. This mutation also demonstrates a pronounced shift from micromolar affinity to nanomolar avidity and the critical role of bivalent engagement. Our structural analysis indicates that TRIM21 undergoes a dynamic rearrangement upon Fc binding, likely influencing its immune function. We propose a novel two-step binding mechanism whereby TRIM21's initial attachment to an Fc site facilitates a conformational change, enhancing its interaction with a second Fc site through increased PRYSPRY domain mobility. This process significantly boosts avidity, which is essential for effective antibody function. Using adeno-associated virus (AAV) as a model, our findings suggest that antibody clustering on the virus is possible and could activate TRIM21's E3 ligase activity, demonstrated as a crucial step in virus neutralization. These insights are vital for advancing antibody engineering and understanding TRIM21’s role in immune responses, offering significant implications for therapeutic applications.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: monoclonal antibodies, FcRn, TRIM21, affinity, avidity, antibody Fc engineering, therapeutic antibodies, immune signaling, viral neutralization
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 15. Juli 2024
• 1. Bericht­erstatter:in: Klein, Christian