Exploring Uncharted Territories in Immunoglobulomics: Comprehensive Profiling of Human Antibody Repertoires in Blood Using Mass Spectrometry

This thesis explores the use of mass spectrometry to understand antibody biology in health and disease, with a focus on antibody repertoires and their roles in immune responses. Antibodies are essential for detecting and neutralizing pathogens and are increasingly used as biopharmaceuticals. However, the diversity of antibodies and their dynamic expression pose challenges for studying the full range of antibody responses. Traditional methods, like genetic sequencing of B cell receptors, do not capture the diversity of secreted antibodies. To address this, I developed and improved mass spectrometry-based Fab clonal profiling, a method that directly analyzes the Fab regions of antibodies in bodily fluids, offering insights into antibody diversity. Chapter 1 introduces the fundamental role of antibodies in immunity, detailing their structure and function. To be able to recognize a vast variety of antigens, the theoretical diversity of antibodies is tremendous, making the mapping of antibody repertoires inherently complex. This chapter describes the development of mass spectrometry-based Fab profiling, which isolates and analyzes antibody clones by cleaving them at the hinge region to separate the Fab and Fc regions, enabling comprehensive Fab repertoire analysis. Chapter 2 investigates the long-term persistence of human IgG1 clones in healthy individuals over one year. The study finds that most abundant IgG1 clones remain stable over time, suggesting that they are produced by long-lived plasma cells and do not undergo further mutation. This highlights the stability of the antibody repertoire in healthy individuals. In Chapter 3 I extend the mass spectrometry-based Fab clonal profiling approach to specifically examine the SARS-CoV-2 specific Fab clonal repertoires in patients infected with different SARS-CoV-2 variants (wild-type, Alpha, Beta, Gamma). The study reveals unique, diverse anti-spike antibody responses, with individual clones showing varying cross-reactivity across different variants. This work demonstrates the potential of Fab clonal profiling for identifying important therapeutic antibodies. Chapter 4 focuses on the anti-citrullinated protein antibody (ACPA) repertoire in rheumatoid arthritis (RA). In RA patients, the ACPA response is polyclonal and highly diverse, with distinct glycovariants detected. This study advances our understanding of autoantibody responses in autoimmune diseases, highlighting the role of glycosylation in autoimmune pathogenesis. Chapter 5 compares two proteases, IgdE and BdpK, used for IgG1 hinge cleavage in Fab profiling. Both proteases produce similar clonal repertoires, but BdpK is more efficient and requires less incubation time. This makes BdpK a preferred choice for high-throughput analysis. Finally, Chapter 6 summarizes the power of mass spectrometry-based Fab clonal profiling in antibody repertoire analysis, emphasizing its potential for therapeutic discovery and understanding immune responses. Future improvements in sensitivity, automation, and throughput will further enhance its applicability in both basic research and clinical settings.