This study established a modified peripheral blood mononuclear cell (PBMC) culture system by incorporating IL-4, IL-21, BAFF, and anti-CD40 agonist antibodies to support B cell survival and activation. It screened 51 monoclonal antibodies with varying clinical immunogenicity rates using IgG secretion as an indicator for immunogenicity risk assessment. Although IgG secretion levels differed among tested antibodies, no significant correlation was observed with clinical immunogenicity ratings. The study highlights challenges in developing B cell-based immunogenicity prediction methods and provides a framework for future optimization.
Literature Overview
The article 'Immunogenicity Risk Assessment of Biotherapeutics Using an Ex Vivo B Cell Assay', published in Antibodies, reviews the application of B cell-based ex vivo assays for immunogenicity risk assessment of biotherapeutic drugs. It discusses limitations of existing methods such as T-cell proliferation assays and MHC-associated peptide proteomics (MAPP) assays, emphasizing the critical role of B cells as professional antigen-presenting cells (APCs) and sources of anti-drug antibodies (ADAs) in immunogenicity evaluation.
Background Knowledge
Monoclonal antibodies (mAbs) have become essential tools for treating cancer, autoimmune diseases, and neurodegenerative disorders due to their high target specificity, long half-life, and low off-target effects. However, mAbs may induce ADA production during clinical use, compromising drug safety, pharmacokinetics, and efficacy. Immunogenicity risks are influenced by multiple factors, including mAb sequence, glycosylation patterns, T/B cell epitopes, human sequence homology, aggregation-prone regions (APRs), and formulation impurities. Patient-related factors such as administration route, dosage, immune status, and HLA haplotypes also impact ADA formation. Current immunogenicity prediction relies on in vitro T-cell proliferation, dendritic cell internalization, and MAPP assays, which cannot evaluate B cell-mediated ADA secretion and may yield false-negative results. Developing an integrated assay to simultaneously assess antigen uptake, T cell activation, and B cell ADA secretion could enhance immunogenicity risk prediction accuracy.
Research Methods and Experiments
The study utilized a modified PBMC culture system supplemented with IL-4, IL-21, BAFF, and anti-CD40 agonist antibodies to support B cell survival and activation. CD8+ T cells were depleted to promote helper T cell functionality, better mimicking in vivo conditions. After 7 days of culture, B cell activation markers (CD80, CD86), immunoglobulin class switching, and proliferation were evaluated by flow cytometry. IgG secretion levels in culture supernatants were quantified using the LEGENDPlex Human Immunoglobulin Isotyping Panel (8-Plex).
Key Conclusions and Perspectives
Research Significance and Prospects
This study represents the first simultaneous evaluation of T cell and B cell immune responses to mAbs within a PBMC culture system, establishing an experimental framework for developing more comprehensive immunogenicity assessment methodologies. Future research directions include optimizing culture conditions to improve correlation between IgG secretion and clinical immunogenicity, exploring mechanisms of naive B cell responses, and implementing this system for medium-to-high throughput screening with significant application potential.
Conclusion
The study developed a PBMC-based B cell ex vivo culture system for immunogenicity risk assessment of monoclonal antibodies. By adding IL-4, IL-21, BAFF, and anti-CD40 agonist antibodies, researchers successfully induced B cell activation, proliferation, and IgG secretion. However, the lack of correlation between IgG secretion levels and clinical immunogenicity ratings indicates the need for further optimization. Experimental findings revealed that high-immunogenicity mAbs (e.g., mAb 1) primarily activate memory B cells with minimal naive B cell responses, potentially limiting predictive capabilities for primary immunogenicity. Future work will focus on improving culture conditions to enhance naive B cell responses, evaluating dual T cell/B cell effects of various mAbs, and establishing a more comprehensive immunogenicity risk assessment workflow. While the current system cannot yet accurately predict clinical immunogenicity, it provides a foundational platform for developing more sensitive and specific detection methods with broad application prospects.
