Monoclonal antibodies (mAbs) stand out as highly specific tools surpassing polyclonal antibodies, finding applications in diagnostics and therapeutics for viral infections, tumors, autoimmune, metabolic, and neurological diseases, as well as transplantation. In immuno-oncology, mAbs and their variants emerge as leading biologic drugs. Since the FDA approval of Orthoclone OKT3 in 1986, over 100 mAbs have been FDA-approved. The COVID-19 pandemic underscores their crucial role in therapeutics, emphasizing the rapid development of biologics. Additionally, mAbs hold promise for prophylactic use, particularly in passive immunization against pathogens like respiratory syncytial virus without available vaccines. The versatility of mAbs positions them as powerful, targeted agents in diverse medical contexts.
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Fig. 1 Single B cell screening technologies using conventional cell culture equipment (Pedrioli A., Oxenius A. 2021).
Advancements in mAb Technologies
The journey of monoclonal antibodies (mAbs) began with the groundbreaking hybridoma technology, a pioneering method that fundamentally reshaped the field of mAb discovery. As time has progressed, this innovative technique has laid the groundwork for numerous significant advancements.
Conventional Cell-Culture-Based Technologies
B Cell Immortalization: Hybridoma technology, despite its historical significance, presents drawbacks such as low throughput and lengthy procedures. Alternative protocols, incorporating electrofusion or fusogenic viruses, aim to overcome these limitations. However, challenges persist, including low-yield antibody production and chromosomal instability.
Memory B Cell and ASC Culture Techniques: Advances in defining specific culture conditions for primary B cells without hybridoma or Epstein-Barr virus (EBV) immortalization have been made. High-throughput human memory B cell cultures, relying on CD40 ligand (CD40L) and cytokines, enable the retrieval of important broadly neutralizing antibodies. Murine B cell screening protocols, such as the Nojima culture, provide efficient and cost-effective alternatives.
Membrane-Bound BCR Staining: Utilizing membrane-bound antibodies (BCR) for antigen-specific memory B cell enrichment involves sorting stained cells by fluorescence-activated cell sorting (FACS). While this method offers high-throughput advantages, potential false-positive binding remains a challenge.
Microfluidic-Based Technologies
Single B Cell Screening Methodologies: Miniaturized platforms based on microfluidic technologies offer improved ex vivo B cell culture and high-throughput identification of antigen-specific B cells. Systems like the Berkeley Lights Beacon, microfluidic chambers, micro-engraved systems, and microcapillary arrays provide open assays, while closed assays, such as droplet-based systems, offer spatial confinement of individual B cells.
B Cell Replica Methodologies: The B cell replica method combines natively paired VH-VL single cell BCRneg ASC-derived sequences with high throughput. By cloning and expressing a B cell-derived library before antigen-specific screening, this approach provides an elegant solution to interrogate natively paired antibodies in a host cell line.
Single B Cell Repertoire Analysis and Clonal Expansion-Guided Identification: Leveraging single-cell transcriptomic techniques like 10x Chromium, this methodology identifies antigen-specific B cells based on clonal expansion. While offering insights into clonal frequency and somatic hypermutations, its complex and time-consuming nature poses challenges.
The field of monoclonal antibody discovery has witnessed remarkable advancements, evolving from classical hybridoma technology to sophisticated microfluidic-based technologies. Despite challenges, these methodologies contribute significantly to the development of therapeutic and diagnostic biologics. As the landscape continues to evolve, the integration of artificial intelligence in antibody discovery campaigns may further streamline the process. Research focused on isolating natively paired VH-VL mAbs and target-agnostic screenings remains crucial, ensuring continuous progress in immunology.
References
- Pedrioli A. Oxenius A. Single B cell technologies for monoclonal antibody discovery. Trends in Immunology. 2021, 42(12): 1143-1158.
- Bayer V. An Overview of Monoclonal Antibodies. Semin Oncol Nurs. 2019, 35(5): 150927.
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