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Conventional Technologies for Monoclonal Antibody (mAb) Generation
Biointron2024-12-26Read time: 5 mins
Image credit: TheScientist/ALPANA MOHTA
The discovery of monoclonal antibodies (mAbs) for therapeutic and research applications has been a cornerstone of modern biotechnology. Two conventional approaches, hybridoma technology and single B cell screening, have historically driven the field, offering distinct strengths and limitations. In addition, advancements in in vitro surface-display technologies have expanded the scope of antibody discovery, enabling high-throughput selection of candidates with desirable properties.
Hybridoma Technology
Introduced in 1975 by Cesar Milstein and Georges Köhler, hybridoma technology was instrumental in the approval of the first FDA-approved antibody therapy, Muromonab-CD3, in 1986. The process involves:
B Cell Fusion: Murine B cells are fused with immortalized myeloma cells using electrofusion or chemical treatment.
Clone Selection: Resulting hybridoma cells are screened for IgG secretion and desirable binding activity, typically using enzyme-linked immunosorbent assays (ELISA).
Therapeutic Development: Selected clones are expanded, and their antibodies are purified for further evaluation.
Hybridoma technology retains native pairing of the immunoglobulin heavy and light chains (VH/VL), ensuring functional antibodies. However, its reliance on fusion efficiency is a notable limitation, with only one antibody-secreting hybridoma generated per 100,000 cells. This technique is predominantly used with murine B cells but has been adapted for other species, including transgenic mice engineered to produce human antibodies.
Despite its limitations, hybridoma technology remains a widely used and reliable approach, particularly for generating therapeutic IgG candidates. Dozens of FDA-approved mAbs on the market today originated from this methodology.
Single B cell screening offers a more direct and efficient route to antibody discovery. This method isolates individual antigen-specific B cells using techniques such as fluorescence-activated cell sorting (FACS). The workflow includes:
B Cell Purification: Antigen-specific B cells are labeled and isolated based on markers for positive and negative selection.
Gene Amplification: RNA from selected cells is converted to complementary DNA (cDNA), and the VH/VL genes are amplified using reverse transcription-polymerase chain reaction (RT-PCR).
Antibody Production: Amplified genes are cloned into expression systems, and candidate antibodies are expressed and purified for evaluation.
This approach retains the natural VH/VL pairing while circumventing the inefficiencies of hybridoma-based fusion. Innovations such as microfluidic platforms have further miniaturized and accelerated single B cell workflows. For example, FACS-sorted B cells can be compartmentalized in microdroplets, enabling high-throughput screening of antigen-specific antibodies.
Single B cell screening has been particularly valuable in infectious disease research, facilitating the discovery of neutralizing antibodies against pathogens such as HIV and RSV. This method excels in isolating rare antibody variants with high affinity and specificity, making it a cornerstone of vaccine development and therapeutic discovery.
Surface-display technologies, including phage and yeast display, provide an alternative to traditional immune repertoire-based approaches. These methods involve converting immune repertoires into libraries displayed on the surface of microorganisms or particles. The process typically includes:
Library Construction: VH/VL genes are cloned into surface-display systems.
Panning and Selection: Libraries are screened iteratively to enrich clones with optimal binding characteristics, such as high affinity and specificity.
Antibody Development: Selected clones are expressed, purified, and characterized for therapeutic potential.
Surface-display technologies are particularly advantageous for their scalability and ability to conduct iterative selection. This enables the refinement of antibodies for complex criteria, such as improved binding under specific conditions. Notably, phage display facilitated the development of adalimumab (Humira), one of the best-selling drugs globally.
Unlike immune repertoire-based methods, surface display can utilize naïve antibody libraries, which are valuable in urgent situations, such as emerging infectious disease outbreaks. Libraries with high diversity (>10¹⁰ variants) enable the isolation of high-affinity candidates even in the absence of prior immune stimulation, as demonstrated during the SARS-CoV-2 pandemic.
Challenges and Considerations in mAb Discovery
While these technologies provide robust solutions, each comes with challenges:
Immunodominance: Immune repertoires may bias responses toward epitopes with limited therapeutic relevance. Antigen design and careful adjuvant selection can mitigate this issue.
Donor Variability: Human immune responses vary significantly, potentially complicating screening efforts. Prescreening donor samples for neutralizing activity can improve outcomes.
Efficiency and Labor: Hybridoma fusion inefficiency and the labor-intensive nature of single B cell screening highlight the need for continued process innovation.
Conclusion
Conventional mAb generation technologies, including hybridoma technology, single B cell screening, and surface-display systems, remain vital to therapeutic antibody discovery. Each method offers unique advantages, from the robustness of hybridoma-derived antibodies to the speed and precision of single B cell screening and the scalability of surface-display approaches. As these technologies continue to evolve, they are poised to drive the next generation of therapeutic breakthroughs.
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