Antibody drugs, due to their high specificity and significant therapeutic effects, possess superior pharmacokinetic properties, making them a crucial player in the biopharmaceutical market. According to Frost & Sullivan’s statistical predictions, the global antibody drug market is expected to reach $443.1 billion by 2030. As the demand for antibody drugs continues to grow, the development of antibody discovery and screening technologies becomes imperative. Currently, widely used antibody technologies include hybridoma technology, phage display technology, and B cell cloning technology. Each technology has its strengths, and in this issue, we will focus on discussing single B cell cloning technology.
1. What is Single B Cell Cloning Technology?
Single B cell cloning technology refers to the utilization of the unique characteristics of a B cell, which contains only one functional heavy chain variable region DNA sequence and one light chain variable region DNA sequence. Additionally, a single B cell produces only one specific antibody. The process involves isolating specific B cells from the immune animal tissue or peripheral blood. Subsequently, through single-cell PCR technology, the DNA sequences of the variable regions of both heavy and light chains are amplified from B cells secreting a single antibody. Finally, the obtained monoclonal antibody with biological activity is expressed in mammalian cells. Currently, single B cell cloning technology has been widely applied in areas such as pathogenic microbial infections, tumors, autoimmune diseases, and organ transplantation, demonstrating unique advantages and promising applications.
2. What are the advantages of Single B Cell Cloning Technology?
Traditional hybridoma technology for antibody development has a long development cycle, typically around 8 months, including the immunization process. Moreover, due to limitations in the fusion rate of hybridomas, only a small fraction of B cells in the entire B cell population undergo complete fusion, resulting in a limited number of positive clones. This makes it unsuitable for comprehensive screening in large antibody libraries. As for phage display technology, despite its large library capacity, the pairing of heavy chain variable regions (VH) and light chain variable regions (VL) typically relies on random combinations, leading to mostly non-natural VH-VL antibody pairings.
In contrast, Single B Cell Cloning Technology, as a new generation antibody development technology following hybridoma technology and phage display technology, comes with several advantages such as a shorter development cycle, a higher number of positive clones, being fully human-sourced, and requiring fewer cells. Most importantly, antibodies obtained through Single B Cell Cloning Technology ensure the natural pairing of light and heavy chains, a crucial aspect in antibody development.
3. What is the process of Single B Cell Cloning Technology?
The entire process of Single B Cell Cloing Technology consists of five main steps, including B cell collection, single B cell screening, antibody gene sequencing and analysis, recombinant antibody expression, and antibody function validation.”
Figure 1. the process of single B cell antibody technology
B cell collection typically originates from peripheral blood, and B cells can be obtained by centrifuging and separating the white cell layer after venous blood collection.
Single B cell screening, also known as individual B cell isolation or positive clone screening, refers to the separation of individual B cells from a mixed population of B cells and is the most critical step in Single B Cell Technology. Depending on the type of sample, single B cell isolation can be categorized as random isolation and antigen-specific isolation. Random isolation involves the separation of B cells without considering antigen specificity, and common methods include microscopic manipulation, laser-capture microdissection, and fluorescence-activated cell sorting. This type of isolation is suitable for samples with high concentrations of antigen-specific antibodies (such as blood samples from vaccine recipients or patients). Antigen-specific isolation involves the separation of B cells specific to a particular antigen. Common methods include fluorescence-labeled antigen multiparameter cell sorting, antigen-labeled magnetic bead separation, microengraving, and cell microarray chips. This type of isolation is suitable for situations where the specific antibody content, such as anti-tumor antibodies or autoimmune antibodies, is relatively low.
After obtaining individual B cells, it is necessary to sequence and analyze their antibody genes. As mentioned earlier, this step can be accomplished by amplifying the DNA sequences of the variable regions of both heavy and light chains using single-cell PCR technology, followed by sequencing. Once sequencing is complete, bioinformatics analysis of the sequencing data is performed, including gene sequence alignment, mutation identification, and affinity assessment.
Following antibody gene sequencing and analysis in the previous step, antibodies with favorable affinities are typically selected for recombinant antibody expression. The specific criteria for selection depend on the experimental design. In the field of biopharmaceutical research, mammalian cell expression systems are commonly used for the expression of recombinant antibodies.
After the recombinant expression of antibody genes, it is necessary to further validate their functionality by determining their biological activity and antigen-binding capability. Common validation methods include flow cytometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry.
4. DIMA’s Rabbit Monoclonal Antibody Discovery Platform Based on Single B Cell Technology
DIMA’s Rabbit Monoclonal Antibody Discovery Platform, based on its innovative single B cell cloning technology, involves the in-vitro culture of individual B cells before cloning the genes for antibodies. In contrast to using single-cell PCR technology, DIMA clones the antibody genes from hundreds of B cells derived from a single parental B cell. This approach significantly enhances the success rate of cloning, allowing DIMA to obtain a greater number of positive lead antibody sequences from immunized animals. The platform, leveraging the advantages of rabbit monoclonal antibodies, offers a one-stop service from antigen synthesis to antibody functional validation, featuring a short time cycle, direct access to antibody gene sequences, and functional protein antigens.
Platform process
Figure 2. The process of DimAb® platform
Cases Display
Development of anti-BCMA therapeutic mAbs for CAR-T application
Using a single B platform to develop a CAR-T cell therapeutic solution for a GPCR target
Additionally, targeting popular drug targets, DIMA has also established a B Cell Seed Library. The DimAb® B Cell Seed Library is constructed from B cells isolated from immunized rabbits, which have been pre-validated through ELISA and FACS, demonstrating specific binding to antigen proteins. The DimAb® B Cell Library is primarily used for screening lead antibody molecules. The screened lead molecules exhibit diverse CDR sequences and favorable drug-like properties. For target points with corresponding seed libraries, lead antibodies can be obtained in as little as 35 days.”