Antibody drugs play an important role in the biopharmaceutical market due to their high specificity, remarkable efficacy, and superior pharmacokinetics. According to Frost & Sullivan**, the global antibody drug market is expected to grow to $443.1 billion by 2030. With the gradual expansion of the demand for antibody drugs, the development of antibody discovery and screening technologies is imperative. At present, the most common applications of antibody technology include hybridoma technology, bacteriophage display technology and B cell cloning technology. Each technology has its own merits, and in this issue, we will focus on single B cell antibody technology.
Single-cell antibody technology refers to the use of a B cell containing only one functional heavy chain variable region DNA sequence and a light chain variable region DNA sequence, and a B cell produces only one specific antibody characteristics, to isolate specific B cells from immunized animal tissues or peripheral blood, and then through single-cell PCR technology from B cells secreting a single antibody to amplify the heavy chain variable region and light chain variable region DNA sequence, and finally express in mammalian cells to obtain biologically active monoclonal antibodies. At present, single B cell antibody technology has been widely used in pathogenic microbial infection, tumors, autoimmune diseases and organ transplantation, and has shown unique advantages and good application prospects.
Traditional hybridoma antibodies have a long development cycle, including immunization, which usually takes about 8 months. Moreover, due to the limitation of hybridoma fusion rate, only a small fraction of B cells in the entire B cell population are completely fused, and few positive clones are obtained, which is not suitable for comprehensive screening of large antibody libraries. For phage display technology, despite its large library capacity, the pairing of heavy chain variable region (VH) and light chain variable region (VL) usually relies on random combinations, and most of the formation is unnatural VH-VL antibody pairing. As a new generation of antibody development technology after hybridoma technology and phage display technology, single B cell antibody technology has the advantages of long development cycle, many positive clones, fully human origin, and less cell size. Most importantly, antibodies obtained through single-cell antibody technology ensure a natural pairing of light and heavy chains, which is critical in antibody development.
There are five main steps in the whole process of single B cell antibody technology, including B cell enrichment, single B cell screening, antibody gene sequencing and analysis, recombinant antibody expression, and antibody function verification.
figure 1. the process of single b cell antibody technology
B cells are enriched, B cells are generally ** in peripheral blood, and B cells can be obtained by venous blood collection and centrifugation to separate the leukocyte layer.
Single B cell screening, also known as single B cell isolation or positive clone screening, refers to the isolation of single B cells from a mixed B cell population and is the most critical step in single B cell technology. Depending on the sample type, single B cell isolation can be divided into random isolation and antigen-specific isolation. Random isolation refers to the isolation of B cells, and the commonly used isolation methods include micromanipulation, laser capture microsection, and fluorescence-activated cell sorting. This type of isolation is suitable for samples with high concentrations of antigen-specific antibodies (e.g., blood samples from vaccine recipients or patients). Antigen-specific isolation refers to the isolation of antigen-specific B cells, and commonly used isolation methods include fluorescently labeled antigen multi-parameter cell fractions, antigen-labeled magnetic bead sorting, microengraving method, and cell microarrays. This type of isolation is suitable for situations where the content of specific antibodies such as anti-tumor antibodies and autoimmune antibodies is low.
After obtaining a single B cell, its antibody genes are sequenced and analyzed. As mentioned earlier, this step can be performed by single-cell PCR technology to amplify the heavy and light chain variable region DNA sequences and then perform sequencing. After sequencing, the sequencing data can be analyzed by bioinformatics, including gene sequence alignment, mutation identification, and affinity evaluation.
Through the sequencing and analysis of antibody genes in the previous step, the antibody genes with better affinity are usually selected for recombinant antibody expression. The specific choice needs to be based on your own experimental design. In the field of biopharmaceutical research, the expression system commonly used for recombinant antibody expression is the mammalian cell expression system.
After the recombinant expression of antibody genes, it is necessary to further verify its function to determine its biological activity and antigen binding ability, and commonly used verification methods include flow cytometry, enzyme-linked immunosorbent and immunohistochemistry.
The innovative rabbit monoclonal antibody discovery platform based on single B cell technology focuses on the in vitro culture of single B cells before cloning antibody genes. In contrast to the traditional use of single-cell PCR, the antibody genes of hundreds of B cells cloned by Dima are derived from a single parental B cell. This approach significantly increases the success rate of cloning, enabling Dymad to obtain more positive lead antibody sequences from immunized animals. Normally, the total number of B cells in a rabbit can reach 3-5x10 8, and the total number of secretion-specific antibody B cell clones can be obtained from it can reach tens of thousands. The platform leverages the advantages of rabbit monoclonal antibodies to provide one-stop services from antigen synthesis to antibody functional validation, with a short time period and direct access to antibody gene sequences and functional protein antigens.
Platform process
figure 2. the process of dimab® platform
Case Study
BCMA-lead antibody molecule development and CAR-T application.
GPRC5D lead antibody molecule development and CAR-T application.
In addition, for popular drug targets, Dima has also prepared a B cell seed bank. The DIMAB B Cell Seed Bank is constructed from B cells isolated from immunized rabbits that have been pre-validated by ELISA and FACS to specifically bind to antigenic proteins. The DIMAB B cell bank is primarily used to screen lead antibody molecules. The screened lead molecules have diverse CDR sequences and good druggability. At present, there are targets in the corresponding seed bank, and the lead antibody can be obtained in 35 days at the earliest. By the end of 2023, in the past five years, Denma has completed the preparation of 407 new target B cell seed banks and the development of 275 drug target lead antibody molecules. In addition to rabbit monoclonal antibodies, Dima has also built a development platform for murine monoclonal antibodies, sheep monoclonal antibodies and nanobodies.