Antibody screening technologies applied in innovative antibody discovery: PII
|11.7.2021||Posted by tactical33 under Advertising & Marketing|
According to the source and use of antibody genes, antibody libraries are divided into immune antibody library and general antibody library. The antibody genes of the immune antibody library are derived from immunized human or animal B cells (lymph nodes, spleen, and peripheral blood lymphocytes). The general antibody library is divided into natural antibody library, fully synthetic antibody library, and semi-synthetic antibody library. According to the different forms of antibody molecules, the antibody library can be divided into Fab antibody library, scFv antibody library, and VHH antibody library. The VHH antibody library is also called single-domain antibody (sdAb) library.
The antibody genes of the natural antibody library are derived from unimmunized human or animal B cells. According to the clonal selection theory of antibody production, after the fetal organs mature, clonal selection will bring about the bias of B cell cloning. There are a large number of B1 and B2 cell clones in fetal liver and bone marrow without clonal selection, with higher diversity of antibodies, so it is the best choice for constructing a natural antibody library.
Natural antibody libraries and synthetic antibody libraries have their own pros and cons. The antibody genes of the natural antibody library are derived from natural B cells, and the epitope diversity is relatively limited, which also affects the affinity of the antibodies to be screened. However, due to the in vivo evolution process, indicators such as stability, solubility, and expression levels perform better. In contrast, the design diversity of the fully synthetic antibody library reaches more than 1020. Even if the bacterial transformation step is restricted during the antibody library construction, the diversity of antibody recognition epitopes far exceeds that of the natural antibody library. However, this kind of artificially designed sequence diversity without in vivo evolution often shows abnormal protein modification or abnormal amino acid clusters, low expression levels, and easy degradation. Therefore, the screened antibodies need to be optimized to be more druggable.
The semi-synthetic antibody library is designed to integrate the advantages of the natural antibody library and the fully synthetic antibody library and overcome their shortcomings. The FR1-CDR1-FR2-CDR2-FR3 gene fragments of the antibody variable region are all derived from natural B cells, while the CDR3 and FR4 regions are artificially synthesized. The amino acid composition and ratio of each position in the CDR3 region are also designed by bioinformatics technology. Therefore, the semi-synthetic antibody library not only reduces the proportion of artificial design, but also ensures sufficient antibody diversity.
Comparison of antibody libraries based on different display technologies
According to different display technologies, antibody libraries are divided into three categories: phage display library, cell-free molecular display library, and cell surface library. The cell-free molecular display antibody library can be divided into ribosome display, mRNA display, and DNA display libraries. According to different host cells, cell surface display libraries can be divided into bacterial surface display, fungal surface display, yeast surface display, and mammalian cell surface display libraries.
Due to the limitation of transformation efficiency, cell culture density, and cell culture volume, the upper storage capacity of the cell surface display antibody library (especially the mammalian cell surface display antibody library) is relatively small (generally less than 10).
The construction of the cell-free molecular display library only requires the preparation of corresponding DNA templates in vitro, and the steps of transforming bacteria or cells are completely unnecessary. Therefore, the upper limit of the storage capacity is the largest, even greater than 1016. The antibody library stored in the form of DNA needs to undergo PCR amplification, in vitro transcription and in vitro translation before screening to achieve the correlation between antibody genotype (RNA or DNA) and phenotype (protein).
Due to the smaller size of the bacterial host and phage particles, the large culture density, and small volume, and the efficiency of bacterial transformation is also higher than that of yeast and mammalian cells. The storage capacity of the phage display antibody library is between the cell surface display antibody library and the cell-free molecule display antibody library, and the upper limit is generally 10^11-10^12, which is sufficient to meet the application requirements of immune antibody libraries and general antibodies.