Antibody screening technologies applied in innovative antibody discovery: PI

Antibody discovery includes two procedures, antibody screening and optimization. Antibody screening is to search for high-quality antibody candidates from the beginning, and antibody optimization is to make necessary improvements to existing antibodies. Antibody discovery is the only way to develop monoclonal antibody (mAb) drugs, bispecific antibody drugs (bsAb), ADC drugs, and Car-T cell therapy drugs. According to the clonal selection theory, animal immunity enriches dominant B cell clones in vivo, while affinity panning or cell sorting enriches dominant antibody clones in vitro. Natural immunogens such as viruses or cells are more likely to be enriched in the body for B cells that recognize natural epitopes than artificially prepared immunogens such as polypeptides or recombinant proteins. If an artificially prepared immunogen is used to immunize animals, B cell clones that are enriched and recognize unnatural epitopes will be inevitably produced, because unnatural epitopes are more immunogenic. The key indicator for screening functional antibodies is more than antibody affinity. Affinity panning or cell sorting based on antigen/antibody interaction should avoid over-enrichment of high-affinity antibodies, and should take into account the diversity of antibody affinity and antibody sequence, and avoid losing rare functional antibody clones. Hybridomas and antibody libraries are the most generally applied antibody screening technologies. After continuous improvement of the gene cloning technology of a single B cell, it has also begun to be used for antibody screening. Hybridomas include mouse, rat, hamster, and rabbit hybridoma technology. Antibody libraries include phage display antibody library (https://www.creative-biolabs.com/phage-display-library-screening.html), cell-free molecule display antibody libraries (ribosome display, mRNA display, and DNA display antibody library), and cell surface display antibody libraries (bacterial surface display, fungal surface display, yeast surface display, and mammalian cell surface display antibody library). HybridomasrnSo far, the hybridoma technology is quite mature. Hybridomas are hybrid cells screened in a limited medium after fusion of B cells with antibody secretion ability and infinite proliferative myeloma cells in vitro, which have both antibody secretion ability and infinite in vitro proliferation ability. Hybridomas only have the in vivo enrichment process produced by immunity without in vitro enrichment process. The supernatant of hybridoma cell culture can be directly detected to complete the preliminary screening. Mouse hybridomas can directly prepare mouse ascites purified monoclonal antibodies, while rat, hamster, and rabbit hybridomas cannot, which need to be cultured in vitro to purify monoclonal antibodies (mAbs). All hybridomas can clone antibody genes from primary cloned or subcloned cells, carry out molecular transformation, and then express recombinant monoclonal antibodies. After the monoclonal antibodies are purified, the functional antibodies can be screened and identified through meticulous functional evaluation. The efficiency of cell electrofusion to prepare hybridomas is very high. However, since each hybridoma cell is the product of the fusion of B cells and myeloma cells, it is unstable during cell division and easily loses the ability to produce antibodies. Therefore, subcloning to isolate a hybridoma (https://www.creative-biolabs.com/Hybridoma-Services.html) clone that stably secretes antibodies is necessary. During subcloning, it is easy for the positive clones to turn negative in the initial screening. Therefore, completing gene cloning before subcloning can avoid losing the antibody genes of the positive clones in the initial screening. The time window before subcloning is very short and the number of cells is small, which limits the flux of gene cloning and molecular conversion before subcloning and reduces the successful screening rate of functional antibodies. Therefore, the use of hybridoma approach to screen functional antibodies usually requires immunization of multiple animals and sometimes repeated immunizations due to fusion or screening failure. This is especially true when developing antibody drugs against difficult targets.