We have developed a simple yet powerful antigen-specific memory B-cell (MBC) bulk selection technique for rapid and effective identification of monoclonal IgGs from immunized mice. Therefore, mild yet efficient methods to isolate the AgSC from immunized animals are needed to interrogate the immune repertoire deep and wide. While flow cytometry works well for single B-cell cloning, its use for bulk isolation is limited by its speed and the harsh effect that electromagnetic field can have on cell integrity at high sorting speed 22, leading to significant loss of cell quantity and quality 23. We hypothesized that increasing the frequency of the parental cognate chain pairings by constructing and panning immunized library generated from antigen-specific B cells (AgSC), could possibly provide greater success in mining the antigen-specific repertoire.Īntigen-specific B cells are commonly isolated by fluorescent activated cell sorting (FACS) 6, 7, 8, 9, 10, 11, 12, 20, 21. Despite such combinatorial pairings, parental cognate pairing is probably retained at a low frequency 18. However, during construction of immune display libraries, cognate chain pairs from the small percentage of antigen-specific cells undergo extensive combinatorial pairing with the vast repertoire of chains from non-antigen-specific cells 19. A popular approach to deep-screen immune repertoire is to make immunized phage libraries, several of which have been reported to be a good source of potent antibodies 16, 17, 18.
Therefore, platforms that rely on immunization of animals, or use of convalescent patient B cells, deal with a huge abundance of irrelevant cells, leading to high attrition between screening and identification of antigen-specific hits. This creates a formidable challenge even for the most advanced screening platforms to mine the entire immune repertoire. The frequency of antigen-specific cells, even after a robust immune reaction, is only between 0.01–0.1% of total B cells 13, 14, 15. However, antibody discovery from immunized animals or convalescent patients, is an intricate process due to the scarcity of antigen-specific cells in a vast diversity of the overall antibody repertoire. The revelation from Dennis Burton’s group that antibodies derived by B-cell cloning had greater therapeutic potency compared to those derived from combinatorial libraries from HIV convalescent patients 3, led to a surge in efforts to discover therapeutic antibodies from immunized animals or convalescent patients 4, 5, 6, 7, 8, 9, 10, 11, 12. As of now there are 129 antibody therapeutics approved or under review in the US and EU 1, 2. Monoclonal antibodies as a promising therapeutic modality are now widely accepted. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells.
Using five different antigens, we show hit rates of 51–88%, compared to about 5% with conventional methods. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. Identifying these cells is akin to finding needle in a haystack. Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells.
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