Targeted fusion of antibody-secreting cells: Unlocking monoclonal antibody production with hybridoma technology

Hybridomas, the first method for creating monoclonal antibodies (mAbs), were reported 50 years ago. This approach, which transformed biomedical research and laid the foundation for many of the current therapeutic, diagnostic, and research reagent applications of mAbs, is still used today, despite reported low fusion yields between short-lived B cells and immortal myeloma cells. To improve hybridoma production yields and accelerate development of new mAbs, we addressed two key limitations: 1) random pairing between myeloma cells and antibody-producing cells, and 2) low efficiency of the polyethylene-glycol-mediated fusion process. We first characterized and isolated antibody-secreting cells (ASCs) from the spleen of immunized mice before cell fusion to increase the probability of successive pairing between the most suitable cell fusion partners and favor the generation of functional hybridomas. Specifically, we developed an optimized workflow combining fluorescence-activated cell sorting with antibody secretion assays, using a panel of five cell-surface markers (CD3, TACI, CD138, MHC-II, and B220) to identify a distinct ASC subset with key characteristics. Such ASCs exhibited a plasmablast phenotype with high MHC-II expression and secreted high levels of antigen (Ag)-specific antibodies in immunized mice. We then implemented a cell electrofusion procedure adapted to low cell numbers (<10⁶ cells), in order to perform the targeted electrofusion of TACI^highCD138^high sorted ASCs. This targeted approach yielded viable hybridomas in 100% of seeded culture wells compared to only 40% for the electrofusion of unsorted cells. In particular, over 60% of hybridomas generated from TACI^highCD138^high sorted ASCs secreted Ag-specific mAbs, including IgGs with high Ag binding affinity (<10^-9 M). These results pave the way for a high-yield mAb production method via cell fusion, with the potential to streamline hybridoma generation and thereby expand access to mAbs.