De novo generation of specific human IgGs by in vitro immunization using autologous proteins containing immunogenic...

In vitro immunization can to used to produce monoclonal antibodies(mAbs), but this technology is limited by poor reproducibility and the requirement of pre-immunized lymphocytes. To improve this approach, we recently developed a method for rapid generation of antigen-specific B cells. Here, we report the application of this system to the production of human IgGs against tumor necrosis factor (TNF). We expressed mutant proteins with site-specific incorporated p-nitrophenylalanine (pNO₂Phe), which stimulated an in vitro immune response in human immune cells. After constructing an antigen-specific antibody library from in vitro immunized B cells identified by fluorescence-activated cell sorting, we demonstrated that many point mutation events of the variable region occurred in our step-by-step co-cultivation system for affinity maturation in vitro. To mimic the class switching, we panned for high-affinity antigen-binding fragments by the phage display method, assembled them and identified hTNF-neutralizing human IgGs. This approach may provide a general method for raising high-affinity monoclonal antibodies against self-proteins. Furthermore, it supports mechanistic understanding in breaking human self-tolerance with pNO₂Phe.