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

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 (pNOPhe)， 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 pNOPhe.