A novel approach for generation of fully human monoclonal antibodies in humanized RAG-hu mice

4090 BACKGROUND: The currently well-established methods for developing fully human mAbs are human hybridoma technology, transgenic mice and phage display. Common limitations of these approaches are either low affinity and cross-reactivity or restricted Ab diversity. The new humanized Rag2-/-gamma c-/- mouse model (RAG-hu) created by intrahepatic injection of CD34 hematopoietic stem cells sustains long-term multi-lineage human hematopoiesis and is capable of mounting immune responses. We reasoned that this model could be suited for generation of antigen specific human B cells and might have considerable promise for development of human therapeutic mAbs against e.g. cancer, autoimmune or infectious diseases. RESULTS: Here we demonstrate that RAG-hu mice produce after immunization abundant antibody-producing human B cells specific for a disease-associated human antigen and that B cells isolated form these mice can be used to generate hybridomas for the production of human mAbs. To assess the capacity of these mice to generate antigen-specific human B cells, they were challenged with either antigenic protein with adjuvant or with plasmid DNA encoding the target antigen. The specific humoral response was assessed by ELISA and FACS. Human B cells were isolated from spleens of immunized RAG-hu mice and used for hybridoma generation. Our results show that both protein and genetic immunizations are capable of generating antigen-specific human B cells in RAG-hu mice and that specific titer rises depending on boost injection and lasts for at least 20 weeks. Moreover, we were able to generate hybridoma lines from extracted human CD19 B cells secreting fully human mAbs with high binding specificity. CONCLUSION: The humanized RAG-hu mouse model has been proven suitable for generation and future immortalization of human B cells specific for a disease-associated human antigen. The considerable advantage of this approach is the possibility to generate a more diverse panel of mAbs with a greater range of characteristics, because the use of entire human B cells repertoire as well as other cellular components of the human immune system and other biomolecules required for antibody development. Therefore, the humanized RAG-hu mouse model holds great promise for immunotherapy development.