Combination of hu-PBL-SCID mice andscFv phage display library: An effective alternative for hu-mAb production

The mammalian immune system recognizes and responds to a wide spectrum of molecules, and its diversity is facilitated by V-(D)-J recombination during ontogeny. The central function of the immune system is to protect the animal from traumas, invasion of foreign agents and aberrations within the animal itself. One of the major components, which help the immune system to fulfill its functions, is the B cell. Each B cell clone expresses or produces a specific immunoglobulin molecule (Ig) that can recognize a distinct molecule or a class of related molecules. The development of the mouse hybridoma technique to produce unlimited amounts of specific mouse antibodies (Ab) in 1975 has opened doors to a wide spectrum of Ab applications, which has facilitated scientific research. Especially, the generalization of using Ab in human immunotherapy is closer than ever. The immunogenicity of the Ab molecules has, however, made human, not mouse Ab an ideal candidate for clinical uses. Currently, four approaches are studied and used to produce specific Ab for therapeutic purposes. The first two approaches are based on the availability of a vast array of ready-made specific mouse hybridomas. Chimeric human/mouse Ab is generated by replacing mouse Ig constant (C) regions by the human counterparts. Furthermore, human framework regions (FR) are grafted into the chimeric human/mouse Ab by polymerase chain reactions (PCR) to produce humanized Ab that retains the original specificity. Over the last 10 years, the generation of complete human Ab (hu-Ab) has been achieved by transgenic and recombinant approaches. Specific human immune responses (hu-IR) can be induced in transgenic mice that are carrying human Ig transgenes. Hybridomas can be easily generated from these mice and used to produce highly specific hu-Ab. Phage display library techniques have been developed and used to express large repertoires of hu-Ig genes, and specific hu-Ig genes can, in turn, be isolated from this library by simple panning steps.; We previously demonstrated that, with appropriate immunization procedures, both primary and secondary hu-IR could be established in severe combined immunodeficient mice that had been engrafted with human peripheral blood lymphocytes (hu-PBL-SCID mice). These results prompted us to establish an efficient system to generate specific human monoclonal Ab (hu-mAb). By combining this animal model with a single chain antibody Fv (scFv) phage display technique, we were able to generate panels of hu-scFv specific to either self-Ag (i.e. hu-TNF-α) or foreign Ag (i.e. respiratory syncytial virus). Our results show that: (1) hu-PBL-SCID mice can be effectively used to generate large hu-Ig gene repertoires for the cloning of highly specific hu-mAb. (2) The combination of hu-PBL-SCID mice and scFv-library can be applied widely to generate large repertoires of hu-Ab against both foreign and self-Ag.; The work presented in this thesis provides a significant step toward the generalization of specific hu-Ab production for both research and clinical purposes.