Construction And Selection Of The Human Naturally Immunized Fab Antibody Phage Display Library From Patients With Colorectal Cancer

Construction and selection of the human naturally immunized Fab antibody phage display library from patients with colorectal cancer ABSTRACT BACKGROUND / OBJECTIVES Nearly 30 years, the occurrence of colorectal cancer (CRC) is rising, and it has becoming one of the most common malignant tumors which threaten the human health. At present, the incidence of CRC has been on the second status in weatern country, and on the fourth in Shanghai, China. It has been clear that the prognosis of CRC is related to early diagnosis and treatment. Since the development and evolution of the CRC is one kind of complicated procedures involving mult-genes, mult-factors and mult-steps, there have been no highly peculiar molecu-pathologic changs and tomor markers. Last decade, the technique of immunology and molecular biology has been extensively applied in diagnosis and treatment of tumor and promoted the study on CRC. In seventies, Kohler and Milstein prepared the monoclonal antibody (McAb) of hybrideoma by means of cell fusion and applied it into prevention, diagnosis and treatment of human diseases. But a major focus of cancer immunology is also on the isolation of antibodies that react selectively with human tumor cells without heterology to human because the antibodies could have important applications for targeting diagnostic and therapeutic agents to tumors and for identifying tumor antigens. The established approach has been to generate large panels of monoclonal antibodies from mice immunized with human tumor cells and to screen the antibodies for reactivity against the tumor. Despite the enormous effort expended on this approach, few antibodies that react preferentially with human tumors, and none that react specifically with one type of tumor (such as colorectal cancer), have been reported. Further attempts to isolate more specific and high affinity antibodies will require improved methods of generating and selecting antibodies against human tumors. One is the introduction of method for synthesizing virtually the entire repertoire of a person抯 antibody genes of variable regions by PCR technique and for expressing the encoded antibodies on the surface of a phage vector. Phage display technique, which has become an increasingly important tool in biotechnology, provides a new way for antibody development. The resulting phage antibody library can be panned to select and clone rare antibodies on the basis of their binding specificities. It can resolve the problems of generating human McAb by hybridoma approach, such as low coefficient of hybridoma cells, difficult establishment of the cells lines, instability and low output of McAb, and no freely immuning to human, et al. Human can be immunized to many antigens (including tumor antigens) but only local lymph nobles, a productive source of antibody producing cells, is readily available. As part of the antibody fragments, Fab is one third of whole antibody made of heavy chain VH-CH1 (Fd fragment) and integrated light chain(K or X). Fab fragment has the similar binding activity as the antibody and has some advantages of low molecule weight, weak heterology, strong infiltration and prokaryotic expression. In this project, by using the leading biotechnology of phage display antibody library, we constructed the naturally immunized phage display libraries expressing Fab antibodies derived from local c...