Construction and application of libraries of artificial antibodies

Cell fusion technology has been utilized widely for preparation of monoclonal antibodies that are specific for particular antigens (Köhler and Milstein, 1975). Three steps are required for preparation of such antibodies: immunization of an animal with the antigen; fusion of spleen cells from the immunized animal with tumor cells; and selection of hybrid cells that can proliferate and produce antibodies with the desired antigen specificity. Two conditions must be fulfilled for success: a certain amount of the purified antigen must be available; and the antigen should exhibit immunogenicity in the injected animal. The entire procedure for preparation of monoclonal antibodies is laborious and takes several months. Therefore, if libraries of antibodies, in which some antibodies can specifically bind to an antigen, can be ready-made and can be applied to any antigen, they would unquestionably contribute greatly to progress in various fields of biology (Milstein, 1990; Winter and Milstein, 1991). Since the libraries should be equivalent in size to the antibody repertoire in vivo (> 10⁸ independent clones), a system of gene expression in E. coli would be optimal because of the ease of handling. After success was achieved in the expression of Fab and Fv in E. coli, this project has become realistic (Better et al., 1988; Skerra and Plückthun, 1988). Moreover, development of the polymerase chain reaction (PCR) has supplied a method that allows recovery in vitro of virtually all the variable heavy (V_H) and variable light (V_L) genes that are expressed in animals (Orlandi et al., 1989; Saiki et al., 1988; Sastry et al., 1989). Amplified V_H and V_L genes have been combined randomly and expressed in E. coli (Huse et al., 1989; Ward et al., 1989). Furthermore, the application of phage display systems to artificial antibodies seems to increase the number of antibodies that feasibly can be handled in practice and to provide a procedure for the easy screening of libraries (Clackson et al., 1991; Kang et al., 1991a; McCafferty et al., 1990; Parmley and Smith, 1988; Smith, 1985).