Antibodies are glycoproteins produced by B cells, and have the function to recognize molecules (antigens) such as proteins and bind thereto. Antibodies are produced in response to various internal and external stimuli (antigens), and cooperate with immunocompetent cells to eliminate bacteria and viruses that invaded the body, thereby playing an important role in the biological defense mechanism of vertebrates. A single type of B cell can produce only a single type of antibody, and a single type of antibody can recognize only a single type of antigen. In the human body, several millions to several hundred millions of types of B cells produce different antibodies to cope with numerous kinds of antigens. These are collectively called immunoglobulin, which is one of the most abundant proteins in the blood and constitutes 20% by weight of the total plasma proteins. Antibodies are utilized, based on their antigen specificities, as molecular-targeted agents and diagnostic agents.
A naturally-occurring antibody molecule forms a Y-shaped basic structure by association of two each of two types of polypeptide chains, the L chain (light chain) and the H chain (heavy chain). The lower half of the Y shape is composed of the Fc region, and the upper half of the Y shape, that is, the V-shaped portion, is composed of two identical Fab regions. The distal half of Fab is called the variable region (V region), and shows diversity in its amino acid sequence so that the antibodies can bind to various antigens. The variable regions in the L chain and the H chain are called VL and VH, respectively. On the other hand, the Fe-side half of Fab is called the constant region (C region), and shows less variability in its amino acid sequence. The constant regions in the L chain and the H chain are called CL and CH1, respectively. Each region in VL and VH that directly contacts with an antigen has especially high diversity in its amino acid sequence, and called the complementarity-determining region (CDR). The other part is called the framework region (FR). Each of VL and VH has 3 CDRs (CDR1 to CDR3), and 4 FRs (FR1 to FR4) surrounding these. The sequence diversity of CDR1 and CDR2 in naive B cells that have not been stimulated with an antigen is derived from the genomic DNA sequence (germline sequence). On the other hand, sequences of CDR3 are newly formed by recombination reaction of the genomic DNA that occurs in the process of differentiation of B cells, and hence its diversity is larger than those of the other CDRs. Moreover, in contrast to L-chain CDR3 formed by single recombination reaction (V-J recombination), H-chain CDR3 is formed through two times of recombination reaction (V-D recombination and D-J recombination), so that the H chain has a larger diversity in the same CDR3. The 6 CDRs form a single continuous surface involved in antigen binding in the distal part of Fab. In naturally-occurring antibodies, the functional and structural unit involved in antigen binding is Fab. While each of VL, VH, CL and CH1 constituting Fab forms an independent domain in terms of the spatial structure, they achieve high stability due to interactions among the 4 domains. Although the Fc region is not directly involved in binding of the antibody to an antigen, it is involved in various effector functions (e.g., the antibody-dependent cell-mediated cytotoxicity function).
Attempts are being made to prepare a gene library that expresses antibodies, in order to screen a gene encoding an antibody that specifically binds to a target antigen. In such a method, in order to obtain an antibody that specifically binds to a target antigen, an antibody library is expressed from DNA encoding the antibody library, and the expressed antibody library is brought into contact with the target antigen to select antibodies that specifically bind to the target antigen, followed by amplification of the DNAs encoding the selected antibodies. This cycle is repeated for screening antibodies. Since each antibody selected by such a screening method using a display technology is accompanied by information on the gene encoding its amino acid sequence, the selected antibody can be immediately prepared in a large amount by genetic engineering based on the genetic information encoding the antibody. Further, the amino acid sequence can also be easily determined by analysis of the genetic information.
An example of the antibody screening method using a display technology is phage display reported in 1985 by G. Smith et al. (Non-patent Document 1). Phage display is a technique in which a foreign protein is expressed as a fusion protein with mainly a coat protein of a filamentous phage, and the polynucleotide encoding the foreign protein of interest is selected. This method is widely used for, for example, selection of an antibody that specifically binds to an antigen molecule (Non-patent Documents 2 and 3). However, construction of a phage library requires the step of transformation of E. coli, and this step limits the size of the library. That is, due to the limit of efficiency of transformation of E. coli, construction of a library having a diversity of, for example, more than 1010 requires several ten times to several thousand times of transformation of E. coli by electroporation. Therefore, construction of a library larger than this is considered unrealistic. Further, since translation into a protein is dependent on E. coli, the efficiency of expression of a protein harmful to E. coli as a host is remarkably low.
On the other hand, cell-free display systems represented by ribosome display are techniques wherein proteins synthesized by a cell-free translation system are associated with polynucleotides encoding the proteins. Screening of an antibody using this system has been reported (Patent Document 1). However, what was actually prepared in this report is a single-chain antibody (scFv) wherein the heavy-chain variable region (VH) is linked to the light-chain variable region (VL) through a linker peptide, and no specific method for preparing Fab is disclosed. That is, it has been thought that Fab, which is constituted by two peptide chains and has a larger molecular weight than scFv, is difficult to be handled in ribosome display because, for example, ribosome display is generally a technique wherein a single molecule of RNA is coordinated with a single molecule of protein utilizing the 3′-end of the RNA, and synthesis of a full-length peptide chain becomes more difficult as the molecular weight of the protein to be synthesized increases (Non-patent Document 4). Further, since Fab is double-chained, the screening efficiency might decrease due to occurrence of not only cis-association of the H chain and the L chain displayed on the same RNA, but also trans-association of the H chain and the L chain on different RNAs.
As an attempt to use Fab in a cell-free display system, Yanagawa et al. reported a method wherein the H chain and the L chain constituting Fab are bicistronically expressed in a water-in-oil emulsion, and the Fab is associated with the DNA encoding it utilizing the interaction between streptavidin and biotin (Non-patent Document 4). However, since this method employs a water-in-oil emulsion, there are still problems in, for example, that the size of the library which can be handled is limited; that, since Fab synthesized in a compartment is once dissociated from DNA and mRNA and only one molecule of Fab is linked to the DNA, the remaining numerous Fab molecules cause competition; that the enrichment ratio is about 100, which is not very high; and that operations in the experiment require high skill.
Further, there is a known method called look-through mutagenesis for increasing the affinity of a target-substance-binding protein to its target substance, in which a library is prepared by introduction of a single amino acid substitution into a target-substance-binding site in a target-substance-binding protein, and the library is used to perform first screening, followed by combining the obtained single amino acid substitutions and performing second screening to screen mutant proteins having improved affinity to the target substance (Patent Document 2). However, in this method, the sequences obtained by the first screening are cloned, and mutant proteins are expressed from the obtained clones to confirm their affinity to the target substance, followed by performing the second screening. This is very laborious.