An antibody is generally composed of two heavy (H) chains and two light (L) chains. A single H chain and single L chain are linked via a disulfide bond to form a H chain-L chain pair, and two such pairs are linked via two disulfide bonds between the H chains to form an antibody. Bispecific antibodies (BsAbs), also called bifunctional antibodies, are multivalent antibodies with specific binding sites for two antigenic determinants. They can react with two types of antigens. BsAbs can be produced using hybrid hybridomas, or more specifically quadromas, which are fusions of two different types of monoclonal antibody-producing cells (U.S. Pat. No. 4,474,893; R. Bos and W. Nieuwenhuitzen Hybridoma (1992) 11(1): 41-51). BsAbs can also be generated by linking Fab (antigen-binding) fragments or Fab′ fragments of two types of monoclonal antibodies, using chemical techniques (M. Brennan et al. Science (1985) 229(1708): 81-3) or genetic engineering. In addition, BsAbs can be produced by covalently linking two complete monoclonal antibodies (B. Karpovsky et al. J. Exp. Med. (1984) 160(6): 1686-701).
Problems underlying BsAb production methods include the possibility of generating ten different types of antibody molecules due to the random combination of immunoglobulin H chains and L chains (M. R. Suresh et al. Methods Enzymol. (1986) 121: 210-28). Of these ten types of antibodies produced by quadromas, the only antibodies with the desired dual specificity are those with the correct L and H chain combination and which are composed of two L chain/H chain pairs with different binding specificities. Therefore, antibodies with the desired specificity must be selectively purified from the ten types of antibodies produced by quadromas. Purification is generally performed using affinity chromatography, but this method is laborious and low yielding (Y. S. Massimo et al. J. Immunol. Methods (1997) 201: 57-66).
Methods that overcome such problems and give higher BsAb yields include, for example, methods of chemically linking antibody fragments such as Fab′-thionitrobenzoic acid derivative and Fab′-thiol (SH) (Brennan et al. Science (1985) 229: 81). Furthermore, methods for more conveniently obtaining Fab′-SH fragments able to be chemically linked include methods for producing these fragments from hosts such as E. coli using genetic recombination techniques (Shalaby et al. J. Exp. Med. (1992) 175: 217-25). Genetic recombination techniques can also be used to obtain BsAbs composed of humanized antibody fragments. Diabodies (Db) are BsAbs constructed from the gene fusion of two types of fragments, and they comprise an L chain variable region (VL) connected to a H chain variable region (VH) by a linker that is too short to allow pairing between the two (P. Holliner et al. Proc. Natl. Acad. Sci. USA (1993) 90: 6444-8; EP No. 404,097; WO93/11161). An example of such a Db that has been further improved is a single-chain Db (WO 03/087163). However, antibody fragments have a shorter serum half-life when compared to full-length antibodies, and do not have effector functions like complete antibodies do. Therefore, in some cases, full-length antibodies are more suitable for diagnosis and therapy.
Methods for efficiently linking generated antibody H chains into heterodimers include a method for introducing a sterically complementary mutation into the CH3 domain (a portion of the constant region) in the multimerized domain of an antibody H chain (Ridgway et al. Protein Eng. (1996) 9: 617-21). H chains produced by this method may still form pairs with the wrong L chains. Japanese Patent Kohyo Publication No. (JP-A (Kohyo)) 2001-523971 (unexamined Japanese national phase publication corresponding to a non-Japanese international publication) describes a method for generating multi-specific antibodies which share common light chains binding to heteromeric polypeptides with antibody-binding domains. However, when any two such antibodies are selected, they rarely share the same L chain, and the method is difficult to perform. Therefore, one of the present inventors proposed a method of screening for a common L chain that corresponds to an arbitrary different H chain and exhibits high affinity (PCT/JP04/000496).
BsAbs having specific binding capacities for two different antigens are useful as targeting agents in clinical fields such as in vitro and in vivo immunodiagnosis, therapy, and immunoassays. For example, they can be used as vehicles to link enzymes to carriers by designing a BsAb so that one of its arms binds to an epitope of an enzyme reaction non-inhibiting portion of an enzyme to be used in an enzyme immunoassay, and the other arm binds to a carrier for immobilization (Hammerling et al. J. Exp. Med. (1968) 128: 1461-73). Another example is antibody-targeted thrombolytic therapy. This therapy examines the use of antibodies that transport enzymes such as urokinase, streptokinase, tissue plasminogen activator, prourokinase, and their precursor proteins, in a manner specific to fibrin in thrombi (T. Kurokawa et al. Bio/Technology (1989) 7: 1163; Japanese Patent Application Kokai Publication No. (JP-A (Kokai)) H05-304992 (unexamined, published Japanese patent application)). Furthermore, there have also been reports of using BsAbs as mouse/human-chimeric bispecific antibodies applicable in cancer targeting (JP-A (Kokai) H02-145187), and in cancer therapy and diagnosis for various tumors (see for example, JP-A (Kokai) H05-213775; JP-A (Kokai) H10-165184; JP-A (Kokai) H11-71288; JP-A (Kohyo) 2002-518041; JP-A (Kohyo) H11-506310; Link et al. Blood (1993) 81: 3343; T. Nitta et al. Lancet (1990) 335: 368-71; L. deLeij et al. Foundation Nationale de Transfusion Sanguine, Les Ulis France (1990) 249-53; Le Doussal et al. J. Nucl. Med. (1993) 34: 1662-71; Stickney et al. Cancer Res. (1991) 51: 6650-5), mycotic therapy (JP-A (Kokai) H05-199894), immune response induction (JP-A (Kohyo) H10-511085; Weiner et al. Cancer Res. (1993) 53: 94-100), induction of killer T-cell function (Kroesen et al. Br. J. Cancer (1994) 70: 652-61; Weiner et al. J. Immunol. (1994) 152: 2385), immunoanalysis (M. R. Suresh et al. Proc. Natl. Acad. Sci. USA (1986) 83: 7989-93; JP-A (Kokai) H05-184383), immunohistochemistry (C. Milstein and A. C. Cuello Nature (1983) 305:537), and such.
Specific antibodies for a certain antigen can be produced via genetic engineering, by obtaining the nucleotide sequences of the H and L chain variable regions which determine the antigen specificity of antibodies (J. Xiang et al. Mol. Immunol. (1990) 27: 809; C. R. Bebbington et al. Bio/Technology (1992) 10: 169). Methods for obtaining antigen-specific H and L chains include methods that utilize phages or phagemids using E. coli as the host (W. D. Huse et al. Science (1989) 246: 1275; J. McCafferty et al. Nature (1990) 348: 552; A. S. Kang et al. Proc. Natl. Acad. Sci. USA (1991) 88: 4363). In these methods, antibody libraries are constructed by generating Fabs, or by generating fusion proteins between a phage coat protein and Fab or a single-strand Fv. Finally, antigenic affinity is examined to select antigen-specific antibodies and their genes from these antibody libraries.