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  Process for producing humanized chimera antibodyUSPTO Application #: 20060058512Title: Process for producing humanized chimera antibody Abstract: A humanized chimera antibody, a pharmaceutical composition comprising a humanized chimera antibody and a pharmaceutically acceptable carrier, and a method of treating cancer which comprises administering to a patient a pharmaceutically acceptable amount of the humanized chimera antibody, are disclosed. (end of abstract)
Agent: Nixon & Vanderhye, PC - Arlington, VA, US Inventors: Kenya Shitara, Nobuo Hanai, Mamoru Hasegawa, Hiromasa Miyaji, Yoshihisa Kuwana USPTO Applicaton #: 20060058512 - Class: 530387300 (USPTO) Related Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Proteins, I.e., More Than 100 Amino Acid Residues, Blood Proteins Or Globulins, E.g., Proteoglycans, Platelet Factor 4, Thyroglobulin, Thyroxine, Etc., Globulins, Immunoglobulin, Antibody, Or Fragment Thereof, Other Than Immunoglobulin Antibody, Or Fragment Thereof That Is Conjugated Or Absorbed, Chimeric, Mutated, Or Recombined Hybrid (e.g., Bifunctional, Bispecific, Rodent-human Chimeric, Single Chain, Rfv, Immunoglobulin Fusion Protein, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20060058512. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present application is a divisional of application Ser. No. 10/166,626, filed Jun. 12, 2002 (allowed), which was a divisional of application Ser. No. 09/225,322, filed Jan. 5, 1999 (now U.S. Pat. No. 6,437,098), which was a divisional of application Ser. No. 08/454,680, filed May 31, 1995 (now U.S. Pat. No. 5,866,692), which was a divisional of application Ser. No. 08/408,133, filed Mar. 21, 1995 (now U.S. Pat. No. 5,750,078), which was a continuation of application Ser. No. 08/292,178, filed Aug. 17, 1994 (abandoned), which was a continuation of application Ser. No. 07/947,674, filed Sep. 17, 1992 (abandoned), which claims benefit of Japan Hei 3-238375, filed Sep. 18, 1991, the entire contents of each of which is hereby incorporated by reference. FIELD OF THE INVENTION [0002] This invention relates to a process for the production of humanized chimera antibody. In contrast to mouse monoclonal antibody, humanized chimera antibody does not cause formation of anti-mouse immunoglobulin antibody in the body of a patient. Thus, side effects are reduced or eliminated and half life in blood increases when the chimera antibody is used. Therapeutic effects which are superior to those obtained in the case of using mouse monoclonal antibody can be obtained in the treatment of human cancers and the like. BACKGROUND OF THE INVENTION [0003] It is known that, when mouse antibodies are administered to humans, they are recognized as foreign substances and cause formation of anti-mouse immunoglobulin antibodies in the human body, and the thus formed antibodies react with the administered mouse antibodies. As a result, side effects occur (J. Clin. Oncol., 2, 881 (1984); Blood, 65, 1349-1363 (1985); J. Natl. Cancer Inst., 80, 932 (1988); Proc. Natl. Acad. Sci. U.S.A., 82, 1242 (1985)), the antibodies are cleared away quickly (J. Nucl. Med., 26, 1011 (1985); Blood, 65, 1349-1363 (1985); J. Natl. Cancer Inst., 80, 937 (1988)) and effects of the antibodies are reduced (J. Immunol., 135, 1530 (1985); Cancer Res., 46, 6489 (1986)). When mouse monoclonal antibody is converted into humanized chimera antibody, human anti-mouse, immunoglobulin antibody form in minimal amounts if at all, and the half life of the chimera antibody in human blood is six times as long as that of mouse monoclonal antibody (Proc. Natl. Acad. Sci. U.S.A., 86, 4220 (1989)). In addition, it is probable that the Fc region of mouse antibody does not fully activate human complement and human effector cells, i comparison with the Fc region of human antibody. For example, the antitumor activity of mouse monoclonal antibody to ganglioside GD.sub.2, which is effected via human effector cells, is improved when the monoclonal antibody is converted into chimera antibody that has the human antibody Fc region (J. Immunol., 144, 1382-1386 (1990)). [0004] Ganglioside is one of the animal cell membrane-constituting glycolipids and is composed of a sugar chain as a hydrophilic side chain, sphingosine as a hydrophobic side chain and fatty acids. It is known that expression of ganglioside varies depending on the type of cells, organs and animal species. In addition, it has been revealed that quantity and quality of the expressed ganglioside change during the canceration process of cells (Cancer Res., 45, 2405 (1985)). For example, it has been reported that gangliosides GD.sub.2, GD.sub.3, GM.sub.2 and the like which hardly exist in normal cells were found in the cells of neuroblastoma, lung small cell carcinoma and melanoma belonging to neuroectodermal-origin tumor which is said to be highly malignant (J. Exp. Med., 155, 1133 (1982); J. Biol. Chem., 257, 12752 (1982); Cancer Res., 47, 225 (1987); ibid., 47, 1098 (1987); ibid., 45, 2542 (1985); Proc. Natl. Acad. Sci. U.S.A., 80, 5392 (1983)). [0005] Ganglioside GD.sub.3 has been found most frequently in melanoma cells among the neuroectodermal-origin tumors, and anti-ganglioside GD.sub.3 monoclonal antibodies (to be referred to as "anti-GD.sub.3 monoclonal antibody" hereinafter) belonging to the mouse IgM class and IgG class have been reported (Int. J. Cancer, 29, 269 (1982); J. Biol. Chem., 257, 12752 (1982); Cancer Res., 47, 225 (1987); Acta Neuropathol., 79, 317 (1989); Proc. Natl. Acad. Sci. U.S.A., 77, 6114 (1980); J. Exp. Med., 155, 1133 (1982); Proc. Natl. Acad. Sci. U.S.A., 81, 5767 (1984)). [0006] KM-641 (FERM BP-3116) disclosed in EP-A-0 493 686 is an anti-GD.sub.3 monoclonal antibody belonging to the mouse IgG3 class, which reacts not only with ganglioside GD.sub.3 but also with ganglioside 3',8'-LD1 and is possessed of a broad range of antitumor spectrum. In addition, KM-641 has stronger binding activities to antigens than anti-GD.sub.3 monoclonal antibody R24 which has been disclosed in J. Exp. Med., 155, 1133 (1982) and it shows strong antitumor activities. [0007] The mouse monoclonal antibody R24 to the ganglioside GD.sub.3 was once used for the treatment of melanoma, but the administered mouse monoclonal antibody R24 did not fully exert its effect due to the formation of anti-mouse immunoglobulin antibody in the patient's body (Eur. J. Cancer Clin. Oncol., 24, suppl 2, s 65 (1988)). [0008] Consequently, the use of chimera antibody for anti-GD.sub.3 monoclonal antibody would be advantageous in that anti-mouse immunoglobulin antibody does not form in the body, side effects are reduced or eliminated, its half life in blood is prolonged and its antitumor effector effect increases, and thus therapeutic effects of the chimera antibody which are superior to those of mouse monoclonal antibody can be obtained in the treatment of human cancers and the like. [0009] Several processes for the production of humanized chimera antibodies are known. Humanized chimera antibody, in which constant regions of the heavy chain (to be referred to as "H chain" hereinafter) and the light chain (to be referred to as "L" chain hereinafter) of mouse monoclonal antibody are converted into human constant regions, is produced in animal cells making use of recombinant DNA techniques. Examples of such processes include a process in which humanized chimera antibody is produced using chromosomal DNA as a gene which encodes mouse H chain variable region (to be referred to as "V.sub.H" hereinafter) and L chain variable region (to be referred to as "V.sub.L" hereinafter) (Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81, 6851 (1984); Neuberger et al., Nature, 314, 268 (1985); Nishimura et al., Cancer Res., 47, 999 (1987); Dorai et al., J. Immunol., 139, 4232 (1987); Kameyama et al., FEBS letter, 244, 301 (1989)) and another process in which humanized chimera antibody is produced using cDNA (Gillies et al., J. Immunol. Methods, 125, 191 (1989); Liu et al., published International Application in Japan No. 2-501886). Cloning and base sequence determination of hybridoma cell chromosomal DNA which encodes mouse V.sub.H and V.sub.L require much time and labor in comparison with those of cDNA that encodes mouse V.sub.H and V.sub.L. Consequently, the process in which cDNA is used for the production of humanized chimera antibody is more desirable than the chromosomal DNA process. [0010] Gillies et al. have succeeded in expressing humanized chimera antibody in animal cells, making use of an expression vector for animal cells having inserted therein a humanized chimera H chain gene obtained by linking mouse V.sub.H-encoding cDNA with human C.sub.H-encoding chromosomal DNA, and a humanized chimera L chain gene obtained by linking mouse V.sub.L-encoding cDNA with human C.sub.L-encoding chromosomal DNA (J. Immunol. Methods, 125, 191 (1989)). However, when an attempt was made to prepare chimera antibodies from several types of antibodies, a problem was found that there were certain chimera antibodies whose L chains could not be expressed without converting leader sequences. In addition, humanized chimera antibody can be produced more simply when cDNA which encodes human C.sub.H and C.sub.L is used instead of the human C.sub.H- and C.sub.L-encoding chromosomal DNA. [0011] In published International Application in Japan No. 2-501886, Liu et al. discloses a process for the expression of humanized chimera antibody in animal cells, which comprises using an expression vector for animal cells having inserted therein a chimera H chain cDNA obtained by linking mouse V.sub.H-encoding cDNA with human C.sub.H-encoding cDNA and a chimera L chain cDNA obtained by linking mouse V.sub.L-encoding cDNA with human C.sub.L-encoding cDNA. According to this process, however, it is necessary to alter the J.sub.H portion of the V.sub.H-encoding cDNA and the J.sub.L portion of the V.sub.L-encoding cDNA by means of mutation, because the cDNA which encodes mouse V.sub.H or V.sub.L is linked with the human C.sub.H- or C.sub.L-encoding cDNA at the J region in the mouse variable region. In addition, with regard to the chimera L chain prepared using mouse Jk5, leucine which is one of the amino acids of the framework 4 is changed to isoleucine when made into humanized chimera antibody. Although amino acid sequence of complementarity-determining region (to be referred to as "CDR" hereinafter) is especially important for antigen-antibody binding, the amino acid sequence of the framework is also an important factor. For example, Riechmann et al. have prepared CDR graft antibody by grafting a rat antibody CDR into a human antibody framework and reported that binding activity of the antibody was reduced by the framework conversion and the antibody activity increased when amino acid sequence of the framework was partially changed (Nature, 333, 323 (1983)). Consequently, there is a possibility that the binding activity of humanized chimera antibody is undesirably reduced when the antibody is produced by the mouse Jk5-aided process disclosed by Liu et al. [0012] In view of the above, when any mouse antibody is converted into humanized chimera antibody, it has been desired to simply and easily produce humanized chimera antibody in which amino acids of the mouse antibody variable region remain completely unchanged. OBJECTS OF THE INVENTION [0013] An object of the present invention is to provide a process for the production of humanized chimera antibody by which the chimera antibody is produced easily without changing any of the amino acids of its mouse antibody variable region. Another object of the present invention is to provide a humanized chimera antibody to ganglioside GD.sub.3 and a process for the production of such antibody. SUMMARY OF THE INVENTION [0014] The present invention relates to a process for producing humanized chimera antibody which comprises the steps of: [0015] (1) constructing a cassette vector by inserting a cDNA coding for human antibody C.sub.H into an expression vector for animal cell use and establishing a cloning site in the upstream region C.sub.H of said cassette vector for inserting a cDNA which encodes nonhuman animal V.sub.H; [0016] (2) digesting a cDNA coding for nonhuman animal antibody V.sub.H with restriction enzymes; [0017] (3) inserting said cDNA coding for nonhuman animal antibody V.sub.H into the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5'-end side of said human antibody C.sub.H and a base sequence corresponding to the 3'-end side of said nonhuman animal antibody V.sub.H and is possessed of restriction enzyme recognition sites on both of its ends, thereby constructing a humanized chimera antibody H chain expression vector in which said cDNA coding for human antibody C.sub.H and said cDNA coding for nonhuman animal antibody V.sub.H are linked together through said synthetic DNA; [0018] (4) constructing a cassette vector by inserting a cDNA coding for human antibody C.sub.L into an expression vector for animal cell use and establishing a cloning site in the upstream region of the C.sub.L of said cassette vector for inserting a cDNA which encodes nonhuman animal antibody V.sub.L; [0019] (5) digesting a cDNA coding for nonhuman animal antibody V.sub.L with restriction enzymes; [0020] (6) inserting said cDNA coding for nonhuman animal antibody V.sub.L into the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5'-end side of said human antibody C.sub.L and a base sequence corresponding to the 3'-end side of said nonhuman animal antibody V.sub.L and is possessed of restriction enzyme recognition sites on both of its ends, thereby constructing a humanized chimera antibody L chain expression vector in which said cDNA coding for human antibody C.sub.L and said cDNA coding for nonhuman animal antibody V.sub.L are linked together through said synthetic DNA; [0021] (7) introducing these expression vectors into host cells to obtain a transformant; and [0022] (8) culturing said transformant in an appropriate culture medium, thereby allowing the transformant to produce and accumulate a humanized chimera antibody, and collecting said humanized chimera antibody from the resulting culture broth. [0023] The cassette vector to be used in the present invention is a vector which is obtained by inserting a cDNA that encodes a constant region of human antibody into an expression vector for animal cell use, in which a cloning site is located in the upstream region of the constant region for inserting a cDNA that encodes a variable region of nonhuman animal antibody. An expression vector for humanized chimera antibody can be constructed easily by inserting a variable region of nonhuman animal antibody into the cloning site of the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5'-end side of a constant region of human antibody and a base sequence corresponding to the 3'-end side of a variable region of nonhuman animal antibody and is possessed of restriction enzyme recognition sites on its both ends. [0024] The present invention also relates to a humanized chimera antibody obtained by the above-described process, a pharmaceutical composition comprising the humanized chimera antibody and a pharmaceutically acceptable carrier, and a method of treating cancer which comprises administering to a patient a pharmaceutically acceptable amount of said humanized chimera antibody. BRIEF DESCRIPTION OF THE DRAWINGS [0025] FIG. 1 shows a restriction enzyme cleavage map of a 9.3 kb XbaI fragment of KM50 cell chromosomal DNA [0026] FIG. 2 shows a construction scheme for plasmid pKMB11. [0027] FIG. 3 shows a construction scheme for plasmid pKMD6. Continue reading... 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