MXPA99010364A - Cachexia remedy - Google Patents

Abstract

A cachexia remedy containing an active ingredient comprising a substance inhibiting the binding of a parathyroid hormone-related peptide and a receptor thereof. Examples of the inhibiting substance include antagonists against parathyroid hormone-related peptide receptors, parathyroid hormone-related peptide antibodies, antibody fragments, and modified antibodies.

Description

and intensive about the discovery of said therapeutic agent. As a result, they found that the development of a substance that can inhibit the binding between PTHrP and a receptor thereof could achieve that object. This discovery leads to the fulfillment of the invention. That is, the present invention relates to a therapeutic agent for cachexia comprising, as an active ingredient, a substance capable of inhibiting the binding between PTHrP and a receptor "thereof" In the present invention, the term "cachexia" "encompasses cancer-induced cachexias." The present invention relates to a therapeutic agent for "cachexia comprising, as an active ingredient, a XX-* - substance capable of inhibiting the link between the protein - ''? related to parathyroid hormone (hereinafter referred to as "PTHrP") and a receptor thereof (hereinafter referred to as "PTHrP receptor"). As used herein, the term "PTHrP receptor" refers to any receptor that binds with PTHrP (such as > * those described ^ in Japanese national phase open publication number 6-506598), regardless of whether the PTHrP receptor is present in a target organ (bone, kidney) or not. As used herein, the term "a substance capable of inhibiting the binding between PTHrP and a receptor thereof (a PTHrP receptor)" refers to any substance that can bind PTHrP to prevent binding of PTHrP to a PTHrP receptor. PTHrP, such as for example an anti-PTHrP antibody; any substance that can bind on a PTHrP receptor to prevent binding of the PTHrP receptor on PTHrP, such as an antagonist against a PTHrP receptor (a PTHrP antagonist), specifically a peptide having a replacement or removal of minus an amino acid residue in the PTHrP peptide or a partial sequence of "PTHrP peptide; or a combination thereof. The anti-PTHrP antibody includes antibodies of known types, such as, for example, humanized antibody, a human antibody (W096 / 33735) or a chimeric antibody (Japanese open patent application number 4-228089), and the exemplary antibody used in the present invention (antibody number 23-57-137-1). The antibody can be of the polyclonal or monoclonal type, but is preferably of the monoclonal type. The PTHrP antagonist includes a polypeptide or a substance of low molecular weight. The PTHrP antagonist includes a substance that binds to a PTHrP receptor in an antagonistic manner against PTHrP, such as a polypeptide having an antagonistic activity against PTHrP as described in Japanese Laid Open Patent Application No. 7-165790; peptides (UNITED STATES OF AMERICA), 1995, 16 (6) 1031-1037; Biochemistry (UNITED STATES OF AMERICA) Apr. 281992, 31 (16) 4026-4033; and Japanese Open National Phase Number 5-509098. These polypeptides can have removal, replacement, addition or insertion of at least one amino acid residue, insofar as they can present an equivalent level of antagonist activity against PTHrP, which are also encompassed in the PTHrP antagonists of the present invention . Next, the present invention will be described in detail by the use of an anti-PTHrP antibody as the "Substance Able to inhibit the binding between PTHrP and a PTHrP receptor." 1. Anti-PTHrP antibody The anti-PTHrP antibody used in the present invention can be any insofar as it can have a therapeutic effect for cachexia, regardless of its source, type, (monoclonal or polyclonal) and configuration. The anti-PTHrP antibody employed in the present invention can be produced by any method known as polyclonal or monoclonal antibody. Preferably, the anti-PTHrP antibody is a monoclonal antibody derived from a mammal. The monoclonal antibody of a mammal includes those produced from a hybridoma and those produced by a genetic engineering technique from a host transformed with a recombinant expression vector carrying a gene for the antibody. The antibody used in the present invention is an antibody that can bind PTHrP in order to prevent the binding of PTHrP to a PTH / PTHrP receptor., thus blocking the signal transduction of PTHrP and thereby inhibiting the biological activity of PTHrP. A specific example of said antibody is antibody number 23-57-137-1 which can be produced with a hybridoma clone number 23-57-137-1. Hybridoma clone number 23-57-137-1 has been designated "mouse-mouse hybridoma number 23-57-137-1" and deposited in accordance with the terms of the Budapest Treaty on August 15, 1996 in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (National Institute of Bioscience and Human Technology, Agency of Science and Technology Industry), Japan (1-3, Higashi 1-chome, Tsu uba-shi, Ibaraki, Japan) under accession number FERM BP-5631. 2. Hybridoma producing antibody Hybridoma producing monoclonal antibody can be produced basically by any known technique. That is, PTHrP is used as an antigen for immunization in accordance with a conventional method of immunization. The resulting immunocytes are fused to know cells of origin by means of a conventional cell fusion method, and cells that produce monoclonal antibodies are screened from the fused cells by means of a conventional screening method. More specifically, the monoclonal antibody production cell can be prepared in the following manner. First, a human PTHrP, which is used as a sensitizing antigen for the production of the antibody, is prepared by expression of the PTHrP / amino acid gene sequence presented in Suva, LJ et al., Science (1987) 237, 893 That is, a nucleotide sequence encoding PTHrP is inserted into any known expression vector, and a suitable host cell is transformed with the expression vector. The PTHrP protein is then isolated and purified from the transformed host cell or from a culture supernatant of the transformed host cell by any known method. Second, the purified PTHrP protein is used as a sensitizing antigen. Alternatively, a peptide of 34 amino acids from the N-terminal region of PTHrP can be used as the sensitizing antigen, which can be chemically synthesized. The mammal immunizing with the sensitizing antigen is not particularly limited. However, the mammal is preferably selected taking into account the compatibility with the cell of origin used for cell fusion.

Generally, a rodent can be used (for example, mouse, rat, hamster, rabbit or monkey). Mammalian immunization with the sensitizing antigen can be carried out in accordance with any known method, for example, by injection of the mammalian sensitization antigen intraperitoneally or subcutaneously. More specifically, the sensitizing antigen is diluted with a phosphate buffered saline (PBS) or normal saline and suspended in said saline, the resulting solution is then mixed with an appropriate amount of an adjuvant (eg, adjuvant). complete Freud) to provide an emulsion. The emulsion is injected into a mammal at intervals of 4 to 21 days. In immunization, the sensitization antigen can be fixed on any suitable vehicle. After immunization, the serum antibody level is checked. When it is confirmed that the level of serum antibodies reaches the desired level, immunocytes are isolated from the mammal which are then subjected to cell fusion. A preferred immunocyte is a spleen cell. The cell of origin used for cell fusion (ie, the counterpart of cell fusion with the immunocyte) is a myeloma cell of a mammal. The myeloma cell is from any known cell line, and, for example P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.l (Current Topics in Microbiology and Immunology (Current Issues in Microbiology and Immunology) (1978) 81, 1-7), NS-1 (Kohler, G. Y Milstein, C. Eur. J. Immunol. (1976) 6. 511-519), MPC-11 (Margulies, DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (from St. Groth S.F. Et al., J. Immunol. (1980) 35, 1-21), S194 (Trowbridge, I.S., J. Exp. Med. (1978) 148, 313-323) or R210 (Galfre, G. et al., Nature (1979) 277, 131-133). The fusion of immunocyte cells with the myeloma cell is carried out basically in accordance with any method known as for example the method of Milstein et al.

(Kohler, G. and Milstein, C, Methods Enzymol, (1981) 73, 3-46) can be used preferably. More specifically, a cell fusion is carried out, for example, in a conventional nutrient culture medium in the presence of a cell fusion promoter. The cell fusion promoter can be propylene glycol (PEG) or a Sendai virus (Japan agglutination virus MHVJ). If desired, for the purpose of improving the melting efficiency, an additive such as dimethyl sulfoxide may also be incorporated. The ratio between the immunocytes and the myeloma cells for cell fusion can be any. For example, immunocytes are used in an amount 1-10 times higher than myeloma cells. The culture medium used for cell fusion is, for example, an RPMl 1640 medium or a MEM medium suitable for the growth of the myeloma cell line, or another medium conventionally used for the culture of said cells. If desired, a serum supplement, such as for example fetal calf serum (FCS) can be added to the culture medium. Cell fusion is carried out, with mixing of the immunocytes and the myeloma cells of given amounts in the culture medium, adding a PEG solution (eg, average molecular weight: about 1000-6000) (previously heated to a temperature of about 37 ° C), usually at a concentration of 30-60% (w / v) and then mixing the resulting solution, thus obtaining fusion cells (hybridomas) subsequently, an appropriate culture medium is added to the solution of culture, and centrifuged in order to remove the supernatant. This procedure is repeated several times in order to remove the cell fusion promoter or the like which are undesirable for the growth of the hybridomas from the culture medium. The obtained hybridomas can be selected by culture in a conventional selection medium, such as a hypoxactin-aminopterin-thymidine (HAT) medium.

The culture of the hybridomas in a HAT medium is carried out for a period of time sufficient to cause the death of the cells other than the desired hybridomas. (ie, cells that do not fuse), usually for several days to several weeks. Subsequently, a conventional limitation dilution method is carried out in order to screen and monoclonal the hybridomas that are secreting the desired antibody. Alternatively, a human antibody having a binding activity against PTHrP can be prepared by sensitizing a human lymphocyte with PTHrP in vitro, and then subjecting the sensitized lymphocyte to cell fusion on a human derived myeloma cell capable of infinite growth (publication Japanese Patent No. 1-59878). Alternatively, a human antibody against PTHrP can be prepared by injecting PTHrP as an antigen to a transgenic animal having the full repertoires of the human antibody genes to provide a cell that produces anti-PTHrP antibody, and immortalizing the cells, such as so that the human antibody can be produced from immortalized cells (international publications number W094 / 25585, WO 93/12227, WO 92/03918 and WO 94/02602). The hybridoma producing monoclonal antibody prepared according to the aforementioned can be subcultured in a conventional culture medium and stored under liquid nitrogen for a long period of time. For the production of a monoclonal antibody from the hybridoma, a method that includes the hybridoma culture method according to a conventional method and the collection of the monoclonal antibody from the culture supernatant, or a method that includes the injection of the hybridoma to a mammal compatible with the hybridoma for the growth of the hybridoma in the body of the mammal and the collection of the hybridoma from the ascites of the mammal can be used. The above method is suitable for the production of high purity antibody, while the latter method is suitable for the production of antibody in large quantities. 3. Recombinant antibody In the present invention, a monoclonal antibody of recombinant type can also be used, which can be produced by cloning an antibody gene from the hybridoma by integrating the antibody gene into a suitable vector, introducing the vector into a host, and producing the antibody from the host in accordance with a conventional genetic recombination technique (see, for example, Vandamme, A.M. et al., Eur. J. Biochem. (1990) 192, 767-775). More specifically, an mRNA encoding a variable region (V) of an anti-PTHrP antibody is isolated from the hybridoma that produces anti-PTHrP antibody. The isolation of the mRNA is carried out by the preparation of total RNA by any method known as for example, guanidium ultracentrifugation method (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299) and AGPC method (Chomczynski , P. et al., Anal. Biochem. (1987) 162, 156-159), and then producing the desired mRNA from the total RNA using a Set of elements for mRNA Purification (Pharmacia) or the like. Alternatively, the mRNA can also be prepared directly using the QuickPrep mRNA Purification Kit (Pharmacia). Next, the cDNA for the antibody V region is synthesized from mRNA with reverse transfectase. The synthesis of the cDNA is carried out using a set of elements for synthesis of first-strand cDNA of reverse transcriptase AMV (Seikagaku Corporation) or similar. The cDNA can also be synthesized or amplified by means of the 5 '-RACE method (Frohman, MA et al., Proc. Nati. Acad. Sci, USA (1988) 85, 8998-9002; Belyavsky, A. Et al., Nucleic Acids Res. (1989) 17, 2919-2932) 5'- Amplifier FINDER RACE (Clontech) in combination with a chain reaction method. of polymerase, or similar. A DNA fragment of interest is isolated, which is purified from the resulting polymerase chain reaction product and then ligated onto a vector DNA to provide a recombinant vector. The recombinant vector is introduced into a host cell such as E. coli. and a colony containing a desired recombinant vector is selected. The nucleotide sequence of the DNA of interest in the recombinant vector is confirmed by, for example, the dideoxynucleotide chain termination method. Once the DNA encoding the V region of anti-PTHrP antibody is obtained, the DNA is integrated into an expression vector containing DNA encoding the constant region (C) of the antibody. For the production of the anti-PTHrP antibody, employed in the present invention, the antibody gene is integrated into an expression vector such that the antibody gene can be expressed under the control of expression control regions (e.g., enhancer , promoter). A host cell is transformed with the expression vector in order to express the antibody. In the expression of the antibody gene, the DNA encoding a heavy chain (H) and the DNA encoding a light (L) chain of the antibody can be integrated into several separate expression vectors, and then a host cell is cotransformed with the resulting recombinant expression vectors. Alternatively, both the DNA encoding a heavy chain and the DNA encoding an antibody light chain can be integrated together into a "unique" expression vector, and then a host cell is transformed with the resulting recombinant expression vector (W094 / 11523 In the production of the recombinant antibody, in addition to the aforementioned host cells, a transgenic animal can also be used as a host.For example, the antibody gene is inserted into a predetermined site of an encoding gene of an inherently produced protein. in the milk of an animal (eg, goat beta-casein) in order to provide a fusion gene A DNA fragment containing the fusion gene introduced into the antibody gene is injected into a goat embryo, and the embryo is then introduced into a goat.The goat that has the embryo carries a transgenic goat.The antibody of interest is secreted in the milk of the transgenic goat or a baby of it. For the purpose of increasing the amount of milk containing antibody, an appropriate hormone can be administered to the transgenic goat (Ebert, K. M. et al., Bio / Technology (1994) 12, 699-702). 4. Modified Antibody In the present invention, for the purpose of reducing heterogeneity against a similar human body, an artificially modified recombinant antibody, including a chimeric antibody and a humanized antibody, can be employed. These modified antibodies can be prepared by any known method. A chimeric antibody that can be employed in the present invention can be prepared by ligating the DNA encoding the V region of antibody prepared according to the aforementioned with DNA encoding a human antibody C region by integrating the binding product into a vector of expression, and introducing the resulting recombinant expression vector into a host for the purpose of producing the chimeric antibody. A humanized antibody is also known as a "human reformed antibody", wherein the regions of complementarity determination (CDRs) of an antibody of a non-human mammal (eg, a mouse) are grafted onto the regions of a human antibody. The general genetic recombination procedure for the production of said humanized antibodies is also known (EP 125023; WO 96/02576). Specifically, a DNA sequence in which CDRs of mouse antibody are ligated through framework regions (FRs) is designed, and synthesized with a polymerase chain reaction method employing several oligonucleotides as primers that were designed to have regions that are connected to the terminal regions of CDRs and FRs. The resulting DNA is ligated onto the DNA encoding the human antibody C region, and the ligand product is integrated into an expression vector. The resulting recombinant expression vector is introduced into a host, thereby producing the humanized antibody (EP 239044, WO 96/02576). The FRs linked through the CDRs are selected such that the CDRs can form a satisfactory antigen binding site. If necessary, amino acid (s) in the FRs of the antibody V region can be replaced in such a way that the CDRs of the human reformed antibody can form an appropriate antigen binding site (Sato, K. Et al., Cancer Res. (1993) 53, 851-856). The C region of the chimeric or humanized antibody can be any C region of human antibody; such as Cganima1, Cgamma2, Cgamma3 or Cgamma4 for the H chain, and Ccappa or Clambda for the L chain. The C region of human antibody can be modified with the purpose of improving the stability of the antibody or ensuring stable production of the antibody. The chimeric antibody is composed of V regions derived from a non-human mammalian antibody and C regions derived from a human antibody. The humanized antibody consists of CDRs derived from a non-human mammalian antibody and FRs and C regions derived from a human antibody. The humanized antibody is especially useful as an active ingredient for the therapeutic agent of the present invention, since the antigenicity of the antibody against a human body is reduced. A specific example of the humanized antibody employed in the present invention is humanized antibody number 23-57-137-1, wherein the CDRs are derived from mouse antibody number 23-57-137-1; and the L chain is composed of the CDRs linked through 3-FRs (FR1, FR2, and FR3) derived from the human antibody HSU 03868 (GEN-BANK, Deftos, M. et al., Scand. J. Immunol., 39, 95-103, 1994) and an FR (RF4) derived from the human antibody S25755 (NBRF-PDB); and the H chain is composed of the CDRs linked through the FRs derived from the human antibody S31679 (NBRF-PDB, Cuisinier, AM et al., Eur. J. Immunol., 23, 110-118, 1993) wherein a part of the amino acid residues in the FRs is replaced in such a way that the reformed humanized antibody can present an antigen binding activity. The E. coli strains containing the plasmids having the DNA encoding the H chain and the L chain of the humanized antibody number 23-57-137-1, respectively, are designated Escherichia coli JM109 (hMBClHcDNA / pUC19) (for the H chain) and Escherichia coli JM109 (jMBClLqlambda / pUC19) (for the L chain), respectively. These strains have been deposited in accordance with the terms of the Budapest Treaty on August 15, 1996 at the National Institute of Bioscience and Human-technology, Agency of Industrial Science and Technology, Japan (1-3, Higashi 1-chome, Tsukuba- shi, Ibaraki, Japan), under accession number FERM BP-5629 for Escherichia coli JM109 (hMBClHcDNA / pUC19), and under accession number FERM BP-5630 for Escherichia coli JM109 (hMBClLqlambda / pUC19). 5. Antibody Variants The antibody employed in the present invention can be any fragment thereof or a modified product of the fragment, to the extent that it can bind on PTHrP and inhibit the activity of PTHrP. For example, the antibody fragment includes Fab, F (ab ') 2, Fv, or a single Fv chain (scFv) composed of an H chain Fv fragment or an L chain Fv fragment linked through a suitable linker . Specifically, such antibody fragments can be produced by dissociating the antibody with an enzyme (eg, papain, pepsin) in antibody fragments, either by constructing a gene encoding the antibody fragment and by inserting the gene into the antibody. an expression vector and by introducing the resulting recombinant expression vector into a suitable host cell, thereby obtaining the expression of the antibody fragment (see, for example, Co, MS, et al., J. Immunol. (1994), 152, 2968-2976; Better, M. & Horwitz, A.H., Methods in Enzymology (1989), 178, 476-496, Academic Press Inc .; Plueckthun, A. & Skerra, A., Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc .; La oyi, E., Methods in _Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669; and Bird, R. E. Et al., TIBTECH (1991) 9, 132-137). A scFv can be produced by joining the V region of H chain to the V region of L chain via a linker, preferably a peptide linker (Huston, JS et al., Proc. Nati, Acad. Sci. USA (1988) 85, 5879-5883). The H chain V region and the L chain V region in scFv can be derived from any of the antibodies described herein. The peptide linker linking the V regions can be any single chain peptide, for example, 12-19 amino acid residues. DNA encoding scFv can be prepared by first amplifying DNA encoding the H chain V region and DNA encoding the L chain V region of the antibody separately by using a DNA fragment encoding the entire region of the H chain or a portion thereof that includes the V region of a DNA fragment that encodes the entire region of the L chain or a portion thereof that includes the V region as temperate and primer pairs that define the terminal ends of the fragments of DNA; and then amplifying the DNA encoding the peptide linker using a DNA fragment encoding the peptide linker as annealed and a pair of primers that define the terminal ends of the DNA fragment such that each terminal end of the linker of peptide is linked to the V region of H chain and V region of L chain, respectively. Once the DNA encoding scFv is prepared, an expression vector carrying the DNA and a host transformed with the expression vector can be prepared by conventional methods. The scFv can be produced from the transformed host in any conventional method. The antibody fragments employed in the present invention can be produced by preparing genes for the fragments and expressing the genes in suitable hosts in accordance with that described above. These antibody fragments are also encompassed in the "Antibody" of the present invention. As a modified form of the aforementioned antibodies, for example, an anti-PTHrP antibody conjugated with any molecule can also be used (polyethylene glycol). Said modified antibodies are also encompassed in the "antibody" of the present invention. The modified antibodies can be prepared by chemical modifications of the antibodies. The technique of chemical modification suitable for this purpose have already been established in the art. 6. Expression and production of recombinant antibody or modified antibody The antibody gene constructed in accordance with that described above can be produced and expressed by known methods. For expression in a mammalian cell, a conventionally useful promoter, the antibody gene can be expressed in a poly (A) signal (located downstream of the 3 'end of the antibody gene) are operably linked. For example, as a useful promoter / enhancer system, an initial human cytomegalovirus / enhancer immediate initial promoter system may be employed. Other promoter / enhancer systems, for example, virus derivatives (e.g., retroviruses, polyoma viruses, adenoviruses and simian viruses 40 (SV40)) and mammalian cell derivatives (e.g. human elongation factor 1 alpha ( HEFlalfa)), can also be used for the expression of the antibody in the present invention. When it is used an SV40 promoter / enhancer system, gene expression can be easily carried out by the method of Mulligan et al. (Nature (1979) 277, 108). When the promoter system of HEFlalfa / enhancer is used, gene expression can be easily performed by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5422). For E. coli expression, a conventional useful promoter, a signal sequence for the secretion of the antibody of interest and the antibody gene can be linked in an operable manner. As a promoter, a lacZ promoter or an araB promoter can be employed. When the lacZ promoter is employed, gene expression can be carried out through the method of Ward et al (Nature (1098) 341, 544-546; FASBE J. (1992) 6, 2422-2427), whereas when an araB promoter is used, the gene expression can be carried out through the method of Better et al. (Better et al., Science (1988) 240, 1041-1043). As regards the sequence of signals for the secretion of the antibody, when the antibody of interest must be secreted in a periplasmic space of E. Coli, a pelB signal sequence can be used (Lei, SP et al., J. Bacteriol. (1987) 169, 4379). The antibody secreted in the periplasmic space is isolated and then redoubled in such a way that the antibody takes an appropriate configuration. The origin of replication derived from viruses (for example, SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV)) or the like can be used. In order to increase the number of gene copies in the host cell system, the expression vector may also contain a selective marker gene, such as an aminoglycoside phosphotransferase (APH) gene, a thymidine kinase gene * (TK). ), a xanthin-guanin phosphoribosyltransferase gene from E. Coli (Ecogpt) and a dihydrofolate reductase (dhfr) gene. For the production of the antibody employed in the present invention, any expression system can be employed, including eukaryotic cell systems and prokaryotic cells. The eukaryotic cell includes established cell lines of animals (e.g., mammals, insects, molds and fungi, yeast). The prokaryotic cell includes bacterial cells such as E. coli cells. It is preferred that the antibody employed in the present invention be expressed in a mammalian cell, such as CHO, Cos, myeloma, BHK, Vero cell and HeLa. The transformed host cell is then cultured in vitro or in vivo in order to produce the antibody of interest. The culture of the host cell can be carried out by any known method. The culture medium that can be used here can be the DMEM medium, MEM, RPMl 1640 or IMDM. The culture medium may contain a serum supplement, such as for example fetal calf serum (FCS). 7. Isolation and Purification of Antibody The antibody expressed and produced in accordance with that described above can be isolated from the cells or from the host animal body and purified until uniformity is obtained. The isolation and purification of the antibody employed in the present invention can be carried out on an affinity column. Examples of "A" protein column include Hyper D, POROS and Sepharose F.F. (Pharmacia) Other methods conventionally used for the isolation and purification of an antibody can also be used; therefore the method is not particularly limited. For example, several chromatographs employing columns including the aforementioned affinity column, filtration, ultrafiltration, salt removal and dialysis, may be employed singly or in combination in order to isolate and purify the antibody of interest (Antibodies A Laboratory Manual, Ed. Harlow, David, Lane, Cold Spring Harbor Laboratory, 1988) (antibodies, a laboratory manual). 8. Determination of antibody activities Determination of antibody binding activity (Antibodies A Laboratory Manual, Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988) or inhibitory activity against a ligand receptor (Harada, A. et al., International Immunology (1993) 5, 681-690) of the antibody employed in the present invention can be carried out by any known method. As a method for determining the antigen binding activity of the anti-PTHrP antibody used in the present invention, it is possible to use, for example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody technique. For example, when an enzyme immunoassay is employed, a sample solution containing the anti-PTHrP antibody (for example, a culture supernatant of cells that produce anti-PTHrP antibodies, or the anti-PTHrP antibody per in purified form) to a plate in which it was previously coated with PTHrP (1-34). A secondary antibody labeled with an enzyme (eg, alkaline phosphatase) is also added to the plate. The plate is incubated and washed. A substrate for the enzyme (eg, p-nitrophenylphosphoric acid) is added to the plate, and the absorbance of the solution in the plate is measured in order to evaluate the antigen binding activity of the antibody. To confirm the activity of the antibody employed in the present invention, an antibody neutralizing activity (e.g., anti-PTHrP antibody) is determined. 9. Routes of administration and pharmaceutical preparations The therapeutic agent of the present invention can be used for the treatment or improvement of cachexia. The cachexia to be treated or improved by the present invention can be of any type, including cancer-induced type. Examples of cancer-induced cachexia include those described in J. Urol. (UNITED STATES OF AMERICA) Mar 1995, 153 (3 Pt 1) p.854-857; Langenbecks Arch. Chir. Suppl II Verh Dtsch Ges Chir (GERMANY) 1990, pages 261-265; Oncology (SWITZERLAND) 1990, 47 (1), pages 87-91; Int. J. Pancreatol. (UNITED STATES OF AMERICA) Aug-Nov 1990, 7 (1-3) pages 141-150; J. Nati. Cancer Inst. (UNITED STATES OF AMERICA) Dec. 19, 1990, 82 (24) pages 1922-1926. Examples of cachexia other than cancer-induced cachexia include the cachexia described in JPEN J. Parenter. Enteral Nutr. (UNITED STATES OF AMERICA) Nov-Dec 1990, 14 (6) pages 605-609; Chest (UNITED STATES OF AMERICA) Nov 1990, 98 (5) pages 1091-1094; Bone Marrow Transplant (ENGLAND) July 1990, 6 (1) pages 53-57. The therapeutic agent comprising the anti-PTHrP antibody as the active ingredient of the present invention can be administered orally or parenterally, but preferably parenterally. The therapeutic agent can be in any dosage form, such as for example a transpulmonary agent (e.g., an agent administered with the aid of a device such as a nebulizer), a nasogastric agent, a transdermal agent (e.g., ointment, cream) or good. an injection. Examples of injection include an intravenous injection (eg drops), an intramuscular injection, an intraperitoneal injection and a subcutaneous injection for systemic or topical administration. The route of administration can be appropriately selected according to the patient's range and the conditions of the diseases. An effective single dose can be selected within a range of 0.001 to 1, 000 mg per kg of body weight. Alternatively, the dose to a patient may be selected within a range of 0.01 to 100,000 mg / kg of body weight. However, the dose of the therapeutic agent it forms, the anti-PTHrP antibody of the present invention is not specifically limited to the aforementioned ranges. The therapeutic agent can be administered to a patient at any stage, including before or after the development of cachexia. Alternatively, the therapeutic agent can be administered at the stage at which the development of weight loss in the patient is predicted. The therapeutic agent comprising the anti-PTHrP antibody as the active ingredient of the present invention can be formulated by any conventional method (Remington's Pharmaceutical Science, latest edition, Mark Publishing • Company, Easton, USA). The formulation may further comprise pharmaceutically acceptable carriers and additives. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium arginate, water soluble dextran, sodium carboxymethylstarch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and acceptable surfactants as pharmaceutical additives. In practical use, the additive is appropriately selected from the aforementioned members either unitarily or in combination according to the dosage form used, but is not limited thereto. For example, an injection can be used which is prepared by dissolving the anti-PTHrP antibody in purified form in a solvent (eg, normal saline, regulator, grape sugar solution) and then by adding the an adsorption prevention agent (e.g., Tween 80, Tween 20, gelatin, human serum albumin). The therapeutic agent of the present invention can also be in a reconstitutional, lyophilized form, which is dissolved before use. For the preparation of the lyophilized dosage form, an excipient such as for example sugar alcohol (for example, mannitol, grape sugar) and a sugar can be incorporated. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphic illustration of the therapeutic effect of an anti-PTHrP antibody on cachexia. Figure 2 is a graphic illustration of the therapeutic effect of an anti-PTHrP antibody on cachexia. Figure 3 is a graphic illustration of the therapeutic effect of an anti-PTHrP antibody on cachexia. Figure 4 is a graphic illustration of the therapeutic effect of an anti-PTHrP antibody on cachexia. Figure 5 is a graphic illustration of the measurement of results of the antigen binding activity. Figure 6 is a graphic illustration of the measurement results of the antigen binding activity. Figure 7 is a graphic illustration of the measurement results of the antigen binding activity. Figure 8 is a graphic illustration of the measurement results of the antigen binding activity. Figure 9 is a graphic illustration of the measurement results of the antigen binding activity. Figure 10 is a graphic illustration of the measurement results of the antigen binding activity. Figure 11 is a graphic illustration of the measurement results of the antigen binding activity. Figure 12 is a graphic illustration of the measurement results of the antigen binding activity. Figure 13 is a graphic illustration of the neutralizing activity of a humanized antibody.

Figure 14 is a graphic illustration of the neutralizing activity of a humanized antibody. Figure 15 is a graphic illustration of the neutralizing activity of a humanized antibody. Figure 16 is a graphic illustration of the therapeutic effect of a humanized antibody on cachexia.

Figure 17 is a graphic illustration of the therapeutic effect of a humanized antibody on cachexia.

Figure 18 is a graphic illustration of the therapeutic effect of a humanized antibody on cachexia.

Figure 19 is a graphic illustration of the therapeutic effect of a humanized antibody on cachexia.

PREFERRED MODE OF THE INVENTION Next, the present invention will be described in more detail with reference to the following reference examples and examples that should not be considered as limiting the technical scope of the invention. (EXAMPLE 1) Pharmacological test using an animal model of cachexia Using an animal model of cachexia (a nude mouse that received a human tumor implant), a murine monoclonal antibody against PTHrP was examined to determine its therapeutic effect on cachexia. As an animal model of cachexia, a nude mouse was used that received an implant with OCC-1 human oral cavity carcinoma (acquired at the Central Institute for Experimental Animáis). It is known that a nude mouse receiving an implant with OCC-1 human oral cavity carcinoma shows an increased level of calcium in the blood as the volume of the tumor increases and develops cachexia symptoms such as weight loss and decreased movements . In this test, the improvement of these symptoms of cachexia induced by OCC-1 human oral cavity carcinoma by the murine monoclonal antibody was evaluated in relation to the level of calcium in the blood, body weight and effect on the prolongation of survival time . OCC-1 human oral cavity carcinoma was implanted in vivo using nude BALB / c-nu / nu mice (Japan CLEA Co., Inc.). For evaluation of the pharmacological effect, nude BALB / c-nu / nu male mice of 6 weeks of age were acquired (Japan CLEA Co., Inc.) and said mice were acclimated for 1 week which resulted in 7 week old mice age, which were provided for use in the evaluation. The cachexia model mice were prepared and divided into groups as follows. The OCC-1 implanted human oral cavity carcinoma of the naked mouse was removed and finally cut into 3 mm cubes. The resulting tumor blocks were implanted subcutaneously in each of the mice in the lateral region at the rate of one piece per mouse. Ten days after the implant, when it was confirmed that the volume of the tumor in each of the mice was sufficiently large, the mice were divided into groups in such a way that blood calcium levels, body weights and tumor volumes were averaged. of the mice in the individual groups, which were provided for use as cachexia model animals. The examination of the therapeutic effect on cachexia was carried out in the following manner. (1) Observation of survival time In the examination of the effect on prolongation of survival time, a mouse monoclonal antibody was administered to the mice of a test group twice a week, and the survival time of each was observed. one of the mice. A single dose of a hypercalcemia treatment agent, pamidronate (pamidronate disodium); Aredia), was administered to the mice of another test group through the tail vein in a dose amount of 15 mg / kg. As a control in this test, a phosphate buffered saline (PBS) was administered to the mice in a control group via the tail vein twice a week in a dose amount of 0.2 mol / mouse. The results appear in figure 1. (2) Observation of calcium level in the blood The mouse monoclonal antibody against PTHrP was administered to the cachexia model mice of a test group twice at two-day intervals through the tail vein in a dose amount of 10 μg or 100 μm per mouse for each administration. A single dose of an existing intercalcemia treatment agent, pamidronate (pamidronate disodium; Aredia) was administered to the mice of another test group via the tail vein in a dose amount of 15 mg / kg. As a control in this test, a phosphate buffered saline (PBS) was administered to the mice in a control group via the tail vein twice at two-day intervals in a dose amount of 0.2 ml / mouse for each administration. (3) Determination of the level of calcium in the blood One and four days after the administration of the mouse monoclonal antibody, the level of calcium in the blood of each of the mice was determined in order to evaluate the pharmacological efficacy of the antibody . The level of calcium in the blood was determined as the ionized calcium level in whole blood, drawing blood from each of the mice through the orbit using a hematocrit tube and applying the blood to a 643 Automatic Ca / pH Analyzer ( Automatic Ca / pH Analyzer) (CIBA-CORNING). The body weight of each of the mice was taken daily up to four days after the administration of the antibody. The results appear in Figures 2 and 3. (4) Determination of tumor volume The tumor volume was determined four days after administration of the antibody, by measuring the longest axis (a mm) and the shortest axis ( b mm) of the tumor and applying both measured values to the Galant equation (ab2 / 2). The results appear in Figure 4. As is apparent from these results, even when the mice that received the antibody in a 10 μg dose amount had blood calcium levels equivalent to the levels of the mice that received pamidronate, an inhibition of weight loss was observed in the mice that received antibody, since the weight loss was not as remarkable as in the case of the mice that received pamidronate. Mice that received the antibody in a dose amount of 100 μg did not show the increase in calcium level in the blood and an inhibition of weight loss was observed to a higher degree, in comparison with the mice that received pamidronate and the control mice. In the case of the mice that received the anti-PTHrP neutralizing antibody in a dose of 100 μg twice a week, a significant degree of prolongation of survival time was observed in comparison with the mice that received pamidronate and the control mice ( p = 0.0003: range test log). As a result, it was found that the mouse monoclonal antibody neutralizing against PTHrP has excellent effects that existing hypercalcemia treatment agents can not show, such as for example the prevention of weight loss and the prolongation of survival time. These results demonstrate that the antibody employed in this test is useful as a therapeutic agent for cachexia associated with malignancy. (EXAMPLE 2) Pharmacological test using animals with hypercalcemia and cachexia models Using a cachexia model animal (a nude mouse that received a human tumor implant), a humanized antibody version "q" against PTHrP was examined to determine its therapeutic effect on cachexia. As a model animal, a nude mouse was used that received an OCC-1 human oral cavity carcinoma implant (purchased from the Central Institute for Experimental Animáis). It is known that a nude mouse that receives an implant with OCC-1 human oral cavity carcinoma has an increased calcium level in the blood as the volume of the tumor increases and develops cachexia symptoms such as weight loss and decreased movement . In this test, the improvement of said symptoms of cachexia induced by OCC-1 human oral cavity carcinoma was evaluated by means of the humanized antibody version "q" in relation to the level of calcium in the blood, body weight and effect on the prolongation of the survival time. The OCC-1 human oral cavity carcinoma subculture was carried out in vivo using nude BALB / c-nu / nu mice (Japan CLEA Co., Inc.). For the evaluation of the pharmacological effect, nude BALB / c-nu / nu male mice of 6 weeks of age (Japan CLEA Co., Inc.) were purchased, which were acclimated for a week to obtain mice of 7 weeks of age, which were supplied for use in the evaluation. The cachexia model mice were prepared and divided into groups as follows. OCC-1 human oral cavity carcinoma was removed from the naked mouse, and then finely cut into a 3 mm cube. The resulting tumor blocks were implanted subcutaneously in each of the mice in the lateral region at the rate of one piece per mouse. Ten days after the implant, when it was confirmed that the tumor volume in each of the mice was sufficiently large, the mice were divided into groups in such a way that the levels of calcium in the blood, body weights and tumor volumes of the mice in the individual groups were averaged, which were provided for use as cachexia model animals.

The examination of the therapeutic effect on cachexia was carried out in the following manner. (1) Observation of survival time In the examination of the effect on the prolongation of the survival time, a humanized antibody version "q" was administered to the mice of a test group twice a week, and the time of survival of each of the mice. As a control of this test, a phosphate buffered saline (PBS) was administered to the mice in a control group via the tail vein twice a week in a dose amount of 0.1 ml / mouse. The results appear in Figure 16. (2) Observation of the calcium level in the blood The humanized antibody version "q" was administered to the cachexia model mice of a test group twice at two-day intervals through of the tail vein in a dose amount of either 10 μg or 100 μg per mouse for each administration. As a control in this test, a phosphate-buffered saline (PBS) was administered to mice in a control group via the tail vein twice at two-day intervals in a dose amount of 0.1 ml / mouse for each administration. (3) Determination of calcium level in the blood One and four days after the first administration of the humanized antibody version "q", the level of calcium in the blood of each one of the mice was determined in order to evaluate the pharmacological efficacy of the antibody. The level of calcium in the blood was determined as the level of ionized calcium in whole blood, taking blood from each of 5 mice through the orbit using a hematocrit tube and applying the blood to 643 Automatic Ca / pH Analyzer automatic Ca / pH) _ (CIBA-CORNING). The body weight of each of the mice was weighed daily up to four days after administration of the antibody. The results appear in figures 17 and • 18. (4) Determination of tumor volume The volume of the tumor was determined four days after the first administration of the antibody, by means of the measurement of the longest axis (a mm) and the shortest axis (b mm) of the tumor and applying both measured values to the Galant equation [ab2 / 2] the results appear in figure 19. • As it is apparent from of these results, the times that received the humanized version of antibody "q" in a doses of either lOμg or lOOμg did not show the increase in calcium level in the blood and it was observed that the weight loss in the mice that received the antibody was inhibited, since the weight loss was not so remarkable that in the case of control mice. In mice that received the humanized version of antibody "q" in a dose of lOOμg twice a week, a significant degree of prolongation of survival time was observed in comparison with the control mice (p = 0.0108: logarithmic range test). The efficacy of the humanized version of "q" antibody on model animals with catexia associated with malignancy was similar to the efficacy of the mouse monoclonal antibody tested above. These results demonstrate that the antibody employed in this test is useful as a therapeutic agent for cachexia associated with malignancy. [Reference example 1] Preparation of hybridomas producing anti-PTHrP mouse monoclonal antibody (1-34) Hybridomas capable of producing a monoclonal antibody against human PTHrP (1-34) (SEQ ID No.75), No. 23- 57-154 and number 23-57-137-1, were prepared in accordance with the method reported by Kanji Sato et al. (Sato, K. Et al., J. bone Mine, Res. 8, 849-860, 1993). The immunogen used was PTHrP (1-34) (Peninsula), which was conjugated with a protein carrier thyroglobulin with carbodiimide (Dojinn). PTHrP conjugated with ticloglobulin (1-34) was dialyzed in order to obtain a solution with a protein concentration of 2μg / ml. The resulting solution was mixed with Freud's adjuvant (Difco) in a mixing ratio of 1: 1 to provide an emulsion.

This emulsion was injected into 16 female BALB / C mice 11 times dorsally-subcutaneously or intraperitoneally in a dose of 100 μg / mouse in each injection, thus immunizing the mice. For the preparation immunization, a complete Freud adjuvant was used; while for reinforcement immunization, a Freud -incomplete adjuvant was employed. For each of the immunized mice their antibody titer in the soil was determined in the following manner. That is, each of the mice has blood removed through the vein of the tail, and the antiserum is separated from the blood. The antiserum was diluted with an RIA regulator and mixed with PTHrP labeled with -125.I (1-34) in order to determine the binding activity. Mice in which it was confirmed that they had a sufficiently high titer received an injection of PTHrP (1-34) without carrier protein intraperitoneally in a dose of 50μg / mouse in the case of final immunization. Three days after the final immunization, the mouse was sacrificed and the spleen was removed. Spleen cells were subjected to cell fusion with mouse myeloma cell line P3x63Ag8U.l in accordance with any known conventional method employing 50% polyethylene glycol 4000. The fused cells prepared in this way were seeded in each well of 85 96-well plates at the rate of 2xl04 / well. The hybridomas were screened in HAT medium in the following manner. Screening of the hybridomas was carried out by determining the presence of recognition of PTHrP in the culture supernatant in which cell growth was observed in HAT medium by a solid phase RIA method. Hybridomas were collected from wells in which the binding capacity with PTHrP recognition antibodies was confirmed. Hybridomas obtained in this way were suspended in RPMI-1640 medium containing 15% FCS supplemented with an OPI complement (Sigma), followed by unification of the hybridomas by a limiting dilution method. Thus, it was possible to obtain two types of hybridoma clones, number 23-57-154 and number 23-57-137-1, both with a strong binding capacity with PTHrP (1-34). Hybridoma clone number 23-57-137-1 was designated "mouse-mouse hybridoma number 23-157-137-1", and was deposited in accordance with the terms of the Budapest Treaty on August 15, 1996 before the National Institute of Bioscience and Human-technology (National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology), Japan (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) under the FERM access number BP-5631. [REFERENCE EXAMPLE 2] Cloning of DNA encoding the V region of mouse monoclonal antibody against human PTHrP (1-34). The cloning of DNA encoding the V region of a mouse monoclonal antibody against human PTHrP (1-34), number 23-57-137-1 was carried out in the following manner. (1) Preparation of mRNA mRNA was prepared from hybridoma number 23-157-137-1 using Quick Prep mRNa Purification Kin (Set of elements for Quick Prep mRNA Purification) (Pharmacia biotech). That is, hybridoma cells number 23-57-137-1 were completely homogenized with an extraction regulator and mRNA was isolated and purified therefrom an oligo (dT) -cellulose column in accordance with the instructions in the column . The resulting solution was subjected to ethanol precipitation in order to maintain the mRNA in the form of a precipitate. The mRNA precipitate was dissolved in an elution buffer. (2) Production and amplification of cDNA for gene coding for the V region of the H chain of mouse (i) Cloning of cDNA for the V region of the H chain of antibody number 23-57-137-1 A gene coding was cloned a H chain V region of the mouse monoclonal antibody against human PTHrP by means of a 5'-RACE method (Frohman, MA et al., Proc. Nati. Acad. Sci. USA, 85, 8998-9002, 1988; Belyabsky, A. et al., Nucleic Acids Res. 17, 2919-2032, 1989). The 5 '-RACE method was carried out using a set of 5'-Ampli FINDER RACE (CLONETECH) elements in accordance with the included instructions - in the set of elements. In this method, the primer used for cDNA synthesis was the MHC2 primer (SEQ ID No. 1) which is capable of hybridizing to the C chain region of the mouse H chain. The mRNA prepared above (approximately 2 μg), which was annealed for cDNA synthesis, was mixed with the MHC2 primer (10 pmol). The resulting mixture reacted with reverse transcriptase at 52 ° C for 30 minutes in order to achieve reverse transcription of the mRNA in cDNA. The resulting reaction solution was added to 6N NaOH to hydrolyze the remaining RNA there (at a temperature of 35 ° C for 30 minutes), and then subjected to ethanol precipitation for the purpose of isolating and purifying the cDNA in the form of a precipitate. The purified cDNA was ligated into Amplifier FINDER Anchor (SEQ ID No. 42) at the 5 'end by reaction with T4 RNA ligase at a temperature of 37 ° C for 6 hours, and additionally at room temperature for 16 hours. As initiators for the amplification of the ANDc by means of a polymerase chain reaction method, the Anchor primer (SEQ ID No. 2) and the MHC-G1 primer (SEQ ID No. 3) (ST Jones, et al. ., Biotechnology, 9, 88 1992).

The polymerase chain reaction solution consd (per 50μl) of 10mM Tris-HCl (pH 8.3), 50mM KCl, 0.25M dNTPs (dATP, dGTP, dCTP, dTTP), 1.5mM MgCl2, 2.5 TaKaRa Taq units (Takara Shuzo Co., Ltd.), 10 pmoles of Anchor initiator, lμl of the reaction mixture of the cDNA with which it had been ligated in initiator MHC-G1 and the amplifier primer FINDER Anchor, on which a layer of mineral oil (50μl) was placed. The polymerase chain reaction was carried out in a Thermal Cycler Model 480J (Perkin Elmer) for 30 cycles under the following conditions: 94 ° C for 45 seconds; 60 ° C for 45 seconds, and 72 ° C for 2 minutes. (ii) Cloning of cDNA for the V region of L chain of antibody number 23-57-137-1 A gene coding for the L chain V region of the mouse monoclonal antibody against human PTHrP was cloned by the method 5'- RACE (Frohman, MA et al., Proc. Nati, Acad. Sci. USA, 85, 8998-9002, 1988; Belyavsky, A. et al., Nuclic Res 17, 2919-2932, 1989). The 5 '-RACE method was carried out using the set of elements 5' -Ampli Finder RACE (Clonetech) in accordance with the instructions included in the set of elements, in this use, an oligo-dT_ primer was used as initiator for synthesize cDNA. The mRNA prepared above (approximately 2 μg) that was annealed for the synthesis of cDNA was mixed with the oligo-dT primer. The resulting mixture reacted with reverse transcriptase to a 52 ° C film for 30 minutes in order to effect the reverse transcription of the nm into cDNA. The resulting reaction solution was added with 6N NaOH in order to hydrolyze any remaining RNA here (A - a temperature of 65 ° C for 30 minutes) the resulting solution was subjected to precipitation to isolate and purify the cDNA as a precipitate. The cDNA synthesized in this way was ligated onto Amplifier FINDER Ancor at the 5 'end by reaction with T4 RNA ligase at a temperature of 37 ° C for 6 hours and additionally at room temperature for 16 hours. An MLC polymerase chain reaction primer (SEQ ID No.4) was defined based on the conserved sequence of the C chain region of lambda L chain of mouse and then synthesized by using the DNA / RNA synthesizer 394 (ABI). The polymerase chain reaction solution comprised (per lOOμL) 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl2, 2.5 units of AmpliTaq (PERKIN ELMER), 50 pmoles of Anchor initiator (SEQ ID No. 2), and 2.5 units of AmpliTaq (PERKIN ELMER), 50 pmoles of Anchor primer (SEQ ID No. 2), and lμl of the reaction mixture of the cDNA to which MLC (SEQ ID No.4) and Amplifier FINDER Ancor were linked, on which was placed a layer of mineral oil (50μl). The polymerase chain reaction was performed in a thermal model cycler 480J (Perkin Elmer) for 35 cycles under the following conditions: 94 ° C for 45 seconds; 60 ° C for 45 seconds; and 72 ° C for 2 minutes. (3) purification and fragmentation of polymerase chain reaction products Each of the DNA fragments amplified by the polymerase chain reaction methods described above was separated by agarose gel electrophoresis on a 3% GTG agarose (FMC Bio Products). For each of the H chain V region and the L chain V region, an agarose gel segment containing a DNA fragment of about 550 pairs of gel bases was removed. Each of the segments of the gel was subjected to purification of the DNA fragment of interest using the set of GENECLEAN II elements (BIO0101) in accordance with the instructions included in the set of elements. The purified DNA was precipitated with ethanol, and the DNA pellet was dissolved in 20 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. A portion (lμl) of the DNA solution was digested with an Xmal restriction enzyme (New England Biolabs) at a temperature of 37 ° C for one hour and was further digested with an EcoRI restriction enzyme (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour. The digestion solution was extracted with phenol and chloroform and then precipitated with ethanol in order to collect the DNA. In this way, two DNA fragments were obtained which contained a gene coding for an H chain V region of 5. mouse and a gene coding for a V region of mouse L chain, respectively, both had an EcoRI recognition sequence at the 5 'end and an Xmal recognition sequence at the 3' end. they contained a gene that encoded the mouse H chain V region, and a gene encoding the V region of the mouse L chain, respectively, were ligated onto a pUC19 vector that had been digested with EcoRI and Xmal at a temperature of 16 ° C for one hour. hour by using a set of elements for binding of ADN ver.2 (Takara Shuzo Co., Ltd.) in accordance with the instructions included in the set of elements. A portion (10 μl) of the ligand mixture was added to 100 μl of a solution containing competent cells of E. coli, JM 109 (Nippon Gene Co., Ltd.). The mixture of cells' rested on ice for 15 minutes, at a temperature of 42 ° C for 1 minute, and then for 1 minute on ice,. The resulting cell mixture was added with 300μl of a SOC medium (Molecular Cloning: A Laboratory Manual (molecular cloning: a laboratory manual), Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) and then incubated at a temperature of 37 ° C for 30 minutes. The resulting cell solution was placed in dishes on an LB agar medium or on a 2xYT agar medium (Molecular Cloning: A Laboratory Manual (Molecular Cloning: a laboratory manual), Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) containing either 100 or 50 μg / ml ampicillin, 0.1 mM IPTG and 20 μg / ml X-gal, and then incubated at a temperature of 37 ° C overnight. In this way, E. coli transformant was prepared. The transformants were cultured at a temperature of 37 ° C overnight in 2 ml of LB medium or 2xYT containing either 100 or 50 μg / ml ampicillin. The cell fraction was applied to a plasmid extractor PI-100S (Kurabo Industries, Ltd.) or QIAprep (QIAGEN) to provide plasmid DNA. The plasmid DNA obtained in this way was sequenced. (4) Sequencing of gene encoding the V region of mouse antibody The nucleotide sequence of the cDNA encoding the region carried in the plasmid was determined in a DNA sequencer 373A (ABI, Perkin Elmer) using the set of elements for sequencing of colorant terminator cycle (Perkin Elmer). In this sequencing, an M13 M4 primer (Takara Shuzo Co., Ltd.) (SEQ ID No.5) and an M13 RV primer (Takara Shuzo Co., Ltd.) (SEQ ID No.6) were employed, and I confirm the nucleotide sequence in both directions. The plasmid containing a gene coding for a V region of H chain of mouse derived from hybridoma number 23-57-137-1 was designated "MBC1H04", and the plasmid containing a gene encoding the V region of L chain of mouse Hybridoma derivative number 23-57-137-1 was designated "MBC1L24". The nucleotide sequences (including the corresponding amino acid sequences) of the DNA encoding the V region of H chain derived from mouse antibody number 23-157-137-1 in the plasmid MBC1H04 and the gene encoding the V region of the L chain derived from mouse antibody number 23-57-137-1 in the plasmid MBC1H24 appear in SEQ ID No.57 and 65, respectively. Both polypeptides for the H chain V fragment and for the L chain V region fragment initiated from the 58th nucleotide (with coding glutamine) in the DNA sequences illustrated in SEQ ID No. 57 and 65, respectively. The amino acid sequences of the polypeptides for the H chain V region and the L chain V region also appear in SEQ ID Nos. 46 and 45, respectively. The E. coli strain containing the plasmid MBC1H04 and the E. coli strain containing the plasmid MBC1L24 were designated "Escherichia coli JM109 (MBC1H04)" and "Escherichia coli JM109 (MBC1L24)", respectively. These strains of E. coli have been deposited in accordance with the terms of the Budapest treaty in the National Institute of Bioscience and Human-technology, Agency of Industrial Science and Technology (National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology), Japan (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) on August 15, 1996, under accession number FERM BP-5628 for Escherichia coli JM109 (MBC1H04) and FERM BP-5627 for Escherichia coli JM109 (MBC1L24), respectively. (5) Determination of CDRs of mouse monoclonal antibody Number 23-57-137-1 against PTHrP The V region of the H chain and the V region of the L chain have similar general structures, in which there are 4 regions of structure (FRs) linked through 3 hypervariable regions (that is, regions that determine complementarity; CDRs). The amino acid sequences of the FRs are relatively well conserved whereas the amino acid sequence of the CDRs have an extremely important variability (Kabat, E.A. et al., "Sequence of Proteins of Immunological Interest" (Sequences of Proteins of Immunological Interest), US Dept.

Helath and Human Services, 1983). Taking these facts into account, amino acid homology of regions was determined V of the mouse monoclonal antibody against human PTHrP with reference to the database of amino acid sequences for antibodies established by Kabat et al. Thus, the CDRs of the V regions were determined in accordance with that indicated in Table 1. The amino acid sequences for CDRs 1-3 in the V region of the L chain are illustrated in SEQ ID Nos. 59 to 61, respectively; and the amino acid sequences for CDRs 1-3 in the H chain V region are illustrated in SEQ ID Nos. 62 to 64, respectively. Table 1 Region V SEQ ID CDR1 CDR2 CDR3 Region V of chain H 57 31-35 50-66 99-107 Region V of chain L 65 23-34 50-60 93-105 [EXAMPLE OF REFERENCE 3]. Chimeric antibody construction (1) Construction of an H chain of chimeric antibody (i) Construction of a H chain V region To ligate on an expression vector carrying Cgammal genomic DNA of human H chain C region, it was modified by means of a polymerase chain reaction method the cloned DNA encoding the mouse H chain V region, a back-initiator MBCl-Sl (SEQ ID No. 7) was designed to hybridize with a DNA sequence encoding the region 5 'of the leader sequence for the V region and to have both a Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196, 947-950 / 1987) and a HindIII recognition sequence. . A forward initiator MBCl-a (SEQ ID No. 8) was designed to hybridize to a DNA sequence encoding the 3 'region of the J region and to have both a donor splice sequence and a recognition sequence. of BamHl. The polymerase chain reaction was carried out using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a regulator. The polymerase chain reaction solution consisted (per 50 μl) of 0.07 μg of a plasmid MBC1H04 as a templated DNA, 50 pmoles of MBCl-a and 50 pmoles of MBCl-Sl as primers, 2.5U of TaKaRa Ex Taq and 0.25 M dNTPs in the regulator, over which a layer of 50μl of mineral oil was placed. The polymerase chain reaction was continued for 30 cycles under the following conditions: 94 ° for one minute; 55 ° for one minute, 72 ° for 2 minutes. The DNA fragments amplified in this way by the polymerase chain reaction method were separated by agarose gel electrophoresis in 3% A Sieve GTG Agarose (FMC Bio Products). Then, a segment of agarose gel containing a DNA fragment of 437 base pairs was removed, and the DNA fragment was purified using a GENECLEAN II Kit (BIO101) in accordance with the instructions included in the set of elements. The purified DNA was collected by ethanol precipitation and then dissolved in 20μl of a solution containing 10mM Tris-HCl (pH 7.4) and 1mM EDTA. A portion (lμl) of the resulting DNA solution was digested with BamHl restriction enzymes and HindIII (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour. The digestion solution was extracted with phenol and chloroform and then precipitated with ethanol in order to collect the DNA of interest. / The obtained HindIII-BamHI DNA fragment containing a gene coding for the H chain V region of mouse was subcloned into pUC19 vector that had been digested with HinlII and BamHl. The resulting plasmid was sequenced in a 373 A DNA sequencer (Perkin Elmer) using M13 M4 primer and M13 RV primer as primers and a set of elements for dye terminator cycle sequencing (Perkin Elmer). As a result, a correct nucleotide sequence gene coding for the H chain region V of mouse derived from hybridoma number 23-57-137-1 and presenting a HindIII recognition sequence and a Kozak sequence in its 5 'region was carried. and a BamHI recognition sequence was obtained in its 3 'region, which was designated "MBClH / pUC19". (ii) Construction of an H chain V region for the chimeric mouse-human H chain cDNA To ligate the Cgammal cDNA of the human H chain C region, the DNA encoding the polymerase chain was encoded by a polymerase chain reaction. V chain region H of mouse constructed in accordance with that described above. A kickback initiator MBC1HVS2 (SEQ ID No. 9) was designed for the H chain V region to cause the replacement of the second amino acid (asparagine) of the sequence that it determines in the front of the leader sequence for the V chain region H for glycine and for having a Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196, 947-950, 1987) and HindIII and EcoRI recognition sequences. A forward primer MBC1HVR2 (SEQ ID No.10) for the H chain V region was designed to hybridize to the DNA sequence encoding the 3 'region of the J region, to encode the 5' region of the region C and to have Apal and Smal recognition sequences. The polymerase chain reaction was carried out using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a regulator. The polymerase chain reaction solution consisted (by 50μl) of 0.6μg of plasmid MBClH / pUC19 as templated DNA, 50 pinoles of MBC1HVS2 and 50 pmoles of MBC1HVR2 as primers, 2.5 U of TaKaRa Ex Taq and 0.25 mM of dNTPs in the regulator, on which a layer of 50μl of mineral oil was placed. The polymerase chain reaction was continued for 30 cycles under the following conditions: 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute. The DNA fragments amplified by the polymerase chain reaction were separated by agarose gel electrophoresis in a Sea Ke GTG Agarose at l%? (FMC _Bio Products). Afterwards, a segment of ag rose gel containing a DNA fragment of 456 base pairs was removed and the DNA fragment was purified using GENECLEAN II Git (BIO101) in accordance with the instructions included in the set of elements. The purified DNA was pre-precipitated with ethanol and then dissolved in 20μl of a solution containing 10mM Tris-HCl (pH 7.4) and 1mM EDTA. The resulting DNA solution (lμg) was digested with EcoRI and Smal restriction enzymes (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for one hour. The digestion solution was extracted with phenol and chloroform and then precipitated with ethanol to collect the DNA. The EcoRI-Smal DNA fragment obtained, which contained a gene coding for the V region of the H chain of mice, was subcloned into pUC19 vector which had been digested with EcoRI and Smal. The resulting plasmid was sequenced in a DNA sequencer 373 A (Perkin Elmer) using the M13 M4 primer and M13 RV primer, and set of elements for color terminator cycle sequencing (Perkin Elmer). As a result, a plasmid containing a gene coding for the H chain V region of mouse derived from hybridoma number 23-57-137-1 of correct nucleotide sequence and having EcoRI and HindIII recognition sequences and a Kozak sequence was obtained. in its 5 'region and Apal and Smal recognition sequences in its 3' region, which was designated "MBClHv / pUC19". , (iii) Construction of Expression Vector for H Chain of Chimeric Antibody cDNA containing the Cgammal DNA of the H chain C region of human antibody was prepared in the following manner. MRNA was prepared from a CHO cell into which both a DHFR-alphaE-RVh-PM-1-f expression vector (see WO 92/19759) encoding genomic DNA from the H chain V region had been introduced. antibody humanized IgGl C region H chain human antibody (Takahashi et al., Cell 29, 671-679, 1982) and an expression vector RVl-PMla (see WO 92/19759) encoding region genomic DNA Chain V L of humanized PMl antibody and C chain Kappa chain L chain of human antibody. Using the mRNA, cDNA containing the H region of humanized PMl antibody and Cgammal from the C region of human antibody was cloned by means of an RT-PCR method, and then subcloned into a pUC19 plasmid at the HindIII-BamHI site. After sequencing, a plasmid having the correct nucleotide sequence was obtained, which was designated "pRVh-PMlf-cDNA." A DHFR-alphaE-RVh-PM-1-f expression vector where both the HindIII site between the SV40 promoter and a DHFR gene and an EcoRI site between the EF-alpha promoter and a H chain V region gene of humanized PMl antibody had been removed, it was prepared for the construction of an expression vector Or for cDNA containing the gene of region V of the H chain of humanized PMl antibody and the Cgammal gene of region C of human antibody. The obtained plasmid (pRVh-PMlf-cDNA) was digested with BamHl, with ends flattened with Klenow fragment and further digested with HindIII thus obtaining a HinlII, Ba Hl fragment with flat ends. The HinlII-Ba HI fragment with flat ends were joined in the expression vector with removal of the EcoRI site and removal of the aforementioned HindIII site DHFR-alphaE-Rvh-PMl-f which had been digested with HindIII and BamHl. Thus, an RVh-PMlf-cDNA expression vector containing cDNA encoding the V region of humanized PMl antibody H chain and the Cgammal gene of human antibody C region. The RVh-PMlf-cDNA expression vector containing the cDNA encoding the H chain V region of humanized PMl antibody and Cgammal of human antibody C region was Directed with Apal and BamHl, and a DNA fragment containing the H chain C region was collected therefrom. The resulting DNA fragment was introduced into the aforementioned plasmid MBClHv / pUC19 which had been digested with Apal and BamHl. The plasmid prepared in this way was designated "MBClHcDNA / pUC19" .- This plasmid contained cDNA encoding the H chain V region of mouse antibody and Cgammal of human antibody C region, and had EcoRI and HindIII recognition sequences in its 5'-region and a sequence of BamHl recognition in its 3 'region. The plasmid MBClHcDNA / pUC19 was digested with EcoRI and BamHl in order to provide a DNA fragment comprising the nucleotide sequence encoding the / H chain of Chimeric Antibodies. The resulting DNA Fragment was introduced into a pCOSI expression vector that had been digested with EcoRI and BamH1, thus providing an 'expression vector for the chimeric antibody, which was designated "MBClHcDNA / pCOSl". Here, the expression vector pCOSl was constructed using HEF-PMh-ggammal (see WO 92/19659) by removing antibody genes there by EcoRI and Smal digestion, and then ligating it on the EcoRI-Notl-BamHI adapter (Takara Shuzo Co., Ltd.). For the preparation of a plasmid for expression in a CHO cell, the plasmid MBClHcDNA / pUC19 was digested with EcoRI and BamHI in order to obtain a DNA fragment containing a gene for the H chain of chimeric antibody. The DNA fragment was then introduced into a pCHO1 expression plasmid that had been digested with EcorI and BanHI to provide an expression plasmid for the chimeric antibody, which was designated "MBClHcDNA / pCHO1". Here, the expression vector pCHOl was constructed using DHFR-alphaE-rVH-PMl-f (see number 92/19659) by removing an antibody gene therefrom by digestion with EcoRI and Smal and then ligating it over a Ecorl-NótI-BamHI adapter (Takara Shuzo Co., Ltd.). (2) Construction of a human L chain C region (i) preparation of cloning vector. To construct a pUC19 vector containing a gene for human L chain C region, a pUC19 vector was prepared with HinlII site removal. The vector pUC19 (2μg) was digested in 20μl of a reaction solution containing 20mM Tris-HCl (pH 8.5), 10mM MgCl2, 1mM DTT, 100mM KCl, 8 U of HindIII (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for one hour. The resulting digestion solution was extracted with phenol and chloroform, and then subjected to ethanol precipitation to collect the DNA of interest. The collected DNA was reacted in 50 μl of a reaction solution containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, 1 mM DTT, 100 mM NaCl, 0.5 mM dNTPs and 6 U fragment Klenow (GIBCO BRL) ambient temperature for 20 minutes, thereby flattening the terminal ends of the DNA. This reaction mixture was extracted with phenol and chloroform and then subjected to ethanol precipitation in order to collect the vector DNA. The vector DNA collected in this way reacted in lOμl of a solution. of reaction containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 5% (volume / volume) of polyethylene glycol-8000 and 0.5 U of T4 DNA ligase (GIBCO BRL) at a temperature of 16 ° C for 2 hours, to cause a self-binding of the vector DNA. The reaction solution (5μl) was reacted with 100μl of a solution containing competent cells of E.coli, JM109 (Nippon Gene Co., Ltd) and the resulting solution was allowed to stand on the wire for 30 minutes at a temperature of 42 ° C. for 1 minute, and then on ice for 1 minute. SOC culture medium (500μl) was added to the reaction solution and then incubated at a temperature of 37 ° C for one hour. The resulting solution was placed on a plate in 2 × YT agar medium (containing 50 μg / ml ampicillin) that had been applied with X-gal IPTG on its surface (Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989), and then cultured at a temperature of 37 ° C overnight, thus obtaining a transformant. The transformant was cultured in a 2xYT medium (20 ml) containing ampicillin (50 μg / ml) at a temperature of 37 ° C overnight. From the cell fraction of the culture medium, a plasmid DNA was isolated and purified using the Plasmid Mini Kit (QIAGEN) in accordance with the instructions included in the Element Set. The purified plasmid was digested with HindIII. The plasmid which was confirmed to have a HindIII site removal was designated "pUC19 deltaHindIII". (ii) Construction of DNA encoding the C chain region of human L chain Lambda It is known that the C region of human antibody L chain has at least 4 isotypes including, Mcg + Ke + Oz ~, Mcg ~ Ke "Oz ", Mcg ~ Ke ~ Oz + and Mcg ~ Ke + Oz" (P. Dariavach, et al., Proc. Nati, Acad. Sci. USA, 84, 9074-8078, 1987.) A search was made to determine the presence of lambda chain C homolog region, L chain of human antibody for the C chain region of lambda chain of mouse L chain number 23-57-137-1 from the EMBL database., it was found that the Mcg + Ke + Oz ~ isotype of lambda chain Lambda chain L chain of human antibody (access number X57819) (P. Dariavach, et al., Proc. Nati. Acad. Sci. USA, 84, 9074-9078, 1987) showed the highest degree of homology with the C chain region of lambda chain of mouse L chain number 23-57-137-1, with a homology of 64.4% in terms of amino acid sequence and a homology of 73.4% in terms of nucleotide sequence. Then, a gene coding for the C region of lambda chain L chain of human antibody was constructed by means of a polymerase chain reaction method. The primer for the polymerase chain reaction was synthesized by the use of a DNA / RNA 394 (ABI) synthesizer. The primers synthesized were the following: HLAMB1 (SEQ ID No. 11) and HLAMB3 (SEQ ID No.13), both with a DNA sequence of sense; and HLAMB2 (SEQ ID No.12) and HLAMB4 (SEQ ID No. 14), both with an antisense DNA sequence; each primer contained a complementary sequence of 20-23 base pairs at both terminal ends. The external primers HLABS (SEQ ID No.15) and HLAMR (SEQ ID No.16) presented sequences homologous to the primers HLAMB1 and HLAMB4, respectively. HLAMBS contained EcoRI, HindIII and Blnl recognition sequences, and HLAMBR contained an EcoRI recognition sequence. In the first polymerase chain reaction, the reactions between HLAMB1 and HLAMB2 and between HLAMB3 and HLAMB4 were carried out. After finishing the reactions, both resulting polymerase chain reaction products were mixed in equivalent amounts, and then assembled in the second subsequent polymerase chain reaction. The reaction solution was added with the external initiators HLAMBS and HLAMBR. This reaction mixture was subjected to the third polymerase chain reaction in order to extend the full-length DNA. Each polymerase chain reaction was carried out using TaKaRa Ex Taq (Takara Shuzo Co., Ltd) in accordance with the instructions included in the set of elements. In the first polymerase chain reaction, lOOμl was used either from a reaction solution containing 5 pmol of HLAMB1, 0.5 pmol of HLAMB2 and 5 U of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) or a solution of reaction containing 0.5 pmol of HLAMB3, 5 pmol of HLAMB4 and 5 U of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) on which a layer of 50μl of mineral oil was placed. The polymerase chain reaction was carried out for 5 cycles under the following conditions: 94 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 1 minute. In the second polymerase chain reaction, a mixture of both reaction solutions (50μl each) was used, on which a 50μl mineral oil layer was placed. The polymerase chain reaction was continued for 3 cycles under the following conditions: 94 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 1 minute. In the third polymerase chain reaction, the reaction solution to which the external initiators HLAMBS and HLAMBR (50 pmol each) were added was used. The polymerase chain reaction was carried out for 30 cycles under the following conditions: 94 ° C for 1 minute, 70 ° C for 1 minute and 72 ° C for one minute. The DNA fragment obtained by the third polymerase chain reaction was subjected to electrophoresis on a 3% low melting point agarose gel (NuSieve GTG Agarose, FMC) and separated and purified from the gel using GNECLEAN II Kit (BIO101) in accordance with the instructions included in the set of elements. The obtained DNA fragment was digested in a reaction solution (20μl) containing 50mM Tris-HCl (pH 7.5), 10mM MgCl2, 1mM DDT, 100mM NaCl and 8 U EcoRI (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for one hour. The solution in digestion was extracted with phenol and chloroform, and the DNA was collected from there by ethanol precipitation. The DNA was dissolved in a solution (8μl) containing 10 mM Tris-HCl (pH 7.4) and one mM EDTA. The above prepared plasmid pUC19 deltaHindIII (O.dμg) was digested with EcoRI in the same manner as above. The solution of. digestion was subjected to phenol / chloroform extraction and then ethanol precipitation, thus obtaining a digested pUC19 deltaHindIII plasmid. The digested plasmid reacted in a reaction solution (50μl) containing 50 mM Tris-HCl (pH 9.0), 1 mM MgCl2 and alkaline phosphatase (E. coli C75, Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 30 minutes to dephosphorylate the plasmid (ie, treat with BAP). The reaction solution was subjected to extraction with phenol / chloroform, and the DNA was collected therefrom by ethanol precipitation. The DNA obtained in this way was dissolved in a solution (10 μl) containing lOmM of Tris-HCl (pH 7.4) and 1 mM of EDTA.

The plasmid treated with BAP pUC19. deltaHindIII (lμl) was ligated onto the polymerase chain reaction product obtained above (4μl) using a set of elements for binding with DNA ver.2 (Takara Shuzo Co., Ltd.). the resulting plasmid was introduced into a competent cell of E. coli, JM109, to provide a transformant. The transformant was grown overnight in a 2xYT medium (2 ml) containing 50μg / ml ampicillin. From the cell fraction, the plasmid was isolated using QIAprep Spin Plasmid Kit (QIAGEN). The obtained plasmid was sequenced for the cloned DNA portion. Sequencing was carried out in a 373 A DNA sequencer (ABI) using the M13 M4 primer and the M13 RV primer (Takara Shuzo Co., Ltd.). As a result, it was found that the cloned DNA showed a removal of 12 base pairs. The plasmid was designated "Clambdadelta / pUcl9". Then, to compensate for the removed portion, the primers HCLMS (SEQ ID No. 17) and HCLMR (SEQ ID No. 18) were synthesized again, and a correct DNA was reconstructed using these primers by a polymerase chain reaction method . In the first polymerase chain reaction, the plasmid Clambdadelta / pUC19 having the DNA removal there was used as a hardened, and the reaction was carried out with each of the first sets of HLAMBS and HACLMS and HCLMS and HLAMB.

The polymerase chain reaction products were purified separately. In the second polymerase chain reaction, the polymerase chain reaction products were assembled together. In the third polymerase chain reaction, the reaction product of the second polymerase chain reaction was added with external primers HLAMBS and HLAMB4 and amplified in order to obtain full-length DNA. In the first polymerase chain reaction, a reaction solution (100μl) containing O.lμg of Clambdadelta / pUC19 was used as tempering, either 50 pmoles of each of the initiators HLAMBS and HCKMR or 50 pmoles of each. of the HCLMS and HLAMB4 and 5U primers from TaKaRa Ex Taq (Takara Shuzo Co., Ltd.), on which a layer of 50μl of mineral oil was placed. The polymerase chain reaction was continued for 30 cycles under the following conditions: 9 ° C for one minute, 60 ° C for one minute and 72 ° C for 1 minute. The polymerase chain reaction products of the first polymerase chain reaction, HLAMB-HCLMR (236 base pairs) and HCLMS-HLAMB4 (147 base pairs), were subjected to electrophoresis separately on a low-point agarose gel. 3% fusion in order to isolate DNA fragments, the DNA fragments were collected and purified from the gels using GENECLEAN II Kit (BIO101). In the second polymerase chain reaction, 20 μl of a reaction solution containing 40 ng of each of the purified DNA fragments and 1 U of TaKaRa Ex Taq (Takara Shuzo, Co., Ltd.) were used on which a layer of 25μl of mineral oil was placed. The polymerase chain reaction was continued for 5 cycles under the following conditions: 94 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 1 minute. In the third polymerase chain reaction, lOOμl of a reaction solution containing 2μl of a reaction solution obtained through the second polymerase chain reaction, 50 pmol of each of the initiators HLAMBS and HLAMB4 y.5 was used. _U of TaKaRa Ex Taq (Takara Shuzo, Co., Ltd.) on which a layer of 50μl of mineral oil was placed. The polymerase chain reaction continued for 30 cycles under the following conditions: 94 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for one minute, thus obtaining a DNA fragment of 357 base pairs (the third polymerase chain reaction product). The DNA fragment was subjected to electrophoresis on a 3% low melting point agarose gel in order to isolate the DNA fragment. The resulting DNA fragment was collected and purified using GENECLEAN Kit (BIO101). A portion (O.lμg) of the DNA fragment obtained in this way was digested with EcoRI, and then subcloned into the pUC19deltaHindIII plasmid that had been treated with BAP. The resulting plasmid was introduced into a competent E. coli cell, JM109, to form a transformant. The transformant was grown overnight in 2 ml of a 2xYT medium containing 50 μg / ml ampicillin. From the cell fraction, the plasmid was isolated and purified using QIAprep Spin Plasmin Kit (QIAGEN). The purified plasmid was sequenced in a 373 A DNA sequencer (ABI) using M13 M4 primer and M13 RV primer (Takara Shuzo Co., Ltd.) the plasmid which was confirmed to have the correct nucleotide sequence without any removal was designated "Clambda / pUC19". (iii) Construction of a gene encoding the human L chain Kappa chain C region A DNA fragment coding for the L chain Kappa chain C region was cloned from the HEF-PMlk-gk plasmid (WO 92/19759) by a polymerase chain reaction method. A forward initiator, HKAPS (SEQ ID No.19) was designed to contain recognition sequences of EcoRI, HindIII and Blnl, and an Hkappa backscatter (SEQ ID No. 20) was designed to contain an EcoRI recognition sequence. These primers were synthesized in a DNA / RNA 394 (ABI) synthesizer. A polymerase chain reaction was carried out using lOOμl of a reaction solution containing O.lμg of a HEF-PMlk-gk plasmid as quenched, 50 pmoles of each of the HKAPS and HKAPA primers and 5U of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.), on which was placed a layer of 50μl of mineral oil. The polymerase chain reaction was performed for 30 cycles under the following conditions: 94 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 1 minute, thus providing a polymerase chain reaction product of 360 pairs of bases. The DNA fragment was isolated and purified by 3% low-melting agarose electrophoresis, and then harvested and purified using GENECLEAN II Kit (BIO101). The DNA fragment obtained in this way was digested with EcoRI, then cloned into p-p1919 deltaHindIII pásmid which had been treated with BAP. The resulting plasmid was introduced into a complete E. coli cell, JM109, to form a transformant. The transformed was grown overnight in 2 ml of a 2xYT medium containing 50μg / ml ampicillin. From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QIAGEN). The purified plasmid was sequenced in a DNA sequencer 373 A (ABI) using an M13 M4 primer and an M13 RV primer (Takara Shuzo Co., Ltd.). The plasmid which was confirmed to have the correct nucleotide sequence was designated "CKAPPA / pUC19". (3) Construction of an L chain expression vector of chimeric antibody. An expression vector was constructed for the L chain of chimeric antibody number 23-57-137-1. A gene encoding the V region of L chain number 23-57-137-1 was ligated onto the HindIII-BInl site (located just in front of the human antibody C region) of each of the plasmids Clambda / pUC19 and Ckappa / pUC19, thus obtaining pUC19 vectors containing the DNA encoding the V region of L chain of antibody number 23-57-137-1 and either of the C chain region of lambda chain L or of the C region of Kappa region of L chain, respectively 1. Each of the resulting vectors was then digested with EcoRI in order to separate the L chain gene from the L chain of chimeric antibody. The gene was subcloned into an HEF expression vector. That is, a DNA fragment encoding an L chain V region of antibody number 23-57-137-1 was cloned from the MBC1L24 plasmid by a polymerase chain reaction method. The primers used in the polymerase chain reaction method were separately synthesized using a DNA / RNA 394 (ABI) synthesizer. A backscatter MBCCHL1 (SEQ ID No. 21) was designed to contain a HindIII recognition sequence and a Kozak sequence (Kozak, M. et al., J. Mol.

Biol. 196, 947-950, 1987), and a forward primer MBCCHL3 (SEQ ID No. 22) was designed to contain BglII and RcoRI recognition sequences. The polymerase chain reaction was carried out using lOOμl of a reaction solution containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2, 0.2 mM dNTPs, O.lμg MBC1L24 , 50 pmoles of each of the initiators MBCCHL1 and MBCCHL3 and Iμl of AmpliTaq (PERKIN ELMER), on which a layer of 50μl of mineral oil was placed. The polymerase chain reaction was carried out for 30 cycles under the following conditions: 94 ° C for 45 seconds, 60 ° C for 45 seconds, and 72 ° C for 2 minutes. A 444 base pair polymerase chain reaction product was electrophoresed on a 3% low melting agarose gel., and was collected and purified using GENECLEAN II Kit (BIO101). The purified polymerase chain reaction product was dissolved in 20μl of a solution containing 10mM Tris-HCl (pH 7.4) and 1mM EDTA. The polymerase chain reaction product (1 μl was digested in 20 μl of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, 1 mM DTT, 50 mM NaCl, 8 U HindIII (Takara Shuzo Co., Ltd.) and 8 U of EcoRI (Takara Shuzo CO., Ltd.) at a temperature of 37 ° C for 1 hour.The digestion solution was subjected to extraction with phenol / chloroform, and the DNA of interest was collected from there by ethanol precipitation, DNA was dissolved in 8μl of a solution containing 10mM Tris-HC1 (pH 7.4) and 1mM EDTA, and a pUC19 plasmid was digested ( lμg) with HindIII and EcoRI, and subjected to extraction in phenol / chloroform and then ethanol precipitation The obtained digested plasmid was treated with BAP with alkaline phosphates (E. coli C75; Takara Shuzo Co., Ltd.). The resulting reaction solution was extracted with phenol and chloroform, and the DNA was collected therefrom by ethanol precipitation. The DNA was dissolved in lOμl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The BAP-treated plasmid pUC19 (lμl) was ligated onto the polymerase chain reaction product obtained above (4μl) using 1 DNA Ligation Kit Ver. 2 (Takara Shuzo Co., Ltd.). The resulting plasmid was introduced into a competent cell of E. coli, JM109 (Nippon Gene Co., Ltd.). in the same way as the aforementioned in order to form a transformant. The transformant was plated on a 2xYP agar medium containing 50μg / ml ampicillin and cultured at 37 ° C overnight. The resulting transformant was then cultured at a temperature of 37 ° C overnight in 2 ml of a 2xYT medium containing 50 μg / ml ampicillin. From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QUIAGEN). After determination of the nucleotide sequence, the plasmid which was confirmed to have the correct nucleotide sequence was designated "CHL / pUC19". Each of the plasmids Clambda / pUC19 and Ckapa / pUC19 (1 μg each) was digested in 20 μl of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl 2, 1 mM DTT, 100 mM of KCl, 8U of HindIII (Takara Shuzo Co., Ltd.) and 2 U of Blnl (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour. The digestion solution was extracted with phenol and chloroform, and the DNA was collected there by ethanol precipitation. The DNA was treated with BAP at a temperature of 37 ° C for 30 minutes. The reaction solution was extracted with phenol and chloroform, and the DNA was collected by ethanol precipitation. The DNA was dissolved in lOμl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The CH1 / pUC19 plasmid containing DNA encoding the V chain region V of number 23-57-137-1 (8μg) was digested * with HindIII and BlnI in the same manner as mentioned above to provide a DNA fragment 409 Base pairs. The DNA fragment was subjected to electrophoresis on a 3% low melting point agarose gel, and then collected and purified using GENECLEAN II Kit (BIO101) from the gel. The DNA was dissolved in lOμl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. DNA for L chain V region DNA (4μl) was subcloned in Iμl of each plasmid treated with Clambda BAP / pUC19 and Ckapa / pUC19, and then introduced into a competent cell of E. coli, JM109, to form a transformant. The transformant was grown overnight in 3 ml of a 2xYT medium containing 50μg / ml ampicillin. From the cell fraction, the plasmid was isolated and purified using QIAprep Spin Plasmid Kit (QIAGEN). The two plasmids prepared in this way were designated "MBCÍL (lambda) / pUC19" and "MBC1L (K) / pUC19", respectively. Each of the plasmids MBC1L (lambda) / pUC19 and MBC1L (Kappa) / pUC19 was digested with EcoRI and then subjected to electrophoresis on a 3% low melting point agarose gel. A DNA fragment of 743 base pairs was isolated and purified from the gel using GENECLEAN II KIT (BI0101), and then dissolved in 10 ml of a solution containing 10 mM tris HCl (pH 7.4) and one mM of EDTA. An expression vector was digested (plasmid HF-PMlk-gk) (2.7μg) with EcoRI and then extracted with phenol and chloroform, and the DNA was collected therefrom by ethanol precipitation. The DNA fragment was treated with BAP, and then subjected to electrophoresis on a 1% low melting point agarose gel. From the gel, a 6561 base pair DNA fragment was isolated and purified using GENECLEAN II Kit (BIO101). The purified DNA fragment was dissolved in lOμg of a solution containing 10 mM Tris-HCl (pH 7.4), and one Mm of EDTA. The HEF vector treated with BAP (2μl) was ligated onto an EcoRI fragment (3μl) of each of the plasmids MBC1L (lambda) / pUC19 and MBC1L (Kappa). The ligand product was introduced into a competent cell of E. coli JM109, to form a transformant. The transformant was cultured in 2ml of a 2xYT medium containing 50μg / ml of ampicillin. From the cellular fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QUIAGEN). The purified plasmid was digested in 20 μl of a reaction solution containing 20 mM tris-HCL (pH 8.5), 10 Mm MgCl 2, 1 mM DTT, 100 mM KCl, 8U HindIII (Takar Shuzo, CO., Ltd.) and 2 U of Pvul (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour. This reaction provided digestion fragments of 5104/2195 base pairs whether the fragment was inserted in the correct orientation or provided digestion fragments of 4378/2926 base pairs if the fragment was inserted in the reverse orientation. The plasmid which was confirmed to have the fragment in the correct orientation was designated "MBC1L (lambda) / neo" for the plasmid MBCÍL (lambda) / pUCl9 or "MBC1L (kappa) / neo" for the plasmid MBC1L (kappa) / pUC19. (4) COS-7 Cell Transfection To evaluate the antigen binding activity and the neutralizing activity of the chimeric antibodies, the expression plasmids prepared above were separately expressed transiently in a COS-7 cell. The transient expression of the chimeric antibodies was carried out using each of the combinations of plasmids MBClHcDNA / pCOSl and MBC1L (lambda) / neo and plasmids MBClHcDNA / pCOSl and MBCIKL (kappa) / neo, by co-transfection of a cell COS-7 with plasmids by electroporation using a gene driver (Bio Rad). That is, the plasmids (10 μg each) were added to a suspension of COS 7 cells (0.8 ml; lxlO7 cells / ml) in PBS (-). The resulting solution was applied with pulses to an electrostatic capacity of 1500V and 2μF in order to cause electroporation. After 10 minutes of a recovery period at room temperature, cells subjected to electroporation were suspended in DMEM medium (GIBCO) containing 2% ultraflute fetal calf serum IgG (GIBCO), and then cultivated using a culture dish of cm in a C02 incubator. After culturing for 72 hours, a culture supernatant was collected and centrifuged to remove cell debris and provided for use as a sample for the subsequent ELISA. In this procedure, the purification of the chimeric antibody from the COS-7 cell culture supernatant was carried out using AffiGel Protein A MAPSII Kit (Bio Rad) • in accordance with the instructions included in Element Set 5. (5) ELISA (i) Determination of Antibody Concentration An ELISA plate for determining the antibody concentration was prepared in the following manner. Each well one 96-well ELISA plate (Maxisorp, NUNC) was covered • with lOOμl of a coating buffer (0.1 M NaHC03, 0.02% NaN3) supplemented with Iμg / ml goat antihuman IgG antibody (TAGO), and then blocked with 200 μl of a dilution buffer [50 mM Tris -HCl, 1 mM MgCl2, 0.1 M NaCl, 0.05% Tween 20, 0.02% NaN3, 1% bovine serum albumin (BSA); pH 7.2]. each dish well received each of the serial dilutions of the COS-7 cell culture supernatant wherein each of the chimeric antibodies had been expressed, or each received the serial dilutions of each of the chimeric antibodies per se in a purified form. The plate was incubated at room temperature for one hour and washed with PBS-Tween 20. Each plate well received then lOOμl of a goat antihuman IgG antibody solution conjugated with alkaline phosphates (TAGO). Then the plate was incubated at room temperature for 1 hour and washed with PBS-Tween , each well received 1 mg / ml of a substrate solution ("SIGMA 104" p-nitrophenylphosphoric acid, SIGMA), the solution was measured in its absorbance at 405 ml using a microplate reader (Bio Rad) to determine the concentration of antibodies. In this determination, purified HuIgGlla bda (the binding site) was used as a standard. (ii) determination of antigen binding capacity An ELISA plate for the determination of antigen binding capacity was prepared in the following manner. Each well of a 96-well ELISA plate was coated with 100μl of a coating buffer supplemented with lμg / ml of PTHrP (1-34) (Peptide Research Institute), and then blocked with 200μl of a dilution buffer. To each well was added the serial dilutions of the culture supernatant of COS-7 cells in which chimeric antibodies had been expressed, or each of the serial dilutions of each of the chimeric antibodies per se in purified form was added. . After incubation of the plate at room temperature and then washing with PBS-Tween 20, each well of the plate received lOOμl of an alkaline phosphatase-conjugated goat antihuman IgG (TAGO) antibody solution. After incubation of the plate at room temperature and after washing with PBS-Tween 20, each well of the plate received 1 mg / ml of a substrate solution ("SIGMA 104", p-Nicrophenylphosphoric acid, SIGMA). The solution was measured in its absorbance at 405 nm using a microplate reader (Bio Rad). As a result, the chimeric antibodies were found to have a binding capacity with human PTHrP (1-34) and the cloned mouse antibody V regions had the correct structures (Figure 5). It was also found that there were no differences in the binding capacity with PTHrP (1-34) between the chimeric antibody with chain C chain lambda chain L and the chimeric antibody with chain C chain Kappa region L. Therefore , the L chain C region of the humanized antibody was constructed using the L chain of humanized antibody. (6) Establishment of CHO cell line capable of stable production of chimeric antibodies To establish a cell line capable of stably producing chimeric antibodies, the expression plasmids prepared above were introduced into CHO cells (DXB11). For the establishment of a cell line capable of producing the chimeric antibodies in a stable manner, use any of the following combinations of the CHO cell expression plasmids:? MBClHcDNA / pCHOl and MCB1L (lambda) / neo; and MBClHcDNA / pCHOl and MBC1L (Kappa) / neo. A CHO cell was cotransfected with the plasmids by electroporation using a Gene Pulser (Bio Rad) in the following manner. The expression vectors were separately dissociated with a restriction enzyme Pvul to provide linear DNAs. The resulting DNAs were extracted with phenol and chloroform and collected by ethanol precipitation. The plasmid DNAs prepared in this way were subjected to electroporation. That is, each of the plasmid DNAs (10 μg each) was added to 0.8 ml of a cell suspension of CHO cells in PBS (-) (I x 10 7 cells / ml). The resulting solution was applied with pulses to an electrostatic capacity of 1500 V and 25 μF. After 10 minutes of recovery period at room temperature, the cells subjected to electroporation were suspended in a MEM-alpha medium (GIBCO) containing 10% fetal calf serum serum (GIBCO). The resulting suspension was cultured using 3 96-well plates (Falcon) in a C02 incubator. The day after the start of the culture, the medium was replaced by a selective medium (ribonucleoside or deoxyribonucleoside-free MEM-alpha medium (GIBCO) containing 10% fetal calf serum (GIBCO) and 500 mg / ml GENETICIN (G418Sulfate; GIBCO)). From the culture medium, cells in which the antibody gene was introduced were selected. The selective medium is replaced by a fresh medium. Approximately 2 weeks after replacing the medium, the cells were observed under the microscope. When favorable cell growth was observed, the cells were determined in the amount of antibodies produced by ELISA as mentioned above. Between the cells, the cells that produced a greater amount of antibodies were screened. The culture of the established cell line capable of stable production of the antibodies in a roller bottle was then increased by using either a ribonucleoside or deoxyribonucleoside-free MEM medium containing 2% fetal calf serum of ultra-low IgG. On day 3 and day 4 of the culture, the supernatant, d.e. culture was collected and then filtered using a 2. Oμm filter (Millipore) in order to remove cell debris therefrom. Purification of the chimeric antibodies from the CHO cell culture supernatant was carried out using a POROS protein (PerSeptive Biosystems) column in ConSep LC100 (Millipore) in accordance with the instructions contained in the Element Set. Purified chimeric antibodies were provided for use as samples for the determination of neutralizing activity and for the examination of therapeutic efficacy in hypercalcemic model animals. The concentration and antigen binding activity of the purified chimeric antibodies was determined using the same ELISA system as mentioned above. [Reference example 4] Construction of humanized antibody (1) Construction of H chain of humanized antibody (i) Construction of region V of humanized chain H An H chain of humanized antibody number 23-57-137-1 was produced by the technique of CDR graft by means of a polymerase chain reaction method. In the case of the production of an H chain of humanized antibody number 23-57-137-1 (version "H") having FRs derived from human antibody S31679 (NBRF-PDB; Culsinier, AM Et al., Eur. J Immunol., 23, 110-118, 1993), the following 6 polymerase chain reaction primers were used: DCR graft initiators: MBC1HGP1 (SEQ ID No. 23) and MBC1HGP3 (SEQ ID No. 24) ( both containing a sense DNA sequence) and MBC1HGP2 (SEQ ID No. 25) and MBC1HGP $ (SEQ ID No. 26) (both containing an antisense DNA sequence), all of which contain a complementary sequence of 15-21 base pairs at both terminal ends thereof; and external primers: MBC1HVS1 (SEQ ID No.27) and MBC1HVR1 (SEQ ID No.28) having a homology with the CDR graft primers MBC1HGP1 and MBC1HGP4, respectively. The CDR graft initiators MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4 were separated on a denatured urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989), and extracted there by means of a crushing and soaking method (Molecular Cloning: A Laboratory Manual (molecular cloning: laboratory manual), Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) of the Following way . Each of the CDR graft initiators (1 nmol) was separated on a 6% denatured polyacrylamide gel in order to provide DNA fragments. From the resulting DNA fragments, a fragment having a desired length was identified on a thin plate of silica gel by UV ray irradiation and then collected therefrom by means of a crush and soak method. The resulting DNA was dissolved in 20μl of a solution containing 10mM Tris-HCl (pH 7.4) and 1mM EDTA. The polymerase chain reaction was carried out using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.). The polymerase chain reaction solution (100μl) contained 1μl of each of the aforementioned CDR graft initiators MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4, 0.25mM dNTPs and 2.5U of TaKaRa Ex Taq in the regulator. The polymerase chain reaction was carried out for 5 cycles under the following conditions: 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute. The resulting reaction solution received the external primers MBC1HVS1 and MBC1HVR1 (50 pmol each). Using this reaction mixture, the polymerase chain reaction continued for 30 additional cycles and under the same conditions. The DNA fragment amplified in this way was separated by agarose gel electrophoresis in a Nu Sieve GTG agarose (FMC Bio. Products) at 4%. An agarose segment containing a DNA fragment of 421 base pairs was removed, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) in accordance with the instructions contained in the set of elements. The DNA fragment purified in this way was precipitated with ethanol and then dissolved in 20 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and one mM EDTA. The resulting polymerase chain reaction mixture was used for the subcloning of the DNA fragment in the pUC1 plasmid that had been digested with BamHI and HindIII, and the frequency of nucleotides of the resulting plasmid was subsequently determined. A plasmid having the correct nucleotide sequence was designated "hMBCHv / pUC19". (ii) Construction of a H chain V region for humanized H chain cDNA To be ligated onto cGNA for humanized H chain C region Cgammal, the DNA for the humanized H chain V region constructed in the previous step was modified by means of a polymerase chain reaction method. For the polymerase chain reaction method, a backtrace initiator MBC1HVS2 was designed to anneal to the coding sequence of the 5 'region of the leader sequence for the V region and to have a Kozak consensus sequence (Kozak et al. , J. Mol. Biol. 196, 947-950, 1987) and HindIII and EcoRI recognition sequences; and a forward primer MBC1HVR2 was designed to hybridize both to the DNA sequence encoding the 3 'region of the J region and to the DNA sequence encoding the 5' region of the C region and to have the recognition sequences Apal and Smal. The polymerase chain reaction was carried out using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and an adjuvant regulator. The polymerase chain reaction solution consisted of 0.4μg of hMBCHv / pUC19 as DNA annealing, 50 pmoles of each of MBC1HVS2 and MBC1HVR2 as primers, 2.5 U of TaKaRa Ex Taq and 0.25 mM of dNTPs in the regulator. The polymerase chain reaction was carried out for 30 cycles under the following conditions: 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 1 minute. The DNA fragment amplified in this way was separated by agarose gel electrophoresis in a Nu Sieve GTG Agarose (FMC Bio. Products) at 3%. A gel segment containing a DNA fragment of 456 base pairs was removed, and the DNA fragment was purified there using GENECLEAN II Kit (BIO 101) in accordance with the instructions found in the Element Set. The DNA fragment purified from. this form was precipitated with ethanol and then dissolved in 20μl of a solution containing 10mM Tris-HCl (pH 7.4) and 1mM EDTA. The polymerase chain reaction solution obtained in this way was used for the subcloning of the DNA fragment in the pUC19 plasmid that had been digested with EcoRI and Smal, and then the resulting plasmid was sequenced. As a result, a plasmid containing DNA encoding the H chain V region of mouse derived from hybridoma Number 23-57-137-1 and containing also EcoRI and HindIII recognition sequences as well as a Kozak sequence in region 5 was obtained. and Apal and Smal recognition sequences in the 3 'region, which was designated "hMBClHv / pUC19". (2) construction of expression vector for H chain of humanized antibody Plasmid RVh-PMlf-cDNA carrying a cDNA sequence for H chain of hPMl antibody was digested with Apal and BamHl in order to obtain a DNA fragment that contains DNA encoding the C region of the H chain. The DNA fragment was introduced into the plasmid hMBClHv / pUC19 that had been digested with Apal and BamHl. The obtained plasmid was designated "hMBClHcDNA / pUC19" this plasmid contained both the DNA encoding the H chain V region of humanized antibody number 23-57-137-1 and the Cgammal DNA of human H chain C region and had EcoRI and HindIII recognition sequences in the 5'region and a BamHl recognition sequence in the 3 'region. The nucleotide sequence and the corresponding amino acid sequence for the "a" version of the humanized H chain carried in the plasmid hMBClHcDNA / pU19 appear in SEQ ID No. 58 and SEQ ID No. 56, respectively. Plasmid hMBClHcDNA / pUC19 was digested with EcoRI and BamHI to provide a DNA fragment containing DNA encoding the H chain. The DNA fragment was introduced into the pCOSI expression plasmid which had been digested with EcoRI and BamHI. as a result, an expression plasmid was obtained for a humanized antibody, which was designated "hMBClHcDNA / pCOSl". In order to produce a plasmid used for expression in a CHO cell, a hBClHcDNA / pUC19 plasmid was digested with EcoRI and BamHl in order to provide a DNA fragment containing DEN encoding the H chain. The DNA fragment was introduced into the pCHO1 expression vector that had been digested with EcoRI and BamHl. As a result, an expression plasmid was obtained for the humanized antibody, which was designated "hMBClHcDNA / pCHOl". (3) Construction of hybrid L chain V region (i) Preparation of FR1 hybrid antibody, 2 / F3,4 A gene was constructed for FR hybrid L chain having both FRs of a humanized antibody and FRs of an antibody of mouse (chimeric), and each region was evaluated to determine humanization. In this step, a hybrid antibody having FR1 and FR2 both derived from a human antibody and FR3 and FR4 both derived from a mouse antibody was prepared by using the AflII restriction site located on CDR2. Plasmids MBC1L (lambda) / neo and hMBClL (lambda) / neo (lOμg each) were digested separately in lOOμl of a reaction solution containing 10 mM Tris-HCl (pH 7.5), mM MgCl2, one mM DTT, 50 mM NaCl, 0.01% (weight / volume) of BSA and 10 U of AflII (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour. The reaction solutions were subjected to electrophoresis in an agarose gel with a low melting point of 2%, thus providing DNA fragments of 6282 base pairs (known as "cl") and 1022 base pairs (known as "cl") "c2") from the plasmid MBC1L (lambda) / neo or DNA fragments of 6282 base pairs (known as "hl") and 1022 base pairs (known as "h2") from the plasmid hMBClL (lambda) ) / neo. These DNA fragments were collected and purified from the gels using GENECLEANII Kit (BIO101). The cl and hl fragment (lμg) was treated with BAP. The DNA fragment was extracted with phenol and chloroform, collected by ethanol precipitation, and dissolved in lOμl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The cl and hl DNA fragments treated with BAP (lμl each) were ligated onto the h2 and c2 DNA fragments (4μl each), respectively, (at a temperature of 4 ° C overnight). Each of the binding products were introduced into a competent cell of E. coli, JM109, in order to form a transformant. The transformant was cultured in 2 ml of a 2xYT medium containing 50 μg / ml ampicillin. From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid kit (QIAGEN). The purified plasmid was digested in 20 μl of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, 1 mM DTT, and or 2 U ApaLI (Takara Shuzo Co., Ltd.) or 8U of BamHl (Takara Shuzo Co., Ltd.) and HindIII (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour. If cl-h2 had a correct binding, this digestion reaction provided fragments of 5560/12246/498 base pairs (by ApaLI digestion) or fragments of 7134/269 base pairs (by Ba Hl / HindI II digestion). Based on this consideration, the desired plasmids were identified. The expression vector encoding the L chain of hybrid FR1, human 2 / mouse FR3.4 antibody was designated "h / mMBClL (lambda) / neo". On the other hand, a clone for hl-cl could not be obtained so, recombination was carried out in a pUC vector, and then the resulting recombinant product was cloned into an HEF vector. In this procedure, the plasmid hMBClLalambda / pUC19, which contained DNA encoding a V region of L chain of humanized antibody without any amino acid replacement, and the plasmid hMBClLdlambda / pUC19 containing DNA encoding the V region of L chain of humanized antibody with an amino acid replacement at position 91, amino acid tyrosine in FR3 (ie, the 87th amino acid according to the description of Kabat) by isoleucine, were used as tempers. The plasmids MBC1L (lambda) / pUC19, hMBClLalambda / pUC19 and hMBClLdlambada / pUC19 (10 μl each) were digested separately in 30μl of a reaction solution containing 10mM Tris-HCl (pH 7.5), 10mM MgCl2, 1 mM DTT, 50 mM NaCl, 0.01% (weight / volume) of BSA, 16U of HindIII and 4 U of AflII at a temperature of 37 ° C for 1 hour. The reaction solutions were subjected separately to electrophoresis on an agarose gel with a low melting point of 2%, thus providing a DNA fragment of 215 base pairs from the plasmid MBC1L (lambda) / pUC19 (known as "c2"). "), and a DNA fragment of 3218 base pairs from each of the plasmids - hMBClLalambda / pUC19 and hMBClLdlambda / pUC19 (known as" hal '"and" hdl' ", respectively). These DNA fragments were collected and purified using GENECLEANII kit (BIO101). Each of the hal 'and hdl' fragments was ligated onto the c2'-fragment and then introduced into a competent cell of E. coli, JM109, to form a transformant. The transformant was cultured in 2 ml of a 2xYT medium containing 50 μg / ml ampicillin. From the cellular fraction, the plasmid was purified using a QIAprep Spin Plasmid Kit (QIAGEN). The plasmids prepared in this way were "designated" m / hMBClLalambda / pUC19"for the plasmid containing the hal 'and" m / hMBClLdlambda / pUC19"fragment in the case of the plasmid containing the hdl' fragment. m / hMBClLalambda and m / hMBclLdlambda / pUC19 was digested with EcoRI The 743 bp DNA fragment was subjected to electrophoresis on a 2% low melting agarose gel, and then harvested and purified using GENECLEANII kit (BIO101). The resulting DNA fragment was dissolved in 20μl of a solution containing 10mM Tris-HCl (pH 7.4) and one mM of EDTA, each of the DNA fragments (4μl each) was ligated onto the treated HEF vector. with BAP obtained above (lμl) The binding product was introduced into a competent cell of E. coli, JM109, in order to form a transformant.The transformant was cultured in 2 ml of a 2xYT medium containing 50μg / ml of ampicillin, from the cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QUIAGEN). Each of the purified plasmids was digested in 20 μl of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl 2, 1 mM DTT, 100 M KCl, 8 U HindIII (Takara Shuzo Co., Ltd.) and 2U of Pvul (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour. Plasmid DNA was identified based on the expectation that if the DNA fragment were inserted into the plasmid in the correct orientation, this digestion would provide a 5104/2195 base pair digestion fragment, whereas if the DNA fragment was inserted in the plasmid in the reverse orientation, this digestion would provide a digestion fragment of 4378/2926 base pairs. The plasmids obtained in this way were expression vectors encoding the L chain of hybrid FRl, 2 mouse / FR3, 4 human antibody, which were the expression vectors designated "m / hMBClLalambda / neo" and? M / hMBClLdlambda / neo ", respectively, (ii) Preparation of FR1 / FR2 hybrid antibody A FR1 / FR2 hybrid antibody was prepared in the same manner as indicated above using a SnaBI restriction site located on CDR1, the plasmids MBC1L (lambda) / neo and h / mMBClL (lambda) / neo (lOμg each) were separately digested in 20μl of a reaction solution containing 10mM Tris-HCl (pH 7.9), 10mM MgCl 2, 1mM DTT, 50mM NaCl, 0.01% (weight / volume) of BSA and 6U of SnaBl (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour The resulting reaction solutions were further digested in 50μl of a reaction solution containing 20mM of Tris-HCl (pH 8.5), 10 mM MgCl, 1 mM DTT, 100 mM KCl, 0.0 1% (weight / volume) of BSA and 6U of Pvul at a temperature of 37 ° C for 1 hour. The resulting reaction solutions were subjected separately to electrophoresis on a 1.5% low melting point agarose gel, thus providing DNA fragments of 4955 base pairs (ml) and 2349 base pairs (m2) from the MBC1L plasmid (lambda) / neo and DNA fragments of 4955 base pairs (hml) and 2349 base pairs (hm2) from the plasmid h / mMBClL (lambda) / neo. These DNA fragments were collected and purified from the gels using GENECLEANII Kit (BIO101). Each of the DNA fragments obtained was dissolved in 40 μl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The ml and hml fragments (lμl each) were ligated onto the hm.2 and m2 fragments (4μl each), respectively. Each of the resulting binding products was introduced into a competent E. coli cell, JM109, to form a transformant. The transformant obtained was cultured in 2 ml of a 2xYT medium containing 50 μg / ml of ampicillin. From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QIAGEN). Each of the purified plasmids was digested in 20μl of a reaction solution containing 10mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT and either 8U of Apal (Takara Shuzo Co., Ltd.) or 2U of ApaLI (Takara Shuzo Co., Ltd.) at a temperature of 37 ° C for 1 hour. If the fragments were ligated correctly, the digestion reaction provided a fragment of 7304 base pairs (by digestion with Apal) or fragments of 5560/1246/498 base pairs (by ApaLI digestion) for ml-hm2, and provided fragments of 6538/766 base pairs (by Apal digestion) or fragments of 3535/2025 / 1246/498 base pairs (by ApaLI digestion) for hml-m2. Based on this consideration, the plasmids were identified. As a result, an expression vector encoding an L chain of human FR1 hybrid antibody / mouse FR2, 3.4 (designated "HmmMBClL (lambda) / neo") and an expression vector encoding an antibody L chain was obtained. hybrid mouse FR1 / human F2 / mouse F3.4 (designated "mhmMBClL (lambda)"). (4) L chain construction of humanized antibody An L chain of humanized antibody number * 23-57-137-1 was prepared by the CDR graft technique by means of the polymerase chain reaction method. For the preparation of an L chain of humanized antibody number 23-57-137-1 (version "a") containing FRl, FR2 and FR3 derived from human antibody HSU03868 (GEN-BANK, Deftos M. et al., Scand. J. Immunol., 39, 95-103, 1994) and FR4 derived from human antibody S25755 (NBRF-PDB), six polymerase chain reaction primers were amplified. The six primers were the following: CDR graft initiators MBC1LGP1 (SEQ IN No.29), and MBC1LGP3 (SEQ ID No. 30), both with a sense DNA sequence, CDR graft initiators MBC1LGP2 (SEQ ID No 31) and MBC1LGP4 (SEQ ID No. 32), both with an antisense DNA sequence, all of which had a complementary sequence of 15-21 base pairs at both terminal ends; and the external primers MBC1LVS1 (SEQ ID No.33) and MBC1LVR1 (SEQ ID No. 34) having a homology with the CDR graft primers MBC1LGP1 and MBC1LGP4, respectively. The CDR graft initiators MBC1LGP1, MBC1LGP2, MBC1LGP3 and MBC1LGP4 were separated on a denatured urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Manual (molecular cloning: a laboratory manual), Sambrook et al., Cold Spring Harbor Laboratory Press , 1989), and extracted from a segment by a crushing and soaking method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Each of the CDR graft initiators (1 mmol) was separated with 6% denatured polyacrylamide gel. The identification of the DNA fragment of a desired length was carried out on a thin plate of silica gel by irradiation of UV rays. The desired DNA fragment was picked up from the gel by means of a crush and soak method. The collected DNA fragment was dissolved in _20μl of a solution containing 10mM Tris-HCl (pH 7.4) and 1mM EDTA. The polymerase chain reaction was carried out using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and an adjuvant regulator. The solution in polymerase chain reaction consisted of 100 μl of each of the CDR graft initiators, MBC1LGP1, MBC1LGP2, MBC1LGP3, and MBC1LGP4, 0.25 mM dNTPs, 2.5 U of Takara Ex Taq in the regulator. Polymerase chain reaction was carried out for 5 cycles under the following conditions: 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute.The resulting reaction mixture was added with 50 pmoles of each of the external primers MBC1LVS1 and MBC1LVR1 By using this reaction mixture, the polymerase chain reaction was continued for an additional 30 cycles under the same conditions The fragment of -ADN amplified in this way was separated by agarose gel electrophoresis in a Nu Sieve GTG 3% agarose (FMC Bio Products) An agarose segment containing a 421 base pair DNA fragment was removed, and purified a DNA fragment there by using GENECLEANII Kit (BIO101 ) from compliance with the instructions included in the set of elements. The polymerase chain reaction mixture obtained in this way was used for the subcloning of the DNA fragment in the pUC19 plasmid which had been digested with Ba Hl and HindIII. The resulting plasmid was sequenced. The plasmid prepared in this way was designated "hMBCL / pUC19". In this plasmid, however, the amino acid at position 104 (corresponding to the amino acid at position 96 according to the description of Kabat) of CDR4 was replaced by _ arginine. For the correction of this amino acid in tyrosine, a correction initiator MBC1LGP10R (SEQ ID No. 35) was designed and synthesized. The polymerase chain reaction was carried out using TaKaRa Ex Taq Takara Shuzo CO., Ltd.) and an adjuvant regulator. The polymerase chain reaction solution consisted (per lOOμl of 0.6μg of plasmid hMBCL / pUC19 as templated DNA, 50 pmoles of each of the primers MBC1LVS1 and MBC1LGP10R, 2.5U of TaKaRa (Takara Shuzo Co., Ltd.) and 0.25 mM dNTPs in the regulator, over which a layer of mineral oil (50μl) was placed.The polymerase chain reaction was carried out for 30 cycles under the following conditions: 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 1 minute The DNA fragment amplified in this way was separated by agarose gel electrophoresis in 1 Nu Sieve GTG 3% agarose (FMC Bio Products) A gel segment containing 1 fragment of 421 base pairs of DNA was removed, the DNA fragment was purified there using GENECLEANII Kit (BIO101) in accordance with the instructions included in the set of elements.The mixture of the polymerase chain reaction prepared in this way was used for the subclo Nation of the DNA fragment in the plasmid pUC19 that had been digested with Ba Hl and HindIII. The plasmid was sequenced using an M13 M4 primer and an M13 RV primer. As a result, it was confirmed that the plasmid had the correct sequence. The plasmid was then digested with HindIII and BlnI, and a DNA fragment of 416 base pairs was separated by electrophoresis in 1% agarose gel. The DNA fragment was purified using GENECLEAN Kit (BIO101) in accordance with the instructions included in the set of elements, and then it was introduced into the plasmids Clambda / pUC19 which had been digested with HindIII and Blnl. The resulting plasmid was designated "hMClLalambda / pUC19". This plasmid was digested with EcoRI in order to provide a DNA fragment encoding a humanized L chain. The DNA fragment was introduced into the plasmid pCOSl in such a way that the initiation codon for the humanized L chain was located downstream of the EFlalfa promoter. The plasmid obtained in this way was designated "hMBClLalambda / pCOSl". The DNA sequence (including the corresponding amino acid sequence) of the "a" version of the humanized L chain appears in SEQ ID No.66. the amino acid sequence of the "a" version also appears in SEQ ID No. 47. A "b" version of the humanized L chain was prepared by the use of a mutagenesis technique by a polymerase chain reaction method. Version "b" was designed in such a way that the amino acid at position 43 glycine (which corresponds to amino 43 according to the description of Kabat) was replaced by proline and the amino acid of position 49 lysine (corresponding to amino acid 49). in accordance with the description of Kabat) was replaced by aspartic acid in the "a" version. The polymerase chain reaction was carried out using a plasmid hMBClLalambda / pUC19 as annealed with a mutagenic primer MBC1LGP5R (SEQ ID No. 36), and an initiator MBC1LVS1. The DNA fragment obtained was deferred with BamHI and HindIII, and the digestion fragment was subcloned into the BamHI-HindIII site of pUC19. After sequencing, the plasmid was digested with HindIII and AflII, and the resulting digestion fragment was ligated onto the plasmid hMBClLalambda / pUC19 that had been digested with HindIII and AflII. The plasmid obtained in this way was designated "hMBClLblambda / pUC19". This plasmid was digested with EcoRI in order to provide a DNA fragment containing DNA encoding the humanized L chain. The DNA fragment was introduced into the pCOSl plasmid in such a way that the initiation condom for the humanized L chain is downstream of the EFlalfa promoter. The plasmid obtained in this way was designated "hMBClLbla bda / pCOSl". A "c" version of humanized L chain was prepared using the mutagenesis technique by a polymerase chain reaction method. The "c" version was designed in such a way that the amino acid at position 84, serine (corresponding to amino acid 80 according to the description of Kabat) was replaced by proline. The polymerase chain reaction was performed using a plasmid hMBClLalambda / pUC19 as annealed with a mutagenic primer MBC1LGP6S (SEQ ID No. 37) and an M13 RV primer. The obtained DNA fragment was digested with BamHl and HindII and then subcloned into pUC19 which had been digested with BamHI and HindII. After sequencing, the plasmid was digested with BstPI and Aor51HI, and the resulting DNA fragment was ligated onto the plasmid hMBClLalambda / pUC19 which had been digested with BstPI and Aor51HI. The plasmid obtained in this way was designated "hMBClLclambda / pUC19". This plasmid was digested with EcoRI in order to provide a DNA fragment containing DNA encoding the humanized L chain. The fragment was introduced into the EcoRI site of the plasmid pCOSl in such a way that the initiation codon for the humanized L chain was located downstream of the EFlalfa promoter. The plasmid obtained in this way was designated "hMBClLclambda / pCOSl". Versions "d", "e" and "f" of the humanized L chain were also prepared using a mutagenesis technique by means of a polymerase chain reaction method. The "d", "e" and "f" versions were designed in such a way that the amino acid at position 91, tyrosine (corresponding to amino acid 87 according to the description of Kabat) was replaced by isoleucine in the "a" versions , "b" and "c", respectively. For each of the versions "d", "e" and "f", a polymerase chain reaction was carried out using each of the plasmids hMBClLalambda / pCOSl (for version "d"), hMBClLblambda / pCOSl ( for the "e" version) and hMBClLclambda / pCOSl (for the "f" version), respectively, as annealed, a mutagenic primer MBC1LGP11R (SEQ ID No. 38) and an M-Sl primer (SEQ ID No. 44). The DNA fragment obtained in this way was digested with BamHl and HindIII and then subcloned into pUC19 which had been digested with BAMHI and HindIII. After sequencing, the plasmid was digested with HindIII and BlnI, and the resulting digestion fragment was ligated onto the plasmid Clambda / pUC19 which had been digested with HindIII and BlnI. The plasmids obtained in this way were designated respectively "hMBClLdlambda / pUC19" (for version "d"), "hMBClLelambda / pUC19" (for version "e"), and "hMBClLflambda / pUC19" (for version "f" ). Each of these plasmids was digested with EcoRI to provide a DNA fragment containing DNA encoding the humanized L chain. The DNA fragment was introduced into the EcoRI site of the plasmid pCOSl in such a way that the initiation codon for the humanized L chain was located downstream of the EFlalfa promoter in the plasmid. The plasmids obtained in this way were designated respectively, "hMBClLdlambda / pCOSl" (for version "e"), "hMBClLelambda / pCOSl" (for version "e") and "hMBClLflambda / pCOSl" (for version "f" ).

Versions "g" and "h" of the humanized L chain were also prepared using a mutagenesis technique by means of a polymerase chain reaction method. The "g" and "h" versions were designed in such a way that the amino acid at position 36, histidine (corresponding to amino acid 36 according to Kabat's description) was replaced by tyrosine in "a" and "d" versions. ", respectively. The polymerase chain reaction was carried out using a mutagenic initiator MBC1LGP9R (SEQ ID No.39), M13 RV primer and the hMBClLalambda / pUC19 plasmid as annealed. An additional polymerase chain reaction was performed using the polymerase chain reaction product thus obtained and the M13 M4 primer as initiator and plasmid hMBClLalambda / pUC19 as annealed. The obtained DNA fragment was digested with HindIII and BlnI and then subcloned into the plasmid Clambda / pUC19 which had been digested with HindII and BlnI. Using this plasmid as an annealing, a polymerase chain reaction was carried out with the primers MBC1LGP13R (SEQ ID No. 40) and MBC1LVS1. The obtained polymerase chain reaction fragment was digested with Apal and HindIII and then introduced into any of the plasmids hMBClLalambda / pUC19 and hMBClLdlamda / pUC19 that had been digested with Apal and HindilII. The obtained plasmids were sequenced. Plasmids that were confirmed as containing the correct sequence were designated "hMBClLglambda / pUC19" (for version "g") and hMBClLhlambda / pUC19"(for version" h "). Each of these plasmids was digested with EcoRI with the In order to provide a DNA fragment containing DNA encoding the humanized L chain, the DNA fragment was introduced into the EcoRI site of the pCOSl plasmid in such a way that the initiation codon for the humanized L chain was located downstream of the EFlalfa promoter. The plasmids obtained in this way were respectively designated "hMBClLglambda / pCOSl" (for the "g" version and "hMBlLhlambda / pCOSl" (for the "h" version). The versions "i", "j", "k" , "1", "m", "n" and "o" of the humanized L chain were also prepared using a mutagenesis technique by means of a polymerase chain reaction method. using the plasmid hMBClLgla mbda / pUC19 as annealed with a mutagenic primer MBC1LGP14S (SEQ ID No. 41) and a VIRV (lambda) primer (SEQ ID No.43). The resulting DNA fragment was digested with Apal and Blnl and then subcloned into the plasmid hMBClLglambda / pUC19 which had been digested with Apal and Blnl. The obtained plasmid was sequenced, and the clone in which the mutation was introduced for each version was selected. The plasmid obtained in this way was designated "hMBClLx? / PUC19 (x = i, j, k, 1, m, n or o)". This plasmid was digested with EcoRI to provide a DNA fragment containing DNA encoding the humanized L chain. The DNA fragment was "introduced into the EcoRI site of the pCOSl plasmid in such a way that the initiation codon for the humanized L chain was located downstream of the EFlá promoter.The plasmid obtained in this way was designated" hMBClLx? / PCOSl "( x = i, j, k, 1, m, not well o) The DNA sequences (including the corresponding amino acid sequences) of the "j", "1", "m" and "o" versions appear in SEQ ID Nos: 67, 68, 69 and 70, respectively The amino acid sequences of these versions also appear in SEQ ID Nos: 48, 49, 50 and 51, respectively The versions "p", "q", "r" , "s" and "t" humanized chain L were designed in such a way that the amino acid in position 87 (tyrosine) was replaced by isoleucine in the versions "i", "j", "k", "1" and " These versions were prepared using the Aor51MI restriction site of FR3 and replacing this site with each of the versions "i", "j", "m", "1" or "o" r the site of the "h" version. That is to say, a restriction fragment Aor51HI (514 base pairs) containing CDR3, a portion of FR3 and all FR4, of a hMBClLx? / pCOSl expression plasmid (x = i, j, m, l or o) was removed. On the removed site, an Aor51HI restriction fragment (514 base pairs) was ligated into the hMBClLh? / PCOS expression plasmid, which contained "CDR3 and a portion of FR3 and all of FR4, such that the amino acid of position 91, tyrosine (corresponding to amino acid 87 according to the description of Kabat) was replaced by isoleucine, a clone was selected from each of versions "i", "j", "m", "1" and "or" in which the amino acid of the position, 91, tyrosine (which corresponds to amino acid 87 according to the Kabat description) was replaced by isoleucine, these modified versions corresponding respectively to the "i", "j" versions , "m", "1" and "o" were designated version "P" / ?? q "/ \? Rr / t lr" and "t", respectively. The obtained plasmid was designated "hMBClLx? / PCOSl (x = p, q, s, rot.) The DNA sequences (including the corresponding amino acids) of the" q ", lt 'versions appear in SEQ ID Nos: 71, 72 , 73 and 74, respectively The amino acid sequences of these versions also appear in SEQ ID Nos: 52, 53, 54 and 55, respectively The plasmid hMBClLq? / PCOSl was digested with HindIII and EcoRI and was then subcloned in the plasmid pUC19 which had been digested with Hindi and EcoRl The plasmid obtained in this way was designated "hMBClLq? / pUC19. The positions of the amino acids replaced in the individual versions of the humanized L chain appear in Table 2 below. Table 2 Amino acid positions replaced in the sequence lists (the amino acid numbering corresponds to the Kabat description) Versions 3 43 45 47 49 80 87 a - b D c d I e D I f I g Y h Y i Y K. j Y K D k Y K V 1 Y K V D m Y D n Y V or Y V D P Y K I q Y K D D I r Y D I s Y K V D I t Y V D I In Table 2, the block letters represent the following amino acids: Y: tyrosine; P: proline; K: lysine, V: valine; D: aspartic acid; and I: isoleucine. E. coli strains each containing the plasmids hMBClHcDNA / pCU19 and hMBClLq? / PUC19 were designated "Escherichia coli JM109 (hMBClHcDNA / pUC19)" and Escherichia coli JM109 (hMBClLq? / PUC19), respectively, which were deposited from in accordance with the terms of the Budapest Treaty before the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, (National Institute of Bioscience and Technology Humana, Industrial Science and Technology Agency), Japan, (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) on August 15, 1996, under accession number No. FERM BP-5629 for Escherichia coli JM109 (hMBClHcDNA / pUC19), and FERM BP -5630 for Escherichia coli JM109 (hmBClLq? / PUC19). (5) Transfection in COS-7 cell For the evaluation of antigen binding activity and the neutralizing activity of hybrid antibodies and humanized antibodies No. 23-57-137-1, expression plasmids were transiently expressed prepared above in COS-7 cells. For the transient expression of the L-chain hybrid antibodies, each of the following combinations of plasmids was co-transfected into a COS-7 cell by electroporation using a Generator (Bio Rad): hMBClHcDNA / pCOSl and h / mMBClL (? ) / neo; hMBClHcDNA / pCOSl and m / hMBClLa? / neo; hMBClHcDNA / pCOSl and m / hMBClLd? / neo; hMBClHcDNA / pCOSl and hmmMBClL (?) / neo; and hMBClHcDNA / pCOSl and mhmMBClL (?) / neo. That is, a suspension of cells (0.8 ml) of COS-7 cellsa in PBS (-) (lxlO7 cells / ml) was added with each combination of the plasmid DNAs (10 μg each). The resulting solution was applied with pulses to an electrostatic capacity of 1,500V and 25 μF. After 10 minutes of recovery period at room temperature, the cells subjected to electroporation were suspended in DMEM medium containing 2% ultra low-level IgG calf serum (GIBCO), and then cultured using a 10 cm culture dish in a C02 incubator. After culturing for 72 hours, a culture supernatant was collected which was centrifuged in order to remove cell debris. Solutions prepared in this way were provided for use in the ELISA assay below. For the transient expression of humanized antibodies No. 23-57-137-1, the combination of hMBClHcDNA / pCOSl and hMBClLx? / PCOSl (x = at) plasmids were cotransfected in a COS-7 cell using Gene Pulser (Bio Rad ) in the same way as described in the case of the hybrid antibodies above. The culture supernatants were prepared and provided for use in the ELISA assay below.

The purification of hybrid antibodies and humanized antibodies from the culture supernatants of COS-7 cells was carried out using a set of AffiGel Protein A MAPSII elements (Bio Rad) in accordance with the instructions included in the set of elements . (6) ELISA (i) Determination of antibody concentration An ELISA plate was prepared for the determination of the antibody concentration in the following manner. Each well of a 96-well ELISA plate (Maxisorp, NUNC) was coated with 100 μl of a coating buffer (0.1 M NaHC03, 0.02% NaN3) containing 1 μg / ml goat antihuman IgG antibody (TAGO), and then blocked with 200 μl of a regulator of dilution [50 mM Tris-HCl, 1 mM MgCl2, 0.1 M NaCl, 0.05% Tween 20, 0.02% NaN3, 1% bovine serum albumin (BSA); pH 7.2]. Each of the wells received each of the serial dilutions of the COS cell culture supernatant where each of the hybrid antibodies and humanized antibodies was expressed, or each of the serial dilutions of each was received. of the hybrid antibodies and humanized antibodies in a purified form. The plate was incubated at room temperature for 1 hour and then washed with PBS-Tween 20. Subsequently, each well received 100 μl of goat antihuman IgG antibody conjugated with alkaline phosphatase (TAGO). The dish was incubated at room temperature for 1 hour and washed with PBS-Tween 20. Subsequently, each of the wells received 1 mg / ml of a substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, SIGMA). 5 The solution in each well was measured in its absorbance at 405 nm using a microplate reader (Bio Rad) in order to determine the concentration of antibodies. In this determination, Hu IgGl was used? purified (the binding site) as standard. 10 (ii) Determination of antigen binding capacity • An ELISA plate was prepared to determine antigen binding capacity in the following manner. Each well of a 96-well ELISA plate (Maxisorp, NUNC) was coated with 100 μl of a coating buffer containing 1 μg / ml of human PTHrP (1-34) and then blocked with 200 μl of a dilution regulator. Subsequently, each well received each of the serial dilutions of the supernatant of • culture of COS-7 cells in which each of the hybrid antibodies and humanized antibodies was expressed, or received each of the serial dilutions of each of the hybrid antibodies and humanized antibodies in a purified form. The plate was incubated at room temperature and washed with PBS-Tween 20. Subsequently, each well received 00 μl of goat antihuman IgG antibody. conjugated with alkaline phosphatase (TAGO). The plate was incubated at room temperature and washed with PBS-Tween 20. Subsequently, each well received 1 mg / ml of a substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, SIGMA). The solution was measured in its absorbance at 405 nm using a microplate reader (Bio Rad). (7) Confirmation of activities (i) Evaluation of the humanized H chain An antibody having an "a" version of humanized H chain and a chimeric L chain was found to have the same level of PTHrP binding activity as a chimeric antibody (see figure 6). This result suggests that the "a" version achieved the humanization of the H chain V region to a sufficient degree to evaluate the humanization. Accordingly, version "a" of the humanized H chain was supplied for use as an H chain of humanized antibody in the following experiments, (ii) Hybrid antibody activity (ii-a) hybrid antibody FRl, 2 / FR3, 4 When the L chain was h / mMBClL (?), No antigen binding activity was observed. In contrast, when the L chain was either m / hMBClLa? or m / hMBClLd ?, the same level of antigen binding activity was observed as the level observed in the case of chimeric antibody No. 23-57-137-1 (Figure 7). These results suggest that there is no problem regarding FR3 and FR4, but that there are amino acid residue (s) that must be replaced in FR1 and FR2 for the preparation of a humanized antibody, (ii-b) FR1 / FR2 hybrid antibody When the L chain was mhmMBClL (?), No antigen binding activity was observed. In contrast, when the L chain was hmmMBClL (?), The same level of antigen binding activity was observed as the level observed in the case of the chimeric antibody No. 23-57-137-1 (FIG. 8). These results suggest that there is no problem regarding FRl but that there are amino acid residue (s) that must be replaced in FR2 for the preparation of a humanized antibody. (iii) Humanized antibody activity Humanized antibodies were determined, each having the "a" to "t" versions of L chain in the antigen binding activity. As a result, it was found that humanized antibodies that have versions "j", "1", "m", "or", "q", "r", "s" and "t" of L chain presented the same level of binding activity with PTHrP than the level presented by the chimeric antibody (figures 9 to 12). (8) Establishment of a CHO cell line capable of stable antibody production For the establishment of a cell line capable of stable production of humanized antibodies, each of the expression plasmids prepared above was introduced into a CHO cell (DXB11) . That is, the establishment of a cell line capable of stably producing a humanized antibody was carried out using each of the following combinations of plasmids as expression vectors for a CHO cell: hMBClHcDNA / pCHOl and hMBClLm? / PCOSl; hMBClHcDNA / pCHOl and hMBClLq? / pCOSl; and hMBClHcDNA / pCHOl and hMBClLr? / pCOSl. The plasmids were co-transfected into a CHO cell by electroporation using Gene Pulser (Bio Rad). Subsequently, the expression vectors were separately dissociated with the restriction enzyme Pvul to provide fragments of linear DNA. The resulting DNA fragments were extracted with phenol and chloroform and then precipitated with ethanol. The DNA fragments prepared in this way were used in the subsequent electroporation. Plasmid DNA fragments (10 μg each) were added to 0.8 ml of a cell suspension of CHO cells in PBS (-) (Ix107 cells / ml). The resulting solution was applied with pulses of an electrostatic capacity of 1500 V and 25 μF. After 10 minutes of recovery period at room temperature, cells treated in this way were suspended in a MEM-alpha medium (GIBCO) containing 10% fetal calf serum (GIBCO), and then cultured in a C02 incubator using 96-well plates (Falcon).

The following day after the start of the culture, the medium was replaced by a selective media containing ribonucleoside or deoxyribonucleoside-free MEM-alpha containing 10% fetal calf serum (GIBCO) and 500 mg / ml GENETICIN (G418Sulfate; GIBCO). From the culture medium, cells in which the antibody gene was introduced were selected. The culture medium was replaced by a fresh medium. Approximately 2 weeks after the replacement of the medium. The cells were observed through the microscope. When favorable cell growth was observed, the cells were determined based on the amount of antibodies produced by conventional ELISA for the determination of antibody concentration in accordance with what was mentioned above. Among the cells, those that produced a greater amount of antibodies were screened. The culture of the established cell line stabilized antibody production layers was increased in a roller bottle using a ribonucleoside or deoxyribonucleoside-free MEM-alpha medium containing fetal calf serum of 2% ultra low IgG. On day 3 and day 4 of the culture, the culture supernatant was collected, which was filtered using a 0.2μm filter (Millipore) in order to remove cell debris therefrom. Purification of the humanized antibodies from the culture supernatant of the CHO cells was carried out using a POROS Protein A (PerSeptive Biosystems) column in ConSep LC100 (Millipore) in accordance with the attached instructions. Humanized antibodies were provided for use in the determination of neutralization activity and examination of pharmacological efficacy in hypercalcemic model animals. The concentration and antigen binding activity of the purified humanized antibodies were determined by the ELISA system in accordance with the above. [Reference Example 5] determination of the neutralizing activity determination of the neutralizing activity of the mouse antibodies, the chimeric antibodies and the humanized antibodies was carried out using the cells of a rat myeloma cell line ROS17 / 2.8- 5. ROS17 / 2.8-5 cells were cultured in a Ham's F-12 medium (GIBCO) containing 10% fetal calf serum (GIBCO) in a C02 incubator. ROS17 / 2.8-5 cells were seeded in each well of a 96-well plate at a rate of 10 4 cells / lOOμl / well and cultured for 1 day. After finishing the culture, the culture medium was replaced by the Ham's medium = F-12 (GIBCO) containing 4 M of Hydrocortisone and 10% of fetal calf serum. After cultivation for 3 to 4 days, the cultured cells were washed with 260μl of Ham's F-12 medium = (GIBCO) and then 80μl of Ham's F-12 medium containing 1 mM of isobutyl-1-methyl xanthine ( IBMX, SIGMA), 10%. of fetal calf serum and 10 mM of HEPES. The resulting mixture was incubated at a temperature of 37 ° C for 30 minutes. The culture media of the mouse antibodies, the chimeric antibodies and the humanized antibodies to be tested for neutralizing activity were previously serially diluted in the following groups: [10 μg / ml, 3.3 μg / ml, 1.1 μg / ml and 0.37 μg / ml], [10 μg / ml, 2 μg / ml, 0.5 μg / ml and 0.01 μg / ml] and [10 μg / ml, 5 μg / ml, 1.25 μg / ml, 0.63 μg / ml and 0.31 μg / ml ] Each of the diluted antibody sample solutions were mixed with an equivalent amount of 4 ng / ml of PTHrP - (1-34). The resulting mixed solution (80μl) was added to each well. In each well, the final concentration of each antibody was returned * from the above-mentioned concentration of the antibody, and consequently the concentration of PTHrP (1-34) became 1 ng / ml. After treatment at room temperature for 10 minutes, the culture supernatant was removed and the residue was washed with PBS 3 times. Subsequently, cAMP was extracted in the cells with 100μl of 0.3% HCl -95% ethanol and then evaporated using a water jet aspirator in order to remove the HCl-ethanol. The residue was dissolved in 120μl of an EIA regulator attached to cAMP (CAYMAN CHEMICAL 'S) in order to extract cAMP from there. CAMP was determined by using the cAMP EIA Kit (CAYMAN CHEMICAL 'S) in accordance with the instructions included in the Element Set. As a result, it was found that, among the humanized antibodies that have the same level of antigen binding activity, those that have "q", "r", "s" and "t" versions of L chain (where the tyrosine in the position 91 was replaced by isoleucine) showed the neutralizing activity closest to the neutralizing activity of the chimeric antibody, and those with a "q" version of L chain presented the highest neutralizing activity (figures 13 to 15). Industrial Application In accordance with what has been described above, the present invention offers a therapeutic agent for the treatment of cachexia comprising, as an active ingredient, a substance capable of inhibiting the binding between PTHrP and a receptor thereof. In pharmacological efficacy tests that use cachexia model animals, this substance can prevent weight loss and prolong the survival time compared to a control. Therefore, the substance is useful for the treatment of cachexia.UENCES 2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAATAGCCCT TGACCAGGCA 20 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CTGGTTCGGC CCACCTCTGA AGGTTCCAGA ATCGATAG_38_(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = - "Synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GAGATCCCGGG CCAGTGGATA GACAGATG 28 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs - (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GGATCCCGGG TCAGRGGAAG GTGGRAACA 29 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GTTTTCCCAG TCACGAC 17 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: • (A) LENGTH: 17 base pairs 5 (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CAGGAAACAG CTATGAC 17 (2) INFORMATION FOR SEQ ID NO : 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs 15 (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other acid nucleic (A) DESCRIPTION: / desc = "synthetic DNA" 20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GTCTAAGCTT CCACCATGAA ACTTCGGGCT C 31 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs 25 (B) TYPE: nucleic acid (C) CHAIN TYPE: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TGTTGGATCC CTGCAGAGAC AGTGACCAGA 30 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE; other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GTCTGAATTC AAGCTTCCAC CATGGGGTTT GGGCTG 36 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 TTTCCCGGGC CCTTGGTGGA GGCTGAGGAG ACGGTGACCA G 41 (2) INFORMATION FOR SEQ ID NO: 11: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 109 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION : / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11; GTCTGAApc AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA CCCCACGGTC ACCCTGTTCC 60 CGCCCTCCTC TGAGGAGCTC CAAGCCAACA AGGCCACACT AGTGTGTCT 109 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 110 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGTTTGGTGG TCTCCACTCC CGCCTTGACG GGGCTGCCAT CTGCCTTCCA GGCCACTGTC 60 ACAGCTCCCG GGTAGAAGTC ACTGATCAGA CACACTAGTG TGGCCTTGTT 110 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13; GGAGTGGAGA CCACCAAACC CTCCAAACAG AGCAACAACA AGTACGCGGC CAGCAGCTAC 60 CTGAGCCTGA CGCCCGAGCA GTGGAAGTCC CACAGAAG 98 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 106 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single - (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TGTTGAATTC TTACTATGAA CATTCTGTAG GGGCCACTGT CTTCTCCACG GTGCTCCCTT 60 CATGCGTGAC CTGGCAGCTG TAGCTTCTGT GGGACTTCCA CTGCTC 106 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GTCTGAATTC AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA 43 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deSc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TGTTGAATTC TTACTATGAA 20 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CAACAAGTAC GCGGCCAGCA GCTACCTGAG CCTGACGCC 39 (2) INFORMATION FOR SEQ ID NO: 18: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION : / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GTAGCTGCTG GCCGCGTACT TGTTGTTGCT CTGTTTGGA 39 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GTCTGAATTC AAGCTTAGTC CTAGGTCGAA CTGTGGCTGC ACCATC 46 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: TGTTGAATTC TTACTAACAC TCTCCCCTGT TGAA 34 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GTCTAAGCTT CCACCATGGC CTGGACTCCT CTCTT 35 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: TGTTGAATTC AGATCTAACT ACTTACCTAG GACAGTGACC TTGGTCCC 48 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 128 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Synthetic DNA" (XI) DESCRI PC SECONSE IA: SEQ ID NO: 23: GTCTAAGCTG CCACCATGGG GTTTGGGCTG AGCTGGGTTT TCCTCGTTGC TCG? TAAGA 60 GGTGTCCAGT GTCAGGTGCA GCTGGTGGAG TCTGGGGGAG GCGTGGTCCA GCCTGGGAGG 120 TCCCTGAG 128 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 125 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) 'DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24; ACCATTAGTA GTGGTGGTAG TTACACCTAC TATCCAGACA GTGTGAAGGG GCGATTCACC 60 ATCTCCAGAG ACAATTCCAA GAACACGCTG TATCTGCAAA TGAACAGCCT GAGAGCTGAG 120 GACAC 225 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 132 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25; CTACCACCAC TACTAATGGT TGCCACCCAC TCCAGCCCCT TGCCTGGAGC CTGGCGGACC 60 CAAGACATGC CATAGCTACT GAAGGTGAAT CCAG4GGCTG CACAGGAGAG TCTCAGGGAC 120 CTCCCAGGCT GG 132 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 110 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TGTTGGATCC CTGAGGAGAC GGTGACCAGG GTTCCCTGGC CCCAGTAAGC AAAGTAAGTC 60 ATAGTAGTCT GTCTCGCACA GTAATACACA GCCGTGTCCT CAGCTCTCAG 110. (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear • ( ii) TYPE OF MOLECULE: other nucleic acid 5 (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GTCTAAGCTT CCACCATGGG GTTTGGGCTG 30 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: 10 (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 15 ( A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: TGTTGGATCC CTGAGGAGAC GGTGACCAGG 30 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: 20 (A) LENGTH: 133 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 25 (A) DESCRIPTION: / desc = "AD Synthetic N "(XI) SEQUENCE DESCRITION: SEQ ID NO: 29: ACAAAGC? C CACCATGGCC TGGACTCCTC TCTTCTTCTT CT? GTTCTT CATTGCTCAG 60 GTTCTTTCTC CCAGCTTGTG CTGACTCAAT CGCCCTCTGC CTCTGCCTCC CTGGGAGCCT 120 CGGTCAAGCT CAC 133 (2) INFORMATION FOR SEQ ID NO: 30: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) DESCRIPTION .DE. SEQUENCE: SEQ ID NO: 30; AGCAAGATGG AAGCCACAGC ACAGGTGATG GGATTCCTGA TCGCTTCTCA GGCTCCAGCT 60 CTGGGGCTGA GCGCTACCTC ACCATCTCCA GCCTCCAGTC TGAGGATGAG GCTGACTA 118 [2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 128 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31 CTGTGGCTTC CATCTTGCTT AAGTTTCATC AAGTACCGAG GGCCCTTCTC TGGCTGCTGC 60 TGATGCCATT CAATGGTGTA CGTACTGTGC TGACTACTCA AGGTGCAGGT GAGCTTGACC 120 GAGGCTCC 128 (2 ) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 114 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE MOLECULE: another nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CCCTCACAAA 60 TTGTTCCGGA ATTGTATCAC CCACACCACA GTAATAGTCA GCCTCATCCT CAGA 114 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: ACAAAGCTTC CACCATG 17 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: "nucleic acid (C) CHAIN TYPE: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CTTGGATCCG GGCTGACCT 19 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 75 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (i) DESCRIPTION OF SEQUENCE: HE Q ID NO: 35: CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CGTACACAAA 60 TTGTTCCTTA ATTGT 75 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" ((xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36; 5 AAAGGATCCT TAAGATCCAT CAAGTACCGA GGGGGCTTCT CTG 43 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid 10 (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: 15 ACAAAGCTTA GCGCTACCTC ACCATCTCCA GCCTCCAGCC TGAGGA 46 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 111 base pairs (B) TYPE: nucleic acid 20 (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: 25 CTTGGATCCG GGCTGACCTA GGACGGTCAG TpGGTCCCT CCGCCGAACA CGTACACAAA 60 TTGTTCCTTA ATTGTATCAC CCACACCACA GATATAGTCA GCCTCATCCT C 111 • (2) INFORMATION FOR SEQ ID NO: 39: 5 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear 10 __ '"(ii) TYPE OF MOLECULE : another nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: CTTCTCTGGC TGCTGCTGAT ACCATTCAAT 'GGTGTACGTA CT 42 (2) INFORMATION FOR SEQ ID NO: 40: 15 (i) ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear 20 (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION : / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: CGAGGGCCCT TCTCTGGCTG CTGCTG 26 (2) INFORMATION N FOR SEQ ID NO: 41: 25 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) TYPE MOLECULE: another nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: GAGAAGGGCC CTARGTACST GATGRAWCTT AAGCA 35 (2) INFORMATION FOR SEQ ID NO: 42:, (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: CACGAATTCA CTATCGATTC TGGAACCTTC AGAGG 35 (2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GGCTTGGAGC TCCTCAGA 18 (2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear 10 (ii) TYPE OF MOLECULE: other nucleic acid • (A) DESCRIPTION: / desc = "Synthetic DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: GACAGTGGTT CAAAGTTTTT. 20 (2) INFORMATION FOR SEQ ID NO: 45: 15 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TYPE: amino acid • (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein 20 - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: Gln Leu Val Leu Thr Gln Ser Ser Be Wing Ser Phe Ser Leu Gly 1 5 10 15 Wing Being Wing Lys Leu Thr Cys Thr Leu Being Ser Gln His Ser Thr 20 25 30 . Tyr Thr lie Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro Lys 35 40 45 25 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Ala Asp Arg 65 70 75 Tyr Leu Ser Be Asn He Gln Pro Glu Asp Glu Wing Met Tyr 80 85 90 He Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly Gln Pro 110 115 (2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS: 10 (A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 46 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly 1 5 10 - 15 Gly Ser Leu Lys Leu Ser Cys Wing Wing Ser Gly Phe Thr Phe Ser 20 25 30 • Ser Tyr Gly Met Ser Trp He Arg Gln Thr Pro Asp Lys Arg Leu 35 40 45 Glu Trp Val Wing Thr He Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr 50 55 60 20 Pro Asp Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Wing 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp 80 85 90 Thr Wing Met Phe Tyr Cys Wing Arg Gln Thr Thr Met Thr Tyr Phe 95 ICO 105 Wing Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Wing 25 110 H5 (2) INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: • (A) LENGTH: 116 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47 Gln Leu Val Leu Thr Gln Ser Pro Be Wing Being Wing Being Leu Gly 10 1 5 10 15 Wing Being Val Lys Leu Thr Cys Thr Leu Being Ser Gln His Being Thr 20 25 30 Tyr Thr He Glu Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 15 65 70 75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 110 115 20 (2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TYPE: amino acid 25 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 í Gln Leu Val Leu Thr Gln Ser Pro Be Wing Being Wing Being Leu Gly 1 5 10 15 Wing Being Val Lys Leu Thr Cys Thr Leu Being Ser Gln His Being Thr 20 25 30 Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 65 70 75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115 (2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 49: Gln Leu Val Leu Thr Gln Ser Pro Be Wing Being Wing Being Leu Gly 1 5 10 15 Wing Being Val Lys Leu Thr Cys Thr Leu Being Ser Gln His Being Thr, 20 25 30 Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 65 70 75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115 (2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50 : Gln Leu Val Leu Thr Gln Ser Pro Be Ala Be Ala Be Leu Gly 1 5 10. 15 Wing Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 65 70 75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115 (2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids ( B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 51: Gln Leu Val Leu Thr Gln Ser Pro Be Wing Being Wing Being Leu Gly 1 5 10 15 Wing Being Val Lys Leu Thr Cys Thr Leu Being Ser Gln His Being Thr 20 25 30 Tyr Thr He Glu Trp Tyr Gln Gln Glp Pro Glu Lys Gly Pro Arg 35 '40"45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 65 70 75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115 (2) INFORMATION FOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein * (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 52 ' Gln Leu Val Leu Thr Gln Ser Pro Be Wing Being Wing Being Leu Gly 1 5 10 15 Wing Being Val Lys Leu Thr Cys Thr Leu Being Ser Gln His Being Thr 20 25 30 Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 65 70 75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90 He Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95. 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115 (2) INFORMATION FOR SEQ ID NO: 53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 53: Gln Leu Val Leu Thr Gln Ser Pro Be Wing Being Wing Being Leu Gly 1 5 10 _,, 15 Wing Being Val Lys Leu Thr Cys Thr Leu Being Ser Gln His Being Thr 20 25 30 Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 65 70 75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90 He Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95 ICO 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115 (2) INFORMATION FOR SEQ ID NO: 54: • (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 15 (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54; Gln Leu Val Leu Thr Gln Ser Pro Be Wing Being Wing Being Leu Gly • 1 5 10 15 Wing Being Val Lys Leu Thr Cys Thr Leu Being Ser Gln His Being Thr 20 25 30 Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 20 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 65 70"75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90, He Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val | 25 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115 (2 ) INFORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: pro tein (xi) DESCRIPTION OF SEQUENCE : SEQ ID NO: 55; Gln Leu Val Leu Thr Gln Ser Pro Be Wing Being Wing Being Leu Gly 1 5 10 15 Wing Being Val Lys Leu Thr Cys Thr Leu Being Ser Gln His Being Thr 20 - .25 30 Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Ser Gly Wing Glu Arg 65 70 75 Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp Glu Wing Asp Tyr 80 85 90 He Cys Gly Val Gly Asp Thr He Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115 (2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 amino acids (B) TI PO: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 56: Gln Val Gln Leu Val Glu Ser Gly Gly Val Val Gln Pro Gly 1 5 10 15 Arg Ser Leu Arg Leu Ser Cys Wing Wing Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met Ser Trp Val Arg Gln Wing Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Wing Thr He Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr 50 55 60 Pro Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 '85 90 Thr Wing Val Tyr Tyr Cys Wing Arg Gln Thr Thr Met Thr Tyr Phe 95' ICO 105 Wing Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 (2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57; ATG AAC TTC GGG CTC AGC TTG ATT TTC CTT GCC CTC ATT TTA AAA 45 Met Asn Phe Gly Leu Ser Leu He Phe Leu Ala Leu lie i PU Lys -15 -10 -5 GGT GTC CAG TGT GAG "GTG CAA CTG GTG GAG TCT GGG GGA" GAC TTA 90 Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu 1 5 10 GTG AAG CCT GGA GGG TCC CTG AAA CTC TCC TGT GCA GCC TCT GGA 135 Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Wing Wing Ser Gly 15 20 25 pc ACT pc AGT AGC TAT GGC ATG TCT TGG ATT CGC CAG ACT CCA 180 Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp He Arg Gln Thr Pro 30 35 40 GAC AAG AGG CTG GAG TGG GTC GCA ACC ATT AGT AGT GGT GGT AGT 225 Asp Lys Arg Leu Glu Trp Val Wing Thr He Ser Ser Gly Gly Ser 45 50 55 TAC ACC TAC TAT CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC 270 Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr He Ser 60 65 70 AGA GAC AAT GCC AAG AAC ACC CTA CT CTG CAG ATG AGC AGT CTG 315 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu 75 80 '- 85 AAG TCT GAG GAC ACA GCC ATG TTT TAC TGT GCA AGA CAG ACT ACT 360 Lys Ser Glu Asp Thr Wing Met Phe Tyr Cys Wing Arg Gln Thr Thr 90 95 100 ATG ACT TAC TIT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC 405 Met Thr Tyr Phe Wing Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 105 110 115 TCT GCA 411 Ser Wing (2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear; i i) T IO OF MOLECULE: cDNA to mRNA (xi) DESCRI SEQUENCE PC: SEQ I D NO: 5Í • ATG GGG Tp GGG CTG AGC TGG GTT TTC CTC GTT GCT CTT TTA AGA 45 Met Gly Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg -15 -10 -5 GGT GTC CAG TGT CAG GTG CAG CTG GTG GAG TCT GGG GGA GGC GTG 90 Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Val Gly Gly Val 1 5 10 GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GCC GCC TCT GGA 135 Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Wing Ser Gly 15 20 25 pc ACC AGT AGC TAT GGC ATG TCT TGG GTC CGC CAG GCT CCA 180 10 Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro 30 35 40 GGC AAG GGG CTG GAG TGG GTG GCA ACC Ap AGT AGT GGT GGT AGT 225 Gly Lys Gly Leu Glu Trp Val Wing Thr He Ser Ser Gly Gly Ser 45 50 55 TAC ACC TAC TAC CAC GAC AGT GTG AAG GGG CGA pC ACC ATC TCC 270 Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr He Ser 60 65 70 15 AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG 315 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 75 80. 85 AGA GCT GAG GAC ACG GCT GTG TAT TAC TGG GCG AGA CAG ACT ACT 360 Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys Wing Arg Gln Thr Thr 90 95 100 ATG ACT TAC TIT GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC 405 Met Thr Tyr Phe Wing Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 20 105 110 115 TCC TCA 411 Ser Ser (2) INFORMATION FOR SEQ ID NO: 59: (i) SEQUENCE CHARACTERISTICS: 25 (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: Lys Ala Ser Gln Asp Val Asn Thr Ala Val Ala 1 5 10 (2) INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids 10 (B) TYPE: amino acid (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: Ser Ala Ser Asn Arg Tyr Thr 1 5. 15 (2) INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid 20 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: Gln Gln His Tyr Ser Thr Pro Phe Thr i 1 5 (2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 62: Pro Tyr Trp Met GIn 1 5 [2) INFORMATION FOR SEQ ID NO: 63: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: Ser He Phe Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys Phe Lys Gly 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 64 Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr '1 5 10 (2) INFORMATION FOR SEQ ID NO: 65: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs ( B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65 ATG GCC TGG ACT CCT CTC pc pc pc pr GGT cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CA Cp GTG CTC ACT CAG TCA TCT TCA GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ala Ser 1. 5 10 15 pC TCC CTG GGA GCC TCA GCA AAA CTC ACG TGC ACC pG AGT AGT 135 Phe Ser Leu Gly Ala Be Ala Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC Ap GAA TGG TAT CAG CAÁ CAG CCA CTC 180 • Gln His Ser Thr Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Leu 30 35 40 AAG CCT AAT TAT GTG ATG GAT CTT AAG CA GAT GGA AGC CAC 225 Lys Pro Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 20 - - - 45 50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pC TCT GGA TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Ser Ser 60 65 70 GGT GCT GAT CGC TAC CTT AGC Ap TCC AAC ATC CAG CCA GAA GAT 315 Gly Wing Asp Arg Tyr Leu Ser He Be Asn He Gln Pro Glu Asp 75 80 85 GAA GCA ATG TAC ATC TGT GGT GTG GGT GAT ACA Ap AAG GAA CAA 360 25 Glu Ala Met Tyr He Cys Gly Val Gly Asp Thr He Lys Glu Gln 90 95. 100? T GTG TAT Gp pc GGC GGT GGG ACC AAG GTC ACT GTC CTA GGT 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 105. 110 H5 CAG CCC 411 Gln Pro (2) INFORMATION FOR SEQ ID NO: 66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 405 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double ( D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 66: ATG GCC TGG ACT CCT CTC pc pc pc TGG G? cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CAG CTT GTG CTG ACT CAG TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Wing Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC pG AGT AGT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT AC TAC ACC Ap GAA TGG CAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr He Glu Trp His Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT CGG TAC pG ATG AAA CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Being Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA Ap GM GM CM 360 Glu Wing Asp Tyr Tyr Cys Gly Val Gly Asp Thr He Lys Glu Gln 90 95 100 Tp GTG TAC GTG pC GGC GGA GGG ACC AM CTG ACC GTC CTA GGT 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 (2) INFORMATION FOR SEQ ID NO: 67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE:, double (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: ATG GCC TGG ACT CCT CTC pc pc pc pT crr CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CAG CTT GTG CTG ACT CM TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Be Wing Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC MG CTC ACC TGC ACC TTC.AGT ACT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC Ap GM TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 MG GGC CCT MG TAC CTG ATG GAT CTT MG CM GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Ser Be Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA Ap AAG GM CM 360 Glu Ala Asp Tyr Tyr Cyr Gyr Gly Val Gly Asp Thr He Lys Glu Gln 90 95 100 Tp GTG TAC GTG pC GGC GGA GGG ACC AM CTG ACC CTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 10 CAG CCC 411 Gln Pro (2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs 15 (B) TYPE: nucleic acid (C) CHAIN TYPE: double ( D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG GCC TGG ACT CCT CTC pc pc pc pr Gp cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CAG CTT GTG CTG ACT CM TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Be Wing Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC MG CTC ACC TGC ACC p AGT AGT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Ser * Ser 25 15 20 25 CAG CAC AGT ACG TAC ACC Ap GM TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr He Glu Trp Tyr Gln Gln Gl n Pro Glu 30 35 40 • MG GGC CCT MG TAC GTG ATG GAT Cp AAG CM GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pc TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Ser Be Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT TAC TAC TGT GGT GTG GGT GAT ACA Ap MG GM CM 36 0 10 Glu Wing Asp Tyr Tyr Cys Gly Val Gly Asp Thr He Lys Glu Gln 90 95 100 • Tp GTG TAC GTG pc GGC GGA GGG ACC AM CTG ACC CTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 * - Gln Pro 15 (2) INFORMATION FOR SEQ ID NO: 69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) ) TYPE OF CHAIN: double 20 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 69: ATG GCC TGT ACT CCT CTC pc pc pc pr CTT cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CAG CTT GTG CTG ACT CM TCG CCC TCT GCC TCT 90 25 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Be Wing Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC MG CTC ACC TGC ACC? G AGT AGT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC Ap GM TGG TAT CAG CAG CAG CCA GAG 180 Gln Hi s Be Thr Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu 30 35. 40 MG GGC CCT AGG TAC CTG ATG GAT CTT MG CM GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Ser Ser 60 65 70 GGG GCT CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT TAC TAC TGT GGT GTG GGT GAT ACA Ap GM GM CM 360 Glu Wing Asp Tyr Tyr Cys Gly Val Gly Asp- Thr He Lys Glu Gln 90 95 100 Tp GTG TAC GTG pC GGC GGA GGG ACC AM CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro (2) INFORMATION FOR SEQ ID NO: 70: (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: ATG GCC TGG ACT CCT CTC pc pc pc pr Gp cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys' Ser -15 -10 -5 GGT TCT pC TCC CAG CTT GTG CTG ACT CM TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Be Wing Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC MG CTC ACC TGC ACC p AGT AGT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC Ap GM TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 MG GGC CCT AGG TAC GTG ATG GAT CTT MG CM GAT GGA AGC CAC. 225 Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Ser Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT TAC TAC TGT GGT GTG GGT GAT ACA Ap MG GM CM 360 Glu Wing Asp Tyr Tyr Cys Gly Val Gly Asp Thr He Lys Glu Gln 90 95 ICO Tp GTG TAC GTG pC GGC GGA GGG ACC AM CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro 20 (2) INFORMATION FOR SEQ ID NO: 71: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid 25 (C) TYPE OF CHAIN : double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71 ATG GCC TGG ACT CCT CTC pc pc pc tp Gp cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CAG CTT GTG CTG ACT CM TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Be Wing Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC MG CTC ACC TGC ACC p AG AGT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Be Ser 15 20 25 CAG CAC AGT ACG TAC ACC Ap GM TGG TAT CAG CAG CAG CCA GAG 10 180 Gln His Ser Thr Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 MG GGC CCT MG TAC CTG ATG GAT CTT AAG CM GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 -50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pc TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp A .rg Phe Ser Gly Ser Ser Ser 15 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT TAC ATC TGT GGT GTG GGT GAT ACA A MG GM CM 360 G lu Ala Asp Tyr He Cys Gly Val Gly Asp Thr He Lys- Glu Gln 90 95 ICO Tp GTG TAC GTG pC GGC GGA GGG ACC AM CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro (2) INFORMATION FOR SEQ ID NO: 72: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO; 72 ATG GCC TGG ACT CCT CTC ire pc pc pr Gp cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CAG Cp GTG CTG ACT CM TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Wing Ser 10 1 5 10 • GCC TCC CTG GGA GCC TCG GTC MG CTC ACC TGC ACC p AGT AGT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC Ap GM TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 MG GGC CCT AGG TAC CTG ATG GAT CTT MG CM GAT GGA AGC CAC 225 15 Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pc TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Ser Be Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 20 75 80 85 GAG GCT TAC ATC TGT GGT CTG GGT GAT ACA Ap AAG G M CM 360 Glu Wing Asp Tyr He Cys Gly Val Gly Asp Thr He Lys Glu Gln 90 95 100 Tp GTG TAC GTG pc GGC GGA GGG ACC AM CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 25 Gln Pro (2) INFORMATION FOR SEQ ID NO: 73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: ATG GCC TGG ACT CCT CTC pc pc pc pr Gp cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CAG Cp GTG CTG ACT CM TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Wing Ser 1 '5 10 - GCC TCC CTG GGA GCC TCG GTC MG CTC ACC TGC ACC? G AGT AGT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 15 20 25 * CAG CAC AGT ACG TAC ACC Ap GM TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 MG GGC CCT AAG TAC GTG ATG GAT Cp MG CA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 '55 20 AGC ACA GGT GAT GGG Ap CCT GAT CGC pc TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Be Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA Ap GM GM CM 360 Glu Wing Asp Tyr He Cys Gly Val Gly Asp Thr He Lys Glu Gln 25 90 95 100 Tp GTG TAC GTG pC GGC GGA GGG ACC AM CTG ACC GTC XTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 HO H5 CAG CCC 411 Gln Pro (2) INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN : double * (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74 ATG GCC TGG ACT CCT CTC pc pc pc tp Gp cp CAT TGC TCA 45 Met Wing Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT pC TCC CAG Cp GTG CTG ACT CM TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Be Wing Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC MG CTC ACC TGC ACC p AG AGT 135 Wing Ser Leu Gly Wing Ser Val Lys Leu Thr Cys Thr Leu Be Ser 15 20"25 CAG CAC AGT ACG TAC ACC Ap GM TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr He Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC GTG ATG GAT CTT MG CM GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG Ap CCT GAT CGC pC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly He Pro Asp Arg Phe Ser Gly Ser Be Ser 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Wing Glu Arg Tyr Leu Thr He Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA Ap MG GM CM 360 Glu Ala As p Tyr He Cys Gly Val Gly Asp Thr He Lys Glu Gln 90 95 100 i GTG TAC GTG pc GGC GGA GGG ACC AM CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro (2) INFORMATION FOR SEQ ID NO: 75: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: line_al (ii) TYPE OF MOLECULE : peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15 Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu He Wing Glu 20 25 30 He His Thr Ala

Claims (1)

1. CLAIMS A therapeutic agent for the treatment of cachexia comprising, as an active ingredient, a substance capable of inhibiting the link between the protein related to parathyroid hormone (PTHrP) and a receptor thereof. The therapeutic agent for the treatment of cachexia of claim 1, wherein the substance is an antagonist against PTHrP. The therapeutic agent for the treatment of cachexia according to claim 1, wherein the substance is an anti-PTHrP antibody. The therapeutic agent for the cachexia treatment of claim 1, wherein the substance is a fragment of an anti-PTHrP antibody and / or a modified fragment thereof. The therapeutic agent for the treatment of cachexia of claim 3 or of claim 4, wherein the antibody is a humanized or chimeric antibody. The therapeutic agent for the treatment of cachexia of claim 5, wherein the humanized antibody is a humanized antibody number 23-57-137-1. The therapeutic agent for the treatment of cachexia of claim 3 or of claim 4, wherein the antibody is of the monoclonal type. The therapeutic agent for the treatment of cachexia of any of claims 1 to 7, wherein cachexia is a cachexia induced by cancer.