CA2401357A1

CA2401357A1 - Tissue degradation inhibitor agent

Info

Publication number: CA2401357A1
Application number: CA002401357A
Authority: CA
Inventors: Hidemi Saito; Toshiaki Tsunenari; Etsuro Onuma; Koh Sato
Original assignee: Individual
Current assignee: Chugai Pharmaceutical Co Ltd
Priority date: 2000-02-28
Filing date: 2000-08-30
Publication date: 2001-09-07
Also published as: WO2001064249A1

Abstract

A tissue decomposition inhibitor which contains a substance inhibiting the binding of a parathyroid hormone-associated peptide to its receptor.

Description

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DESCRIPTION
TISSUE DEGRADATION INHIBITING AGENT
TECHNICAL FIELD
The present invention relates to a tissue degradation inhibiting agent which comprises a substance inhibiting the binding of parathyroid hormone-related peptide (PTHrP) to its receptor.
BACKGROUND ART
Parathyroid hormone-related peptide (hereinafter, PTHrP) is a protein produced by a tumor and is a major causative substance of humoral hypercalcemia of malignancy.
PTHrP induces humoral hypercalcemia of malignancy (hereinafter referred to as "HHM") by promoting bone resorption and renal tubular calcium reabsorption. Currently, calcitonin and bisphosphonate having an inhibiting agenty action on bone resorption are used to treat HHM.
However, since the progression of HHM is so rapid as to significantly deteriorate the QOL
(Quality of Life) of patients with terminal cancer, development of a more effective therapeutic agent on the basis of any cause of the disease has been awaited.
An antibody against parathyroid hormone-related peptide (hereinafter referred to as an "anti-PTHrP antibody") is superior to bisphosphonate because the anti-PTHrP
antibody produces an effect for HHM immediately after administration, while the bisphosphonate need days to produce an effect. Further, the anti-PTHrP antibody is also useful as a therapeutic agent for cachexia seen in patients with terminal cancer (Japanese Patent Application Laying-Open (kokai) No. 11-80025).

Cancer cachexia is one of the paraneoplastic syndromes, which are characterized by a sharp body weight loss, and is often found in many patients with advanced cancer. The body weight loss has been thought to be caused by, for example, a reduced dietary intake due to loss of appetite. Development of cancer cachexia not only largely affects survival time, but also significantly deteriorates the QOL of patients, and adversely affects, for example, treatment with a chemotherapeutic agent. Accordingly, controlling cachexia is of extreme importance as a part of cancer treatment.
It is known that HHM is induced in a model rat transplanted with a human lung cancer strain LC-6. In this model rat, blood calcium level is improved using a bisphosphonate, which is the existing HHM therapeutic agent, or the like.
However, administration of the bisphosphonate to a HHM model rat showing a symptom of cachexia, such as a sharp decrease in body weight, results in only a slight recovery in the body weight.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a tissue degradation inhibiting agent which comprises, as an active ingredient, a substance inhibiting the binding of PTHrP to its receptor.
As a result of thorough studies to address the above-mentioned problems, the present inventors have completed the present invention on the basis of the finding that a substance inhibiting the binding of parathyroid hormone-related peptide to its receptor can inhibit tissue degradation.
Specifically, the present invention is a tissue degradation inhibiting agent which comprises, as an active ingredient, a substance inhibiting the binding of parathyroid hormone-related peptide to its receptor. Examples of the substance include an antagonist against the parathyroid hormone-related peptide receptor, an anti-parathyroid hormone-related peptide antibody (for example, a humanized or chimeric monoclonal antibody), and a fragment and/or a modified form of the antibody. An example of a humanized antibody is a humanized #23-57-137-1 antibody or the like. Further, an example of a tissue is a muscle tissue or adipose tissue. Examples of the above tissue degradation include those caused by cancer cachexia, septicemia, severe injury or muscular dystrophy. The inhibiting agent is also effective for patients having, for example, a blood cytokine level (at least one of those selected from the group consisting of, for example, IL-6, G-CSF, IL-11 and LIF) higher than the normal level.
In particular, the inhibiting agent of the present invention is effective, even in the presence of inflammatory cytokines, such as IL-6, IL-11 and LIF, which are believed to involve tissue degradation.
A detailed description of the present invention will now be given.
This specification includes part or all of the contents disclosed in the specification and drawings of Japanese Patent Application No. 2000-52414, of which the present application claims priority.
The present invention is a tissue degradation inhibiting agent which comprises, as an active ingredient, a substance inhibiting the binding of parathyroid hormone-related peptide (PTHrP) to its receptor (PTHrP receptor).
The present inventors have surmised that the anti-PTHrP antibody may play some role in weight fluctuation based on findings that the antibody has a drug effect of normalizing calcium level as well as of recovering weight. That is, the present inventors have postulated that body weight loss resulting from cachexia is caused by distruction of tissues. Thus, the present inventors have completed the present invention on the basis of the finding that the anti-PTHrP antibody inhibits disruption of tissues.
In the present specification, the term "PTHrP receptor" refers to, for example, a receptor binding to PTHrP described in Japanese Patent Application Laying-Open (kohyo) No.
6-506598. The term "PTHrP receptor" can refer to a PTHrP receptor which may be or not present on a target organ (for example, bone or kidney).
The term "a substance inhibiting the binding of PTHrP to the PTHrP receptor"
refers to either one of, or both (1) a substance which inhibits the binding of PTHrP to the PTHrP receptor by binding of itself to PTHrP (for example, an anti-PTHrP
antibody), and (2) a substance which inhibits the binding of PTHrP to the PTHrP receptor by binding of itself to the PTHrP receptor [for example, an antagonist against the PTHrP receptor (which is also referred to as a PTHrP antagonist), specifically, PTHrP peptide having substitution and/or deletion of at least one amino acid, and a partial sequence of PTHrP peptide, etc.].
The anti-PTHrP antibody used in the present invention is not limited based on its origin, type (monoclonal, polyclonal) and form, so far as the anti-PTHrP
antibody has an inhibitory effect on tissue degradation by binding to PTHrP.
Examples of the anti-PTHrP antibody include a humanized antibody, a human antibody (W096/33735), a chimeric antibody (Japanese Patent Application Laying-Open (kokai) No. 4-228089), and a mouse antibody (for example, #23-57-137-1 antibody produced by hybridoma #23-57-137-1). Note that the antibody may be a polyclonal antibody, but a monoclonal antibody is preferable.

Examples of the PTHrP antagonist include a polypeptide and a low molecular compound, but are not limited thereto. Specific examples of a substance binding antagonistically to the PTHrP receptor against PTHrP include polypeptides having PTHrP
antagonist activity described in Japanese Patent Application Laying-Open (kokai) No. 7-165790, Japanese Patent Application Laying-Open (kohyo) No. 5-509098, Peptides (The United States) 1995, 16 (6) 1031-1037, and Biochemistry (The United States) Apr. 28, 1992, 31 (16) 4026-4033. Moreover, the PTHrP antagonist according to the present invention also includes the polypeptide which has deletion, substitution, addition and/or insertion of at least one amino acid and has an equivalent PTHrP antagonist activity.
The present invention is illustrated below with an anti-PTHrP antibody as an example of "a substance inhibiting the binding of PTHrP to the PTI3rP
receptor".
1. Anti-PTHrP antibody The anti-PTHrP antibody used in the present invention can be produced by any known method as a polyclonal or monoclonal antibody. Preferably, the anti-PTHrP antibody used in the present invention is a monoclonal antibody derived from, particularly, a mammal.
The mammal-derived monoclonal antibody includes those produced by a hybridoma and those produced by the host transformed with the expression vector carrying the gene cording the antibody which is constructed using genetic engineering techniques. The antibody can bind to PTHrP to inhibit binding of PTHrP to the PTH/PTHrP receptor, thereby blocking the signal transduction of PTHrP and consequently inhibiting the biological activity of PTHrP
A specific example of such the antibody is #23-57-137-1 antibody which is produced by a hybridoma clone #23-57-137-1, or the like.
The hybridoma clone #23-57-137-1 was designated as "mouse-mouse hybridoma #23-57-137-1" and deposited under the terms of the Budapest Treaty on August 15, 1996 with the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology, Japan (1-l, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) under the accession No. FERM BP-5631.
2. Antibody-producing hybridoma A monoclonal antibody-producing hybridoma can be produced as follows. Namely, in this process PTHrP is used as a sensitizing antigen in accordance with a standard immunization method. The resulting immunocytes are fused to known parent cells by a standard cell fusion method, and monoclonal antibody-producing cells are screened from the fused cells by a standard screening method.
First, human PTHrP, which is used as a sensitizing antigen for producing the antibody, is prepared by expressing the PTHrP gene/amino acid sequence disclosed in Suva, L.
J. et al., Science (1987) 237, 893. That is, the gene encoding PTHrP is inserted into a known expression vector system, and a suitable host cell is transformed with the expression vector.
The target PTHrP protein is then purified from the transformed host cell or from the culture supernatant of the transformed host cell by any known method.
Then, the purified PTHrP protein is used as a sensitizing antigen.
Alternatively, the N-terminal 34 peptides of PTHrP may be chemically synthesized and used as the sensitizing antigen.
The mammal to be immunized with the sensitizing antigen is not particularly limited.
However, the mammal is preferably selected taking into consideration compatibility with the parent cell used for cell fusion. Generally, a rodent (e.g., mouse, rat, hamster), rabbit, monkey or the like may be used.

The immunization of the animal with the sensitizing antigen can be performed in accordance with any known method. For example, a general technique is performed by injecting the sensitizing antigen to a mammal intraperitoneally or subcutaneously.
Specifically, the sensitizing antigen is diluted with or suspended in an appropriate amount of phosphate-buffered saline (PBS), physiological saline or the like. The resulting dilution or suspension is then mixed with an appropriate amount of a common adjuvant (e.g., Freund's complete adjuvant), if necessary. The mixture is emulsified and administered to a mammal several times at intervals of 4 to 21 days. For the immunization with the sensitizing antigen, a suitable Garner may be used together with it.
After the immunization, the serum antibody level is checked. When the serum antibody level is confirmed to have reached a desired level, immunocytes are isolated from the mammal and then subjected to cell fusion. A particularly preferable immunocyte is a spleen cell.
The parent cell used for fusion with the above immunocyte (i.e., the counterpart in the cell fusion with the immunocyte) is a myeloma cell derived from a mammal.
The myeloma cell preferably used herein is of any known cell line, and, for example, P3 (P3x63Ag8.653) (J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J.
Immunol. (1976) 6, 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S.
F. et al., J.
Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323) or 8210 (Galfre, G. et al., Nature (1979) 277, 131-133).
Cell fusion of the above immunocyte to the myeloma cell is basically performed in accordance with any known method, such as the method of Milstein et al.
(Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).
More specifically, the cell fusion is performed, for example, in a conventional nutrient culture medium in the presence of a cell fusion promoter. The cell fusion promoteing reagent may be polyethylene glycol (PEG), a Sendai virus (Hemagglutinating Virus of Japan; HVJ), or the like. If desired, for the purpose of improving the fusion efficiency, an auxilliary agent such as dimethyl sulfoxide may be used for incorporation.
The ratio between the immunocytes and the myeloma cells used for the cell fusion may be optionally set. For example, the immunocytes are used preferably in the number of 1-10 times larger than the myeloma cells. The culture medium used for the cell fusion is, for example, RPMI 1640 medium or MEM medium, which is suitable for the growth of the above-mentioned myeloma cell lines, or another medium normally used for the culture of such cell lines. Further, a serum supplement, such as feral calf serum (FCS), may be added to the culture medium.
The cell fusion is performed by fully mixing given amounts of the immunocytes and the myeloma cells in the above culture medium, adding a PEG solution (e.g., mean molecular weight: about 1000-6000) (which has been preheated to about 37°C) to the mixture usually to a concentration of 30-60% (w/v), and then mixing the resulting solution, thereby producing the desired fusion cells (hybridomas). Subsequently, an appropriate culture medium is added to the culture solution successively, and the solution is centrifuged to remove the supernatant.
This procedure is repeated several times to remove substances such as the cell fusion promoting agent that are undesirable for the growth of the hybridomas, from the culture medium.
The thus obtained hybridomas can be selected by culturing in a standard selective medium, such as Hypoxanthine-Aminopterin-Thymidine (HAT) medium. The culturing of the hybridomas in HAT medium is performed for a period of time long enough to cause the death of the cells other than the desired hybridomas (i.e., cells that fail to fuse), usually a period of several days to several weeks. Subsequently, a standard limiting dilution method is performed for screening and mono-cloning of the hybridomas that are producing the desired antibody.
In addition to preparing the hybridomas by immunizing a non-human mammal with the antigen as described above, a human lymphocyte may be sensitized with PTHrP in vitro, followed by fusion of the sensitized lymphocyte to a desired human-derived myeloma cell capable of infinite growth, thereby producing a human antibody having a binding activity against PTHrP (Japanese Patent Publication No. 1-59878). Alternatively, a human antibody against PTHrP may be prepared by administering PTHrP as an antigen to a transgenic animal that has the entire repertories of human antibody genes to produce an anti-PTHrP antibody-producing cell, and then immortalizing the cells, thus producing the human antibody from the immortalized cell (International Patent Publication Nos. WO 94/25585, WO
93/12227, WO
92/03918 and WO 94/02602).
The monoclonal antibody-producing hybridoma prepared as described above can be subcultured in a standard culture medium and stored under liquid nitrogen for a long period.
Examples of a method that may be employed to obtain a monoclonal antibody from the hybridoma include a method that involves culturing the hybridoma in accordance with a standard technique and collecting the monoclonal antibody from the culture supernatant, or that involves administering the hybridoma to a mammal compatible with the hybridoma to grow the hybridoma in the mammal and collecting the hybridoma from the ascites of the mammal. The former method is suitable for producing the antibody in high purity, while the latter method is suitable for producing the antibody in a large amount.
3. Recombinant antibody In the present invention, a recombinant-type monoclonal antibody may be used, which can be produced by cloning an antibody gene from the hybridoma, integrating the antibody gene into a suitable vector, introducing the vector into a host, and then producing the antibody from the host according to a standard genetic recombination technique (see, for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).
Specifically, mRNA encoding a variable (V) region of an anti-PTHrP antibody is isolated from the anti-PTHrP antibody-producing hybridoma. The mRNA is isolated by preparing total RNA by any known method, such as a guanidium ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299) or an AGPC method (Chomczynski, P et al., Anal. Biochem. (1987) 162, 156-159), and then producing the desired mRNA from the total RNA using an mRNA Purification Kit (Pharmacia) or the like.
Alternatively, the mRNA may also be prepared directly using a QuickPrep mRNA
Purification Kit (Pharmacia).
Next, cDNA for the antibody V-region is synthesized from the mRNA with a reverse transcriptase. The synthesis of the cDNA is performed using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like. The cDNA may also be synthesized and amplified by, for example, the 5'-RACE method (Frohman, M.A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002;
Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using a 5'-Ampli FINDER RACE Kit (CLONETECH) in combination with the PCR method.
A DNA fragment of interest is purified from the resulting PCR product and then ligated to a vector DNA to obtain a recombinant vector. The recombinant vector is introduced into E. coli or the like, and then the resulting colony is selected, thereby preparing the desired recombinant vector. The nucleotide sequence of the DNA of interest is confirmed by a known method, for example, a dideoxynucleotide chain termination method.
Once DNA encoding the anti-PTHrP antibody V-region is obtained, the DNA is integrated into an expression vector containing DNA encoding a desired antibody constant (C) region.
For the production of the target anti-PTHrP antibody used in the present invention, the antibody gene is integrated into an expression vector so that the antibody gene can be expressed under the control of expression control regions (e.g., enhancer and promoter). A
host cell is transformed with the expression vector to express the antibody.
To express the antibody gene, DNA encoding the heavy (H) chain and DNA
encoding the light (L) chain of the antibody may be integrated separately into expression vectors, and then a host cell is co-transformed with the resulting recombinant expression vectors. Alternatively, both the DNA encoding the H-chain and the DNA encoding the L-chain of the antibody may be integrated together into a single expression vector, and then a host cell may be transformed with the resulting recombinant expression vector (WO
94/11523).
To produce the recombinant antibody, besides the above-mentioned host cells, a transgenic animal may also be used as a host. For example, the antibody gene is inserted into a gene encoding a protein inherently produced in the milk of an animal (e.g., goat a -casein) to obtain a fusion gene. A DNA fragment containing the antibody gene-introduced fusion gene is injected into an embryo of a goat, and the embryo is then introduced into a female goat. The female goat having the embryo bears a transgenic goat. The antibody of interest is secreted in the milk from the transgenic goat or a progeny thereof. For increasing the amount of the desired antibody-containing milk from the transgenic goat, an appropriate hormone may 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, for example, reducing the heteroantigenicity for a human body, an artificially modified genetic recombinant antibody may be used, such as a chimeric antibody or a humanized antibody. These modified antibodies can be prepared by the following known methods.
A chimeric antibody useful in the present invention can be prepared by ligating the DNA encoding the antibody V-region prepared as set forth above to a DNA
encoding a human antibody C-region, integrating the ligation product into an expression vector, and introducing the resulting recombinant expression vector into a host to produce the chimeric antibody.
A humanized antibody is also referred to as a "reshaped human antibody", in which the complementarity determining regions (CDRs) of an antibody of a non-human mammal (e.g., a mouse) are grafted to those of a human antibody. The general genetic recombination procedures for producing such humanized antibody are also known (EP 125023; WO
96/02576).
Specifically, a DNA sequence which has been designed to have mouse antibody CDRs ligated through framework regions (FRs) of a human antibody is amplified by the PCR
method using as primers several oligonucleotides which have been prepared to have regions overlapping the terminal regions of both the CDRs and the FRs. The resulting DNA is ligated to DNA encoding human antibody C-region, and the ligation product is integrated into an expression vector. The resulting recombinant expression vector is introduced into a host, thereby producing the humanized antibody (EP 239400, WO 96/02576).
The FRs of the human antibody ligated through the CDRs are selected so that the CDRs can form a suitable antigen binding site. If necessary, an amino acids) in the FRs of the antibody V-region may be replaced so that the CDRs of the reshaped human antibody can form a suitable antigen binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).
The C-region of the chimeric or humanized antibody may be any human antibody C-region, such as C Y 1, C Y 2, C ?' 3 or C Y 4 for the H-chain, and C r~ or C
~l for the L-chain. The human antibody C-region may be modified for improving the antibody or the 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 is composed of CDRs derived from a non-human mammalian antibody and FRs and C-regions derived from a human antibody. The humanized antibody is useful as an active ingredient for the inhibiting agent of the present invention, because the antigenicity of the antibody against a human body is reduced.
A specific example of the humanized antibody usable in the present invention is humanized #23-57-137-1 antibody, in which the CDRs are derived from mouse-derived #23-57-137-1 antibody, the L-chain is composed of the CDRs ligated through three FRs (FR1, FR2 and FR3) derived from human antibody HSU 03868 (GEN-BANK, Deftos, M. et al., Scand. J. Immunol., 39, 95-103, 1994) and a FR fragment (FR4) derived from human antibody S25755 (NBRF-PDB), and the H-chain is composed of the CDRs ligated through FRs derived from human antibody S31679 (NBRF-PDB, Cuisinier, AM et al., Eur.
J.
Immunol. 23, 110-118, 1993) in which a part of the amino acid residues in the FRs is substituted so that the reshaped humanized antibody can exhibit an antigen-binding activity.
The E. coli strain JM 109 (hMBC 1 HcDNA/pUC 19) having a plasmid containing DNA encoding the H-chain and the E. coli strain JM 109 (hMBC 1 Lq ~l /pUC 19) having a plasmid containing DNA encoding the L-chain of the humanized #23-57-137-1 antibody were deposited under the terms of the Budapest Treaty on August 15, 1996 with the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology, Japan (1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) under the accession Nos. FERM BP-5629 and FERM BP-5630, respectively.

5. Modified Antibody The antibody used in the present invention may be a fragment of an antibody or a modified form of the fragment, as long as it binds to PTHrP to inhibit the activity thereof.
Examples of such a fragment of an antibody include Fab, F(ab')2, Fv, or a single chain Fv (scFv) composed of a H-chain Fv fragment and a L-chain Fv fragment linked together through a suitable linker. Specifically, such antibody fragments can be produced by cleaving the antibody with an enzyme (e.g., papain, pepsin) into antibody fragments, or by constructing a gene encoding the antibody fragment and inserting the gene into an expression vector and introducing the resulting recombinant expression vector into a suitable host cell, thereby expressing the antibody fragment (see, for example, Co, M. S., 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.; Lamoyi, 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).
An scFv can be produced by linking the H-chain V-region to the L-chain V-region of antibodies through a linker, preferably a peptide linker (Huston, J. S. et al., Proc. Natl.
Acad. Sci. USA (1988) 85, 5879-5883). The H-chain V-region and the L-chain V-region in the scFv may be derived from any one of the antibodies described herein. The peptide linker which binds the V-regions may be, for example, any single chain peptide of 12-19 amino acid residues.
DNA encoding the scFv can be prepared by amplifying in PCR two template sequences which are the entire sequences or partial sequences of DNA encoding the H-chain or the H-chain V-region and DNA encoding the L-chain or the L-chain V-region of the antibody, wherein the partial sequence is corresponding to a desired amino acid sequence region encoded in the DNAs using two primer pairs that are located on the terminal sides of the template sequences respectively; and subsequently further amplifying both the resulting amplified products and DNA encoding the peptide linker as template sequence using primer pairs which is designed to be enabled to ligate the terminal ends of the peptide linker to the H-chain and the L-chain respectively, during amplification.
Once the DNA encoding the scFv is prepared, an expression vector carrying the DNA and a host transformed with the expression vector can be prepared by standard methods.
The scFv can be produced from the transformed host by a standard method.
The fragments of the antibody may be produced by preparing genes and expressing the genes in suitable hosts as described above. The antibody fragments are also encompassed in the "antibody" of the present invention.

As a modified form of the above-mentioned antibodies, for example, an anti-PTHrP
antibody conjugated to any molecule (e.g., polyethylene glycol (PEG)) may also be used.
Such modified antibodies are also encompassed in the "antibody" of the present invention.
The modified antibodies can be prepared by chemical modification of the antibodies. The chemical modification techniques 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 as described above can be expressed and obtained by known methods. For the expression in a mammalian cell, a conventional useful promoter, the antibody gene to be expressed and a poly(A) signal (located downstream of the 3' end of the antibody gene) may be operably linked and expressed. For example, as a promoter/enhancer system, a human cytomegalovirus immediate early promoter/enhancer system may be used.
Other promoter/enhancer systems usable in the expression of the antibody used in the present invention include those derived from virus promoters (e.g., retrovirus, polyoma virus, adenovirus and sinuan virus 40 (SV40)) and those derived from mammalian cells (e.g., human elongation factor 1 a (HEFT cx ).
When an SV40 promoter/enhancer system is used, the gene expression may be performed readily by the method of Mulligan et al. (Nature (1979) 277, 108).
When a HEF1 a promoter/enhancer system is used, the gene expression may be performed readily by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322).
For the expression in E. coli, a conventional useful promoter, a signal sequence for secreting an antibody and the antibody gene to be expressed may be operably linked. As such a promoter, lacz promoter or araB promoter may be used. When lacz promoter is used, the gene expression may be performed by the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427). When araB promoter is used, the gene expression may be performed by the method of Better et al. (Better et al., Science (1988) 240, 1041-1043).
Regarding the signal sequence for secretion of the antibody, when the antibody of interest is intended to be secreted in a periplasmic space of the E. coli, pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) may be used. The antibody secreted into the periplasmic space is isolated and then refolded so that the antibody takes an appropriate configuration for use.
Regarding the replication origin, those derived from viruses (e.g., SV40, polyoma virus, adenovirus, and bovine papilloma virus (BPV)) or the like may be used.
To increase the gene copy number in the host cell system, the expression vector may further contain a selective marker gene, such as an aminoglycoside phosphotransferase (APH) gene, a thymidine kinase (TK) gene, an E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene and a dihydrofolate reductase (dhfr) gene.
For the production of the antibody used in the present invention, any expression system, such as eukaryotic and prokaryotic cell systems, may be used. Examples of the eukaryotic cell include established cell lines of animals (e.g., mammals, insects, molds and fungi, yeast). Examples of the prokaryotic cell include bacterial cells such as E. coli cells.
It is preferable that the antibody used in the present invention be expressed in a mammalian cell, such as a CHO, COS, myeloma, BHK, Vero or HeLa cell.
Next, the transformed host cell is cultured in vitro or in vivo to produce the antibody of interest. The culturing of the host cell may be performed by any known method. For example, the culture medium usable herein may be DMEM, MEM, RPMI 1640 or IMDM
medium, and the culture medium may contain a serum supplement, such as fetal calf serum (FCS).

7. Isolation and purification of antibody The antibody expressed and produced as described above may be isolated from the cells or the host animal and purified to uniformity. The isolation and purification of the antibody used in the present invention may be performed on an affinity column.
For instance, examples of a protein A column include Hyper D, POROS and Sepharose F.F.
(Pharmacia). The method is not particularly limited and other methods conventionally used for the isolation and purification of a normal protein may also be employed.
For example, various chromatographs using columns other than the above-mentioned affinity column, filtration, ultrafiltration, salting out and dialysis may be appropriately used singly or in combination to isolate and purify the antibody of interest (Antibodies A
Laboratory Manual.
Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

8. Determination of the activities of the antibody The determination of the antigen-binding activity (Antibodies A Laboratory Manual, Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988) or the inhibiting agenty activity against binding of a ligand receptor (Harada, A. et al., International Immunology (1993) 5, 681-690) of the antibody used in the present invention may be performed by any known methods.
As the method for the determination of the antigen-binding activity of the anti-PTHrP antibody used in the present invention, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or a fluorescent antibody method may be employed. For example, when enzyme immunoassay is employed, a sample containing the anti-PTHrP antibody (e.g., a culture supernatant of anti-PTHrP antibody-producing cells, or the anti-PTHrP antibody in a purified form) is added to a plate on which PTHrP (1-34) is previously coated. A secondary antibody labeled with an enzyme (e.g., alkaline phosphatase) is further added to the plate. The plate is incubated and washed.
A substrate for the enzyme (e.g., p-nitrophenylphosphoric acid) is added to the plate, and the absorbance is measured to evaluate the antigen-binding activity of the antibody.
To confirm the activity of the antibody used in the present invention, a neutralizing activity of the anti-PTHrP antibody is determined.

9. Routes for administration and pharmaceutical preparations The tissue degradation inhibiting agent containing the anti-PTHrP antibody of the present invention as an active ingredient may be administered orally or parenterally, but preferably parenterally. Specifically, the agent may be administered in a transpulmonary dosage form (e.g., which is administered with the help of a device such as a nebulizer), a transnasal dosage form, a transdermal dosage form (e.g., ointment, cream), an injection dosage form, or the like. Examples of an injection dosage form include those for an intravenous injection such as a drip, an intramuscular injection, an intraperitoneal injection and a subcutaneous injection and may be administered systematically or locally. The route of administration may be appropriately selected depending on the age and the symptoms of a patient. An effective single dose may be selected within the range from 0.001 to 1,000 mg per kg of body weight. Alternatively, the dose per patient may be selected within the range from 0.01 to 100,000 mg/body. However, the dosage of the inhibiting agent comprising the anti-PTHrP antibody of the present invention is not particularly limited to these ranges.
Examples of diseases for which the inhibiting agent of the present invention is administered include, but are not limited to, cancer cachexia, septicemia, external injury (e.g., severe and moderate injuries), muscular dystrophy and the like. The inhibiting agent of the present invention may also be administered a patient with two or more of the above diseases or the above diseases complicated with other diseases. Further, the inhibiting agent, for example, may also be administered to a patient with a blood level higher than the normal level with respect to at least one of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), IL-11, leukemia inhibitory factor (LIF) and ai acid glycoprotein (al -AG). The term "normal level" in the present invention means the mean value of the blood levels of 10 to 100 healthy persons, or means the commonly published clinical test values. For example, the clinical test values range from 0.2 to 4.6 pg/mL for IL-6, 7.7 to 38.9 pg/mL
for G-CSF, less than 31.3 pg/mL for IL-11, and 42 to 93 mg/dl for al -AG.
Regarding the timing of administration of the inhibiting agent of the present invention, the inhibiting agent may be administered either before or after the development of the clinical symptoms of the above diseases, or may be administered at a time when body weight loss is predicted.
The inhibiting agent comprising the anti-PTHrP antibody of the present invention as an active ingredient may be formulated by any standard 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 pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, poly(sodium acrylate), sodium arginate, water soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthane gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.
In the practical use, the additive is appropriately selected from, but is not limited to, the above members, either singly or in combination, depending on the employed dosage form of the inhibiting agent of the present invention. For example, for use as an injection formulation, the purified anti-PTHrP antibody is dissolved in a solvent (e.g., physiological saline, a buffer, or a glucose solution) and then an adsorption-preventing agent (e.g., Tween 80, Tween 20, a gelatin, or human serum albumin) is added thereto. The inhibiting agent of the present invention may also be in a reconstitutable freeze-dried form, which is dissolved before use. For the formulation of the freeze-dried dosage form, an excipient such as a sugar alcohol (e.g., mannitol, grape sugar) or a sugar may be incorporated.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 shows the effect of the humanized antibody on body weight and blood ionized calcium level in OCC-1-JCK transplanted mice.
Fig. 2 shows the effect of the humanized antibody on body weight and blood ionized calcium level in Colon 26 transplanted mice.
Fig. 3 shows the effect of the humanized antibody on body weight and blood ionized calcium level in LC-6-JCK transplanted rats.
Fig. 4 shows time course in leukocyte count in LC-6-JCK transplanted rats.
BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be further described by means of examples and reference examples. However, the examples are provided for illustrative purposes only, and are not intended to limit the technical scope of the invention.
[EXAMPLE 1) Test (1) Inhibition of Tissue degradation 1. Preparation of humanized anti-PTHrP antibody In this example, a humanized anti-PTHrP antibody against PTHrP (1-34) was used as an example of the anti-PTHrP antibody.
The humanized anti-PTHrP antibody was prepared by genetic recombination techniques as described in Reference Example 4 (L-chain version q). The obtained humanized anti-PTHrP antibody (hereinafter referred to as "humanized antibody") was added to 20 mmol/L citric acid solution, and 1 mol/L Tris solution was added thereto, and then the solution is adjusted to pH 6.6 (concentration: 6.34 mg/mL). The solution was stored at or below -70°~C . Antibodies to be used herein were confirmed to be intended antibodies by SDS-PAGE for its molecular weight and by binding assay using ELISA for its binding ability.
After thawing the above antibody solution, the solution was diluted to 1 mg/mL
using Dulbecco's PBS (-) (NISSUI PHARMACEUTICAL CO., LTD., code. 05913, hereinafter, "PBS") under sterile condition.
2. Experimental Animal Mice used herein were generally and widely used BALB/cAnNCrj mice (for transplantation with Colon 26) or BALB/cAJcl-nu mice (for transplantation with JCK) transplantable with mouse colon cancer strain Colon 26 or human oral cavity cancer strain OCC-1-JCK. 30 male mice were used for each experiment. 6 week-old mice were selected to be transplanted with tumor cells. In addition, BALB/cAnNCrj mice were purchased from CHARLES RIVER JAPAN, INC., and BALB/cAJcl-nu mice from CLEA
JAPAN, INC.
After 30 purchased mice were acclimatized for about 1 week, 7 mice were randomly chosen, and put in a normal group to which tumors were not transplanted.
Tumors were transplanted to the remaining 23 mice. On day 7 after transplantation, the tumors were confirmed to "take." 20 mice in which tumors took were selected. The mice were divided, mice per group, into a group administered with PBS and a group administered with the humanized antibody of the present invention, while balancing between the groups in body weight as an index.
The rearing conditions for mice were as follows.

Room's temperature: 24 2C

Relative humidity : 55 10%

Ventilation frequency: 10 to 30 times/hour Lighting duration : 14 hours Rearing cage : M-4 cage (215x320x 130 mm, made of polycarbonate, provided with bedding) Rearing density : 5 - 7 mice/cage Feed and feeding CE-2 (CLEA JAPAN, INC.), free feeding method:

Water and supply tap water, free intake of water with a 200 method: mL water supply bottle 3. Experimental method 3-1. Preparation of disease model Tumor strains used: Colon 26 (Mouse colon cancer strain), OCC-1-JCK (human oral cavity cancer strain) Tumor strain source: Colon 26 (Chemotherapy Center, Japanese Foundation for Cancer Research), OCC-1-JCK (Central Institute for Experimental Animals) Subculture of tumor strain: Colon 26 was subcultured in vivo every 2 weeks using BALB/cAnNCrj. OCC-1-JCK was subcultured in vivo every 3 weeks using BALB/cAJcl-nu. Colon 26 and OCC-1-JCK transplanted mouse models were prepared as follows.
Specifically, on a day of transplantation with tumor cells, the mice having in vivo subcultured tumor were sacrificed by cervical-vertebral dislocation. Then, the tumor was excised to prepare a 3 mm block, and then transplanted subcutaneously to a mouse. The tumors were allowed to "take" in the transplanted mice, thereby establishing model mice.
3-2. Measurement of blood ionized calcium level (blood iCa level) Immediately after collecting approximately 60 p1 of blood from the eye pit, the blood iCa level was measured with 643Ca++/pH analyzer (Chiron).
3-3. Measurement of serum biochemical level (glucose, triglyceride and cholesterol levels) Under anesthesia, approximately 1 mL of blood was collected from the descending aorta into a Separapid tube (SEKISUI CHEMICAL Co., Ltd.), and then allowed to stand at room temperature for 30 min or more. The serum was separated by centrifugation (3,000 rpm, 20 min, HTTACHI, OSPR-22), and then frozen and stored at or below -20°C .
The serum biochemical levels (glucose, triglyceride and cholesterol levels) were measured using an autoanalyzer (HITACHI, type 7170, Autoanalyzer).
3-4. Administration of humanized antibody Humanized antibodies were administered intravenously (i.v.) at a dose of 0.1 mg/mouse (a humanized antibody-administered group). Further, Dulbecco's PBS (-) was administered in a PBS administered group. Administration was performed twice in total, on days 7 and 10 after transplantation. No substance was administered in the normal group, which had had undergone no tumor transplantation.
Table 1 summarizes the mouse group configuration and the administration schedule.
Table 1. Group configuration Group Administration Abbreviated Substance Number Administration of Number of soup administered name Dose days of adminis-group animals trations PBS

1 administered10 PBS O.lmL/mouse ou Humanized Days 7 2 and 10 0.1 mg/mouse antibody- Humanized administered10 antibody (PBS solution 0.1 mL/mouse) ou _ 3 Normal- 7 none group The drug e~cacy test is summarized as follows.
Parameters tested: body weight, adipose tissue (periphery of orchis) weight, muscle tissue (gastrocnemius muscle) weight, serum glucose level, serum triglyceride level, serum cholesterol level, blood iCa level, and systemic condition.
For OCC-1-JCK transplanted mice, body weight was measured on days 7, 10 and 13 after tumor transplantation; blood iCa level was measured on days ? and 13;
and other parameters were measured on day 13.
For Colon 26 transplanted mice, body weight was measured on days 7, 10 and 14 after tumor transplantation; blood iCa level was measured on days 7 and 14; and other parameters were measured on day 14.
Evaluation of drug efficacy: The inhibiting agenty effect of administration of the humanized antibody on the decrease in body weight, adipose tissue weight and muscle tissue weight was studied. Further, effects on recovery of blood iCa level, serum glucose level, serum triglyceride level and serum cholesterol level to levels close to normal levels were also studied.
3-S. Examination of effect of humanized antibody Groups subjected to comparison: PBS-administered group and humanized antibody-administered group Hypothesis verified: Compared to the PBS-administered group, the humanized antibody-administered group shows changes in body weight, adipose tissue weight, muscle tissue weight, blood iCa level, serum glucose level, serum triglyceride level, and serum cholesterol level.
Statistical analysis method employed: Body weight, adipose tissue weight, muscle tissue weight, blood iCa level, serum glucose level, serum triglyceride level, and serum cholesterol level were tested by t-test for the normal group and the PBS-administered group.
For the parameters showing significant differences as tested by t-test, we further determined if significant differences exist between the PBS-administered group and the humanized antibody-administered group by employing t-test. The t-test is performed at 5%
two-sided significance level. SAS was used for the analysis.
4. Result a6 4-1. Effect on OCC-1-3CK transplanted mice ( 1 ) Effect on body weight, blood iCa level and systemic condition (Fig. 1 ) Body weight: Compared to the normal group, the PBS-administered group showed a significant decrease in body weight (on days 12 and 13 after tumor transplantation, * : p<0.05 compared to the normal group, tested by t-test). Compared to the PBS-administered group, the humanized antibody-administered group showed a significantly inhibited body weight loss (#: p<0.05 compared to PBS-administered group, tested by t-test), and body weight was maintained to the same degree as that in the normal group (body weight (g) on day 13 after transplantation: 24.64 ~ 0.61 in normal group, 19.37 ~ 0.59 in PBS-administered group, 24.11 ~ 0.47 in humanized antibody-administered group) (Indicated by mean ~ SE. The same indication applies hereinafter.).
Blood iCa level: A significant increase was observed in the PBS-administered group compared to the normal group (on day 13 after tumor transplantation (*)).
Compared to the PBS-administered group, the humanized antibody-administered group showed a significantly inhibited increase in blood iCa level (#) [blood iCa level (mmol/L) on day 13 after transplantation: 1.36 ~ 0.01 in normal group, 2.73 ~ 0.10 in PBS-administered group, 1.62 ~
0.01 in humanized antibody-administered group].
Systemic condition: Compared to the normal group, a deteriorated systemic condition was visually confirmed in the PBS-administered group. In the humanized antibody-administered group, an improvement in the deteriorated systemic condition was found.
(2) Effect on adipose tissue (periphery of orchis) weight and muscle tissue (gastrocnemius muscle) weight (Table 2) W .-.
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(3) Effect on serum biochemical levels (glucose, triglyceride, and cholesterol levels) (Table 2) Significance decreases in glucose, triglyceride and cholesterol levels were observed in the PBS-administered group, compared to the normal group (*). Compared to the PBS-administered group, the humanized antibody-administered group showed the tendency to inhibit decreases of such levels, but showed a significant improvement only in triglyceride level (#).
4-2. Effect on Colon 26 transplanted mice (1) Effect on body weight, blood iCa level and systemic condition (Fig. 2) Body weight: Compared to the normal group, a significant decrease in body weight was observed in the PBS-administered group (on and after day 10 after tumor transplantation, * : p<0.05 compared to the normal group, tested by t-test). Compared to the PBS-administered group, the body weight loss was significantly inhibited in the humanized antibody-administered group (#: p<0.05 compared to the PBS-administered group, tested by t-test). Body weight (g) on day 14 after transplantation was 26.78 ~ 0.38 in the normal group, 19.04 ~ 0.35 in the PBS-administered group, and 21.51 ~ 0.32 in the humanized antibody-administered group.

Blood iCa level: A significant increase was observed in the PBS-administered group compared to the normal group (on day 14 after tumor transplantation (*)).
Compared to the PBS-administered group, the increase in blood iCa level was significantly inhibited in the humanized antibody-administered group (#). Blood iCa level (mmol/L) on day 14 after transplantation was 1.34 ~ 0.01 in the normal group, 1.94 ~ 0.09 in the PBS-administered group, and 1.54 ~ 0.03 in the humanized antibody-administered group.
Systemic condition: Compared to the normal group, a deteriorated systemic condition was visually confirmed in the PBS-administered group. In the humanized antibody-administered group, such a deteriorated systemic condition was somewhat improved.
(2) Effect on adipose tissue (periphery of orchis) weight and muscle tissue (gastrocnemius muscle) weight (Table 3) r.
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(3) Effect on serum biochemical levels (glucose, triglyceride, and cholesterol levels) (Table 3) Compared to the normal group, the PBS-administered group showed a significant decrease in serum glucose level (*), which was not improved by administration with the humanized antibody. The PBS administered group showed a significant decrease in serum triglyceride level, compared to the normal group; and the humanized antibody-administered group showed an inhibited decrease, which was not a significant inhibition, in serum triglyceride level. The serum cholesterol level did not change in the PBS-administered and the humanized antibody-administered groups, and also the normal group.
4-3. Conclusion Cancer cachexia is a clinical syndrome which causes in cancer patients a significant body weight loss due to loss of appetite, hypermetabolism or the like. The serum biochemical characteristics of cancer cachexia include low glucose level and hyperlipidemia.
Some tumors are known to induce cancer cachexia in a tumor transplantation model.
Typical tumor strains include SEKI melanoma (human melanoma), Colon 26 (mouse colon cancer cell line), and OCC-1-JCK (human oral cavity cancer cell line).
Transplantation of such a tumor into a nude mouse results in a significant body weight loss.

On the other hand, a significant body weight loss similar to that in cancer cachexia is also observed in a HHM model. PTHrP has been determined as a causative agent for HHM
(L.J. Suva, et al, Science 237, 893-896, 1987).
Accordingly, the present inventors prepared cancer cachexia models transplanted with either of 2 tumor strains, human oral cavity cancer cell line OCC-1-JCK and mouse colon cancer cell line Colon 26, and examined the effect of humanized antibodies in the models.
It was showed that the body weight (and further the adipose tissue weight and the muscle tissue weight) started to decrease markedly after transplantation, on day 10 in OCC-1-JCK transplanted mice, or on day 7 in Colon 26 transplanted mice. Both types of the transplanted mice also showed in a low glucose state, which was a clinical feature of cancer cachexia. These models exhibited hypercalcemia, suggesting a complication of cancer cachexia with HHM.
When humanized antibodies were administered to the above 2 types of model mice, a significant improvement effect was observed not only in body weight and blood iCa level, but also in adipose tissue weight and muscle tissue weight. Moreover, an improvement effect on low triglyceride level was observed in OCC-1-JCK transplanted mice. That is, mice showed the tendency, though not significant, to improve low glucose level.
Thus, in the present invention, the humanized anti-PTHrP antibody is useful as a therapeutic agent or a preventive agent for tissue degradation associated with various diseases, such as cancer cachexia, because the antibody exhibited a tissue degradation inhibitory effect on adipose tissue and muscle tissue.
(Example 2] Test (2) Inhibition of tissue degradation 1. Humanized anti-PTHrP antibody Humanized anti-PTHrP antibodies used herein were those prepared in Example 1.
2. Experimental animal Rats used herein were nude rats F344/N Jcl-rnu (CLEA JAPAN, INC) transplantable with a human tumor cell line and capable of stably producing a HHM model. 80 rats at 5 weeks old were purchased, and then acclimatized for about 1 week. Next, SS
rats randomly chosen therefrom were transplanted with tumors. The rats were euthanized by ether inhalation.
The rearing conditions for rats were as follows.
Room's temperature : 24 ~ 2°C
Relative humidity : 55 ~ 10%
Ventilation frequency : 10 to 30 times/hour Lighting time : 5:00 to 19:00 Rearing cage and rearing density: 8 to 11 rats were reared per cage (a stainless wire mesh lifting cage with 660 x 310 x 200 mm in dimension) during the acclimatization period and the period ranging from tumor transplantation to the start of the experiment.
During the experiment, one rat was reared per polycarbonate cage (265 x 425 x 160 mm in dimension).
Feed and feeding method: CE-2 (CLEA JAPAN, INC.), free feeding Water and supply method: tap water, free intake of water Routine management: according to management criteria for rearing experimental animals (edited by the Experimental Animal Care Committee of Chugai Pharmaceutical Co., Ltd.) 3. Experimental method 3-1. Preparation of disease model Tumor strains used: LC-6-JCK (human lung large cell carcinoma strain) Tumor strain source: Purchased from Central Institute for Experimental Animals Subculture of tumor strain: Subculturing was performed by transplanting subcutaneously a tumor block and growing the tumor for an in vivo tumor strain into mice (BALB/cA Jcl-nu; CLEA JAPAN, INC.). Subculturing was performed every 3 to 4 weeks.
Preparation of model: Mice having the subcultured tumor strains were sacrificed by cervical-vertebral dislocation. Then, the tumor was excised from the mice to prepare a 3 mm block, and then transplanted subcutaneously to a rat, thereby preparing a model rat.
Transplantation was performed for 55 rats randomly chosen from the 80 rats.
The remaining 25 rats were untreated.
Criteria for model establishment: A rat individual having a visible subcutaneous growth of the transplanted tumor and showing higher blood iCa level than that of a normal group was determined as a HHM model rat. In this experiment, a rat showing an iCa level of 1.80 mmol/L, or more was used.
Grouping method: Blood iCa level was measured on day 49 after tumor transplantation, so as to confirm whether or not a HHM model had been established. HHM
models were classified using the blood iCa level as an index. 24 rats were randomly chosen therefrom and divided evenly into 3 groups (8 rats/group): a saline-administered group, a humanized antibody-administered group, and a tumor-excised group. Further, 8 rats were randomly chosen from the 25 untreated rats and grouped as a normal group.
3-2. Measurement of blood ionized calcium level (blood iCa level) Blood was collected via the tail vein, and then immediately measured using a calcium analyzer (automatic pH/Ca++ analyzer Chiron 634, Bayer Medical).
3-3. Collection of sera and measurement of biochemical levels (glucose, triglyceride, cholesterol and free fatty acid levels), cytokines (human IL-6, human G-CSF, human IL-11, and human LIF) and rat a~-AG
Blood was collected from a rat under anesthesia. Blood collected from the descending aorta into a Separapid tube (SEKISUI CHEMICAL Co., Ltd.) was allowed to stand at room temperature for 30 min or more. The serum was separated by centrifugation (3000 rpm, 20 min, HITACHI, OSPR-22), and then frozen and stored at or below -20~ .
Serum biochemical levels (glucose, triglyceride, cholesterol and free fatty acid levels) were measured using an autoanalyzer (HITACHI, type 7170, autoanalyzer).
Cytokines (human IL-6, human G-CSF, human IL-11 and human LIF) and rat al-AG
were respectively measured using a commercially available EIA kit, Quantikine~
human IL-6 (R&D systems), Quantikine° human G-CSF (R&D systems), Quantikine~ human (R&D systems), LIF/HILDA EASIAiK2 (BIOSOURCE), and Panatest~ A series rat al-AG
(Panafarm Laboratories).
3-4. Measurement of leukocyte count 20 p1 of blood was collected via the tail vein, and then immediately suspended in a cell pack (PL30L). Then, the leukocyte count was measured using Sysmex Microcell counter F-800.
3-5. Measurement of adipose tissue weight and muscle tissue weight Adipose tissues (periphery of orchis) and muscle tissues (gastrocnemius muscle) were excised, and then the weights were measured using 5HIMAZU LIBROR EB-330H with SHIMAZU EP-50 printer.
3-6. Administration of humanized antibody A dose of 3.0 mg/kg of humanized antibodies was administered intravenously (i.v.) (a humanized antibody-administered group). Physiological saline was intravenously administered to a negative control group (a saline-administered group). The administered solutions were used at a dose of 0.1 mL was administered per 100g of body weight. Nothing was administered to the normal group that had not been transplanted with tumors.
Excision of tumor:
The skin at the root portion of the tumor was tied with a vinyl string (Tie band: a band for an sterile bag) so as to be enclosed by the string (a tumor-excised group). A few hours later and on the next day, the string was stretched and tied again. On the next day, the tumors were confirmed as being necrotized.
Test schedule:
On day 49 after tumor transplantation, body weight, blood iCa level and leukocyte count were measured. Based on the measurement results, grouping was performed.
Then, for the saline-administered group and the humanized antibody-administered group, saline or humanized antibodies were administered, respectively. For the tumor-excised group, tumors were excised therefrom. On days 1, 4, 7 and 10 after administration, body weight, blood iCa level and leukocyte count were measured. On day 10 after administration, adipose tissue weight and muscle tissue weight were measured, blood and serum were collected, and then serum biochemical levels and cytokines were measured.
3-7. Method for statistical analysis An unpaired t-test was performed for the differences in the mean values of the normal group and the saline-administered group on each day of measurement. In this test, first, an F-test was performed for variances. When an F-value was 5 % or more, Student's t-test for equal variance was performed, and when an F-value was 5 % or less, Welch's t-test for unequal variance was performed at 5 % two-sided significance level. Next, Dunnett's multiple comparison test was performed at 5 % two-sided significance level, for the differences in the mean values on each day of measurement between the saline-administered group, and the humanized antibody-administered group or the tumor-excised group. SAS
Ver. 6.12 was used for the statistical analysis.
4. Result 4-1. Effect on body weight and blood iCa level (Fig. 3) Body weight (Fig. 3, upper panel): Throughout the days of measurement, the saline-administered group (-~-) showed significant decreases in body weight compared to the normal group (-1-) (p<0.05 compared to the normal group, measured by t-test).
Compared to the saline-administered group, the body weight started to significantly increase in the humanized antibody-administered group (-~-) from day 4 after administration, and showed significant differences up to days 7 and 10 (#: p<0.05 compared to the saline-administered group, measured by Dunnett's multiple comparison test). The tumor-excised group (-~-) showed an equivalent recovery from body weight loss, and showed a significant difference on days 4, 7 and 10 after administration compared to the saline-administered group (#).
Blood iCa level (Fig. 3, lower panel): Throughout the days of measurement, the saline-administered group showed a significantly higher blood iCa level compaired to the normal group. Compared to the saline-adnunistered group, the humanized antibody-administered group showed a significantly inhibited increase in blood iCa level on days 1, 4, 7 and 10 after administration (#). The tumor-excised group also showed a significantly inhibited increase in blood iCa level compared to the saline-administered group on days l, 4, 7 and 10 after administration (#).
4-2: Effect on adipose tissue (periphery of orchis) weight, and muscle tissue (gastrocnemius muscle) weight (Table 4) Adipose tissue weight: The saline-administered group showed a significant decrease compared to the normal group (*). Compared to the saline-administered group, the decrease was significantly inhibited in the humanized antibody-administered group and the tumor-excised group (#).
Muscle tissue weight: The saline-administered group showed a significant decrease compared to the normal group (*). Compared to the saline-administered group, the decrease was significantly inhibited in the humanized antibody-administered group and the tumor-excised group (#).
4-3. Effect on serum biochemical levels (glucose, triglyceride, cholesterol and free fatty acid levels) (Table 4) Compared to the normal group, the saline-administered group showed significant decreases in glucose and free fatty acid levels, and an increase in cholesterol level (*).
Compared to the saline-administered group, the humanized antibody-administered group showed a significant decrease in cholesterol level (#); however, none of the remaining parameters were significantly improved. The tumor-excised group showed improving effects on glucose and cholesterol levels (#).

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4-5. Effect on leukocyte (Fig. 4) Compared to the normal group, the saline-administered group showed a significant increase in the leukocyte count throughout the days of measurement (p<0.05 compared to the normal group, measured by t-test). The humanized antibody-administered group showed a significant decrease in the leukocyte count on day 4 after administration compared to the saline-administered group (# significance level: 5%, measured by Dunnett's multiple comparison), but showed no difference on other days of measurement. On days 1, 4, 7 and after administration, the tumor-excised group showed significant decreases (#).
#: There was a significant difference compared to the saline-administered group.
5. Conclusion The present inventors prepared a cancer cachexia model rat transplanted with the human lung large cell carcinoma strain LC-6-JCK, and studied the effect of the humanized antibody by comparing the model with the tumor-excised model.
After tumor transplantation, LC-6-JCK-transplanted rats developed hypercalcemia with marked body weight loss, thereby establishing a model developing cancer cachexia together with HHM. The model also showed the clinical features of cancer cachexia, such as decreased adipose tissue weight and muscle tissue weight, and lowered glucose level, in addition to production of cytokines derived from tumors and a protein induced therefrom, and increased leukocyte count.
When the humanized antibody was administered to the above model rat, significant improving effects were observed not only in body weight and blood iCa level, but also in adipose tissue weight and in muscle tissue weight. Moreover, a tendency to improve low glucose level, though not a significant effect, was observed. However, no inhibiting agenty effect was observed on production of cytokines derived from tumors and the protein induced therefrom, or on leukocytes.

When tumors were excised, significant improving effects at the level equivalent to that resulting from administration of the antibody were observed on body weight, blood iCa level, adipose tissue weight and muscle tissue weight. Improving effects on low glucose level and on high cholesterol level were observed. Inhibiting agenty effects were also observed on production of cytokines derived from tumors and the protein induced therefrom.
Further, an inhibiting agenty effect was observed on an increase in leukocyte count.
In the present invention, the humanized anti-PTHrP antibody exhibited an inhibiting agenty effect on tissue degradation of the adipose tissue and the muscle tissue, suggesting that the antibody is useful as a therapeutic agent for tissue degradation associated with various diseases, such as cancer cachexia. Based on the fact that production of cytokines derived from tumors was not inhibited by the administration of the antibody, the present inventors have found that even in a state of a high blood level of cytokines, such as IL-6, G-CSF, IL-11, LIF and al-AG, administration of the substance of the present invention that inhibits the binding of PTHrP to its receptor can inhibit tissue degradation associated with various diseases. Particularly, the present inventors have found that administration of the substance of the present invention that inhibits the binding of PTHrP to its receptor can inhibit tissue degradation associated with various diseases, even in the presence of inflammatory cytokines at high concentration, such as IL-6, IL-11 and LIF, which are believed to involve tissue degradation (for example, as described in Clinical Science, 89, 431-439, 1995).
[REFERENCE EXAMPLE 1 ]
Preparation of hybridomas producing anti-PTHrP (1-34) mouse monoclonal antibody Hybridomas capable of producing a monoclonal antibody against human PTHrP (1-34) (SEQ ID NO: 75), #23-57-154 and #23-57-137-1, were prepared as follows (see Sato, K.
et al., J. Bone Miner. Res. 8, 849-860, 1993). The amino acid sequence of the human PTHrP
(1-34) is shown in SEQ ID N0:75.

For use as an immunogen, PTHrP (1-34) (Peninsula) was conjugated with a carrier protein thyroglobulin using carbodiimide (Dojinn). The thycloglobulin-conjugated PTHrP
(1-34) was dialyzed to obtain a solution having a protein concentration of 2 ~.g/ml. The resulting solution was mixed with Freund's adjuvant (Difco) at a mixing ratio of 1:1 to give an emulsion. This emulsion was injected to 16 female BALB/C mice 11 times subcutaneously at the back or intraperitoneally at a dose level of 100 gg/mouse for each injection, thereby immunizing the mice. For the priming immunization, Freund's complete adjuvant was used;
while for the boosting immunization, Freund's incomplete adjuvant was used.
Each of the immunized mice was determined for its antibody titer in the serum in the following manner. That is, each of the mice was blood-drawn via its tail vein, and the anti-serum is separated from the blood. The anti-serum was diluted with a RIA
buffer and mixed with lzsI-labeled PTHrP (1-34) to determine binding activity. The mice that were confirmed to have a sufficiently increased titer were injected with PTHrP (1-34) without a carrier protein intraperitoneally at a dose level of 50 g.g/mouse for the final immunization.
Three days after the final immunization, the mouse is sacrificed and the spleen was removed therefrom. The spleen cells were subjected to cell fusion with mouse myeloma cell line P3x63Ag8U.1 in accordance with a conventional known method using 50%
polyethylene glycol 4000. The fused cells thus prepared were seeded to each well of eighty-five 96-well plates at a density of 2 x 104iwe11. Hybridomas were screened in HAT medium as follows.
The screening of hybridomas was performed by determining the presence of PTHrP-recognition antibodies in the culture supernatant of the wells in which cell growth had been observed in HAT medium, by solid phase RIA method. The hybridomas were collected from the wells in which binding ability to the PTHrP-recognition antibodies had been confirmed. The hybridomas thus obtained was suspended into RPMI-1640 medium containing 15% FCS supplemented with OPI-supplement (Sigma), followed by unification of the hybridomas by limiting dilution method. Thus, two types of hybridoma clones, #23-57-154 and #23-57-137-l, could be obtained, both which had a high binding ability to PTHrP (1-34).
Hybridoma clone #23-57-137-1 was designated as "mouse-mouse hybridoma #23-57-137-1 ", and has been deposited under the terms of the Budapest Treaty on August 15, 1996 at the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology, Japan (1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) under the accession No. FERM BP-5631.
[REFERENCE EXAMPLE 2]
Cloning of DNAs encoding V-regions of mouse monoclonal antibody against human PTHrP
(1-34) Cloning of DNAs encoding the V-regions of a mouse monoclonal antibody against human PTHrP (1-34), #23-57-137-1, was performed in the following manner.
( 1 ) Preparation of mRNA
mRNA from hybridoma #23-57-137-1 was prepared using Quick Prep mRNA
Purification Kit (Pharmacia Biotech). That is, cells of hybridoma #23-57-137-1 were fully homogenized with an extraction buffer, and mRNA was isolated and purified therefrom on an oligo(dT)-Cellulose Spun Column in accordance with the instructions included in the kit. The resulting solution was subjected to ethanol precipitation to obtain the mRNA
as a precipitate.
The mRNA precipitate was dissolved in an elution buffer.
(2) Production and amplification of cDNA far gene encoding mouse H-chain V-region (i) Cloning of cDNA for #23-57-137-1 antibody H-chain V-region A gene encoding H-chain V-region of the mouse monoclonal antibody against human PTHrP was cloned by 5'-RACE method (Frohman, M. A. et al., Proc. Natl.
Acad. Sci.
USA, 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-2932, 1989).
The 5'-RACE method was performed using 5'-Ampli FINDER RACE Kit (CLONETECH) in accordance with the instructions included in the kit. In this method, the primer used for synthesis of cDNA was MHC2 primer (SEQ ID NO: 1) which is capable of hybridizing to mouse H-chain C-region. The above-prepared mRNA (about 2 fig), which was a template for the cDNA synthesis, was mixed with MHC2 primer (10 pmoles). The resulting mixture was reacted with a reverse transcriptase at 52~C for 30 minuets to effect the reverse transcription of the mRNA into cDNA.
The resulting reaction solution was added with 6N NaOH to hydrolyze any RNA
remaining therein (at 65~ for 30 min.) and then subjected to ethanol precipitation to isolate and purify the cDNA as a precipitate. The purified cDNA was ligated to Ampli FINDER
Anchor (SEQ ID NO: 42) at the 5' end by reacting with T4 RNA ligase at 37~C
for 6 hours and additionally at room temperature for 16 hours. As the primers for amplification of the cDNA by PCR method, Anchor primer (SEQ ID NO: 2) and MHC-G 1 primer (SEQ ID
NO:
3) (S.T. Jones, et al., Biotechnology, 9, 88, 1991) were used.
The PCR solution comprised (per 50 p1) 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl2, 2.5 units of TaKaRa Taq (Takara Shuzo Co., Ltd.), 10 pmoles Anchor primer, and 1 p1 of the reaction mixture of the cDNA to which MHC-G1 primer and Ampli FINDER Anchor primer had been ligated, over which mineral oil (50 ~1) was layered. The PCR was performed on Thermal Cycler Model 480J (Perkin Elmer) for 30 cycles under the conditions: 94~ for 45 sec.; 60~
for 45 sec.;
and 72°C for 2 min.

,. "
(ii) Cloning of cDNA for #23-57-137-1 antibody L-chain V-region A gene encoding L-chain V-region of the mouse monoclonal antibody against human PTHrP was cloned by 5'-RACE method (Frohman, M. A. et al., Proc. Natl.
Acad. Sci.
USA, 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-2932, 1989).
The 5'-RACE method was performed using 5'-Ampli Finder RACE Kit (CLONETECH) in accordance with the instructions included in the kit. In this method, oligo-dT
primer was used as the primer for synthesizing cDNA. The above-prepared mRNA (about 2 fig), which was a template for the cDNA synthesis, was mixed with oligo-dT primer. The resulting mixture was reacted with a reverse transcriptase at 52°C for 30 min. to effect the reverse transcription of the mRNA into cDNA. The resulting reaction solution was added with 6N
NaOH to hydrolyze any RNA remaining therein (at 65°C for 30 min.). The resulting solution was subjected to ethanol precipitation to isolate and purified the cDNA as a precipitate. The cDNA thus synthesized was ligated to Ampli FINDER Anchor at the 5' end by reacting with T4 RNA ligase at 37~ for 6 hours and additionally at room temperature for 16 hours.
A PCR primer MLC (SEQ ID NO: 4) was designed based on the conserved sequence of mouse L-chain ~ chain C-region and then synthesized using 394 DNA/RNA
Synthesizer (ABI). The PCR solution comprised (per 100 g.1) 10 mM Tris-HCl (pH
8.3), 50 mM KCI, 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl2, 2.5 units of AmpliTaq (PERKIN ELMER), 50 pmoles of Anchor primer (SEQ ID NO: 2), and 1 ~1 of the reaction mixture of the cDNA to which MLC (SEQ m NO: 4) and Ampli FINDER
Anchor were ligated, over which mineral oil (50 ~,l) was layered. The PCR reaction was performed on Thermal Cycler Model 4803 (Perkin Elmer) for 35 cycles under the conditions: 94°C for 45 sec.; 60~ for 45 sec.; and 72°C for 2 min.

(3) Purification and fragmentation of PCR products Each of the DNA fragments amplified by PCR method described above was separated by agarose gel electrophoresis on a 3% Nu Sieve 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 by was excised from the gel.
Each of the gel segments was subjected to purification of the DNA fragment of interest using GENECLEAN II Kit (8I0101) in accordance with the instructions included in the kit. The purified DNA was precipitated with ethanol, and the DNA precipitate was dissolved in 20 p,1 of a solution containing 10 mM Tris-HCl(pH 7.4) and 1 mM EDTA. An aliquot (1 p1) of the DNA solution was digested with a restriction enzyme XmaI (New England Biolabs) at 37°C
for 1 hour and further digested with a restriction enzyme EcoRI (Takara 5huzo Co., Ltd.) at 37~ for 1 hour. The digestion solution was extracted with phenol and chloroform and then precipitated with ethanol to collect the DNA.
In this manner, two DNA fragments containing a gene encoding mouse H-chain V-region and a gene encoding mouse L-chain V-region, respectively, were obtained, both which had an EcoRI recognition sequence on the 5' end and an XmaI recognition sequence on the 3' end.
The EcoRI-XmaI DNA fragments containing a gene encoding mouse H-chain V-region and a gene encoding mouse L-chain V-region, respectively, were separately ligated to pUC 19 vector that had been digested with EcoRI and XmaI at 16~C for 1 hour using DNA
Ligation Kit ver.2 (Takara Shuzo Co., Ltd.) in accordance with the instructions included in the kit. An aliquot (10 p1) of the ligation mixture was added to 100 p.1 of a solution containing competent cells of E. coli, JM 109 (Nippon Gene Co., Ltd.). The cell mixture was allowed to stand on ice for 15 min., at 42~ for 1 min. and additionally for 1 min. on ice. The resulting cell mixture was added with 300 p1 of SOC medium (Molecular Cloning:
A

Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) and then incubated at 37°C for 30 min. The resulting cell solution was plated on LB agar medium or 2xYT agar medium (Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) containing either 100 or 50 pg/ml of ampicillin, 0.1 mM of IPTG and 20 ~g/ml of X-gal, and then incubated at 37°C overnight. In this manner, E. coli transformants were prepared.
The transformants were cultured at 37~ overnight in 2 ml of LB or 2xYT medium containing either 100 or 50 pg/ml of ampicillin. The cell fraction was applied to Plasmid Extracter PI-100( (Kurabo Industries, Ltd.) or QIAprep Spin Plasmid Kit (QIAGEN) to give a plasmid DNA. The plasmid DNA was sequenced as follows.
(4) Sequencing of genes encoding mouse antibody V-regions The nucleotide sequence of the cDNA coding region carried on the plasmid was determined in DNA Sequencer 373A (ABI; Perkin-Elmer) using Dye Terminator Cycle Sequencing Kit (Perkin-Elmer). M13 Primer M4 (Takara Shuzo Co., Ltd.) (SEQ 117 NO: 5) and M13 Primer RV (Takara Shuzo Co., Ltd.) (SEQ 117 NO: 6) were used as the primers for sequencing, and the nucleotide sequence was confirmed in the both directions.
The plasmid containing a gene encoding mouse H-chain V-region derived from hybridoma #23-57-137-1 was designated as "MBC1H04", and the plasmid containing a gene encoding mouse L-chain V-region derived from hybridoma #23-57-137-1 was designated as "MBC 1 L24". The nucleotide sequences (including the corresponding amino acids sequences) of the gene encoding the mouse #23-57-137-1 antibody-derived H-chain V-region in plasmid MBC1H04 and the gene encoding the mouse #23-57-137-1 antibody-derived L-chain V-region in plasmid MBC1H24 were shown in SEQ. ID Nos: 57 and 65, respectively.
The amino acid sequences of the polypeptides for the H-chain V-region and the L-chain V-region were shown in SEQ. ID NOs: 46 and 45, respectively.
The E. coli strain containing the above plasmid MBC1H04 and the E. coli strain containing the above plasmid MBC1L24 were designated as "Escherichia coli (MBC 1H04)" and "Escherichia coli JM 109 (MBC 1L24)", respectively. These E.
coli strains have been deposited under the terms of the Budapest Treaty at the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology, Japan (1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) on August 15, 1996, under the Accession No. FERM BP-5628 for Escherichia coli JM109 (MBC1H04) and FERM
BP-5627 for Escherichia coli JM 109 (MBC 1 L24), respectively.
(5) Determination of CDRs of mouse monoclonal antibody #23-57-137-1 against human PTHrP
The H-chain V-region and the L-chain V-region have general structures similar to each other, each of which has four framework regions (FRs) linked through three hypervariable regions (i.e., complementarity determining regions; CDRs). The amino acid sequences of the FRs are relatively well conserved, while the amino acid sequence of the CDRs have an extremely high variability (Kabat, E.A. et al., "Sequence of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983).
In view of these facts, the homology in amino acid between the V-regions of the mouse monoclonal antibody against human PTHrP was determined with reference to the database of amino acid sequences of antibodies established by Kabat et al.
Thus, the CDRs of the V-regions were determined as shown in Table 6.
The amino acid sequences of CDRs 1-3 in the L-chain V-region are shown in SEQ
ID Nos: 59 to 61, respectively; and the amino acid sequences of CDRs 1-3 in the H-chain V-a region are shown in SEQ ID Nos: 62 to 64, respectively.
Table 6 V-region SEQ ID NO. CDR 1 CDR2 CDR3 H-chain V-region57 31-35 50-66 99-107 L-chain V-region65 23-34 50-60 93-105 [REFERENCE EXAMPLE 3J Construction of Chimeric Antibody (1) Construction of chimeric antibody H-chain (i) Construction of H-chain V-region To ligate to an expression vector carrying a genomic DNA of human H-chain C-region C r 1, the cloned DNA encoding mouse H-chain V-region was modified by PCR
method. A backward primer MBC 1-S 1 (SEQ ID NO: 7) was designed to hybridize to a DNA sequence encoding the 5' region of the leader sequence of 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 primer MBC1-a (SEQ D7 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 BamHI-recognition sequence. The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a buffer appended thereto. The PCR
solution comprised (per 50 ~l) 0.07 ~,g of plasmid MBC 1 H04 as a template DNA, 50 pmoles of MBC 1-a and 50 pmoles of MBC 1-S 1 as primers, 2.5U of TaKaRa Ex Taq and 0.25 mM
dNTPs in the buffer, over which 50 ~,l of mineral oil was layered. The PCR was run for 30 cycles under the conditions: 9490 for 1 min.; 55~ for 1 min.; 72~ for 2 min.
The DNA
fragments thus amplified by the PCR method were separated by agarose gel electrophoresis on a 3% Nu Sieve GTG Agarose (FMC Bio. Products).

Then, an agarose gel segment containing a DNA fragment of 437 by was excised, and the DNA fragment was purified therefrom using GENECLEAN II Kit (BIO101) in accordance with the instructions included in the kit. The purified DNA was collected by ethanol precipitation, and then dissolved in 20 p1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. An aliquot (1 p1) of the resulting DNA solution was digested with restriction enzymes BamHI and HindIII (Takara Shuzo Co., Ltd.) at 37°C for 1 hour.
The digestion solution was extracted with phenol and chloroform and then precipitated with ethanol to collect the DNA of interest.
The obtained HindIII-BamHI DNA fragment, which containing a gene encoding the mouse H-chain V-region, was subcloned into pUC 19 vector that had been digested with HindIII and BamHI. The resulting plasmid was sequenced on DNA Sequencer 373A
(Perkin-Elmer) using M 13 Primer M4 and M 13 Primer RV as primers and Dye Terminator Cycle Sequencing Kit (Perkin-Elmer). As a result, a plasmid which carried a gene of correct nucleotide sequence encoding the mouse H-chain V-region derived from hybridoma #23-57-137-1 and had a HindIII-recognition sequence and a Kozak sequence on its 5' region and a BamHI-recognition sequence on its 3' region was obtained, which was designated as "MBC lHIpUC 19".
(ii) Construction of H-chain V-region for preparation of cDNA-type of mouse-human chimeric H-chain To ligate to cDNA of the human H-chain C-region C r l, the DNA encoding the mouse H-chain V-region constructed as described above was modified by PCR
method. A
backward primer MBC1HVS2 (SEQ ID NO: 9) for the V-region was designed to cause the replacement of the second amino acid (asparagine) of the sequence encoding the front part of the leader sequence of the H-chain V-region by glycine and to have 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 a DNA sequence encoding the 3' region of the J region, to encoding the 5' region of the C-region and to have ApaI- and SmaI-recognition sequences.
The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a buffer appended thereto. The PCR solution comprised (per 50 p1) 0.6 p.g of plasmid MBC1H/pUCl9 as a template DNA, 50 pmoles of MBC1HVS2 and SO pmoles of MBC1HVR2 as primers, 2.5U of TaKaRa Ex Taq and 0.25 mM of dNTPs in the buffer, over which 50 ~1 of mineral oil was layered. The PCR reaction was run for 30 cycles under the conditions: 94°C for 1 min.; 55°C for 1 min.; 72~ for 1 min. The DNA fragments amplified by the PCR reaction were separated by agarose gel electrophoresis on a 1 % Sea Kem GTG Agarose (FMC Bio. Products). Then, an agarose gel segment containing a DNA
fragment of 456 by was excised and the DNA fragment was purified therefrom using GENECLEAN II Kit (BIO101) in accordance with the instructions included in the kit. The purified DNA was precipitated with ethanol and then dissolved in 20 ~.l of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.
The resulting DNA solution ( 1 p,g) was digested with restriction enzymes EcoRI
and SmaI (Takara Shuzo Co., Ltd.) at 37°C for 1 hour. The digestion solution was extracted with phenol and chloroform and then precipitated with ethanol to collect the DNA. The obtained EcoRI-SmaI DNA fragment, which containing a gene encoding the mouse H-chain V-region, was subcloned into pUCl9 vector that had been digested with EcoRI
and SmaI.
The resulting plasmid was sequenced on DNA Sequencer 373A (Perkin-Elmer) using Primer M4 and M 13 Primer RV, and Dye Terminator Cycle Sequencing Kit (Perkin-Elmer).
As a result, a plasmid which contained a gene of correct nucleotide sequence encoding mouse H-chain V-region derived from hybridoma #23-57-137-1 and had EcoRI- and HindIII-recognition sequences and a Kozak sequence on its 5' region and ApaI- and SmaI-recognition sequences on its 3' region was obtained, which was designated as "MBC 1Hv/pUC
19".
(iii) Construction of expression vector for chimeric antibody H-chain cDNA containing the DNA for human antibody H-chain C-region C ?' 1 was prepared as follows. mRNA was prepared from a CHO cell into which both an expression vector DHFR-DE-RVh-PM-1-f (see WO 92/19759) encoding the genomic DNAs of humanized PM 1 antibody H-chain V-region and human antibody H-chain C-region IgG 1 (N.
Takahashi et al., Cell 29, 671-679, 1982) and an expression vector RV1-PMIa (see WO
92/19759) encoding the genomic DNAs of humanized PMl antibody L-chain V-region and human antibody L-chain r~ chain C-region had been introduced. Using the mRNA, cDNA
containing the humanized PM 1 antibody H-chain V-region and the human antibody C-region C ?' 1 was cloned by RT-PCR method, and then subcloned into plasmid pUC 19 at the HindIII-BamHI site. After sequencing, a plasmid which had the correct nucleotide sequence was obtained, which was designated as "pRVh-PMlf cDNA".
An expression vector DHFR- D E-RVh-PM-1-f in which both a HindIII site located between SV40 promoter and a DHFR gene and an EcoRI site located between EF-1 a promoter and a humanized PM 1 antibody H-chain V-region gene had been deleted, was prepared for the construction of an expression vector for cDNA containing the humanized PM 1 antibody H-chain V-region gene and the human antibody C-region C r 1 gene.
The plasmid obtained (pRVh-PMlf cDNA) was digested with BamHI, blunt-ended with Klenow fragment, and further digested with HindIII, thereby obtaining a blunt-ended HindIII-BamHI fragment. The blunt-ended HindIII-BamHI fragment was ligated to the above-mentioned HindIII site- and EcoRI site-deleted expression vector DHFR- O
E-RVh-PM1-f that had been digested with HindIII and BamHI. Thus, an expression vector RVh-PM 1 f cDNA was constructed which contained cDNA encoding the humanized PM 1 antibody H-chain V-region and the human antibody C-region C 'Y 1.
The expression vector RVh-PM 1 f cDNA containing the cDNA encoding the humanized PM 1 antibody H-chain V-region and the human antibody C-region C Y 1 was digested with ApaI and BamHI, and a DNA fragment containing the H-chain C-region was collected therefrom. The resulting DNA fragment was introduced into the plasmid MBC 1Hv/pUC 19 that had been digested with ApaI and BamHI. The plasmid thus prepared was designated as "MBC IHcDNA/pUC 19". This plasmid contained cDNA encoding the mouse antibody H-chain V-region and the human antibody C-region C Y 1, and had EcoRI-and HindIII-recognition sequences on its 5' region and a BamHI-recognition sequence on its 3' region.
The plasmid MBC 1 HcDNA/pUC 19 was digested with EcoRI and BamHI to give a DNA fragment comprising a nucleotide sequence encoding the chimeric antibody H-chain.
The resulting DNA fragment was introduced into an expression vector pCOS 1 that had been digested with EcoRI and BamHI, thereby giving an expression vector for the chimeric antibody, which was designated as "MBC 1 HcDNA/pCOS 1 ". Here, the expression vector pCOSl was constructed using HEF-PMh-g Y 1 (see WO 92/19759) by deleting therefrom an antibody genes by digestion with EcoRI and SmaI, and then ligating it to EcoRI-NotI-BamHI
Adaptor (Takara Shuzo Co., Ltd.) For preparing a plasmid for the expression in a CHO cell, the plasmid MBCIHcDNA/pUCl9 was digested with EcoRI and BamHI to obtain a DNA fragment containing a gene for the chimeric antibody H-chain. The DNA fragment was then introduced into an expression plasmid pCH01 that had been digested with EcoRI
and BamHI
to give an expression plasmid for the chimeric antibody, which was designated as "MBCIHcDNA/pCH01". Here, the expression vector pCH01 was constructed using DHFR-DE-rvH-PMl-f (see WO 92/19759) by deleting therefrom an antibody gene by digestion with EcoRI and SmaI, and then ligating it to EcoRI-NotI-BamHI
Adaptor (Takara Shuzo Co., Ltd.) (2) Construction of human L-chain C-region (i) Preparation of cloning vector To construct pUC 19 vector containing a gene for human L-chain C-region, a HindIII site-deleted pUC 19 vector was prepared. pUC 19 vector (2 pg) was digested in 20 ~l of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM
DTT, 100 mM KCI, 8 U of HindIII (Takara Shuzo Co., Ltd.) at 37~ for 1 hour. The resulting digestion solution was extracted with phenol and chloroform, and then subjected to ethanol precipitation to collect the DNA of interest.
The DNA collected was reacted in 50 ~,l of a reaction solution containing 50 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 mM NaCI, 0.5 mM dNTPs and 6U of Klenow fragment (GIBCO BRL) at room temperature for 20 min., thereby rendering the terminal ends of the DNA blunt. This reaction mixture was extracted with phenol and chloroform and then subjected to ethanol precipitation to collect the vector DNA.
The vector DNA thus collected was reacted in 10 ~.1 of a reaction solution containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 5 % (v/v) polyethylene glycol-8000 and 0.5 U of T4 DNA ligase (GIBCO BRL) at 16~ for 2 hours, to cause self ligation of the vector DNA. The reaction solution (5 ~1) was added to 100 ~,1 of a solution containing competent cells of E. coli, JM109 (Nippon Gene Co., Ltd.), and the resulting solution was allowed to stand on ice for 30 min., at 42~ for 1 min., and additionally on ice for 1 min. SOC culture medium (500 p1) was added to the reaction solution and then incubated at 37~ for 1 hour. The resulting solution was plated on 2xYT

agar medium (containing 50 ~g/ml of ampicillin) on which X-gal and IPTG had been applied (Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989), and then cultured at 37°C overnight, thereby obtaining a transformant.
The transformant was cultured in 2xYT medium (20 ml) containing ampicillin (50 pg/ml) at 37~ overnight. From the cell fraction of the culture medium, a plasmid DNA
was isolated and purified using Plasmid Mini Kit (QIAGEN) in accordance with the instructions included in the kit. The purified plasmid was digested with HindIII. The plasmid that was confirmed to have a HindIII site-deletion was designated as "pUC 19 D
HindIII" .
(ii) Construction of DNA encoding human L-chain ~1 chain C-region Human antibody L-chain ~l chain C-region is known to have at least four isotypes including Mcg+Ke+Oz-, Mcg-Ke Oz-, Mcg~Ke-Oz+ and Mcg-Ke+Oz- (P. Dariavach, et al., Proc. Natl. Acad. Sci. USA, 84, 9074-9078, 1987). A search was made for a human antibody L-chain ~l chain C-region homologous to the #23-57-137-1 mouse L-chain ~l chain C-region from the EMBL database. As a result, it was found that the isotype Mcg+Ke+Oz~ of the human antibody L-chain ~l chain (Accession No. X57819) (P
Dariavach, et al., Proc. Natl. Acad. Sci. USA, 84, 9074-9078, 1987) showed the highest degree of homology to the #23-57-137-1 mouse L-chain ~1 chain C-region, with a 64.4%
homology in terms of amino acid sequence and a 73.4% homology in terms of nucleotide sequence.
Then, a gene encoding the human antibody L-chain ~ chain C-region was constructed by PCR method. The primers for the PCR were synthesized using 394 DNA/RNA
Synthesizer (ABI). The synthesized primers were as follows: HLAMBI (SEQ ID NO:
11) and HLAMB3 (SEQ ID NO: 13), both having a sense DNA sequence; and HLAMB2 (SEQ

ID NO: 12) and HLAMB4 (SEQ ID NO: 14), both having an antisense DNA sequence;
each primer containing a complementary sequence of 20-23 by on the both terminal ends.
External primers HLAMBS (SEQ ID NO: 15) and HLAMBR (SEQ ID NO: 16) had sequences homologous to the primers HLAMB 1 and HLAMB4, respectively. HLAMBS
contained EcoRI-, HindIII- and BInI-recognition sequences, and HLAMBR
contained an EcoRI-recognition sequence. In the first-round PCR reaction, the reactions between HLAMB 1 and HLAMB2 and between HLAMB3 and HLAMB4 were performed. After the reactions were completed, both of the resulting PCR products were mixed in equivalent quantities, and then assembled in the second-round PCR reaction. The reaction solution was added with the external primers HLAMBS and HLAMBR. This reaction mixture was subjected to the third-round PCR reaction to amplify the full length DNA.
Each PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) in accordance with the instructions included in the kit. In the first-round PCR reaction, 100 p,1 of either a reaction solution containing 5 pmoles of HLAMB1, 0.5 pmole of HLAMB2 and SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) or a reaction solution containing 0.5 pmole of HLAMB3, 5 pmoles of HLAMB4 and SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) was used, over which 50 p1 of mineral oil was layered. The PCR reaction was run for 5 cycles under the conditions: 94°C for 1 min., 60~ for 1 min. and 72°C
for 1 min.
In the second-round PCR reaction, a mixture of both the reaction solutions (50 ~1 each) was used, over which 50 ~l of mineral oil was layered. The PCR reaction was run for 3 cycles under the conditions: 94~ for 1 min., 60~ for 1 min. and 72~ for 1 min.
In the third-round PCR reaction, the reaction solution to which the external primers HLAMBS and HLAMBR (50 pmoles each) were added was used. The PCR reaction was run for 30 cycles under the conditions: 94~ for 1 min., 60~ for 1 min. and 72~
for 1 min.
The DNA fragment obtained by the third-round PCR reaction was subjected to electrophoresis on a 3 % low-melting agarose gel (NuSieve GTG Agarose, FMC), and separated and purified from the gel using GENECLEAN II Kit (BIO 101 ) in accordance with the instructions included in the kit.
The obtained DNA fragment was digested in a reaction solution (20 p1) containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 mM NaCI and 8U of EcoRI
(Takara Shuzo Co., Ltd.) at 37°C for 1 hour. The digestion solution was extracted with phenol and chloroform, and the DNA was collected therefrom by the ethanol precipitation.
The DNA was dissolved in a solution (8 p1) containing 10 mM Tris-HCl (pH 7.4) and 1 mM
EDTA.
The above-prepared plasmid pUC 19 D HindIII (0.8 fig) was digested with EcoRI
in the same manner as set forth above. The digestion solution was subjected to phenoUchloroform extraction and then ethanol precipitation, thereby giving a digested plasmid pUCl9 0 HindIII. The digested plasmid was reacted in a reaction solution (50 p1) containing 50 mM Tris-HCl (pH 9.0), 1 mM MgCl2 and alkaline phosphatase (E.
coli C75;
Takara Shuzo Co., Ltd.) at 37~ for 30 min. to dephosphorylate (i.e., BAP-treat) the plasmid.
The reaction solution was subjected to phenol/chloroform extraction, and the DNA was collected therefrom by ethanol precipitation. The DNA thus obtained was dissolved in a solution (10 ~,1) containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.
The BAP-treated plasmid pUC 19 D HindIII ( 1 p1) was ligated to the above-obtained PCR product (4 p1) using DNA Ligation Kit Ver.2 (Takara Shuzo Co., Ltd.). The resulting plasmid was introduced into a competent cell of E. coli, JM 109, to give a transformant. The transformant was cultured overnight in 2xYT medium (2 ml) containing 50 ~g/ml of ampicillin. From the cell fraction, the plasmid was isolated using QIAprep Spin Plasmid Kit (QIAGEN).
The obtained plasmid was sequenced for the cloned DNA part. The sequencing was performed on 373A DNA Sequencer (ABI) using M13 Primer M4 and M13 Primer RV
(Takara Shuzo Co., Ltd.). As a result, it was found that the cloned DNA had a 12-by deletion therein. The plasmid was designated as "C ~l 0 /pUC 19". Then, for making up for the deleted part, primers HCLMS (SEQ ID NO: 17) and HCLMR (SEQ ID NO: 18) were newly synthesized, and a DNA of correct sequence was reconstructed using these primers by PCR method.
In the first-round PCR reaction, the plasmid C ~l D /pUC 19 having the DNA
deletion therein was used as a template, and the reaction was performed with each of the primer sets of HLAMBS and HCLMS and HCLMS and HLAMB4. The PCR products were purified separately. In the second-round PCR reaction, the PCR products were assembled together. In the third-round PCR reaction, the reaction product of the second-round PCR
reaction was added with external primers HLAMBS and HLAMB4 and amplified to give the full length DNA.
In the first-round PCR reaction, a reaction solution ( 100 p1) containing 0.1 pg of C ~l 0 /pUC 19 as a template, either 50 pmoles of each of the primers HLAMBS
and HCLMR
or 50 pmoles of each of the primers HCLMS and HLAMB4, and SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) was used, over which 50 p1 of mineral oil was layered. The PCR
reaction was run for 30 cycles under the conditions: 94~ for 1 min., 60~ for 1 min. and 72 °C for 1 min.

The PCR products of the first-round PCR reaction, HLAMBS-HCLMR (236 bp) and HCLMS-HLAMB4 (147 bp), were subjected to electrophoresis separately on a 3% low-melting agarose gel to isolate the DNA fragments. The DNA fragments were collected and purified from the gels using GENECLEAN II Kit (BIO101). In the second-round PCR
reaction, 20 p1 of a reaction solution containing 40 ng of each of the purified DNA fragments and 1U of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) was used, over which 25 p1 of mineral oil was layered. The PCR reaction was run for 5 cycles under the conditions: 94~C
for 1 min., 60°C for 1 min. and 72°C for 1 min.
In the third-round PCR reaction, 100 p1 of a reaction solution containing 2 p,1 of the reaction solution obtained by the second-round PCR reaction, 50 pmoles of each of external primers HLAMBS and HLAMB4 and SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) was used, over which 50 p1 of mineral oil was layered. The PCR reaction was run for 30 cycles under the conditions: 94~ for 1 min., 60°rC for 1 min. and 72~ for 1 min., thereby obtaining a DNA fragment of 357 by (the third PCR product). The DNA fragment was subjected to electrophoresis on a 3% low-melting agarose gel to isolate the DNA fragment.
The resulting DNA fragment was collected and purified using GENECLEAN Kit (BIO101).
An aliquot (0.1 pg) of the DNA fragment thus obtained was digested with EcoRI, and then subcloned into plasmid pUC 19 D HindIII that had been BAP-treated.
The resulting plasmid was introduced into a competent cell of E. coli, 3M 109, to form a transformant. The transformant was cultured overnight in 2 ml of 2xYT medium containing 50 pg/ml of ampicillin. From the cell fraction, the plasmid was isolated and purified using QIAprep Spin Plasmid Kit (QIAGEN).
The purified plasmid was sequenced on 373A DNA Sequencer (ABI) using M 13 Primer M4 and M 13 Primer RV (Takara Shuzo Co., Ltd.). The plasmid that was confirmed to have the correct nucleotide sequence without any deletion was designated as "C ~1 /pUC 19".
(iii) Construction of gene encoding human L-chain ~ chain C-region A DNA fragment encoding the L-chain ~c chain C-region was cloned from plasmid HEF-PMIk-gk (WO 92/19759) by PCR method. A forward primer HKAPS (SEQ
ID NO: 19) was designed to contain EcoRI-, HindIII and BInI-recognition sequences, and a backward primer HKAPA (SEQ 117 NO: 20) was designed to contain an EcoRI-recognition sequence. These primers were synthesized on 394 DNA/RNA Synthesizer (ABI).
A PCR reaction was performed using 100 p1 of a reaction solution containing 0.1 pg of plasmid HEF-PMIk-gk as a template, 50 pmoles of each of primers HKAPS and HKAPA
and SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.), over which 50 p,1 of mineral oil was layered. The PCR reaction was run for 30 cycles under the conditions:
94°C for 1 min., 60 °C for 1 min. and 72°C for 1 min., thereby giving a PCR product of 360 bp. The DNA
fragment was isolated and purified by electrophoresis on a 3 % low-melting agarose, and then collected and purified using GENECLEAN II Kit (BIO101).
The thus obtained DNA fragment was digested with EcoRI, and then cloned into plasmid pUC 19 (HindIII that had been BAP-treated. The resulting plasmid was introduced into a competent cell of E. coli, JM 109, to form a transformant. The transformant was cultured overnight in 2 ml of 2xYT medium containing 50 pg/ml of ampicillin.
From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QIAGEN).
The purified plasmid was sequenced on 373A DNA Sequencer (ABI) using M13 Primer M4 and M 13 Primer RV (Takara Shuzo Co., Ltd.). The plasmid that was confirmed to have the correct nucleotide sequence was designated as "C ~ /pUC 19".

(3) Construction of chimeric antibody L-chain expression vector An expression vector for the chimeric #23-57-137-1 antibody L-chain was constructed. A gene encoding #23-57-137-1 L-chain V-region was ligated to the HindIII-BInI site (located just in front of the human antibody C-region) of each of the plasmids C ~1 /pUC 19 and C r~ /pUC 19, thereby obtaining pUC 19 vectors that contained the DNAs encoding the chimeric #23-57-137-1 antibody L-chain V-region and either of the L-chain chain C-region or the L-chain ~ region C-region, respectively. Each of the resulting vectors was then digested with EcoRI to separate the gene for the chimeric antibody L-chain.
The gene was subcloned into HEF expression vector.
That is, a DNA fragment encoding #23-57-137-1 antibody L-chain V-region was cloned from plasmid MBC1L24 by PCR method. Primers used in the PCR method were separately synthesized using 394 DNA/RNA Synthesizer (ABI). A backward primer 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 D7 NO: 22) was designed to contain BgIII- and RcoRI-recognition sequences.
The PCR reaction was performed using 100 ~,l of a reaction solution containing mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.1 ~,g MBC 1 L24, SO
pmoles of each of primers MBCCHL1 and MBCCHL3 and 1 w1 of AmpliTaq (PERKIN
ELMER), over which 50 ~,l of mineral oil was layered. The PCR reaction was run for 30 cycles under the conditions: 94°~ for 45 sec., 60'~ for 45 sec. and 72~
for 2 min.
A PCR product of 444 by was electrophoresed on a 3 % low-melting agarose gel, and collected and purified using GENECLEAN II Kit (BIO101). The purified PCR
product was dissolved in 20 ~.1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.

The PCR product ( 1 ~,1) was digested in 20 ~.1 of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 mM NaCI, 8U of HindIII (Takara Shuzo Co., Ltd.) and 8U of EcoRI (Takara Shuzo Co., Ltd.) at 37~C for 1 hour. The digestion solution was subjected to phenol/chloroform extraction, and the DNA of interest was collected therefrom by ethanol precipitation. The DNA was dissolved in 8 p1 of a solution containing mM Tris-HCl (pH 7.4) and 1 mM EDTA.
In the same manner, plasmid pUCl9 (1 ~.g) was digested with HindIII and EcoRI, and subjected to phenol/chloroform extraction and then ethanol precipitation.
The obtained digested plasmid was BAP-treated with alkaline phosphatase (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 10 ~1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.
The BAP-treated plasmid pUCl9 (1 w1) was ligated to the above-obtained PCR
product (4 ~l) using 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 manner as set forth above, to form a transformant. The transformant was plated on 2xYT agar medium containing 50 ~,g/ml of ampicillin and cultured at 37°C overnight. The resulting transformant was then cultured at 37°C overnight in 2 ml of 2xYT medium containing 50 ~g/ml of ampicillin. From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QIAGEN). After determining the nucleotide sequence, the plasmid that was confirmed to have the correct nucleotide sequence was designated as "CHL/pUC 19".
Each of plasmids C ~ /pUC 19 and C ~c /pUC 19 ( 1 ~.g each) was digested in 20 p1 of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT, 100 mM

The transient expression of the chimeric antibodies was performed using each of the combinations of plasmids MBC 1 HcDNA/pCOS 1 and MBC 1 L ( ~ )/neo and plasmids MBC 1 HcDNA/pCOS 1 and MBC 1 L( r~ )/neo, by co-tansfecting a COS-7 cell with the plasmids by electroporation using Gene Pulser (Bio Rad). That is, the plasmids (10 pg each) were added to a COS-7 cell suspension (0.8 ml; 1 x 107 cells/ml) in PBS(-).
The resulting solution was applied with pulses at an electrostatic capacity of 1,SOOV and 2 ~,F to cause electroporation. After 10 min. of recovery period at room temperature, the electroporated cells were suspended in DMEM medium (GIBCO) containing 2% Ultra Low IgG fetal 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 and centrifuged to remove cell debris, and was 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 performed using AffiGel Protein A MAPSII Kit (Bio Rad) in accordance with the instructions included in the kit.
(5) ELISA
(i) Determination of antibody concentration An ELISA plate for determining antibody concentration was prepared as follows.
Each well of a 96-well ELISA plate (Maxisorp, NLJNC) was coated with 100 p1 of a coating buffer (0.1 M NaHC03, 0.02% NaN3) supplemented with 1 pg/ml of goat anti-human IgG
antibody (TAGO), and then blocked with 200 p,1 of a dilution buffer [50 mM
Tris-HCI, 1 mM
MgCl2, 0.1 M NaCI, 0.05% Tween 20, 0.02% NaN3, 1% bovine serum albumin (BSA);
pH
7.2]. Each well of the plate was added with each of the serial dilutions of the COS-7 cell culture supernatant in which each of the chimeric antibodies had been expressed, or added with each of the serial dilutions of each of the chimeric antibodies per se in a purified form.

The plate was incubated at room temperature for 1 hour and washed with PBS-Tween 20.
Each well of the plate was then added with 100 ~1 of a solution of alkaline phosphatase-conjugated goat anti-human IgG antibodies (TAGO). After the plate was incubated at room temperature for 1 hour and washed with PBS-Tween 20, each well was added with 1 mg/ml of a substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, SIGMA). The solution was measured on its absorbance at 405 nm using Microplate Reader (Bio Rad) to determine the antibody concentration. In this determination, Hu IgGI ~l Purified (The Binding Site) was used as the standard substance.
(ii) Determination of antigen-binding ability An ELISA plate for the determination of antigen-binding ability was prepared as follows. Each well of a 96-well ELISA plate was coated with 100 p1 of a coating buffer supplemented with 1 ~.g/ml of human PTHrP (1-34) (Peptide Research Institute), and then blocked with 200 p1 of a dilution buffer. Each well was added with each of the serial dilutions of the COS-7 cell culture supernatant in which each of the chimeric antibodies had been expressed, or added with each of the serial dilutions of each of the chimeric antibodies per se in a purified form. After the plate was incubated at room temperature and washed with PBS-Tween 20, each well of the plate was added with 100 ~,1 of a solution of alkaline phosphatase-conjugated goat anti-human IgG antibodies (TAGO). After the plate was incubated at room temperature and washed with PBS-Tween 20, each well of the plate was added with 1 mg/ml of a substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, SIGMA). The solution was measured on its absorbance at 405 nm using Microplate Reader (Bio Rad).
As a result, it was found that the chimeric antibodies had an ability to bind to human PTHrP (1-34) and the cloned mouse antibody V-regions had the correct structures (FIG. 5). It was also found that there was no difference in the ability to bind to PTHrP (1-d9 34) between the chimeric antibody with L-chain ~l chain C-region and the chimeric antibody with L-chain ~c chain C-region. Therefore, the humanized antibody L-chain ~l chain was used for construction of the L-chain C-region of the humanized antibody.
(6) Establishment of CHO cell line capable of stable production of chimeric antibodies To establish a cell line capable of producing the chimeric antibodies stably, the above-prepared expression plasmids were introduced into CHO cells (DXB11).
For the establishment of a cell line capable of producing the chimeric antibodies stably, either of the following combinations of the expression plasmids for CHO cell was used: MBC IHcDNA/pCH01 and MBC 1 L( ~1 )/neo; and MBC 1 HcDNA/pCH01 and MBC 1 L( r~ )/neo. A CHO cell was co-transfected with the plasmids by electroporation using Gene Pulser (Bio Rad) as follows. The expression vectors were separately cleaved with a restriction enzyme PvuI to give linear DNAs. The resulting DNAs were extracted with phenol and chloroform and collected by precipitation with ethanol. The plasmid DNAs thus prepared were subjected to electroporation. That is, each of the plasmid DNAs (10 g,g each) was added to 0.8 ml of a cell suspension of CHO cells in PBS(-) (1x10' cells/ml). The resulting solution was applied with pulses at an electrostatic capacity of 1,SOOV and 25 p.F.
After 10 min. of recovery period at room temperature, the electroporated cells were suspended in MEM- cx medium (GIBCO) containing 10% fetal calf serum (GIBCO).
The resulting suspension was cultured using three 96-well plates (Falcon) in a C02 incubator.
On the day following the culturing being started, the medium was replaced by a selective medium [ribonucleoside- or deoxyribonucleoside-free MEM- c~ medium (GIBCO) containing 10% fetal calf serum (GIBCO) and 500 mg/ml of GENETICIN
(G418Sulfate;
GIBCO)]. From the culture medium, cells into which the antibody gene was introduced were selected. The selective medium is replaced by a fresh one. About two weeks after the medium replacement, the cells were observed under a microscope. When a satisfactory cell growth was observed, the amount of the antibodies produced was determined by ELISA as set forth above. Among the cells, those cells which produced a larger amount of antibodies were screened.
Then, the culturing of the established cell line capable of stable production of the antibodies was scaled up in a roller bottle using ribonucleoside- or deoxyribonucleoside-free MEM medium containing 2% Ultra Low IgG fetal calf serum. On day 3 and day 4 of the culturing, the culture supernatant was collected and then filtered on a 0.2-wm filter (Millipore) to remove cell debris therefrom.
Purification of the chimeric antibodies from the CHO cell culture supernatant was performed using POROS Protein A Column (PerSeptive Biosystems) on ConSep LC

(Millipore) in accordance with the instructions included in the kit. The 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 the antigen-binding activity of the purified chimeric antibodies were determined using the same ELISA system as set forth above.
[REFERENCE EXAMPLE 4] Construction of humanized antibody (1) Construction of humanized antibody H-chain (i) Construction of humanized H-chain V-region A humanized #23-57-137-1 antibody H-chain was produced by CDR-grafting technique by means of PCR method. For the production of a humanized #23-57-137-antibody H-chain (version "a") having FRs derived from human antibody S31679 (NBRF-PDB; Cuisinier, A. M. et al., Eur. J. Immunol., 23, 110-118, 1993), the following six PCR
primers were used: CDR-grafting primers: MBC1HGP1 (SEQ ID NO: 23) and MBC1HGP3 (SEQ ID NO: 24) (both containing a sense DNA sequence) and MBC1HGP2 (SEQ ID
NO:

25) and MBC1HGP4 (SEQ ID NO: 26) (both containing an antisense DNA sequence), all of which containing a 15-21 by complementary sequence on both terminal ends thereof; and external primers: MBC1HVS1 (SEQ ID NO: 27) and MBC1HVR1 (SEQ ID NO: 28) having a homology to the CDR-grafting primers MBC1HGP1 and MBC1HGP4, respectively.
The CDR-grafting primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4 were separated on an urea-denatured polyacrylamide gel (Molecular Cloning: A
Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989), and extracted therefrom by crush-and-soak method (Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) in the following manner.
Each of the CDR-grafting primers (1 nmole) was separated on a 6% denatured polyacrylamide gel to give DNA fragments. From the resulting DNA fragments, a DNA
fragment having a desired length was identified on a silica gel thin plate by irradiation of UV
ray and then collected therefrom by crush-and-soak method. The resulting DNA
was dissolved in 20 p1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM
EDTA. The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.). The PCR
reaction solution (100 ~.1) comprised 1 p1 of each of the above-mentioned CDR-grafting primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4, 0.25 mM dNTPs and 2.5U of TaKaRa Ex Taq in the buffer. The PCR reaction was run for 5 cycles under the conditions: 94~C for 1 min., 55~ for 1 min. and 72~C for 1 min. The resulting reaction solution was added with the external primers MBC1HVS1 and MBC1HVR1 (50 pmoles each). Using this reaction mixture, the PCR reaction was run for additional 30 cycles under the same conditions. The DNA fragment thus amplified was separated by agarose gel electrophoresis on a 4% Nu Sieve GTG agarose (FMC Bio. Products).
An agarose segment containing a DNA fragment of 421 by was excised, and the n DNA fragment was purified therefrom using GENECLEANII Kit (BIO101) in accordance with the instructions included in the kit. The DNA fragment thus purified was precipitated with ethanol and then dissolved in 20 ~.l of a solution containing 10 mM Tris-HCI (pH 7.4) and 1 mM EDTA. The resulting PCR reaction mixture was used for subcloning of the DNA
fragment into plasmid pUC 19 that had been digested with BamHI and HindIII, and subsequently the nucleotide sequence of the resulting plasmid was determined.
A plasmid having the correct nucleotide sequence was designated as "hMBCHv/pUCl9".
(ii) Construction of H-chain V-region of Humanized H-chain cDNA
To ligate to cDNA for humanized H-chain C-region C r 1, the DNA for the humanized H-chain V-region constructed in the above step was modified by PCR
method.
For the PCR method, a backward primer MBC1HVS2 was designed to hybridize to the sequence encoding 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 MBC 1HVR2 was designed to hybridize to both the DNA sequence encoding the 3' region of the J region and the DNA sequence encoding the 5' region of the C-region and to have ApaI- and SmaI-recognition sequences.
The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a buffer appended thereto. The PCR reaction solution comprised 0.4 pg of hMBCHv/pUCl9 as a DNA template, 50 pmoles of each of MBC1HVS2 and MBC1HVR2 as primers, 2.5U of TaKaRa Ex Taq and 0.25 mM dNTPs in the buffer. The PCR
reaction was run for 30 cycles under the conditions: 94~C for 1 min., 55~ for 1 min. and 72~ for 1 min.
The DNA fragment thus amplified was separated by agarose gel electrophoresis on a 3% Nu Sieve GTG agarose (FMC Bio. Products).
A gel segment containing a DNA fragment of 456 by was excised, and the DNA

fragment was purified therefrom using GENECLEANII Kit (BIO101) in accordance with the instructions included in the kit. The DNA fragment thus purified was precipitated with ethanol and then dissolved in 20 ~l of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The PCR reaction solution thus obtained was used for subcloning of the DNA
fragment into plasmid pUC 19 that had been digested with EcoRI and SmaI, and then the resulting plasmid was sequenced. As a result, a plasmid was obtained which contained a DNA encoding mouse H-chain V-region derived from hybridoma #23-57-137-1 and also contained EcoRI- and HindIII-recognition sequences and a Kozak sequence on the 5' region and ApaI- and SmaI-recognition sequences on the 3' region, which was designated as "hMBC 1 Hv/pUC 19".
(2) Construction of expression vector for humanized antibody H-chain Plasmid RVh-PM 1 f cDNA carrying a cDNA sequence for hPM 1 antibody H-chain was digested with ApaI and BamHI to give a DNA fragment containing a DNA
fragment containing a DNA encoding the H-chain C-region. The DNA fragment was introduced into plasmid hMBC 1Hv/pUC 19 that had been digested with ApaI and BamHI. The obtained plasmid was designated as "hMBC IHcDNA/pUC 19". This plasmid contained both a DNA
encoding the humanized #23-57-137-1 antibody H-chain V-region and a DNA
encoding the human H-chain C-region C Y 1 and had EcoRI- and HindIII-recognition sequences on the 5' region and a BamHI-recognition sequence on the 3' region. The nucleotide sequence and the corresponding amino acid sequence of the humanized H-chain version "a" carried on the plasmid hMBCIHcDNA/pUCl9 are shown in SEQ ID NO: 58 and SEQ ID NO: 56, respectively.
The plasmid hMBC 1 HcDNA/pUC 19 was digested with EcoRI and BamHI to give a DNA fragment containing a DNA encoding the H-chain. The DNA fragment was introduced into expression plasmid pCOS 1 that had been digested with EcoRI
and BamHI.

As a result, an expression plasmid for a humanized antibody was obtained, which was designated as "hMBCIHcDNA/pCOSI".
To produce a plasmid used for expression in a CHO cell, plasmid hMBC lHcDNA/pUC 19 was digested with EcoRI and BamHI to give a DNA fragment containing a DNA encoding the H-chain. The DNA fragment was introduced into expression vector pCH01 that had been digested with EcoRI and BamHI. As a result, an expression plasmid for the humanized antibody was obtained, which was designated as "hMBC 1 HcDNA/pCH01 ".
(3) Construction of L-chain hybrid V-region (i) Preparation of FR1,2/FR3,4 hybrid antibody A gene for the FR hybrid L-chain having both FRs from a humanized antibody and FRs from a mouse (chimeric) antibody was constructed, and evaluated each region for the humanization. In this step, a hybrid antibody having FRl and FR2 both derived from a human antibody and FR3 and FR4 both derived from a mouse antibody was prepared by utilizing the AflII restriction site located on CDR2.
Plasmids MBC 1 L( ~ )/neo and hMBC 1 L( ~l )/neo ( 10 g.g each) were separately digested in 100 p1 of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 mM NaCI, 0.01 % (w/v) of BSA and 10 U of AflII (Takara Shuzo Co., Ltd.) at 37~ for 1 hour. The reaction solutions were subjected to electrophoresis on a 2% low-melting agarose gel, thereby giving DNA fragments of 6282 by (referred to as "c1" ) and 1022 by (referred to as "c2") from the plasmid MBC1L( ~l )/neo or DNA fragments of 6282 by (referred to as "hl" ) and 1022 by (referred to as "h2") from the plasmid hMBCIL( ~ )/neo.
These DNA fragments were collected and purified from the gels using GENECLEANII Kit (BIO101).

Each of the c 1 and h 1 fragments ( 1 pg each) was BAP-treated. The DNA
fragment was extracted with phenol and chloroform, collected by ethanol precipitation, and then dissolved in 10 p1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.
The BAP-treated c1 and hl DNA fragments (1 p1 each) were ligated to the h2 and c2 DNA fragments (4 ~,l each), respectively, (at 4~ overnight). Each of the ligation products was introduced into a competent cell of E. coli, JM 109, to form a transformant.
The transformant was cultured in 2 ml of 2xYT medium containing 50 pg/ml of ampicillin.
From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QIAGEN).
The purified plasmid was digested in 20 ~1 of a reaction solution containing 10 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and either 2U of ApaLI (Takara Shuzo Co., Ltd.) or 8U of BamHI (Takara Shuzo Co., Ltd.) and HindIII (Takara Shuzo Co., Ltd.) at 37°C
for 1 hour. It was expected that if the cl-h2 was ligated correctly, this digestion reaction would give fragments of 5560/1246/498 by (by the ApaLI digestion) or fragments of 7134/269 by (by the BamHI/HindIII digestion). Based on this expectation, the desired plasmids were identified.
The expression vector encoding the human FR1,2/mouse FR3,4 hybrid antibody L-chain was designated as "h/mMBCIL(~ )/neo". On the other hand, since a clone for the hl-c 1 could not be obtained, recombination on a pUC vector was performed and then the resulting recombinant product was cloned into a HEF vector. In this procedure, plasmid hMBC 1 La ~ /pUC 19, which contained DNA encoding a humanized antibody L-chain V-region without any amino acid replacements, and plasmid hMBCILd ~1 /pUCl9, which contained a DNA encoding a humanized antibody L-chain V-region with an amino acid replacement at the 91-position amino acid tyrosine in FR3 (i.e., the 87th amino acid in accordance with The Kabat's prescription) by isoleucine, were used as templates.
Plasmids MBC 1 L( ~l )/pUC 19, hMBC 1 La ~1 /pUC 19 and hMBC 1 Ld ~l /pUC 19 ( ~,1 each) were separately digested in 30 p.1 of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 mM NaCI, 0.01 % (w/v) of BSA, 16U of HindIII and 4U of AflII at 37~ for 1 hour. The reaction solutions were separately subjected to electrophoresis on a 2% low-melting agarose gel, thereby giving a DNA fragment of 215 by from plasmid MBC1L( ~ )/pUCl9 (referred to as "c2"') and a DNA fragment of 3218 by from each of plasmids hMBC 1 La ~1 /pUC 19 and hMBC 1 Ld ~ /pUC 19 (referred to as "ha 1 "' and "hdl"', respectively). These DNA fragments were collected and purified using GENECLEANII Kit (BIO101).
Each of the ha 1' and hd 1' fragments was ligated to the c2' fragment and then introduced into a competent cell of E. coli, JM 109, to form a transformant.
The transformant was cultured in 2 ml of 2xYT medium containing 50 g,g/ml of ampicillin.
From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit (QIAGEN).
The plasmids thus prepared were designated as "m/hMBC 1 La ~ /pUC 19" for the ha 1' fragment-containing plasmid and "m/hMBC 1 Ld ~1 /pUC 19" for the hd 1' fragment-containing plasmid.
Each of the obtained plasmids m/hMBC 1 La ~l /pUC 19 and m/hMBC 1 Ld ~1 /pUC

was digested with EcoRI. The DNA fragment of 743 by was electrophoresed on a 2% low-melting agarose gel, and then collected and purified therefrom using GENECLEANII Kit (BIO 101 ). The resulting DNA fragment was dissolved in 20 g,1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.
Each of the DNA fragments (4 p1 each) was ligated to the above-obtained BAP-treated HEF vector (1 p1). The ligation product was introduced into a competent cell of E.
coli, JM 109, to form a transformant. The transformant was cultured in 2 ml of 2xYT
medium containing 50 pg/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 20 p1 of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT, 100 mM KCI, 8U of HindIII
(Takara Shuzo Co., Ltd.) and 2U of PvuI (Takara Shuzo Co., Ltd.) at 37~ for 1 hour. It was expected that if the DNA fragment was inserted in the plasmid in a correct orientation, this digestion would give digestion fragments of 5104/2195 bp, whereas if the DNA
fragment is inserted in the plasmid in the reverse orientation, this digestion would give digestion fragments of 4378/2926 bp. The plasmid DNA was identified based on the expectation.
The plasmids thus obtained were expression vectors encoding mouse FR1,2/human FR3,4 hybrid antibody L-chain, which were designated as expression vectors "m/hMBC 1 La ~l /neo"
and "m/hMBC 1 Ld ~ / neo", respectively.
(ii) Preparation of FR1/FR2 hybrid antibody An FR1/FR2 hybrid antibody was prepared in the same manner as set forth above utilizing a SnaBI restriction site located on CDR1.
Plasmids MBC1L( ~1 )/neo and h/mMBCIL( ~ )/neo (10 pg each) were separately digested in 20 p,1 of a reaction solution containing 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT, 50 mM NaCI, 0.01 % (w/v) of BSA and 6U of SnaBI (Takara Shuzo Co., Ltd.) at 37~ for 1 hour. The resulting reaction solutions were further digested in 50 ~1 of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT, 100 mM
KCI, 0.01 % (w/v) of BSA and 6U of PvuI at 37°~ for 1 hour.

The resulting reaction solutions were separately subjected to electrophoresis on a 1.5% low-melting agarose gel, thereby giving DNA fragments of 4955 by (ml) and 2349 by (m2) from the plasmid MBC 1 L( ~ )/neo and DNA fragments of 4955 by (hm 1 ) and 2349 by (hm2) from the plasmid h/mMBC 1L( ~l )/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 p1 of a solution containing 10 mM Tris-HCI (pH
7.4) and 1 mM
EDTA.
The m 1 and hm 1 fragments ( 1 ~,1 each) were ligated to the hm2 and m2 fragments (4 ~,1 each), respectively. Each of the resulting ligation products was introduced into a competent cell of E. coli, JM 109, to form a transformant. The transformant obtained was cultured in 2 ml of 2xYT medium containing 50 ~g/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 20 ~l of a reaction solution containing mM Tris-HCI (pH 7.5), 10 mM MgClz, 1 mM DTT and either 8U of ApaI (Takara Shuzo Co., Ltd.) or 2U of ApaLI (Takara Shuzo Co., Ltd.) at 37~C for 1 hour.
It was expected that if the fragments were ligated correctly, the digestion reaction would give a fragment of 7304 by (by the ApaI digestion) or fragments of 5560/1246/498 by (by the ApaLI digestion) for ml-hm2, and would give fragments of 6538/766 by (by the ApaI
digestion) or fragments of 3535/2025/1246/498 by (by the ApaLI digestion) for hml-m2.
Based on this expectation, the plasmids were identified. As a result, an expression vector encoding a human FRl/mouse FR2,3,4 hybrid antibody L-chain (designated as "hmmMBCIL( ~ )/neo") and an expression vector encoding a mouse FR1/human FR2/mouse FR3,4 hybrid antibody L-chain (designated as "mhmMBC 1 L( ~1 )/neo") were obtained.

(4) Construction of humanized antibody L-chain A humanized #23-57-137-1 antibody L-chain was prepared by CDR-grafting technique by means of PCR method. For the preparation of a humanized #23-57-antibody L-chain (version "a") that contained FR1, 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 525755 (NBRF-PDB), six PCR primers were used.
The six primers were as follows: CDR-grafting primers MBC1LGP1 (SEQ 1T7 NO:
29) and MBC1LGP3 (SEQ 117 NO: 30), both having a sense DNA sequence, CDR-grafting primers MBC1LGP2 (SEQ ID NO: 31) and MBC1LGP4 (SEQ ID NO: 32), both having an antisense DNA sequence, all of which had a 15-21 by complementary sequence on the both terminal ends; and external primers MBC1LVS1 (SEQ ID NO: 33) and MBC1LVR1 (SEQ
ID
NO: 34) having a homology tv the CDR-grafting primers MBC 1 LGP 1 and MBC 1 LGP4, respectively.
The CDR-grafting primers MBC1LGP1, MBC1LGP2, MBC1LGP3 and MBC1LGP4 were separated on a urea-denatured polyacrylamide gel (Molecular Cloning: A
Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) and extracted therefrom by crush-and-soak method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
Each of the CDR-grafting primers (1 nmole each) was separated on a 6%
denatured polyacrylamide gel. The identification of the DNA fragment of a desired length was performed on a silica gel thin plate by irradiation of UV ray. The desired DNA
fragment was collected from the gel by crush-and-soak method. The collected DNA
fragment was dissolved in 20 p1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM
EDTA.

The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a buffer appended thereto. The PCR reaction solution comprised (per 100 p1) 1 ~.1 of each of the CDR-grafting primers MBC 1 LGP l, MBC 1 LGP2, MBC 1 LGP3 and MBC 1 LGP4, 0.25 mM dNTPs, 2.5U of TaKaRa Ex Taq in the buffer. The PCR reaction was run for 5 cycles under the conditions: 94°C for 1 min., 55~ for 1 min. and 72~
for 1 min. The resulting reaction mixture was added with 50 pmoles of each of the external primers MBC1LVS1 and MBC1LVR1. Using this reaction nuxture, the PCR reaction was run for additional 30 cycles under the same conditions. The DNA fragment thus amplified was separated by agarose gel electrophoresis on a 3 % Nu Sieve GTG agarose (FMC
Bio.
Products).
An agarose segment containing a DNA fragment of 421 by was excised, and the DNA fragment was purified therefrom using GENECLEANII Kit (BIO 101 ) in accordance with the instructions included in the kit. The PCR reaction mixture thus obtained was used for subcloning of the DNA fragment into plasmid pUC 19 that had been digested with BamHI
and HindIII. The resulting plasmid was sequenced. The plasmid thus prepared was designated as "hMBCL/pUC 19". In this plasmid, however, the 104-position amino acid (corresponding to the 96th amino acid in accordance with the Kabat's prescription) of CDR4 was replaced by arginine. For the correction of this amino acid to tyrosine, a correction primer MBC1LGP10R (SEQ ID NO: 35) was designed and synthesized. The PCR
reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a buffer appended thereto.
The PCR reaction solution comprised (per 100 ~l) 0.6 ~,g of the plasmid hMBCL/pUC 19 as a template DNA, 50 pmoles of each of the primers MBC1LUS1 and MBC1LGP10R, 2.5U
of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and 0.25 mM dNTPs in the buffer, over which mineral oil (50 L~.1) was layered. The PCR reaction was run for 30 cycles under the conditions: 94~ for 1 min., 55~ for 1 min. and 72~ for 1 min. The DNA fragment thus amplified was separated by agarose gel electrophoresis on a 3% Nu Sieve GTG
agarose (FMC

Bio. Products).
A gel segment containing a DNA fragment of 421 by was excised, and the DNA
fragment was purified therefrom using GENECLEANII Kit (BIO101) in accordance with the instructions included in the kit. The PCR reaction mixture thus prepared was used for subcloning of the DNA fragment into plasmid pUC 19 that had been digested with BamHI and HindIII.
The plasrnid was sequenced using M 13 Primer M4 and M 13 Primer RV. As a result, it was confirmed that the plasmid had the correct sequence. The plasmid was then digested with HindIII and BInI, and a DNA fragment of 416 by was separated by electrophoresis on a 1 % agarose gel. The DNA fragment was purified using GENECLEANII Kit (BIO101) in accordance with the instructions included in the kit, and then introduced into plasmid C ~ /pUC 19 that had been digested with HindIII
and BInI. The resulting plasmid was designated as "hMBC 1 La ~ /pUC 19". This plasmid was digested with EcoRI to give a DNA fragment encoding humanized L-chain. The DNA fragment was introduced into plasmid pCOS 1 so that the initiation codon for the humanized L-chain was located downstream to the EFl c~ promoter. The plasmid thus obtained was designated as "hMBC 1La ~l /pCOS 1". The DNA sequence (including the corresponding amino acid sequence) of the humanized L-chain version "a" is shown in SEQ ID NO: 66. The amino acid sequence of the version "a" is also shown in SEQ ID NO: 47.
A humanized L-chain version "b" was prepared using mutagenesis by PCR method.
The version "b" was designed such that the 43-position amino acid glycine (corresponding to the 43th amino acid in accordance with the Kabat's prescription) was replaced by proline and the 49-position amino acid lysine (corresponding to the 49th amino acid accordance with the Kabat's prescription) by aspartic acid in the version "a". The PCR reaction was performed using plasmid hMBC 1 La ~l /pUC 19 as a template and a mutagenic primer MBC 1 LGPSR
(SEQ ID NO: 36) and a primer MBC 1 LV S 1. The DNA fragment obtained was digested with BamHI and HindIII, and the digestion fragment was subcloned into the BamHI-HindIII
site of pUC 19. After sequencing, the plasmid was digested with HindIII and Af l II, and the resulting digestion fragment was ligated to plasmid hMBC 1 La ~ /pUC 19 that had been digested with HindIII and AfIII.
The thus obtained plasmid was designated as "hMBCILb ~ /pUCl9". This plasmid was digested with EcoRI to give a DNA fragment containing a DNA
encoding the humanized L-chain. The DNA fragment was introduced into plasmid pCOS 1 such that the initiation colon for the humanized L-chain was located downstream to the EF1 cx promoter.
The plasmid thus obtained was designated as "hMBC 1Lb ~. /pCOS 1".
A humanized L-chain version "c" was prepared using mutagenesis by PCR method.
The version "c" was designed such that the 84-position amino acid serine (corresponding to the 80th amino acid in accordance with the Kabat's prescription) was replaced by proline.
The PCR reaction was performed using plasmid hMBC 1 La ~1 /pUC 19 as a template and a mutagenic primer MBC 1 LGP6S (SEQ ID NO: 37) and a primer M 13 Primer RV The DNA
fragment obtained was digested with BamHI and HindIII and then subcloned into pUC 19 that had been digested with BamHI and HindIII.
After sequencing, the plasmid was digested with BstPI and Aor5lHI, and the resulting DNA fragment was ligated to plasmid hMBC 1La ~l /pUC 19 that had been digested with BstPI and Aor5lHI. The plasmid thus obtained was designated as "hMBCILc ~
/pUC 19". This plasmid was digested with EcoRI to give a DNA fragment containing a DNA encoding the humanized L-chain. The fragment was introduced into the EcoRI
site of plasmid pCOS 1 such that the initiation colon for the humanized L-chain was located downstream to the EF1 cx promoter. The plasmid thus obtained was designated as "hMBC 1 Lc ~1 /pCOS 1 ".
Humanized L-chain versions "d", "e" and "f' were also prepared using mutagenesis by PCR method. The versions "d", "e" and "f' were designed such that the 91-position amino acid tyrosine (corresponding to the 87th amino acid in accordance with the Kabat's prescription) was replaced by isoleucine in the versions "a", "b" and "c", respectively. For each of the versions "d", "e" and "f', a PCR reaction was performed using each of plasmid hMBC 1La ~1 /pCOS 1 (for version "d"), hMBC 1 Lb ~l /pCOS 1 (for version "e") and hMBC 1Lc ~/pCOSl (for version "f'), respectively, as a template, a mutagenic primer (SEQ D7 NO: 38) and a primer M-S 1 (SEQ ID NO: 44). The DNA fragment thus obtained was digested with BamHI and HindIII and then subcloned into pUC 19 that had been digested with BamHI and HindIII. After sequencing, the plasmid was digested with HindIII and BInI, and the resulting digestion fragment was ligated to plasmid C ~1 /pUC 19 that had been digested with HindIII and BInI.
The thus obtained plasmids were respectively designated as "hMBC 1 Ld ~. /pUC
19"
(for version "d"), "hMBC 1 Le ~ /pUC 19" (for version "e") and "hMBC 1 Lf ~l /pUC 19" (for version "f'). Each of these plasmids was digested with EcoRI to give a DNA
fragment containing a DNA encoding the humanized L-chain. The DNA fragment was introduced into the EcoRI site of plasmid pCOS 1 such that the initiation codon for the humanized L-chain was located downstream to the EF 1 a promoter of the plasmid. The plasmids thus obtained were respectively designated as "hMBC 1Ld ~l /pCOS 1" (for version "d"), "hMBC 1Le ~ /pCOS 1" (for version "e") and "hMBC 1Lf ~1 /pCOS 1" (for version "f').
Humanized L-chain versions "g" and "h" were also prepared using mutagenesis by PCR method. The versions "g" and "h" were designed such that the 36-position amino acid histidine (corresponding to the 36th amino acid in accordance with the Kabat's prescription) was replaced by tyrosine in the versions "a" and "d", respectively. The PCR
reaction was performed using a mutagenic primer MBC1LGP9R (SEQ ID NO: 39), M13 Primer RV
and plasmid hMBC 1 La ~1 /pUC 19 as a template. An additional PCR was performed using the PCR product thus obtained and M 13 Primer M4 as primers and plasmid hMBC 1 La ~ /pUC 19 as a template. The DNA fragment obtained was digested with HindIII and BInI
and then subcloned into plasmid C ~1 /pUC 19 that had been digested with HindIII and BInI. Using this plasmid as a template, a PCR reaction was performed using primers (SEQ ID NO: 40) and MBC 1 LV S 1. The PCR fragment obtained was digested with ApaI
and HindIII and then introduced into either of plasmids hMBC 1 La ~ /pUC 19 and hMBC 1 Ld ~ /pUC 19 that had been digested with ApaI and HindIII. The plasmids obtained were sequenced. Plasmids that were confirmed to contain the correct sequence were designated as "hMBC 1 Lg ~1 /pUC 19" (for version "g") and "hMBC 1 Lh ~1 /pUC 19" (for version "h"). Each of these plasmids was digested with EcoRI to give a DNA fragment containing a DNA
encoding the humanized L-chain. The DNA fragment was introduced into the EcoRI
site of plasmid pCOS 1 such that the initiation codon for the humanized L-chain was located downstream to the EF1 c~ promoter. The plasmids thus obtained were respectively designated as "hMBCILg~1/pCOSI" (for version "g") and "hMBCILh~1/pCOSl" (for version "h").
Humanized L-chain versions "i", "j ", "k", "1", "m", "n" and "o" were also prepared using mutagenesis by PCR method. The PCR reaction was performed using plasmid hMBCILa~1 /pUCl9 as a template and a mutagenic primer MBC1LGP14S (SEQ ID NO:
41) and a primer V 1 RV ( ~ ) (SEQ ID NO: 43). The resulting DNA fragment was digested with ApaI and BInI and then subcloned into plasmid hMBC 1 Lg ~1 /pUC 19 that had been digested with ApaI and BInI. The obtained plasmid was sequenced, and the clone into which the mutation for each version was introduced was selected. The thus obtained plasmid was g5 designated as "hMBC 1 Lx ~l /pUC 19 (x=i, j, k, l, m, n or o)". This plasmid was digested with EcoRI to give a DNA fragment containing a DNA encoding the humanized L-chain.
The DNA fragment was introduced into the EcoRI site of plasmid pCOS 1 such that the initiation codon for the humanized L-chain was located downstream of the EF1 c~ promoter.
The thus obtained plasrnid was designated as "hMBCILx ~. /pCOSl" (x = i, j, k, l, m, n or o). The DNA sequences (including the corresponding amino acid sequences) of the versions "j ", "1", "m" and "o" are shown in SEQ ID NOs: 67, 68, 69 and 70, respectively. The amino acid sequences of these versions are also shown in SEQ ID Nos: 48, 49, 50 and 51, respectively.
Humanized L-chain versions "p", "q", "r", "s" and "t" were designed such that the 87-position amino acid (tyrosine) was replaced by isoleucine in the versions "i", "j", "m", "1"
and "o", respectively. These versions were prepared utilizing an Aor5lMI
restriction site on FR3 and replacing that site of each of the versions "i", "j", "m", "1" or "o"
by that site of the version "h". That is, an Aor5lHI restriction fragment (514 bp) containing CDR3, a part of FR3 and the entire FR4 were removed from an expression plasmid hMBCILx ~1 /pCOS 1 (x =
i, j, m, l or o). To the removed site, an Aor5lHI restriction fragment (514 bp) in the expression plasmid hMBC 1 Lh ~ /pCOS, which containing CDR3 and a part of FR3 and the entire FR4, was ligated, so that the 91-position amino acid tyrosine (corresponding to the 87th amino acid in accordance with the Kabat's prescription) was replaced by isoleucine. The resulting plasmid was sequenced. A clone of each of the versions "i", "j", "m"
"1" and "o" in which 91-position amino acid tyrosine (corresponding to the 87th amino acid in accordance with the Kabat's prescription) was replaced by isoleucine was selected. These modified versions respectively corresponding to the versions "i", "j ", "m" "1" and "o"
were designated as versions "p", "q", "s", "r" and "t", respectively. The obtained plasmid was designated as "hMBC 1 Lx ~1 /pCOS 1 (x =p, q, s, r or t). The DNA sequences (including the corresponding amino acids) of the versions "q", "r", "s" and "t" are shown in SEQ ID Nos:
71, 72, 73 and 74, respectively. The amino acid sequences of these versions are also shown in SEQ
ID Nos: 52, 53, 54 and 55, respectively.
Plasmid hMBC 1 Lq ~ /pCOS 1 was digested with HindIII and EcoRI and then subcloned into plasmid pUC 19 that had been digested with HindIII and EcoRI.
The plasmid thus obtained was designated as "hMBC 1Lq ~l /pUC 19.
The positions of the replaced amino acids in the individual versions of the humanized L-chain are shown in Table 7 below.
8'7 Table 7 Versions 36 43 45 47 49 80 87 a P D

c P

D I

P I

g I

i Y K

D

k Y K V

D

m D

n Y V

o D

P

q Y K D I

r Y D I

s D

t D I

In Table 7, capital 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 plasmids hMBC 1 HcDNA/pUC 19 and hMBC 1 Lq ~
/pUC 19 were designated as "Escherichia coli JM 109 (hMBC 1 HcDNA/pUC 19)" and "Escherichia coli JM 109 (hMBC 1 Lq ~ /pUC 19)", respectively, which have been deposited under the terms of Budapest Treaty at the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology, Japan (1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) on August 15, 1996, under the accession No. FERM BP-5629 for Escherichia coli JM109 (hMBCIHcDNA/pUCl9), and FERM BP-5630 for Escherichia coli JM 109 (hMBC 1 Lq ~ /pUC 19).
(5) Transfection into COS-7 cell For the evaluation of the antigen-binding activity and the neutralizing activity of the hybrid antibodies and the humanized #23-57-137-1 antibodies, the above-prepared expression plasmids were expressed transiently in COS-7 cells. For the transient expression of the L-chain hybrid antibodies, each of the following combinations of plasmids were co-transfected into a COS-7 cell by electroporation using Gene Pulser (Bio Rad):
hMBCIHcDNA/pCOSl and h/mMBC 1L( ~1 )/neo; hMBC lHcDNA/pCOS 1 and m/hMBC 1La ~l /neo;
hMBC lHcDNA/pCOS 1 and m/hMBC 1 Ld ~ /neo; hMBC lHcDNA/pCOS 1 and hmmMBC 1 L( ~1 )/neo; and hMBC 1 HcDNA/pCOS 1 and mhmMBC 1 L( ~. )/neo. That is, a cell suspension (0.8 ml) of COS-7 cells in PBS(-) (1x10' cells/ml) was added with each combination of the plasmid DNAs ( 10 pg each). The resulting solution was applied with pulses at an electrostatic capacity of 1,500V and 25 gF. After 10 min. of recovery period at room temperature, the electroporated cells were suspended in DMEM medium containing 2%
Ultra Low IgG fetal 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 and centrifuged to remove cell debris. The solutions thus prepared were provided for use in the ELISA below.
For the transient expression of the humanized #23-57-137-1 antibodies, plasmids of hMBC lHcDNA/pCOS 1 and hMBC 1 Lx ~l /pCOS 1 (x = a-t) were co-transfected into a COS-7 cell using Gene Pulser (Bio Rad) in the same manner as described for the above hybrid antibodies. The culture supernatants were prepared and provided for use in the ELISA
below.
The purification of the hybrid antibodies and the humanized antibodies from the COS-7 cell culture supernatants was performed using AffiGel Protein A MAPSII
Kit (Bio Rad) in accordance with the instructions included in the kit.

(6) ELISA
(i) Determination of antibody concentration An ELISA plate for determining antibody concentration was prepared as follows.
Each well of a 96-well ELISA plate (Maxisorp, NUNC) was coated with 100 p1 of a coating buffer (0.1 M NaHC03, 0.02% NaN3) containing 1 pg/ml of goat anti-human IgG
antibody (TAGO) and then blocked with 200 ~c 1 of a dilution buffer [50 mM Tris-HCI, 1 mM MgCl2, 0.1 M NaCI, 0.05% Tween 20, 0.02% NaN3, 1% bovine serum albumin (BSA); pH
7.2].
Each of the wells was added with each of the serial dilutions of the COS cell culture supernatant in which each of the hybrid antibodies and the humanized antibodies was expressed, or added with 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 for 1 hour and washed with PBS-Tween 20. Subsequently, each of the wells was added with 100 p1 of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO). The plate was incubated at room temperature for 1 hour and washed with PBS-Tween 20.
Subsequently, each of the wells was added with 1 mg/ml of a substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, SIGMA). The solution in each well was measured on its absorbance at 405 nm using Microplate Reader (Bio Rad) to determine the antibody concentration. In this determination, Hu IgGI ~ Purified (The Binding Site) was used as the standard substance.
(ii) Determination of antigen-binding ability An ELISA plate for determining antigen-binding ability was prepared as follows.
Each well of a 96-well ELISA plate (Maxisorp, NUNC) was coated with 100 p,1 of a coating buffer containing 1 p,g/ml of human PTHrP (1-34) and then blocked with 200 p.1 of a dilution buffer. Subsequently, each well was added with each of the serial dilutions of the COS-7 cell culture supernatant in which each of the hybrid antibodies and humanized antibodies was expressed, or added with 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 was added with 100 ~.l of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO). The plate was incubated at room temperature and washed with PBS-Tween 20. Subsequently, each well was added with 1 mg/ml of a substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, SIGMA). The solution was measured on its absorbance at 405 nm using Microplate Reader (Bio Rad).
(7) Confirmation of activities (i) Evaluation of humanized H-chain It was found that an antibody having both a humanized H-chain version "a" and a chimeric L-chain exhibited the same level of PTHrP-binding activity as that of a chimeric antibody. This result suggests that the version "a" achieves the humanization of the H-chain V-region in the degree enough to evaluate the humanization. Therefore, the humanized H-chain version "a" was provided for use as a humanized antibody H-chain in the following experiments.
(ii) Activity of hybrid antibodies (ii-a) FR1,2/FR3,4 hybrid antibody When the L-chain was h/mMBC 1L( ~ ), no antigen-binding activity was observed.
In contrast, when the L-chain was either m/hMBC 1 La ~ or m/hMBC 1 Ld ~1, the same level of antigen-binding activity as that of the chimeric #23-57-137-1 antibody was observed (FIG.
7). These results suggest that FR3 and FR4 have no problem as humanized antibodies but FR1 and FR2 contain amino acid residues) that need to be replaced.
(ii-b) FR1/FRZ hybrid antibody When the L-chain was mhmMBCIL ( ~l ), no antigen-binding activity was observed.
In contrast, when the L-chain was hmmMBC 1 L( ~ ), the same level of antigen-binding activity as that of the chimeric #23-57-137-1 antibody was observed (FIG. 8).
These results suggest that FR 1 has no problem as a humanized antibody but FR2 contains amino acid residues) that need to be replaced.
(iii) Activity of humanized antibodies The antigen-binding activity of the humanized antibodies having the L-chain versions "a" to "t", respectively, were determined. As a result, it was found that the humanized antibodies having the L-chain versions "j ", "1" "m", "o", "q", "r", "s" and "t"
exhibited the same levels of PTHrP-binding activity as that of the chimeric antibody.
(8) Establishment of CHO cell line capable of stable production of antibody For establishing a cell line capable of stable production of humanized antibodies, each of the above-prepared expression plasmids was introduced into a CHO cell (DXB11).
That is, the establishment of a cell line capable of stable production of a humanized antibody was performed using each of the following combinations of plasmids as expression vectors for a CHO cell; hMBCIHcDNA/pCH01 and hMBCILm~/pCOSl;
hMBCIHcDNA/pCH01 andhMBCILq~1/pCOSl; andhMBCIHcDNA/pCH01 and hMBC 1 Lr ~ /pCOS 1. The plasmids were co-transfected into a CHO cell by electroporation using Gene Pulser (Bio Rad). Subsequently, the expression vectors were separately cleaved with restriction enzyme PvuI to give linear DNA fragments. The resulting DNA
fragments were extracted with phenol and chloroform and then precipitated with ethanol.
The DNA
fragments thus prepared were used in the subsequent electroporation. That is, the plasmid DNA fragments (10 pg each) were added to 0.8 ml of a cell suspension of CHO
cells in PBS(-( 1 x 107 cells/ml). The resulting solution was applied with pulses at an electrostatic capacity of 1,500V and 25 ~.F. After 10 min. of recovery period at room temperature, the cells thus treated were suspended in MEM- c~ medium (GIBCO) containing 10% fetal calf serum (GIBCO), and then cultured in a C02 incubator using 96-well plates (Falcon).
On the day following the culturing being started, the medium was replaced by ribonucleoside- or deoxyribonucleoside-free MEM- a selective medium containing 10% fetal calf serum (GIBCO) and 500 mg/ml of GENETICIN (G418Sulfate; GIBCO). From the culture medium, cells into which the antibody gene was introduced were selected. The culture medium was replaced by a fresh one. About two weeks after the medium replacement, the cells were observed microscopically. When a satisfactory cell growth was observed, the amount of the antibodies produced was determined by conventional ELISA for determination of antibody concentration as set forth above. Among the cells, those cells which produced a larger amount of antibodies were screened.
The culturing of the established cell line capable of stable production of antibodies was scaled up in a roller bottle using a ribonucleoside- or deoxyribonucleoside-free MEM- cx medium containing 2% Ultra Low IgG fetal calf serum. On each of day 3 and day 4 of the culturing, the culture supernatant was collected and filtered on a 0.2-pm filter (Millipore) to remove cell debris therefrom. The purification of the humanized antibodies from the culture supernatant of the CHO cells was performed using POROS Protein A Column (PerSeptive Biosystems) on ConSep LC 100 (Millipore) in accordance with the appended instructions.
The humanized antibodies were provided for use in the determination of neutralizing activity and examination of pharmacological efficacy in hypercalcemic model animals.
The concentration and the antigen-binding activity of the purified humanized antibodies were determined by the ELISA system as set forth above.
[REFERENCE EXAMPLE 5] Determination of neutralizing activity The determination of neutralizing activity of the mouse antibodies, the chimeric antibodies and the humanized antibodies was performed using rat myeloma cell line ROS 17/2.8-5 cells. The ROS 17/2.8-5 cells were cultured in Ham'S F-12 medium (GIBCO) containing 10% fetal calf serum (GIBCO) in a C02 incubator. The ROS17/2.8-5 cells were seeded into each well of a 96-well plate at a density of 104 cells/100 pl/well and cultured for one day. After the culturing was completed, the culture medium was replaced by Ham'S F-12 medium (GIBCO) containing 4 mM Hydrocortisone and 10% fetal calf serum.
After culturing for three to four days, the cultured cells were washed with 260 ~,1 of Ham'S F-12 medium (GIBCO), and then added with 80 p1 of Ham's F-12 medium containing 1 mM
isobutyl-1-methyl xanthine (IBMX, SIGMA), 10% fetal calf serum and 10 mM
HEPES.
The resulting mixture was incubated at 37~ for 30 min.
The culture mediums of the mouse antibodies, the chimeric antibodies and the humanized antibodies to be tested for neutralizing activity were previously diluted serially in the following dilution series: [10 pg/ml, 3.3 wg/ml, 1.1 g,g/ml and 0.37 pg/ml], [10 pglml, 2 pg/ml, 0.5 pg/ml and 0.01 pg/ml] and [ 10 pg/ml, 5 ~,g/ml, 1.25 pg/ml, 0.63 ~.g/ml and 0.31 ~g/ml]. Each of the diluted antibody sample solutions was mixed with an equivalent amount of 4 ng/ml of PTHrP (1-34). The resulting mixed solution (80 ~,l) was added to each well.
In each well, the final concentration of each antibody became a quarter of the above-mentioned concentration of the antibody, and accordingly the concentration of PTHrP (1-34) became 1 ng/ml. After the treatment at room temperature for 10 min., the culture supernatant was removed and the residue was washed with PBS three times.
Subsequently, cAMP in the cells was extracted with 100 p1 of a 0.3% HCl-95% ethanol and then evaporated using a water jet aspirator to remove the HCl-ethanol. The residue was dissolved in 120 p1 of EIA buffer appended to cAMP EIA Kit (CAYMAN CHEMICAL'S) to extract the CAMP
therefrom. The cAMP was determined using cAMP EIA Kit (CAYMAN CHEMICAL'S) in accordance with the instructions included in the kit. As a result, it was found that, among the humanized antibodies having the same levels of antigen-binding activity as that of the chimeric antibody, those antibodies having L-chain versions "q", "r", "s" and "t" (in which the 91-position tyrosine was replaced by isoleucine) exhibited the similar neutralizing activity to 9~

that of the chimeric antibody, and that antibody having a L-chain version "q"
exhibited the strongest neutralizing activity.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
Industrial Applicability The present invention provides a tissue degradation inhibiting agent which comprises as an active ingredient a substance inhibiting the binding of a parathyroid hormone-related peptide to its receptor. The inhibiting agent of the present invention is useful as a pharmaceutical composition for inhibiting body weight loss associated cancerous cachexia.
Sequence Listing Free Text SEQ ID NO: Synthesized SEQ ID NO: Synthesized SEQ ID NO: Synthesized SEQ 117 NO: Synthesized SEQ ID NO: Synthesized DNA

SEQ ll~ NO: Synthesized SEQ ID NO: Synthesized SEQ ID NO: Synthesized SEQ ID NO: Synthesized SEQ ID NO: Synthesized DNA

SEQ ID NO: Synthesized SEQ ID NO: Synthesized SEQ ID NO: Synthesized SEQ ID NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ a7 NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ ~ NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ n7 NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized SEQ m NO: Synthesized r SEQ B7 NO: 42 Synthesized DNA
SEQ m NO: 43 Synthesized DNA
SEQ m NO: 44 Synthesized DNA

r SEQUENCE LISTING
<110~ CHUGAI PHARMACEUTICAL CO., LTD.
<120~ Tissue degradation inhibiting agent <130~ PH-1015-PCT
<140~ PCT/JP00/05886 <141~ 2000-08-30 <150> JP2000-52414 <151> 2000-02-28 <160~ 75 <170~ PatentIn Ver. 2.0 <210~ 1 <211~ 20 <212~ DNA
<213> Artificial SeQuence <220>
<223~ Synthetic DNA
<400~ 1 aaatagccct tgaccaggca 20 <210> 2 <211~ 38 <212~ DNA
<213> Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 2 ctggttcggc ccacctctga aggttccaga atcgatag 38 <210~ 3 <211~ 28 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 3 ggatcccggg ccagtggata gacagatg 28 <210~ 4 <211~ 29 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 4 ggatcccggg tcagrggaag gtggraaca 29 <210~ 5 <211~ 17 <212~ DNA
<213~ Artificial Sequence <220~
<223> Synthetic DNA
<400~ 5 gttttcccag tcacgac 17 <210~ 6 <211~ 17 <212~ DNA
<213> Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 6 caggaaacag ctatgac 17 <210~ 7 <211~ 31 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 7 gtctaagctt ccaccatgaa acttcgggct c 31 <210~ 8 <211~ 30 <212~ DNA
<213~ Artificial Sequence <220>
<223~ Synthetic DNA
<400~ 8 tgttggatcc ctgcagagac agtgaccaga 30 <210~ 9 <211~ 36 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 9 gtctgaattc aagcttccac catggggttt gggctg 36 <210~ 10 <211~ 41 <212~ DNA
<213~ Artificial SeQUence <220~
<223~ Synthetic DNA
<400~ 10 tttcccgggc ccttggtgga ggctgaggag acggtgacca g 41 <210~ 11 <211> 109 <212~ DNA
<213> Artificial SeQUence <220~
<223~ Synthetic DNA
<400~ 11 gtctgaattc aagcttagta cttggccagc ccaaggccaa ccccacggtc accctgttcc 60 cgccctcctc tgaggagctc caagccaaca aggccacact agtgtgtct 109 <210~ 12 <211~ 110 <212~ DNA
<213~ Artificial Sequence <220~
<223> Synthetic DNA
<400> 12 ggtttggtgg tctccactcc cgccttgacg gggctgccat ctgccttcca ggccactgtc 60 acagctcccg ggtagaagtc actgatcaga cacactagtg tggccttgtt 110 <210~ 13 <211> 98 <212> DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 13 ggagtggaga ccaccaaacc ctccaaacag agcaacaaca agtacgcggc cagcagctac 60 ctgagcctga cgcccgagca gtggaagtcc cacagaag 98 <210~ 14 <211~ 106 <212~ DNA

<213~ Artificial Sequence <220~
<223> Synthetic DNA
<400~ 14 tgttgaattc ttactatgaa cattctgtag gggccactgt cttctccacg gtgctccctt 60 catgcgtgac ctggcagctg tagcttctgt gggacttcca ctgctc 106 <210~ 15 <211~ 43 <212> DNA
<213~ Artificial Sequence <220>
<223~ Synthetic DNA
<400~ 15 gtctgaattc aagcttagta cttggccagc ccaaggccaa ccc 43 <210~ 16 <211~ 20 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA

<400~ 16 tgttgaattc ttactatgaa 20 <210~ 17 <211~ 39 <212~ DNA
<213~ Artificial SeQuence <220>
<223~ Synthetic DNA
<400~ 17 caacaagtac gcggccagca gctacctgag cctgacgcc 39 <210> 18 <211> 39 <212~ DNA
<213~ Artificial SeQuence <220~
<223~ Synthetic DNA
<400~ 18 gtagctgctg gccgcgtact tgttgttgct ctgtttgga 39 <210~ 19 <211~ 46 <212~ DNA

<213~ Artificial SeQUence <220>
<223~ Synthetic DNA
<400~ 19 gtctgaattc aagcttagtc ctaggtcgaa ctgtggctgc accatc 46 <210> 20 <211> 34 <212> DNA
<213> Artificial Sequence <220~
<223> Synthetic DNA
<400~ 20 tgttgaattc ttactaacac tctcccctgt tgaa 34 <210~ 21 <211~ 35 <212~ DNA
<213~ Artificial SeQUence <220~
<223> Synthetic DNA
<400~ 21 gtctaagctt ccaccatggc ctggactcct ctctt 35 <210~ 22 <211~ 48 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 22 tgttgaattc agatctaact acttacctag gacagtgacc ttggtccc 48 <210~ 23 <211> 128 <212> DNA
<213~ Artificial Se4uence <220~
<223> Synthetic DNA
<400~ 23 gtctaagctt ccaccatggg gtttgggctg agctgggttt tcctcgttgc tcttttaaga 60 ggtgtccagt gtcaggtgca gctggtggag tctgggggag gcgtggtcca gcctgggagg 120 tccctgag 128 <210~ 24 <211~ 125 <212~ DNA
<213~ Artificial SeQuence <220~
<223~ Synthetic DNA
<400~ 24 accattagta gtggtggtag ttacacctac tatccagaca gtgtgaaggg gcgattcacc 60 atctccagag acaattccaa gaacacgctg tatctgcaaa tgaacagcct gagagctgag 120 gacac <210> 25 <211~ 132 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 25 ctaccaccac tactaatggt tgccacccac tccagcccct tgcctggagc ctggcggacc 60 caagacatgc catagctact gaaggtgaat ccagaggctg cacaggagag tctcagggac 120 ctcccaggct gg 132 <210~ 26 <211~ 110 <212~ DNA
<2I3~ Artificial SeQUence <220~
<223~ Synthetic DNA
<400~ 26 tgttggatcc ctgaggagac ggtgaccagg gttccctggc cccagtaagc aaagtaagtc 60 atagtagtct gtctcgcaca gtaatacaca gccgtgtcct cagctctcag 110 <210> 27 <211~ 30 <212~ DNA
<213~ Artificial SeQuence <220~
<223~ Synthetic DNA
<400~ 27 gtctaagctt ccaccatggg gtttgggctg 30 <210~ 28 <211~ 30 <212~ DNA
<213~ Artificial SeQuence <220~
<223~ Synthetic DNA
<400~ 28 tgttggatcc ctgaggagac ggtgaccagg 30 <210~ 29 <211~ 133 <212~ DNA
<213~ Artificial Sequence <220>
<223~ Synthetic DNA
<400~ 29 acaaagcttc caccatggcc tggactcctc tcttcttctt ctttgttctt cattgctcag 60 gttctttctc ccagcttgtg ctgactcaat cgccctctgc ctctgcctcc ctgggagcct 120 cggtcaagct cac 133 <210~ 30 <211~ 118 <212~ DNA
<213> Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 30 agcaagatgg aagccacagc acaggtgatg ggattcctga tcgcttctca ggctccagct 60 ctggggctga gcgctacctc accatctcca gcctccagtc tgaggatgag gctgacta 118 <210~ 31 <211> 128 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 31 ctgtggcttc catcttgctt aagtttcatc aagtaccgag ggcccttctc tggctgctgc 60 tgatgccatt caatggtgta cgtactgtgc tgactactca aggtgcaggt gagcttgacc 120 gaggctcc <210~ 32 <211~ 114 <212> DNA
<213> Artificial Sequence <220>
<223~ Synthetic DNA
<400~ 32 cttggatccg ggctgaccta ggacggtcag tttggtccct ccgccgaaca ccctcacaaa 60 ttgttcctta attgtatcac ccacaccaca gtaatagtca gcctcatcct caga 114 <210~ 33 <211> 17 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 33 acaaagcttc caccatg 17 <210> 34 <211~ 19 <212~ DNA
<213> Artificial SeQuence <2Z0~
<223~ Synthetic DNA
<400~ 34 cttggatccg ggctgacct 19 <210> 35 <211~ 75 <212~ DNA
<213> Artificial SeQUence <220~
<223~ Synthetic DNA
<400~ 35 cttggatccg ggctgaccta ggacggtcag tttggtccct ccgccgaaca cgtacacaaa 60 ttgttcctta attgt 75 <210> 36 <211~ 43 <212~ DNA
<213~ Artificial Sequence <220~
<223> Synthetic DNA
<400~ 36 aaaggatcct taagatccat caagtaccga gggggcttct ctg 43 <210~ 37 <211~ 46 <212> DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 37 acaaagctta gcgctacctc accatctcca gcctccagcc tgagga 46 <210~ 38 <211~ 111 <212> DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 38 cttggatccg ggctgaccta ggacggtcag tttggtccct ccgccgaaca cgtacacaaa 60 ttgttcctta attgtatcac ccacaccaca gatatagtca gcctcatcct c 111 <210> 39 <211~ 42 <212~ DNA
<213~ Artificial Sequence <220>
<223> Synthetic DNA
<400> 39 cttctctggc tgctgctgat accattcaat ggtgtacgta ct 42 <210> 40 <211~ 26 <212~ DNA
<213~ Artificial Sequence <220~
<223> Synthetic DNA
<400~ 40 cgagggccct tctctggctg ctgctg 26 <210~ 41 <211~ 35 <212~ DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 41 gagaagggcc ctargtacst gatgrawctt aagca 35 <210~ 42 <211~ 35 <212~ DNA
<213~ Artificial SeQUence <220~
<223~ Synthetic DNA
<400~ 42 cacgaattca ctatcgattc tggaaccttc agagg 35 <210~ 43 <211> 18 <212> DNA
<213~ Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 43 ggcttggagc tcctcaga 18 <210~ 44 <211~ 20 <212~ DNA
<213> Artificial Sequence <220~
<223~ Synthetic DNA
<400~ 44 gacagtggtt caaagttttt 20 <210~ 45 <211~ 118 <212~ PRT
<213~ Mus musculus <400~ 45 Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser Phe Ser Leu Gly Ala Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro Lys Tyr Val Met Asp Leu Lvs Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ser Ile Ser Asn Ile Gln Pro Glu Asp Glu Ala Met Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly Gln Pro <210~ 46 <211~ 118 <212~ PRT
<213~ Mus musculus <400> 46 Glu Val Leu ValGlu Ser Gly AspLeu Val ProGly Gln Gly Lys Gly Ser Leu Leu SerCys Ala Ser GlyPhe Thr SerSer Lys Ala Phe Tyr Gly Met Trp IleArg Gln Pro AspLys Arg GluTrp Ser Thr Leu Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala <210~ 47 <211~ 116 <212~ PRT
<213~ Homo sapiens <400~ 47 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly <210~ 48 <211~ 118 <2I2~ PRT
<213~ Homo Sapiens <400~ 48 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro <210~ 49 <211~118 <212~PRT

<213~Homo Sapiens <400~ 49 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro <210>50 <211~118 <212~PRT

<213~Homo sapiens <400~ 50 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro <210~51 <211>118 <212~PRT

<213~Homo sapiens <400~ 51 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro <210~ 52 <211~ 118 <212~ PRT
<213~ Homo sapiens <400~ 52 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro <210> 53 <211~ 118 <212> PRT
<213~ Homo Sapiens <400> 53 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Va1 Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro <210~ 54 <211~ 118 <212~ PRT
<213~ Homo sapiens <400> 54 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro <210> 55 <211~ 118 <212~ PRT

<213> Homo sapiens <400~ 55 Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro <210~ 56 <211~ 118 <212~ PRT
<213~ Homo Sapiens <400~ 56 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser <210~ 57 <211~ 411 <212~ DNA
<213~ Mus musculus <220~
<221~ CDS
<222~ ( 1) . . (411) <220~
<221> mat_peptide <222~ (58) . . (411) <400~

atgaac ttcggg ctcagc ttgatt ttcctt gcc ctc:att ttaaaa ggt 48 MetAsn PheGly LeuSer LeuIle PheLeu Ala LeuIle LeuLys Gly gtccag tgtgag gtgcaa ctggtg gagtct ggg ggagac ttagtg aag 96 ValGln CysGlu ValGln LeuVal GluSer Gly GlyAsp LeuVal Lys cctgga gggtcc ctgaaa ctctcc tgtgca gcc tctgga ttcact ttc 144 ProGly GlySer LeuLys LeuSer CysAla Ala SerGly PheThr Phe agtagc tatggc atgtct tggatt cgccag act ccagac aagagg ctg 192 SerSer TyrGly MetSer TrpIle ArgGln Thr ProAsp LysArg Leu gagtgg gtcgca accatt agtagt ggtggt agt tacacc tactat cca 240 GluTrp ValAla ThrIle SerSer GlyGly Ser TyrThr TyrTyr Pro gacagt gtgaag gggcga ttcacc atctcc aga gacaat gccaag aac 288 AspSer ValLys GlyArg PheThr IleSer Arg AspAsn AlaLys Asn acccta tacctg caaatg agcagt ctgaag tct gaggac acagcc atg 336 ThrLeu TyrLeu GlnMet SerSer LeuLys Ser GluAsp ThrAla Met ttttac tgtgca agacag actact atgact tac tttget tactgg ggc 384 PheTyr CysAla ArgGln ThrThr MetThr Tyr PheAla TyrTrp Gly caaggg actctg gtcact gtctct gca 411 GlnGly ThrLeu ValThr ValSer Ala <210~ 58 <211~ 411 <212~ DNA
<213~ Homo sapiens <220~
<221~ CDS
<222~ (1) . . (411) <220~
<221~ mat_peptide <222~ (58) . . (411) <400~

atgggg tttggg ctgagc tgggtt ttcctc gtt getctt ttaaga ggt 48 MetGly PheGly LeuSer TrpVal PheLeu Val AlaLeu LeuArg Gly gtccag tgtcag gtgcag ctggtg gagtct ggg ggaggc gtggtc cag 96 ValGln CysGln ValGln LeuVal GluSer Gly GlyGly ValVal Gln cctggg aggtcc ctgaga ctctcc tgtgca gcc tctgga ttcacc ttc 144 ProGly ArgSer LeuArg LeuSer CysAla Ala SerGly PheThr Phe agtagc tatggc atgtct tgggtc cgccag get ccaggc aagggg ctg 192 SerSer TyrGly MetSer TrpVal ArgGln Ala ProGly LysGly Leu gagtgg gtggca accatt agtagt ggtggt agt tacacc tactat cca 240 Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro gac agt gtg aag ggg cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn acg ctg tat ctg caa atg aac agc ctg aga get gag gac acg get gtg 336 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val tat tac tgt gcg aga cag act act atg act tac ttt get tac tgg ggc 384 Tyr Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe Ala Tyr Trp Gly cag gga acc ctg gtc acc gtc tcc tca 411 Gln Gly Thr Leu Val Thr Val Ser Ser <210> 59 <211~ 11 <212~ PRT
<213~ Homo sapiens <400~ 59 Lys Ala Ser Gln Asp Val Asn Thr Ala Val Ala <210~ 60 <211> 7 <212~ PRT
<213~ Homo sapiens <400~ 60 Ser Ala Ser Asn Arg Tyr Thr <210~ 61 <211> 9 <212~ PRT
<213~ Homo Sapiens <400~ 61 Gln Gln His Tyr Ser Thr Pro Phe Thr <210~ 62 <211~ 5 <212~ PRT
<213~ Homo sapiens <400~ 62 Pro Tyr Trp Met Gln <210~ 63 <211~ 16 <212> PRT
<213~ Homo sapiens cag gga acc ctg gtc ac <400~ 63 Ser Ile Phe Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys Phe Lys Gly <210~ 64 <211~ 11 <212> PRT
<213~ Homo sapiens <400~ 64 Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr <210~ 65 <211~ 411 <212~ DNA
<213~ Mus musculus <220>
<221~ CDS
<222~ (1) . . (411) <220~
<221~ mat_peptide <222~ (58) . . (411) <400~ 65 atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly tctttc tcccaa cttgtg ctcact cagtca tcttca gcc tctttc tcc 96 SerPhe SerGln LeuVal LeuThr GlnSer SerSer Ala SerPhe Ser ctggga gcctca gcaaaa ctcacg tgcacc ttgagt agt cagcac agt 144 LeuGly AlaSer AlaLys LeuThr CysThr LeuSer Ser GlnHis Ser acgtac accatt gaatgg tatcag caacag ccactc aag cctcct aag 192 ThrTyr ThrIle GluTrp TyrGln GlnGln ProLeu Lys ProPro Lys tatgtg atggat cttaag caagat ggaagc cacagc aca ggtgat ggg 240 TyrVal MetAsp LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly attcct gatcgc ttctct ggatcc agctct ggtget gat cgctac ctt 288 IlePro AspArg PheSer GlySer SerSer GlyAla Asp ArgTyr Leu agcatt tccaac atccag ccagaa gatgaa gcaatg tac atctgt ggt 336 SerIle SerAsn IleGln ProGlu AspGlu AlaMet Tyr IleCys Gly gtgggt gataca attaag gaacaa tttgtg tatgtt ttc ggcggt ggg 384 ValGly AspThr IleLys GluGln PheVal TyrVal Phe GlyGly Gly accaag gtcact gtccta ggtcag ccc 411 ThrLys ValThr ValLeu GlyGln Pro <210~

<211~411 <212~DNA

<213~Homo sapiens <220>
<221~ CDS
<222~ (1).. (411) <220~
<221~ mat_peptide <222~ (58) . . (411) <400>

atggcc tggact cctctc ttcttc ttcttt gttctt cat tgctca ggt 48 MetAla TrpThr ProLeu PhePhe PhePhe ValLeu His CysSer Gly tctttc tcccag cttgtg ctgact caatcg ccctct gcc tctgcc tcc 96 SerPhe SerGln LeuVal LeuThr GlnSer ProSer Ala SerAla Ser ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agt cagcac agt 144 LeuGly AlaSer ValLys LeuThr CysThr LeuSer Ser GlnHis Ser acgtac accatt gaatgg catcag cagcag ccagag aag ggccct cgg 192 ThrTyr ThrIle GluTrp HisGln GlnGln ProGlu Lys GlyPro Arg tacttg atgaaa cttaag caagat ggaagc cacagc aca ggtgat ggg 240 TyrLeu MetLys LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly attcct gatcgc ttctca ggctcc agc tctggg getgag cgctac ctc 288 IlePro AspArg PheSer GlySer Ser SerGly AlaGlu ArgTyr Leu accatc tccagc ctccag tctgag gat gagget gactat tactgt ggt 336 ThrIle SerSer LeuGln SerGlu Asp GluAla AspTyr TyrCys Gly gtgggt gataca attaag gaacaa ttt gtgtac gtgttc ggcgga ggg 384 ValGly AspThr IleLys GluGln Phe ValTyr ValPhe GlyGly Gly g5 100 105 accaaa ctgacc gtccta ggtcag ccc 411 ThrLys LeuThr ValLeu GlyGln Pro <210~ 67 <211~ 411 <212~ DNA
<213> Homo Sapiens <220~
<221~ CDS
<222~ (1) . . (411) <220~
<221> mat_peptide <222~ (58) . . (411) <400~ 67 atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48 MetAla Trp ThrProLeu PhePhe PhePhe Val LeuHis CysSer Gly tctttc tcc cagcttgtg ctgact caatcg ccc tctgcc tctgcc tcc 96 SerPhe Ser GlnLeuVal LeuThr GlnSer Pro SerAla SerAla Ser ctggga gcc tcggtcaag ctcacc tgcacc ttg agtagt cagcac agt 144 LeuGly Ala SerValLys LeuThr CysThr Leu SerSer GlnHis Ser acgtac acc attgaatgg tatcag cagcag cca gagaag ggccct aag 192 ThrTyr Thr IleGluTrp TyrGln GlnGln Pro GluLys GlyPro Lys tacctg atg gatcttaag caagat ggaagc cac agcaca ggtgat ggg 240 TyrLeu Met AspLeuLys GlnAsp GlySer His SerThr GlyAsp Gly attcct gat cgcttctca ggctcc agctct ggg getgag cgctac ctc 288 IlePro Asp ArgPheSer GlySer SerSer Gly AlaGlu ArgTyr Leu accatc tcc agcctccag tctgag gatgag get gactat tactgt ggt 336 ThrIle Ser SerLeuGln SerGlu AspGlu Ala AspTyr TyrCys Gly gtgggt gat acaattaag gaacaa tttgtg tac gtgttc ggcgga ggg 384 ValGly Asp ThrIleLys GluGln PheVal Tyr ValPhe GlyGly Gly accaaa ctg accgtccta ggccag ccc 411 ThrLys Leu ThrValLeu GlyGln Pro <210~ 8 <211~411 <212~DNA

<213~Homo sapiens <220~
<221~ CDS
<222~ (1) . . (411) <220>
<221~ mat_peptide <222~ (58) . . (411) <400~ 68 atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly tct ttc tcc cag ctt gtg ctg act caa tcg ccc tct gcc tct gcc tcc 96 Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser ctg gga gcc tcg gtc aag ctc acc tgc acc ttg agt agt cag cac agt 144 Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser acg tac acc att gaa tgg tat cag cag cag cca gag aag ggc cct aag 192 Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys tac gtg atg gat ctt aag caa gat gga agc cac agc aca ggt gat ggg 240 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly attcct gatcgc ttctca ggctcc agc tctggg getgagcgc tac ctc 288 IlePro AspArg PheSer GlySer Ser SerGly AlaGluArg Tyr Leu accatc tceage etccag tctgag gat gagget gactattac tgt ggt 336 ThrIle SerSer LeuGln SerGlu Asp GluAla AspTyrTyr Cys Gly gtgggt gataca attaag gaacaa ttt gtgtac gtgttcggc gga ggg 384 ValGly AspThr IleLys GluGln Phe ValTyr ValPheGly Gly Gly accaaa ctgacc gtccta ggccag ccc 411 ThrLys LeuThr ValLeu GlyGln Pro <210~ 69 <211~ 411 <212~ DNA
<213~ Homo sapiens <220>
<221~ CDS
<222~ (1) . . (411) <220~
<221~ mat_peptide <222~ (58) . . (4111 <400~ 69 atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly tct ttc tcc cag ctt gtg ctg act caa tcg ccc tct gcc tct gcc tcc 96 Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agt cagcac agt 144 LeuGly AlaSer ValLys LeuThr CysThr LeuSer Ser GlnHis Ser acgtac accatt gaatgg tatcag cagcag ccagag aag ggccct agg 192 ThrTyr ThrIle GluTrp TyrGln GlnGln ProGlu Lys GlyPro Arg tacctg atggat cttaag caagat ggaagc cacagc aca ggtgat ggg 240 TyrLeu MetAsp LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly attcct gatcgc ttctca ggctcc agctct gggget gag cgctac ctc 288 IlePro AspArg PheSer GlySer SerSer GlyAla Glu ArgTyr Leu accatc tccagc ctccag tctgag gatgag getgac tat tactgt ggt 336 ThrIle SerSer LeuGln SerGlu AspGlu AlaAsp Tyr TyrCys Gly gtgggt gataca attaag gaacaa tttgtg tacgtg ttc ggcgga ggg 384 ValGly AspThr IleLys GluGln PheVal TyrVal Phe GlyGly Gly accaaa ctgacc gtccta ggccag ccc 411 ThrLys LeuThr ValLeu GlyGln Pro <210~ 70 <211~411 <212~DNA

<213~Homo sapiens <220~
<221~ CDS
<222> ( 1) . . (411) <220~
<221~ mat_peptide <222> (58) . . (411) <400~

atggcc tggact cctctc ttcttc ttcttt gttctt cat tgctca ggt 48 MetAla TrpThr ProLeu PhePhe PhePhe ValLeu His CysSer Gly tctttc tcccag cttgtg ctgact caatcg ccctct gcc tctgcc tcc 96 SerPhe SerGln LeuVal LeuThr GlnSer ProSer Ala SerAla Ser ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agt cagcac agt 144 LeuGly AlaSer ValLys LeuThr CysThr LeuSer Ser GlnHis Ser acgtac accatt gaatgg tatcag cagcag ccagag aag ggccct agg 192 ThrTyr ThrIle GluTrp TyrGln GlnGln ProGlu Lys GlyPro Arg tacgtg atggat cttaag caagat ggaagc cacagc aca ggtgat ggg 240 TyrVal MetAsp LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly attcctgat cgc ttctca ggctcc agctct gggget gagcgc tac ctc 288 IleProAsp Arg PheSer GlySer SerSer GlyAla GluArg Tyr Leu accatctcc ~agcctccag tctgag gatgag getgac tattac tgt ggt 336 ThrIleSer Ser LeuGln SerGlu AspGlu AlaAsp TyrTyr Cys Gly gtgggtgat aca attaag gaacaa tttgtg tacgtg ttcggc gga ggg 384 ValGlyAsp Thr IleLys GluGln PheVal TyrVal PheGly Gly Gly accaaactg acc gtccta ggccag ccc 411 ThrLysLeu Thr ValLeu GlyGln Pro <210~71 <211~411 <212~DNA

<213~Homo sapiens <220~
<221> CDS
<222~ ( 1) . . (411) <220~
<221~ mat_peptide <222~ (58) . . (411) <400~ 71 atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48 Met Ala TrpThr ProLeu PhePhe PhePhe ValLeu His CysSer Gly tct ttc tcccag cttgtg ctgact caatcg ccctci gcc tctgcc tcc 96 Ser Phe SerGln LeuVal LeuThr GlnSer ProSer Ala SerAla Ser ctg gga gcctcg gtcaag ctcacc tgcacc ttgagt agt cagcac agt 144 Leu Gly AlaSer ValLys LeuThr CysThr LeuSer Ser GlnHis Ser acg tac accatt gaatgg tatcag cagcag ccagag aag ggccct aag 192 Thr Tyr ThrIle GluTrp TyrGln GlnGln ProGlu Lys GlyPro Lys tac ctg atggat cttaag caagat ggaagc cacagc aca ggtgat ggg 240 Tyr Leu MetAsp LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly att cct gatcgc ttctca ggctcc agctct gggget gag cgctac ctc 288 Ile Pro AspArg PheSer GlySer SerSer GlyAla Glu ArgTyr Leu acc atc tccagc ctccag tctgag gatgag getgac tat atctgt ggt 336 Thr Ile SerSer LeuGln SerGlu AspGlu AlaAsp Tyr IleCys Gly gtg ggt gataca attaag gaacaa tttgtg tacgtg ttc ggcgga ggg 384 Val Gly AspThr IleLys GluGln PheVal TyrVal Phe GlyGly Gly acc aaa ctgacc gtccta ggccag ccc 411 Thr Lys LeuThr ValLeu GlyGln Pro <210~

<211~411 <212~DNA

<213~Homo sapiens <220>
<221~ CDS
<222~ (1) . . (411) <220~
<221> mat_peptide <222~ (58) . . (411) <400~

atggcc tgg actcct ctcttcttc ttc tttgtt cttcat tgctca ggt 48 MetAla Trp ThrPro LeuPhePhe Phe PheVal LeuHis CysSer Gly tctttc tcc cagctt gtgctgact caa tcgccc tctgcc tctgcc tcc 96 SerPhe Ser GlnLeu ValLeuThr Gln SerPro SerAla SerAla Ser ctggga gcc tcggtc aagctcacc tgc accttg agtagt cagcac agt 144 LeuGly Ala SerVal LysLeuThr Cys ThrLeu SerSer GlnHis Ser acgtac acc attgaa tggtatcag cag cagcca gagaag ggccct agg 192 ThrTyr Thr IleGlu TrpTyrGln Gln GlnPro GluLys GlyPro Arg tacctg atg gatctt aagcaagat gga agccac agcaca ggtgat ggg 240 TyrLeu Met AspLeu LysGlnAsp Gly SerHis SerThr GlyAsp Gly ~r , attcct gatcgc ttctca ggc tccagc tctggg gct:gag cgctac ctc 288 IlePro AspArg PheSer Gly SerSer SerGly AlaGlu ArgTyr Leu accatc tccagc ctccag tct gaggat gagget gactat atctgt ggt 336 ThrIle SerSer LeuGln Ser GluAsp GluAla AspTyr IleCys Gly gtgggt gataca attaag gaa caattt gtgtac gtgttc ggcgga ggg 384 ValGly AspThr IleLys Glu GlnPhe ValTyr ValPhe GlyGly Gly accaaa ctgacc gtccta ggc cagccc 411 ThrLys LeuThr ValLeu Gly GlnPro <210> 73 <211~ 411 <212> DNA
<213~ Homo sapiens <220~
<221~ CDS
<222~ (1) . . (411) <220~
<221~ mat_peptide <222~ (58) . . (411) <400~ 73 atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly tct ttc tcc cag ctt gtg ctg act caa tcg ccc tct gcc tct gcc tcc 96 Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agtcag cacagt 144 LeuGly AlaSer ValLys LeuThr CysThr LeuSer SerGln HisSer acgtac accatt gaatgg tatcag cagcag ccagag aagggc cctaag 192 ThrTyr ThrIle GluTrp TyrGln GlnGln ProGlu LysGly ProLys tacgtg atggat cttaag caagat ggaagc cacagc acaggt gatggg 240 TyrVal MetAsp LeuLys GlnAsp GlySer HisSer ThrGly AspGly attcct gatcgc ttctca ggctcc agctct gggget gagcgc tacctc 288 IlePro AspArg PheSer GlySer SerSer GlyAla GluArg TyrLeu accatc tccagc ctccag tctgag gatgag getgac tatatc tgtggt 336 ThrIle SerSer LeuGln SerGlu AspGlu AlaAsp TyrIle CysGly gtgggt gataca attaag gaacaa tttgtg tacgtg ttcggc ggaggg 384 ValGly AspThr IleLys GluGln PheVal TyrVal PheGly GlyGly accaaa ctgacc gtccta ggccag ccc 411 ThrLys LeuThr ValLeu GlyGln Pro <210~ 74 <211~411 <212>DNA

<213~Homo sapiens <220~
<221~ CDS
<222~ (1) . . (411) <220>
<221~ mat_peptide <222~ (58) . . (41 I) <400>

atggcc tggact cctctc ttcttc ttcttt gttctt cattgc tca ggt 48 MetAla TrpThr ProLeu PhePhe PhePhe ValLeu HisCys Ser Gly tctttc tcccag cttgtg ctgact caatcg ccctct gcctct gcc tcc 96 SerPhe SerGln LeuVal LeuThr GlnSer ProSer AlaSer Ala Ser ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agtcag cac agt 144 LeuGly AlaSer ValLys LeuThr CysThr LeuSer SerGln His Ser acgtac accatt gaatgg tatcag cagcag ccagag aagggc cct agg 192 ThrTyr ThrIle GluTrp TyrGln GlnGln ProGlu LysGly Pro Arg tacgtg atggat cttaag caagat ggaagc cacagc acaggt gat ggg 240 TyrVal MetAsp LeuLys GlnAsp GlySer HisSer ThrGly Asp Gly r~

attcctgat cgcttc tca ggctcc agctct gggget gagcgc tacctc 288 IleProAsp ArgPhe Ser GlySer SerSer GlyAla GluArg TyrLeu accatctcc agcctc cag tctgag gatgag getgac tatatc tgtggt 336 ThrIleSer SerLeu Gln SerGlu AspGlu AlaAsp TyrIle CysGly gtgggtgat acaatt aag gaacaa tttgtg tacgtg ttcggc ggaggg 384 ValGlyAsp ThrIle Lys GluGln PheVal TyrVal PheGly GlyGly accaaactg accgtc cta ggccag ccc 411 ThrLysLeu ThrVal Leu GlyGln Pro <210~ 75 <211~ 34 <212> PRT
<213> Homo sapiens <400~ 75 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His Thr Ala

Claims

1. A tissue degradation inhibiting agent which comprises a substance inhibiting the binding of parathyroid hormone-related peptide to its receptor.

2. The inhibiting agent according to claim 1, wherein the substance is an antagonist against the parathyroid hormone-related peptide receptor.

3. The inhibiting agent according to claim 1, wherein the substance is an anti-parathyroid hormone-related peptide antibody.

4. The inhibiting agent according to claim 1, wherein the substance is a fragment and/or a modified form of the anti-parathyroid hormone-related peptide antibody.

5. The inhibiting agent according to claim 3 or 4, wherein the antibody is a monoclonal antibody.

6. The inhibiting agent according to claim 3 or 4, wherein the antibody is a humanized antibody or a chimeric antibody.

7. The inhibiting agent according to claim 6, wherein the humanized antibody is a humanized #23-57-137-1 antibody.

8. The inhibiting agent according to any one of claims 1 to 7, wherein a tissue is a muscle tissue or an adipose tissue.

9. The inhibiting agent according to any one of claims 1 to 8, wherein tissue degradation is caused by cancerous cachexia, septicemia, severe injury or muscular dystrophy.

10. The inhibiting agent according to any one of claims 1 to 9, which is administered to a patient having a blood level of cytokine higher than the normal level.

11. The inhibiting agent according to claim 10, wherein the cytokine is at least one selected from the group consisting of IL-6, G-CSF, IL-11 and LIF.

12. The inhibiting agent according to claim 10, wherein the cytokine is an inflammatory cytokine.