JPH1180025A - Cachexia treatment - Google Patents

Abstract

(57) [Summary] [Problem] To provide a cachexia therapeutic agent. SOLUTION: The therapeutic agent for cachexia contains, as an active ingredient, a substance that inhibits the binding between parathyroid hormone-related peptide and its receptor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a parathyroid hormone-related peptide (Parathyroid hormone related protein (P
THrP)) relates to a therapeutic agent for cachexia, which contains, as an active ingredient, a substance that inhibits binding between the receptor and THrP)).

[0002]

2. Description of the Related Art Cachexia seen in terminal cancer patients is one of the paraneoplastic syndromes of malignant tumors, including anorexia, weight loss,
It causes poor general condition with anemia, water electrolyte abnormality, and immune abnormality as main symptoms. For cancer patients, the onset of cachexia not only results in life-threatening end-of-life symptoms, but also significantly impairs the patient's quality of life (QOL). Have physical and social consequences.

[0003] In recent years, it has been revealed that cachectin, which was considered to be a causative agent of cancer cachexia, is the same factor as tumor necrosis factor (TNF). Subsequently, a similar effect was clarified for cytokines such as interleukin 1 (IL-1), IL-6, LIF, and IFN, indicating that cancer cachexia is a pathological condition in which multiple factors act in combination. Has become apparent.

[0004] It is known that human oral floor cancer-derived OCC-1 cell line produces various humoral factors related to such cancer cachexia, and when OCC-1 cells are transplanted into nude mice, Causes various symptoms such as cachexia (Kajimura N. e
t al., Cancer Chemother. Pharmacol., 1996, 38 Supp
l. pS48-52, Tanaka R. et al., Jpn. J. Clin. Oncolo
gy Apr. 1996, 26 (2) p88-94). This is because the OCC-1 cell line transplanted into nude mice produces various cytokines (G-CSF, IL-6, LIF, IL-11, PTHrP, etc.) as they grow, and these factors combine It is thought to act to cause the above-mentioned symptoms.

[0005] As described above, it is considered that the symptoms of nude mice transplanted with the OCC-1 cell line are very common to those of human terminal cancer patients. However, to date, there is no report on a drug for such cachexia.

[0006]

An object of the present invention is to provide a therapeutic agent for cachexia, which contains, as an active ingredient, a substance that inhibits the binding between PTHrP and its receptor.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to provide such a therapeutic agent, and as a result, have achieved the object with a substance that inhibits the binding of a parathyroid hormone-related peptide to its receptor. And found that the present invention was completed. That is, the present invention is a therapeutic agent for cachexia comprising, as an active ingredient, a substance that inhibits the binding between a parathyroid hormone-related peptide and its receptor. Examples of the cachexia include those derived from cancer.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a parathyroid hormone related protein (PTH).
It is a therapeutic agent for cachexia containing as an active ingredient a substance that inhibits the binding between rP) and its receptor (PTHrP receptor). As used herein, the term `` PTHrP receptor '' refers to a receptor that binds to PTHrP described in, for example, Japanese Patent Publication No. 6-506598,
It may or may not be a PTHrP receptor present on the target organ (eg, bone or kidney).

[0009] The term "substance which inhibits the binding between PTHrP and PTHrP receptor" refers to PTHrP by binding to PTHrP.
A substance that inhibits the binding of PTHrP to the PTHrP receptor (eg, an anti-PTHrP antibody), and a substance that inhibits the binding of PTHrP to the PTHrP receptor by binding to the PTHrP receptor (eg, an antagonist to the PTHrP receptor) PTHrP antagonists), specifically, PTHrP peptides in which at least one amino acid has been substituted or deleted, or PTHrP
P peptide partial sequence).

Examples of the anti-PTHrP antibody include known antibodies such as a humanized antibody, a human antibody (WO96 / 33735) or a chimeric antibody (JP-A-4-28089), and an antibody (# 23-57-137-1 antibody).
The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody. PTHrP antagonists include polypeptides and small molecules, for example, a substance that binds to a PTHrP receptor in an antagonistic manner to PTHrP,
For example, JP-A-7-165790, Peptides (UNITED STATE
S) 1995, 16 (6) 1031-1037, Biochemistry (UNITED ST
ATES) Apr. 281992, 31 (16) 4026-4033, Tokiohei 5-50909
No. 8, a polypeptide having PTHrP antagonist activity. Further, among the polypeptides exemplified above, polypeptides having at least one amino acid deleted, substituted, added, or inserted and having equivalent PTHrP antagonist activity are also included in the PTHrP antagonist of the present invention. In the present invention, an anti-PTHrP antibody will be described as an example of "a substance that inhibits the binding between PTHrP and a PTHrP receptor".

1. Anti-PTHrP Antibody The anti-PTHrP antibody used in the present invention does not matter its origin, kind (monoclonal, polyclonal) and shape as long as it has a therapeutic effect on cachexia.

[0012] The anti-PTHrP antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. Anti-PTHr used in the present invention
As the P antibody, a monoclonal antibody derived from a mammal is particularly preferable. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. This antibody is an antibody that binds to PTHrP, thereby inhibiting the binding of PTHrP to the PTH / PTHrP receptor, blocking PTHrP signal transduction, and inhibiting the biological activity of PTHrP.

[0013] Such antibodies include the # 23-57-137-1 antibody produced by the hybridoma clone # 23-57-137-1. Hybridoma clone # 23-5
7-137-1 was designated as mouse-mouse hybridoma # 23-57-137-1 by the Institute of Biotechnology and Industrial Technology (I 1-3, Higashi 1-3-1, Higashi, Tsukuba, Ibaraki Prefecture) on August 15, 1996. Day, FE
Deposited internationally under the Budapest Treaty as RM BP-5631.

2. Antibody-producing hybridoma A monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, PTHrP is used as a sensitizing antigen, immunized with the conventional immunization method, the obtained immune cells are fused with a known parent cell by a normal cell fusion method, and a monoclonal antibody is obtained by a normal screening method. It can be produced by screening antibody-producing cells.

Specifically, a monoclonal antibody can be prepared as follows. First, human PTHrP, which is used as a sensitizing antigen for obtaining antibodies, was obtained from Suva, LJ et al.,
PTHrP gene disclosed in Science (1987) 237, 893 /
Obtained by expressing the amino acid sequence. That is,
After the gene sequence encoding PTHrP is inserted into a known expression vector system to transform a suitable host cell, the desired PTHrP protein is purified from the host cell or the culture supernatant by a known method.

Next, this purified PTHrP protein is used as a sensitizing antigen. Alternatively, the N-terminal 34 peptides of PTHrP can be prepared by chemical synthesis and can be used as a sensitizing antigen.

As a mammal immunized with a sensitizing antigen,
Although not particularly limited, it is preferable to select in consideration of compatibility with a parent cell used for cell fusion, and it is generally a rodent animal, for example, mouse, rat, hamster, or rabbit. , Monkeys and the like are used.

Immunization of an animal with a sensitizing antigen is performed according to a known method. For example, as a general method,
It is performed by injecting the sensitizing antigen intraperitoneally or subcutaneously into the mammal. Specifically, the sensitizing antigen was changed to PBS (Phospha
diluted with te-Buffered Saline) or saline, etc.
The suspension is mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified, and then administered to the mammal several times every 4 to 21 days. In addition, a suitable carrier can be used at the time of immunization with the sensitizing antigen. After immunization in this way and confirming that the level of the desired antibody in the serum increases, immune cells are collected from the mammal and subjected to cell fusion. Preferred immune cells include spleen cells in particular. Can be

Mammalian myeloma cells are used as the other parent cells to be fused with the immunocytes. This myeloma cell can be used in various known cell lines, for example, P3 (P3x63A
g8.653) (J. Immnol. (1979) 123, 1548-1550), P3x6
3Ag8U.1 (Current Topics in Microbiology and Immunol
ogy (1978) 81, 1-7), NS-1 (Kohler. G. and Milstei
n, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11
(Margulies. DHet al., Cell (1976) 8, 405-41
5), SP2 / 0 (Shulman, M. et al., Nature (1978) 27
6, 269-270), FO (de St. Groth, SF et al., J. I.
mmunol. Methods (1980) 35, 1-21), S194 (Trowbridg
e, ISJ Exp. Med. (1978) 148, 313-323), R210
(Galfre, G. et al., Nature (1979) 277, 131-133)
Etc. are preferably used.

Cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enz.
ymol. (1981) 73, 3-46). More specifically, the cell fusion is carried out in a usual nutrient culture solution, for example, in the presence of a cell fusion promoter. As a fusion promoter, for example, polyethylene glycol (PE
G), Sendai virus (HVJ) and the like are used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency.

The ratio of the use of the immune cells to the myeloma cells can be arbitrarily set. For example, it is preferable that the number of immune cells be 1 to 10 times that of myeloma cells. As the culture solution used for the cell fusion, for example, RPMI1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution,
In addition, a normal culture solution used for this type of cell culture can be used, and a serum replacement solution such as fetal calf serum (FCS) can also be used in combination.

In the cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution (for example, having an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually used for 30 to 30 minutes. A target fused cell (hybridoma) is formed by adding and mixing at a concentration of 60% (w / v). Subsequently, an operation of adding an appropriate culture solution successively and centrifuging to remove the supernatant is repeated to remove a cell fusion agent or the like that is unfavorable for hybridoma growth.

The hybridoma thus obtained is selected by culturing it in an ordinary selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing in the above HAT culture solution requires a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fused cells) to die.
continue. Then, a usual limiting dilution method is performed, and screening and single cloning of a hybridoma producing the desired antibody are performed.

In addition to obtaining the above hybridomas by immunizing non-human animals with an antigen, human lymphocytes can be in vitro
Sensitized to PTHrP, and the sensitized lymphocytes are fused with human-derived myeloma cells having permanent division potential to obtain a desired human antibody having PTHrP binding activity (Japanese Patent Publication No. 1-59878). reference). Furthermore, an anti-PTHrP antibody-producing cell may be obtained by administering PTHrP as an antigen to a transgenic animal having the entire repertoire of the human antibody gene, and a human antibody to PTHrP may be obtained from the immortalized cells. (International Patent Application Publication Number WO 94/2558
No. 5, WO 93/12227, WO 92/03918,
WO 94/02602).

The hybridoma producing the monoclonal antibody thus produced can be subcultured in a usual culture solution, and can be stored for a long time in liquid nitrogen. To obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a conventional method, and a method of obtaining the culture supernatant is used. And the like. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.

3. In the present invention, as a monoclonal antibody, a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing this into a host, and using gene recombination technology is used. (For example, Vandamme, AM et al., Eur. J. Biochem.
(1990) 192, 767-775, 1990).

Specifically, mRNA encoding the variable (V) region of the anti-PTHrP antibody is isolated from the hybridoma producing the anti-PTHrP antibody. mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Bi
ochemistry (1979) 18, 5294-5299), the AGPC method (Chomczy
nski, P. et al., Anal. Biochem. (1987) 162, 156-15
9) Preparation of total RNA by mRNA purification
Prepare the target mRNA using Kit (Pharmacia) or the like. In addition, QuickPrep mRNA Purification Kit (Pharma
mRNA can be directly prepared.

Antibodies from the obtained mRNA using reverse transcriptase
Synthesize cDNA for V region. cDNA synthesis is performed using AMV Reverse T
This is performed using a ranscriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) or the like. To perform cDNA synthesis and amplification, 5'-AmpliFINDER RACE Kit (Clontech
5'-RACE method using PCR (Frohman, MAet a)
l., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-900
2. Belyavsky, A. et al., Nucleic Acids Res. (1989)
17, 2919-2932) can be used.

The desired DNA fragment is purified from the obtained PCR product and ligated to a vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. Then, the base sequence of the target DNA is confirmed by a known method, for example, a dideoxynucleotide chain termination method.

After obtaining the DNA encoding the V region of the desired anti-PTHrP antibody, the DNA is inserted into an expression vector containing the DNA encoding the desired antibody constant region (C region). To produce the anti-PTHrP antibody used in the present invention,
The antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, host cells are transformed with this expression vector to express the antibody.

The expression of the antibody gene may be carried out by separately transforming the DNAs encoding the antibody heavy chain (H chain) or light chain (L chain) into an expression vector and co-transforming the host cell,
Alternatively, host cells may be transformed by incorporating DNAs encoding the H chain and the L chain into a single expression vector (see WO 94/11523).

For the production of recombinant antibodies, not only the above-mentioned host cells but also transgenic animals can be used. For example, an antibody gene is inserted into a gene encoding a protein (such as goat β-casein) that is specifically produced in milk to prepare a fusion gene. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat. A desired antibody is obtained from milk produced by a transgenic goat born from a goat that has received an embryo or a progeny thereof. Hormones may also be used in transgenic goats as appropriate to increase the amount of milk containing the desired antibody produced from the transgenic goats (Ebert, KM et al., Bi
o / Technology (1994) 12, 699-702).

4. Modified Antibodies In the present invention, in addition to the above-mentioned antibodies, genetically modified antibodies artificially modified for the purpose of reducing xenoantigenicity to humans and the like, for example, chimeric antibodies, humanized (Hu
manized) antibodies can be used. These modified antibodies can be produced using known methods. A chimeric antibody can be obtained by ligating the DNA encoding the antibody V region obtained as described above with the DNA encoding the human antibody C region, inserting the DNA into an expression vector, introducing the resultant into a host, and producing the host. Using this known method, chimeric antibodies useful in the present invention can be obtained.

The humanized antibody is also called a reshaped human antibody, which is a complementarity determining region (CDR) of a non-human mammal, for example, a mouse antibody.
etermining region) into a complementarity determining region of a human antibody, and a general gene recombination technique is also known (European Patent Application Publication No. EP 125023, W
O 96/02576).

Specifically, a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody is replaced with a portion overlapping both terminal regions of the CDR and FR. Using several oligonucleotides prepared to have
Synthesized by the R method. The obtained DNA is ligated to a DNA encoding the human antibody C region, then incorporated into an expression vector, and introduced into a host to produce a humanized antibody (EP 239400, WO 96 / No. 02576).

The framework region of a human antibody to be linked via CDR is selected so that the complementarity determining region forms a favorable antigen-binding site. If necessary, the amino acids of the framework regions in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., CancerRe
s. (1993) 53, 851-856).

For the C region of the chimeric antibody and the humanized antibody, those of a human antibody are used.
1, Cγ2, Cγ3, and Cγ4 can be used, and Cκ and Cλ can be used in the L chain. In addition, the human antibody C region may be modified in order to improve the stability of the antibody or its production.

A chimeric antibody comprises a variable region of an antibody derived from a mammal other than human and a constant region derived from a human antibody. On the other hand, a humanized antibody is composed of a complementarity determining region of an antibody derived from a mammal other than human, a framework region and a C region derived from a human antibody. Since the humanized antibody has reduced antigenicity in the human body, it is useful as an active ingredient of the therapeutic agent of the present invention.

The humanized antibody that can be used in the present invention includes a humanized # 23-57-137-1 antibody. Humanized # 23-
The 57-137-1 antibody has the complementarity determining region of the mouse-derived # 23-57-137-1 antibody, and the human antibody HSU03868 (GEN-
BANK, Deftos M et al., Scand. J. Immunol., 39, 95-103,
1994) and three FR fragments (FR1, FR2 and FR3) derived from human antibody S25755 (NBRF-PDB) (FR4) derived from human antibody S25755 (NBRF-PDB).
(NBRF-PDB, Cuisinier AM et al., Eur. J. Immunol., 23,
110-118, 1993), wherein amino acid residues in the framework region are partially substituted so as to have antigen-binding activity.

Escherichia coli having a plasmid containing DNA encoding the L chain or the H chain of the humanized # 23-57-137-1 antibody was obtained from the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (1 Higashi, Tsukuba City, Ibaraki Prefecture). (Chome 1-3) on August 15, 1996
Escherichia coli JM109 (hMBC1HcDNA / pUC19), which is Escherichia coli having a plasmid containing DNA encoding a chain, is referred to as EFERC BP-5629, and is Escherichia coli having a plasmid containing DNA encoding an L chain.
JM109 (hMBC1Lqλ / pUC19) has been internationally deposited under FERM BP-5630 under the Budapest Treaty.

5. Modified Antibody The antibody used in the present invention may be an antibody fragment or a modified product thereof, as long as it binds to PTHrP and inhibits the activity of PTHrP. For example, antibody fragments include Fab, F (a
b ′) ₂ , Fv, or single chain Fv (scFv) in which Fvs of H chain or L chain are linked by a suitable linker. Specifically, an antibody is treated with an enzyme such as papain or pepsin to generate an antibody fragment, or a gene encoding these antibody fragment is constructed and introduced into an expression vector. Expressed (for example, Co, MS et al., J. Immunol. (1994) 152, 2968-
2976, Better, M. & Horwitz, AH Methods in Enzym
ology (1989) 178, 476-496, Academic Press, Inc., P.
lueckthun, A. & Skerra, A. Methods in Enzymology
(1989) 178, 476-496, Academic Press, Inc., Lamoy
i, E., Methods in Enzymology (1989) 121, 652-663,
Rousseaux, J. et al., Methods in Enzymology (198
9) 121, 663-669, Bird, RE et al., TIBTECH (199
1) See 9, 132-137).

The scFv can be obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc.
Natl. Acad. Sci. USA (1988) 85, 5879-5883). sc
The H chain V region and L chain V region in the Fv may be from any of those described herein as antibodies.
As the peptide linker connecting the V regions, for example, any single-chain peptide consisting of 12 to 19 amino acid residues is used.

The DNA encoding the scFv may be any of the DNAs encoding the H chain or H chain V region and the DNA encoding the L chain or L chain V region of the antibody or all or a desired sequence thereof. Using the DNA portion encoding the amino acid sequence as a template, amplify by PCR using primer pairs defining both ends thereof, and then further encode the peptide linker portion, and both ends are linked to the H chain and L chain, respectively. By combining and amplifying pairs of primers defined as follows.

Once the DNA encoding scFv has been prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method. Thus, scFv can be obtained according to a conventional method. Fragments of these antibodies can be produced in the same manner as described above, by obtaining the gene, expressing the gene, and using a host. The “antibody” in the present invention also includes these antibody fragments.

As a modified antibody, an anti-PTHrP antibody bound to various molecules such as polyethylene glycol (PEG) can also be used. The “antibody” in the present invention also includes these modified antibodies. Such modified antibodies are
It can be obtained by chemically modifying the obtained antibody. In addition, a method for modifying an antibody has already been established in this field.

6. Expression and Production of Recombinant Antibody or Modified Antibody The antibody gene constructed as described above can be expressed and obtained by a known method. For mammalian cells,
A useful useful promoter commonly used, an antibody gene to be expressed, and a polyA signal operably linked to the 3 ′ downstream thereof can be expressed. For example, as the promoter / enhancer, human cytomegalovirus early promoter / enhancer (human cytomegalovirus imm
edit early promoter / enhancer).

Other promoters / enhancers that can be used for the expression of antibodies used in the present invention include retroviruses, polyomaviruses, adenoviruses, and the like.
Viral promoters / enhancers such as simian virus 40 (SV40) or mammalian cell-derived promoters / enhancers such as human elongation factor 1α (HEF1α) are exemplified.

When using the SV40 promoter / enhancer, the method of Mulligan et al. (Nature (1979) 277, 10
8) and when using the HEF1α promoter / enhancer, the method of Mizushima et al. (Nucleic Acids R
es. (1990) 18, 5322), gene expression can be easily performed.

In the case of Escherichia coli, the gene can be expressed by operably linking a commonly used useful promoter, a signal sequence for antibody secretion, and the antibody gene to be expressed. Examples of the promoter include a lacz promoter and an araB promoter. la
When using the cz promoter, the method of Ward et al. (Nature
(1098) 341, 544-546; FASEB J. (1992) 6, 2422-242.
7) or when using the araB promoter, the method of Better et al. (Science (1988) 240, 1041-1043)
Can be expressed by

As a signal sequence for antibody secretion,
For production in the periplasm of E. coli, the pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4
379). After separating the antibody produced in the periplasm, the antibody structure is appropriately refolded and used.

The replication origin includes SV40, polyoma virus, adenovirus, bovine papilloma virus (BP
V) and the like. Further, for amplification of the gene copy number in a host cell system, the expression vector is
Aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) as selectable markers
It can contain genes and the like.

For the production of the antibodies used in the present invention,
Any expression system can be used, for example, a eukaryotic or prokaryotic cell system. Eukaryotic cells include, for example, established mammalian cell lines, insect cell lines, animal cells such as eukaryotic fungal cells and yeast cells, and prokaryotic cells include, for example, bacterial cells such as E. coli cells. Preferably, the antibodies used in the present invention are mammalian cells, such as CH
It is expressed in O, COS, myeloma, BHK, Vero, and HeLa cells.

Next, the transformed host cells were in vitro
Alternatively, the desired antibody is produced by culturing in vivo. Culture of the host cell is performed according to a known method. For example,
As a culture solution, DMEM, MEM, RPMI1640, IMDM can be used, and a serum supplement such as fetal calf serum (FCS) can be used in combination.

7. Antibody Separation and Purification The antibody expressed and produced as described above can be separated from cells and host animals and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed using an affinity column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F.
F. (Pharmacia) and the like. In addition, the separation and purification methods used for ordinary proteins may be used, and are not limited at all. For example, an antibody can be separated and purified by appropriately selecting and combining a chromatography column other than the affinity column, a filter, ultrafiltration, salting out, dialysis, and the like (Antibodies A Laboratory Manual. Ed Harlow, David
Lane, Cold Spring Harbor Laboratory, 1988).

8. Confirmation of Antibody Activity Antigen binding activity of the antibody used in the present invention (Antibodies A
Laboratory Manual.Ed Harlow, David Lane, Cold Spr
Ining Harbor Laboratory, 1988), ligand receptor binding inhibitory activity (Harada, A. et al., International Imm.
A known means can be used for measurement of unology (1993) 5, 681-690).

As a method for measuring the antigen-binding activity of the anti-PTHrP antibody used in the present invention, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) or fluorescent antibody Method can be used. For example, when using the enzyme immunoassay, a sample containing an anti-PTHrP antibody is placed on a plate coated with PTHrP (1-34),
For example, a culture supernatant of an anti-PTHrP antibody-producing cell or a purified antibody is added. After adding a secondary antibody labeled with an enzyme such as alkaline phosphatase, incubating and washing the plate, adding an enzyme substrate such as p-nitrophenyl phosphate, and measuring the absorbance, it is possible to evaluate the antigen binding activity. it can. To confirm the activity of the antibody used in the present invention, the neutralizing activity of the anti-PTHrP antibody is measured.

9. Administration method and formulation The therapeutic agent of the present invention is used for the treatment or improvement of cachexia. The type of cachexia does not matter whether it is derived from cancer. For example, as those derived from cancer, J. Urol. (UNITED STATES) Mar 1995, 153 (3 Pt
1) p854-857, Langenbecks Arch. Chir. Suppl II Verh
Dtsch Ges Chir (GERMANY) 1990, p261-265, Oncology
(SWITZERLAND) 1990, 47 (1) p87-91, Int. J. Pancre
atol. (UNITED STATES) Aug-Nov 1990, 7 (1-3) p141-1
50, J. Natl. Cancer Inst. (UNITEDSTATES) Dec 19, 1
990, 82 (24) p1922-1926.

[0058] JPN J. J.
Parenter. Enteral Nutr. (UNITEDSTATES) Nov-Dec 199
0, 14 (6) p605-609, Chest (UNITED STATES) Nov 199
0, 98 (5) p1091-1094, Bone Marrow Transplant. (ENG
LAND) Jul 1990, 6 (1) p53-57.

The therapeutic agent containing the anti-PTHrP antibody of the present invention as an active ingredient can be administered either orally or parenterally, but is preferably administered parenterally. Pulmonary administration using a device such as a nephrizer), nasal administration, transdermal administration (eg, ointment, cream), injection, and the like. Examples of the injection form include systemic or local administration by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like. The administration method can be appropriately selected depending on the age and symptoms of the patient. The effective dose is selected from the range of 0.001 mg to 1000 mg per kg of body weight at a time. Alternatively, a dosage of 0.01 to 100000 mg / body per patient can be selected. However, the therapeutic agent containing the anti-PTHrP antibody of the present invention is not limited to these doses.

The administration may be performed before or after the onset of cachexia, or when the weight loss is predicted. The therapeutic agent containing the anti-PTHrP antibody of the present invention as an active ingredient can be formulated according to a conventional method (Remington's Pharmaceutical Scien
ce, latest edition, Mark Publishing Company, Easto
n, USA) and pharmaceutically acceptable carriers and additives.

Examples of such carriers and pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, Water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, acacia, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA ), Mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like.

The actual additives are selected from the above alone or in appropriate combination depending on the dosage form of the therapeutic agent of the present invention, but are not limited thereto. For example,
When used as an injection preparation, a purified anti-PTHrP antibody is dissolved in a solvent, for example, a physiological saline solution, a buffer solution, a glucose solution, and the like, and an anti-adsorption agent, such as Tween80, Tween2 is added thereto.
What added 0, gelatin, human serum albumin, etc. can be used. Alternatively, it may be freeze-dried to make a dosage form that is dissolved and reconstituted before use,
As an excipient for freeze-drying, for example, sugar alcohols such as mannitol and glucose and sugars can be used.

[0063]

EXAMPLES The present invention will be described more specifically with reference to Examples and Examples. However, the present invention does not limit the technical scope to these examples and the like. [Example 1] Drug efficacy test in cachexia model animal The therapeutic effect of a mouse monoclonal antibody against PTHrP on cachexia was examined using a human tumor-nude mouse transplant system cachexia model animal. Human oral floor cancer OCC-1 as a model animal (purchased from Central Research Institute for Experimental Animals)
Was transplanted into nude mice. Human oral floor cancer OCC-1
In a nude mouse transplanted with, the calcium concentration in the blood increases with an increase in the tumor, and cachectic symptoms such as weight loss and decrease in the amount of exercise occur. The improvement of the cachexia symptom caused by human floor floor cancer OCC-1 was evaluated by the improvement of the mouse monoclonal antibody using blood calcium concentration, body weight and life-prolonging effect as indices.

The passage of human oral floor cancer OCC-1 was BALB / c-nu / n
u In vivo was performed using nude mice (CLEA Japan). For the efficacy evaluation, 6-week-old male BALB / c-nu / nu nude mice (CLEA Japan) were purchased, and after acclimation for 1 week, 7-week-old animals were used. Preparation and grouping of cachexia model animals were performed as follows. That is, the subcultured human oral floor cancer OCC-1 was excised, and a tumor mass finely cut into a 3 mm square block was implanted subcutaneously into the flank of a mouse one by one. After confirming that the tumor volume was sufficiently large on the 10th day after tumor mass transplantation, blood calcium concentration,
The animals were divided into groups using the body weight and the tumor volume as indices so that the indices were averaged to obtain cachexia model animals. Examination of the therapeutic effect on cachexia was performed as follows.

(1) Observation of survival period In examining the survival effect, mouse monoclonal antibody was
A single dose was administered to observe the survival time. Pamidronate (Aledia), already prescribed as a drug for treating hypercalcemia, was administered once at a dose of 15 mg / Kg into the tail vein. As a control, phosphate buffered saline (PBS) was administered into the tail vein twice a week at 0.2 ml / mouse. The result is shown in FIG.

(2) Observation of Blood Calcium Concentration
A mouse monoclonal antibody against PTHrP at 10 μg or 100 μg per animal was administered twice every two days into the tail vein.
Pamidronate (Aledia), already prescribed as a drug for treating hypercalcemia, was administered once at a dose of 15 mg / Kg into the tail vein. As a control, phosphate buffered saline (PBS) was administered twice every two days into the tail vein at 0.2 ml / mouse.

(3) Measurement of Blood Calcium On the 1st and 4th days after the administration of the mouse monoclonal antibody, the blood calcium concentration was measured to evaluate the efficacy of each antibody. Blood calcium concentration was measured from the orbit using a hematocrit tube and 643 automatic Ca / pH analyzer (CIBA-CORNING)
Was used to measure the concentration of ionized calcium in whole blood.
Body weight was measured daily up to 4 days after antibody administration. The results are shown in FIGS.

(4) Measurement of tumor weight The tumor volume was determined on the fourth day after the antibody administration, by the major axis (a mm) of the tumor.
And minor axis (b mm) were measured, Galant formula ab ^2/2
Was calculated as the tumor volume. The result is shown in FIG.

From the above results, with respect to the blood calcium concentration, at the antibody concentration of 10 μg, the weight loss associated with the malignant tumor was suppressed as compared with the pamidronate administration group or the control group, although there was no difference from the pamidronate administration group. . In the group to which the antibody concentration of 100 μg was administered, an increase in blood calcium concentration was suppressed as compared with the pamidronate administration group or the control group, and weight loss was also suppressed as compared with the pamidronate administration group or the control group. In the group to which 100 μg of the anti-PTHrP neutralizing antibody was administered twice a week, the survival time was significantly prolonged (p = 0.0003: Log Rank test) compared to the pamidronate administration group or the control group. From this, the neutralizing mouse monoclonal antibody against PTHrP has effects such as suppression of weight loss and prolongation of survival time which are not present in existing therapeutic agents for hypercalcemia. Therefore, the utility of this antibody as a therapeutic agent for cachexia associated with malignant tumors was demonstrated.

Example 2 Efficacy Test in Hypercalcemia / Cachexia Model Animal Cachexia of humanized antibody version q against PTHrP using a cachexia model animal of human tumor-nude mouse transplantation system The therapeutic effect on quality was examined. Human oral floor cancer OCC-1 as a model animal (purchased from Central Research Institute for Experimental Animals)
Was transplanted into nude mice. Human oral floor cancer OCC-1
In a nude mouse transplanted with, the calcium concentration in the blood increases with an increase in the tumor, and cachectic symptoms such as weight loss and decrease in the amount of exercise occur. The cachexia symptoms caused by human floor floor cancer OCC-1 were converted to humanized antibody version q
Was evaluated using blood calcium concentration, body weight and life-prolonging effect as indices.

The passage of the human floor floor cancer OCC-1 was BALB / c-nu / n
u In vivo was performed using nude mice (CLEA Japan). For the efficacy evaluation, 6-week-old male BALB / c-nu / nu nude mice (CLEA Japan) were purchased, and after acclimation for 1 week, 7-week-old animals were used. Preparation and grouping of cachexia model animals were performed as follows. That is, the subcultured human oral floor cancer OCC-1 was excised, and a tumor mass finely cut into a 3 mm square block was implanted subcutaneously into the flank of a mouse one by one. After confirming that the tumor volume was sufficiently large on the 10th day after transplantation of the tumor mass, the tumor volume, blood calcium concentration and body weight were used as indices to divide the groups so that each index was averaged, and cachexia Model animals were used. Examination of the therapeutic effect on cachexia was performed as follows.

(1) Observation of Survival Time In examining the survival effect, the humanized antibody version q was used for 2 weeks / week.
It was administered into the circumcised vein to observe the survival time. As a control, phosphate buffered saline (PBS) was added at 0.1 ml / m
Ouse was administered into the tail vein twice a week. The result is shown in FIG.

(2) Observation of Blood Calcium Concentration
10 μg or 100 μg of the humanized antibody version q per animal was administered twice into the tail vein two days apart. As a control,
Phosphate buffered saline (PBS) was similarly administered at 0.1 ml / mouse.

(3) Measurement of Blood Calcium On the 1st and 4th days after the first administration of humanized antibody version q, the blood calcium concentration was measured to evaluate the efficacy of the antibody. Blood calcium concentration was collected from the orbit using a hematocrit tube, and measured as whole blood ionized calcium concentration using a 643 automatic Ca / pH analyzer (CHIRON). Body weight was measured daily until day 4. The results are shown in FIGS. 17 and 18. (4) Measurement tumor volume tumor weight, initial dose and on day 4, the tumor major axis (amm) and minor axis (b mm) were measured and calculated by equation ab ^2/2 of the Galant. The result is shown in FIG.

As described above, by administering 10 μg or 100 μg of humanized antibody version q, an increase in blood calcium concentration and a decrease in body weight associated with malignant tumor were suppressed as compared with the control group. In addition, when 100 μg of humanized antibody version q was continuously administered twice a week, the survival time was significantly prolonged compared to the control group (p = 0.0108: Log Rank tes).
t) was observed. The effect of this humanized antibody version q on cachexia model animals associated with malignant tumors was similar to the effect of the mouse monoclonal antibody previously reported. This demonstrated the usefulness of this antibody as a therapeutic agent for hypercalcemia and cachexia associated with malignant tumors.

[Reference Example 1] Preparation of anti-PTHrP (1-34) mouse monoclonal antibody-producing hybridoma Monoclonal antibody-producing hybridomas # 23-57-154 and # 23-57 against human PTHrP (1-34) (SEQ ID NO: 75) -137-1 was produced by Sato Miki et al. (Sat
o, K. et al., J. Bone Miner. Res. 8, 849-860, 199
3). For use as an immunogen, PTHrP (1-3
4) (Peninsula) and thyroglobulin, a carrier protein, were conjugated using carbodiimide (Dojinn). PTHrP bound to thyroglobulin (1-3
4) is dialyzed and adjusted to a protein concentration of 2 μg / ml, and then mixed with Freund's adjuvant (Difco) in 1:
After mixing and preparing the emulsion, 16 female BALB / C mice were immunized subcutaneously on the back or intraperitoneally with 100 μg per animal 11 times. For the first immunization, Freund's complete adjuvant was used, and for the second and subsequent boosters, Freund's incomplete adjuvant was used.

The antibody titer in the serum of the immunized mouse was measured by the following method. That is, blood was collected from the mouse tail vein, and after separation of serum, antiserum diluted with RIA buffer and 125I-labeled PTHrP (1-34) were mixed, and the binding activity was measured. PTHrP (1-34) having no carrier protein bound to the peritoneal cavity of the mouse with an increased antibody titer
Was final immunized with 50 μg per animal.

On the third day of the final immunization, the mice were sacrificed, the spleens were removed, and the spleen cells and the mouse myeloma cell line P3x63Ag8U.
1 was subjected to cell fusion according to a conventional method using 50% polyethylene glycol 4000. 2 × 10 ⁴ /
The cells were seeded in 85 96-well plates based on the number of cells in the wells. Hybridomas were selected using a HAT medium.

Hybridoma screening was performed using HA
The culture supernatant in the well where growth was observed in T medium was
It was performed by measuring the presence or absence of a PTHrP-recognizing antibody by Method A and selecting it. Hybridomas were collected from the wells in which the ability to bind to the antibody was recognized, suspended in RPMI-1640 medium containing 15% FCS and supplemented with OPI-supplement (Sigma), and homogenized by limiting dilution. Was carried out. Clones # 23-57-154 and # 23-57-137-1 having strong binding ability to PTHrP (1-34) were obtained. The hybridoma clone # 23-57-137-1 was a mouse-mouse hybrid
As oma # 23-57-137-1, the Institute of Biotechnology, Institute of Industrial Science and Technology (1-3 1-3, Higashi, Tsukuba, Ibaraki Prefecture)
On August 15, 2008, it was deposited internationally under the Budapest Treaty as FERM BP-5631.

Reference Example 2 Human PTHrP (1-34)
Cloning of DNA Encoding Variable Region of Mouse Monoclonal Antibody Against Human PTHrP (1-34) DNA encoding the variable region of mouse monoclonal antibody # 23-57-137-1 was cloned as follows. (1) Preparation of mRNA The mRNA from hybridoma # 23-57-137-1 was
It was prepared using ep mRNA PurificationKit (Pharmacia Biotech). Ex hybridoma # 23-57-137-1 cells
After homogenizing completely with traction Buffer, mRNA was purified using oligo (dT) -Cellulose Spun Column according to the prescription of the kit, and ethanol precipitation was performed. mR
The NA precipitate was dissolved in Elution Buffer.

(2) Preparation and Amplification of cDNA for Gene Encoding Mouse H Chain V Region (i) Cloning of # 23-57-137-1 Antibody H Chain V Region cDNA H of mouse monoclonal antibody against human PTHrP
The cloning of the gene encoding the chain V region is performed by 5′-
RACE method (Frohman, MA et al., Proc. Natl. Aca
d. Sci. USA, 85, 8998-9002, 1988; Belyavsky, A. et.
al., Nucleic Acids Res. 17, 2919-2932, 1989). 5'-Ampli FINDER RACE in 5'-RACE method
Using kit (CLONETECH), the operation was performed according to the prescription attached to the kit. As a primer used for cDNA synthesis, an MHC2 primer (SEQ ID NO: 1) that hybridizes with a mouse H chain constant region (C region) was used. Using about 2 μg of the mRNA prepared as described above as a template, MH
Add 10pmole of C2 primer, reverse transcriptase,
Reverse transcription to cDNA was performed by reacting for 5 minutes.

Hydrolysis of RNA with 6N NaOH (65 ° C., 30
After that, cDNA was purified by ethanol precipitation. By reacting with T4 RNA ligase at 37 ° C for 6 hours and at room temperature for 16 hours, Ampli was added to the 5 'end of the synthesized cDNA.
FINDER Anchor (SEQ ID NO: 42) was ligated. Using this as a template, primers for amplification by PCR
Primer (SEQ ID NO: 2) and MHC-G1 primer (SEQ ID NO: 3) (ST Jones, et al., Biotechnology,
9,88,1991). The PCR solution contains 10 μl in 50 μl.
mM Tris-HCl (pH 8.3), 50 mM KCl, 0.25 mM dNTPs (dATP, d
GTP, dCTP, dTTP), 1.5 mM MgCl ₂ , 2.5 units of TaKa
Ra Taq (Takara Shuzo), 10 pmole Anchor primer, MHC-G1 primer and Ampli FINDER
Contains 1 μl of reaction mixture of cDNA ligated to Anchor. The solution was overlaid with 50 μl of mineral oil. PCR is Thermal
Use Cycler Mode 1480J (Perkin Elmer) at 94 ° C for 4
Temperature cycling was performed 30 times for 5 seconds, 60 ° C. for 45 seconds, and 72 ° C. for 2 minutes.

(Ii) # 23-57-137-1 cDN of antibody L chain V region
Cloning of A L of mouse monoclonal antibody against human PTHrP
The cloning of the gene encoding the chain V region is performed by 5′-
RACE method (Frohman, MA et al., Proc. Natl. Aca
d. Sci. USA 85, 8998-9002, 1988; Belyavsky, A. et
al., Nucleic Acids Res. 17, 2919-2932, 1989). 5'-AmpliFinder RACE K for 5'-RACE method
Using it (Clonetech), the operation followed the attached prescription. cD
Oligo-dT primer was used as a primer used for NA synthesis. Using about 2 μg of the mRNA prepared as described above as a template, an oligo-dT primer was added thereto.
Reverse transcription to cDNA was performed by reacting for 30 minutes. After hydrolyzing the RNA with 6N NaOH (65 ° C., 30 minutes), the cDNA was purified by ethanol precipitation. The above-described Ampli FINDER Anchor was added to the synthesized cDNA at the 5 'end of T4RN.
Ligation was performed by reacting with A ligase at 37 ° C. for 6 hours and at room temperature for 16 hours.

From the conserved sequence of the mouse L chain λ chain constant region,
The R primer MLC (SEQ ID NO: 4) was designed and 394 DNA / R
It was synthesized using an NA synthesizer (ABI). The PCR solution contains 10 mM Tris-HCl (pH 8.3) in 100 μl thereof,
50 mM KCl, 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP
TP), 1.5 mM MgCl ₂ , 2.5 units of AmpliTaq (PERK
IN ELMER), 50 pmole Anchor primer (SEQ ID NO: 2), and MLC (SEQ ID NO: 4) and Ampli FINDER
Contains 1 μl of reaction mixture of cDNA ligated to Anchor. The solution was overlaid with 50 μl of mineral oil. PCR is Thermal
Using Cycler Model480J (Perkin Elmer) at 94 ° C
The temperature cycle was performed 35 times for 5 seconds, at 60 ° C. for 45 seconds, and at 72 ° C. for 2 minutes.

(3) Purification and Fragmentation of PCR Product The DNA fragment amplified by the PCR method as described above was
Separation was performed by agarose gel electrophoresis using Sieve GTG agarose (FMC Bio. Products). A piece of agarose containing a DNA fragment of about 550 bp in length as an H chain V region and about 550 bp in length of an L chain V region was cut out, and GENECLEAN II Kit (B
DNA fragment was purified using IO101) according to the instructions attached to the kit. After precipitating the purified DNA with ethanol, 10 mM
Tris-HCl (pH 7.4) was dissolved in 20 μl of a 1 mM EDTA solution.
1 μl of the obtained DNA solution was mixed with the restriction enzyme XmaI (New England B
iolabs) for 1 hour at 37 ° C., followed by restriction enzyme Eco
Digested with RI (Takara Shuzo) for 1 hour at 37 ° C. The digestion mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation. Thus at the 5'-end
A DNA fragment containing a gene encoding a mouse H chain V region and an L chain V region having an EcoRI recognition sequence and an XmaI recognition sequence at the 3′-end was obtained.

The EcoRI-Xm containing the genes encoding the mouse H chain V region and L chain V region prepared as described above.
The pUC19 vector prepared by digesting the aI DNA fragment with EcoRI and XmaI was used as a DNA ligation kit ver.2
Using (Takara Shuzo), the reaction was carried out at 16 ° C. for 1 hour according to the attached prescription, and ligated. Next, 10 μl of the above ligation mixture was
100 μl of M109 competent cells (Nippon Gene)
And the cells were placed on ice for 15 minutes, at 42 ° C. for 1 minute, and on ice for 1 minute. Then, 300 μl of SOC medium (Molecular Cloning: A Labgoratory Manual, Sambr
ook, et al., Cold Spring Harbor Laboratory Press, 1
989) and incubate at 37 ° C for 30 minutes.
μg / ml or 50 μg / ml ampicillin, 0.1 mM
LB agar medium or 2 × YT agar medium containing IPTG and 20 μg / ml X-gal (Molecular Cloning: A Labgora)
tory Manual, Sambrook, et al., Cold Spring Harbor L
(Aboratory Press, 1989), and the cells were incubated at 37 ° C. overnight to obtain E. coli transformants.

The transformant was used at 100 μg / ml or 50 μg / ml.
LB medium containing 2 g / ml ampicillin or 2 ×
After culturing overnight at 37 ° C. in 2 ml of YT medium, plasmid extractor PI-100Σ (Kurabo Industries) or QIAprep Spin
Plasmid DNA was prepared using Plasmid Kit (QIAGEN), and the nucleotide sequence was determined.

(4) Determination of the nucleotide sequence of the gene coding for the mouse antibody V region
It was determined by DNA Sequencer 373A (Perkin-Elmer, ABI) using the Terminator Cycle Sequencing kit (Perkin-Elmer). M13 Primer M as a sequencing primer
Using 4 (Takara Shuzo) (SEQ ID NO: 5) and M13 Primer RV (Takara Shuzo) (SEQ ID NO: 6), the sequence was determined by confirming the nucleotide sequence in both directions.

Hybridoma # 23-57-1 thus obtained
The plasmid containing the gene encoding the mouse H chain V region derived from 37-1 was named MBC1H04, and the plasmid containing the gene encoding the L chain V region was named MBC1L24.
Mouse # 2 contained in plasmid MBC1H04 and MBC1L24
The nucleotide sequence (including the corresponding amino acid sequence) of the gene encoding the H chain V region and L chain V region of the 3-57-137-1 antibody
Are shown in SEQ ID NOs: 57 and 65, respectively. The polypeptides of the H chain V region and L chain V region fragments both start from the 58th position (encoding glutamine) of the nucleotide sequence represented by SEQ ID NOS: 57 and 65. These amino acid sequences are
The fragment of the chain V region is shown in SEQ ID NO: 46, and the fragment of the L chain V region is shown in SEQ ID NO: 45.

The plasmids MBC1H04 and MBC1
E. coli having L24 is Escherichiacoli JM109 (MBC1H04
) And Escherichia coli JM109 (MBC1L24) to the Institute of Biotechnology and Industrial Technology (1-3 1-3, Higashi, Tsukuba, Ibaraki, Japan) on August 15, 1996.
FERM BP-562 for E. coli JM109 (MBC1H04)
8.FERM for Escherichia coli JM109 (MBC1L24)
Deposited internationally under the Budapest Treaty as BP-5627.

(5) Determination of CDR of Mouse Monoclonal Antibody # 23-57-137-1 Against Human PTHrP The overall structures of the H chain V region and L chain V region are similar to each other. One framework part consists of three hypervariable regions, the complementarity determining regions (CD
R). The amino acid sequence of the framework is relatively well conserved, while the amino acid sequence of the CDR regions has extremely high variability (Kabat, EAet).
al., `` Sequence of Proteins of Immunological Inte
rest "US Dept. Health and Human Services, 1983).
Based on this fact, the amino acid sequence of the variable region of the mouse monoclonal antibody against human PTHrP was
The CDR regions were determined as shown in Table 1 by applying homology to the database of antibody amino acid sequences prepared by bat et al. In addition, C of the L chain V region
For the amino acid sequences of DR1 to 3,
The amino acid sequences of CDRs 1 to 3 of the H chain V region are shown in SEQ ID NOs: 62 to 64, respectively.

[0092]

[Table 1]

Reference Example 3 Construction of Chimeric Antibody (1) Construction of Chimeric Antibody H Chain (i) Construction of H Chain V Region Cloning to ligate to an expression vector containing genomic DNA of human H chain C region Cγ1 The mouse H chain V region was modified by PCR. Back primer MBC1-S
1 (SEQ ID NO: 7) hybridizes to DNA encoding the 5'-side of the V region leader sequence and has a Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196, 947).
-950, 1987) and the restriction enzyme Hind III. The forward primer MBC1-a (SEQ ID NO: 8) hybridizes to the DNA sequence encoding the 3'-side of the J region, and comprises a splice donor sequence and a restriction enzyme.
It was designed to have a BamHI recognition sequence. PCR, TaKa
Using Ra Ex Taq (Takara Shuzo), 0.07 μg of plasmid MBC1H04 as template DNA, MBC1-a and MBC1-S1 as primers were added to 50 μl each of 50 μl of the reaction mixture, and 2.
Under the conditions containing 5 U of TaKaRa Ex Taq and 0.25 mM dNTP, the upper layer of 50 μl of mineral oil was added using the attached buffer solution, and the temperature cycle was 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes. so
Performed 30 times. The DNA fragment amplified by the PCR method is 3% Nu Sie
ve Separated by agarose gel electrophoresis using GTG agarose (FMC Bio. Products).

A piece of agarose containing a DNA fragment of 437 bp was cut out, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. After recovering the purified DNA by ethanol precipitation, 10 mM Tris-HCl (pH 7.4),
It was dissolved in 20 μl of a 1 mM EDTA solution. Obtained DNA solution 1
μl with restriction enzymes BamHI and Hind III (Takara Shuzo) at 37 ℃
Digested for 1 hour. The digestion mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation.

The Hind III-BamHI DNA fragment containing the gene encoding the mouse H chain V region prepared as described above was
PUC prepared by digestion with Hind III and BamHI
Subcloned into 19 vectors. To confirm the nucleotide sequence of this plasmid, use Dye Terminator with primers M13 Primer M4 and M13 Primer RV as primers.
Cycle Sequencing kit (Perkin-Elmer)
The nucleotide sequence was determined using queuer 373A (Perkin-Elmer). Hybridoma # 23-57-137 having correct nucleotide sequence
-1 and a gene encoding a mouse H chain V region, and a Hind III recognition sequence and a Kozak sequence on the 5'-
The plasmid having a BamHI recognition sequence on the − side was ligated with MBC1H / pUC19
It was named.

(Ii) cDNA type mouse-human chimera H
Construction of H chain V region for preparation of chain In order to ligate with the cDNA of human H chain C region Cγ1, the mouse H chain V region constructed as described above was modified by PCR. Back primer MBC1HVS2 for H chain V region
(SEQ ID NO: 9) converts the second asparagine of the sequence encoding the first of the leader region of the V region to glycine, and obtains a Kozak consensus sequence (Kozak, M. et al., J. Mol.
Biol., 196, 947-950, 1987) and Hind III and Eco.
It was designed to have an RI recognition sequence. The forward primer MBC1HVR2 (SEQ ID NO: 10) for the H chain V region hybridizes to the DNA sequence encoding the 3'-side of the J region, and encodes the sequence 5'-side of the C region, and ApaI and Sma
It was designed to have an I recognition sequence.

PCR was performed using TaKaRa Ex Taq (Takara Shuzo),
In 0 μl of the reaction mixture, 0.6 μg of plasmid MBC1H / pUC19 as template DNA, MBC1HVS2 and MBC1 as primers
HVR2 50pmole each, TaKaRa Ex Taq 2.5U, 0.25
50 μl using the attached buffer under conditions containing mM dNTPs
Was performed 30 times with a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute. DNA amplified by PCR
Fragments were digested with 1% Sea Kem GTG agarose (FMC Bio.Product
Separation by agarose gel electrophoresis using s). Four
A piece of agarose containing a DNA fragment of 56 bp in length was cut out and
The DNA fragment was purified using NECLEAN II Kit (BIO101) according to the instructions attached to the kit. After the purified DNA is precipitated with ethanol, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution 20 μl
Was dissolved.

[0098] 1 µl of the obtained DNA solution was subjected to restriction enzyme EcoRI.
And Smal (Takara Shuzo) at 37 ° C for 1 hour. Extract the digestion mixture with phenol and chloroform,
DNA was recovered by ethanol precipitation. Contains the gene encoding the mouse H chain V region prepared as described above
The EcoRI-SmaI DNA fragment was subcloned into a pUC19 vector prepared by digesting with EcoRI and SmaI. In order to confirm the nucleotide sequence of this plasmid, primers M13 Primer M4 and M13 Primer RV were used as primers in Dye Terminator Cycle Sequencing kit (Perkin-E
lmer) and the nucleotide sequence was determined by DNA Sequencer 373A (Perkin-Elmer). Contains the gene encoding the mouse H chain V region derived from hybridoma # 23-57-137-1 having the correct nucleotide sequence, and EcoRI and Hind
III recognition sequence and Kozak sequence, ApaI and Sm
The plasmid having the aI recognition sequence was designated as MBC1Hv / pUC19.

(Iii) Construction of Expression Vector for Chimeric Antibody H Chain cDNA containing human antibody H chain C region Cγ1 was prepared as follows. That is, the humanized PM1 antibody H chain V
Genomic DNA of IgG1 region and human antibody H chain C region IgG1
(N. Takahashi, et al., Cell 29, 671-679 1982). Expression vector DHFR- △ E-RVh-PM-1-f (WO92 / 19
CHO) into which the expression vector RV1-PM1a (see WO92 / 19759) encoding the genomic DNA of the humanized PM1 antibody L chain V region and human antibody L chain κ chain C region has been introduced.
MRNA was prepared from the cells, and the humanized P was
A cDNA containing the M1 antibody H chain V region and the human antibody C region Cγ1 was cloned and subcloned into HindIII and BamHI sites of pUC19. After confirming the nucleotide sequence, a plasmid having a correct sequence was designated as pRVh-PM1f-cDNA.

The HindIII site between the SV40 promoter and the DHFR gene on DHFR-ΔE-RVh-PM-1-f, and between the EF-1α promoter and the humanized PM1 antibody H chain V region An expression vector lacking a certain EcoRI site was prepared and used for construction of a cDNA expression vector containing a humanized PM1 antibody H chain V region and a human antibody C region Cγ1. After digesting pRVh-PM1f-cDNA with BamHI,
The fragment was blunted with the Klenow fragment and further digested with HindIII to prepare a HindIII-BamHI blunted fragment. This Hind I
The II-BamHI blunted fragment was ligated with the Hind III site and EcoR
The DHFR-ΔE-RVh-PM1-f lacking the I site was ligated to an expression vector prepared by digesting with HindIII and SmaI, and the humanized PM1 antibody H chain V region and human antibody C
Expression vector RVh-P containing cDNA encoding region Cγ1
M1f-cDNA was constructed.

After digesting the expression vector RVh-PM1f-cDNA containing the cDNA encoding the humanized PM1 antibody H chain V region and human antibody C region Cγ1 with ApaI and BamHI,
A DNA fragment containing the chain C region was recovered and introduced into MBC1Hv / pUC19 prepared by digestion with ApaI and BamHI. The plasmid thus prepared was named MBC1HcDNA / pUC19. This plasmid contains cDNA encoding the H chain V region of the mouse antibody and the C region Cγ1 of the human antibody, and contains 5′-
EcoRI and HindIII recognition sequences at the end, BamH at the 3'-end
Has an I recognition sequence.

The plasmid MBC1HcDNA / pUC19 was digested with EcoRI and BamHI, and a DNA fragment containing the nucleotide sequence encoding the H chain of the obtained chimeric antibody was introduced into an expression vector pCOS1 prepared by digesting with EcoRI and BamHI. . The thus obtained chimeric antibody expression plasmid was
It was named C1HcDNA / pCOS1. The expression vector pCOS1
Removes the antibody gene from HEF-PMh-gγ1 (see WO92 / 19759) by digestion with EcoRI and SmaI and gives EcoRI-NotI-Bam
It was constructed by connecting HI Adapter (Takara Shuzo).

To prepare a plasmid to be used for expression in CHO cells, the plasmid MBC1HcDNA / pUC1
9 was digested with EcoRI and BamHI, and the resulting DNA fragment containing the chimeric antibody H chain sequence was introduced into an expression plasmid pCHO1 prepared by digestion with EcoRI and BamHI. The thus obtained chimeric antibody expression plasmid was
It was named C1HcDNA / pCHO1. The expression vector pCHO1
Was obtained from DHFR-ΔE-rvH-PM1-f (see WO92 / 19759)
The antibody gene is deleted by RI and SmaI digestion, and EcoRI-N
It was constructed by connecting otI-BamHI Adapter (Takara Shuzo).

(2) Construction of human L chain constant region (i) Construction of cloning vector To construct a pUC19 vector containing a human L chain constant region, a Hind III site-deleted pUC19 vector was constructed. pUC1
9 2 μg of vector was added to 20 mM Tris-HCl (pH 8.5), 10 mM M
The digestion was carried out at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing gCl ₂ , 1 mM DTT, 100 mM KCl, and 8 U of Hind III (Takara Shuzo). The digestion mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation. The recovered DNA was subjected to 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DT
T, 100 mM NaCl, 0.5 mM dNTP, 6 U Klenow
The reaction was performed at room temperature for 20 minutes in a 50 μl reaction mixture containing the fragment (GIBCO BRL) to blunt the ends.
The reaction mixture was extracted with phenol and chloroform,
Vector DNA was recovered by ethanol precipitation.

The recovered vector DNA was added to 50 mM Tris-HCl (p
H7.6), 10 mM MgCl ₂ , 1 mM ATP, 1 mM DTT, 5% (v / v)
16 ° C. in a 10 μl reaction mixture containing polyethylene glycol-8000, 0.5 U T4 DNA ligase (GIBCO BRL)
For 2 hours to allow self-ligation. Reaction mixture 5 μl
E. coli JM109 competent cells (Nippon Gene) 10
After adding to 0 µl, the mixture was allowed to stand on ice for 30 minutes, then at 42 ° C for 1 minute, and further on ice for 1 minute. SOC medium 500 μl
, And incubated at 37 ° C for 1 hour.
gal and IPTG coated on the surface of 2 × YT agar medium (50 μg /
ml Ampicillin) (Molecular Cloning: A Labgora)
tory Manual, Sambrook, et al., Cold Spring Harbor L
aboratory Press, 1989) and cultured at 37 ° C. overnight to obtain a transformant. The transformant was cultured overnight at 37 ° C. in 20 ml of 2 × YT medium containing 50 μg / ml ampicillin, and plasmid DNA was purified from the cell fraction using a Plasmid Mini Kit (QIAGEN) according to the attached protocol. The purified plasmid was digested with HindIII, and the plasmid confirmed to have deleted the HindIII site was named pUC19ΔHindIII.

(Ii) Construction of Gene Encoding Human L Chain λ Chain Constant Region The human antibody L chain λ chain C region is composed of Mcg + Ke + Oz−, M
cg- Ke- Oz-, Mcg- Ke- Oz +, Mcg-
At least four isotypes of Ke + Oz- are known (P. Darariavach, et al., Proc. Natl. Acad. Sci. U.
SA, 84, 9074-9078, 1987). # 23-57-137-1 Human antibody L chain λ chain C region having homology with mouse L chain λ chain C region
As a result of searching in the BL database, the isotype is Mc
g + Ke + Oz- (accession No. X57819) (P. Dariava
ch, et al., Proc. Natl. Acad. Sci. USA, 84, 9074-90
78, 1987) shows the highest homology with the human antibody L chain λ chain, and the homology with the # 23-57-137-1 mouse L chain λ chain C region is 64.4% in the amino acid sequence and 73.4% in the base sequence. Met.

Thus, the construction of the gene encoding the human antibody L chain λ chain C region was carried out by using the PCR method. Each primer was synthesized using a 394 DNA / RNA synthesizer (ABI). HLAMB1 (SEQ ID NO: 11) and HLAMB3 (SEQ ID NO:
13) has a sense DNA sequence, HLAMB2 (SEQ ID NO: 12) and HLAMB4 (SEQ ID NO: 14) have antisense DNA sequences, and have complementary sequences of 20 to 23 bp at both ends of each primer.

External primers HLAMBS (SEQ ID NO: 15), HL
AMBR (SEQ ID NO: 16) has sequences homologous to HLAMB1 and HLAMB4, respectively, and HLAMBBS is EcoRI, HindIII, Bl
HLAMBR contains an EcoRI recognition sequence, and HLAMBR contains an EcoRI recognition sequence. In the first PCR, HLAMB1-HLAMB2 and HLAMB3-HLAMB4 were reacted. After the reaction, mix them in equal volumes, and
Was assembled. Further, external primers HLAMBS and HLAMBR were added, and the full-length DNA was amplified by the third PCR.

For PCR, TaKaRa Ex Taq (Takara Shuzo) was used.
Performed according to the attached prescription. In the first PCR, 5pmole of H
LAMB1 and 0.5pmole HLAMB2 and 5U TaKaRa Ex Taq
(Takara Shuzo), or 0.5 pmole HLAMB3 and 5 pmole HLAMB4 and 5 U
Using 100 µl of the reaction mixture containing TaKaRa Ex Taq (Takara Shuzo), 50 µl of mineral oil was layered on the top and subjected to a temperature cycle of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute. Performed 5 times.

In the second PCR, the reaction solution was mixed in 50 μl portions,
50 μl of mineral oil was applied to the upper layer three times at a temperature cycle of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute. Third
The PCR was performed 30 times by adding 50 pmole of each of the external primers HLAMBS and HLAMBR to the reaction solution and performing a temperature cycle of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute. Third
3% low melting point agarose gel (NuSiev
e GTG Agarose, FMC) followed by GENECLEANII
Using the Kit (BIO101), the gel was recovered and purified from the gel according to the attached instructions.

The obtained DNA fragment was treated with 50 mM Tris-HCl (pH 7.
5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM NaCl, 8 U EcoR
Digestion was performed at 37 ° C. for 1 hour in a 20 μl reaction mixture containing I (Takara Shuzo). After extracting the digestion mixture with phenol and chloroform and recovering the DNA by ethanol precipitation,
It was dissolved in 8 mM of 1 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution.

0.8 μg of the plasmid pUC19 ΔHind III was similarly digested with EcoRI, extracted with phenol and chloroform, and recovered by ethanol precipitation. Digested plasmid pUC19 ΔHind III was added to 50 mM Tris-HCl (pH 9.0), 1 mM
Dephosphorylation treatment (BAP treatment) was carried out at 37 ° C. for 30 minutes in 50 μl of a reaction mixture containing MgCl ₂ and alkaline phosphatase (E. coli C75, Takara Shuzo). The reaction solution was extracted with phenol and chloroform, DNA was recovered by ethanol precipitation, and then dissolved in 10 mM Tris-HCl (pH 7.4) and 10 μl of 1 mM EDTA solution.

The above plasmid BUC-treated plasmid pUC19Δ
1 µl of Hind III and 4 µl of the above PCR product were combined with DNA Ligation Ki
t Ligation was performed using Ver.2 (Takara Shuzo) and transformed into E. coli JM109 competent cells. 50 transformants obtained
After culturing overnight in 2 ml of 2 × YT medium containing μg / ml ampicillin, a QIAprep Spin Plasmid Kit (QIAG
The plasmid was purified using EN).

With respect to the above plasmid, the nucleotide sequence of the cloned DNA was confirmed. The nucleotide sequence was determined using a 373A DNA sequencer (ABI), and M13 Primer M4 and M13 Pricer RV (Takara Shuzo) were used as primers. As a result, it was found that there was a 12 bp deletion in the cloned DNA. The plasmid containing this DNA was named CλΔ / pUC19. Therefore, primers HCLMS (SEQ ID NO: 17) to supplement that portion, H
CLMR (SEQ ID NO: 18) is newly synthesized and the correct D
The construction of NA was performed.

The plasmid CλΔ containing the deleted DNA in the first PCR
/ PUC19 as template, primers HLAMBS and HCLMR, HCLM
Reaction was performed with S and HLAMB4. Each PCR product was purified and assembled in a second PCR. Further, external primers HLAMBS and HLAMB4 were added, and the full-length D
NA was amplified.

In the first PCR, CλΔ / pUC19 was used as a template.
0.1 μg, primers HLAMBS and HCLMR 50 pmole each,
Alternatively, HCLMS and HLAMB4 50pmole each, 5U TaKaRa
Using 100 µl of the reaction mixture containing Ex Taq (Takara Shuzo), apply 50 µl of mineral oil on the upper layer, and perform 30 cycles of 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C. went.

PCR products HLAMBS-HCLMR (236 bp), HCLMS-HLA
After each MB4 (147 bp) was electrophoresed on a 3% low melting point agarose gel, it was recovered and purified from the gel using the GENECLEANII Kit (BIO101). In the second PCR, each 40 ng of the purified DNA fragment,
Using a 20 μl reaction mixture containing 1 U of TaKaRa Ex Taq (Takara Shuzo), 25 μl of mineral oil was layered on the top and subjected to a temperature cycle of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute. Performed 5 times.

In the third PCR, 2 μl of the second PCR reaction solution, 50 pmole of each of the external primers HLAMBS and HLAMB4, and 5 U of TaKaRa E
Using 100 μl of the reaction mixture containing x Taq (Takara Shuzo), 50 μl of mineral oil was overlaid. PCR was performed at a temperature cycle of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute.
Performed 30 times. 3% DNA fragment of the third PCR product, 357 bp
After electrophoresis on a low melting point agarose gel, GENECLEAN II
It was recovered and purified from the gel using Kit (BIO101).

After 0.1 μg of the obtained DNA fragment was digested with EcoRI, it was subcloned into a BAP-treated plasmid pUC19ΔHindIII. E. coli JM109 competent cells were transformed and 2 × containing 50 μg / ml ampicillin.
After culturing overnight in 2 ml of YT medium, QIAprep Spin
Plasmid was purified using Plasmid Kit (QIAGEN).
The nucleotide sequence of the purified plasmid was changed to M13 Primer M4.
And M13 Primer RV (Takara Shuzo) using a 373A DNA sequencer (ABI). Plasmids confirmed to have the correct nucleotide sequence without deletion were cloned into Cλ /
It was pUC19.

(Iii) Construction of Gene Encoding Human L Chain κ Chain Constant Region L chain κ chain C from plasmid HEF-PM1k-gk (WO92 / 19759)
The DNA fragment encoding the region was cloned using the PCR method. 394 Forward primer HKAPS (SEQ ID NO: 19) synthesized using DNA / RNA synthesizer (ABI) is EcoRI, Hind II
I and BlnI recognition sequences were added to the rear primer HKAPA (SEQ ID NO: 2).
0) was designed to have an EcoRI recognition sequence. Plasmid HEF-PM1k-gk 0.1 μg as template, primer HKAP
50 μl of mineral oil was overlaid using 100 μl of a reaction mixture containing 50 pmole each of S and HKAPA and 5 U of TaKaRa Ex Taq (Takara Shuzo). 1 minute at 94 ° C, 1 minute at 60 ° C, 72
The reaction was carried out at 30 ° C. for 1 minute for 30 cycles. 360 bp PCR
After electrophoresis of the product on a 3% low melting point agarose gel, GE
The gel was recovered and purified using the NECLEANII Kit (BIO101).

After digesting the obtained DNA fragment with EcoRI,
It was cloned into BAP-treated plasmid pUC19 ΔHind III. E. coli JM109 competent cells were transformed into 2 × YT medium 2 containing 50 μg / ml ampicillin.
cultivation overnight, and then use the QIAprep Spin Plasmid K
The plasmid was purified using it (QIAGEN). M13 Primer M4, M13 Primer RV
(Takara Shuzo) using a 373A DNA sequencer (ABI). The plasmid confirmed to have the correct nucleotide sequence was designated as Cκ / pUC19.

(3) Construction of chimeric antibody L chain expression vector A chimeric # 23-57-137-1 antibody L chain expression vector was constructed.
Plasmids # 23-57-137-1 were added to HindIII and BlnI sites immediately before the human antibody constant regions of plasmids Cλ / pUC19 and Cκ / pUC19.
By linking the genes encoding the L chain V region, pUC19 vectors encoding the chimeric # 23-57-137-1 antibody L chain V region and L chain λ chain or L chain κ chain constant region were prepared. The chimeric antibody L chain gene was cut out by EcoRI digestion and subcloned into a HEF expression vector.

That is, plasmids MBC1L24 to # 23-57
The -137-1 antibody L chain V region was cloned using PCR. Each primer was synthesized using a 394 DNA / RNA synthesizer (ABI
Was carried out using The rear primer MBCCHL1 (SEQ ID NO: 21) is composed of a Hind III recognition sequence and a Kozak sequence (Kozak, M.et).
196, 947-950, 1987), the forward primer MBCCHL3 (SEQ ID NO: 22) was designed to have BglII and EcoRI recognition sequences.

The PCR was performed using 10 mM Tris-HCl (pH 8.3), 50 mM KC
l, 1.5 mM MgCl ₂ , 0.2 mM dNTP, 0.1 μg MBC1L24
, MBCCHL1 and MBCCHL3 as primers at 50pmo each
le, containing 100 μl of AmpliTaq (PERKIN ELMER)
Using 50 μl of mineral oil, add 94 μl of reaction mixture
For 30 seconds at 45 ° C., 45 seconds at 60 ° C., and 2 minutes at 72 ° C. After electrophoresing the 444 bp PCR product on a 3% low melting point agarose gel, GENECLEAN II kit
(BIO101), recovered and purified from the gel, 10 mM Tris-HC
l (pH 7.4) and dissolved in 20 μl of a 1 mM EDTA solution. 1 μl of the PCR product was added to 10 mM Tris-HCl (pH 7.5), 10 mM MgC
_{l 2, 1mM DTT, 50mM NaCl} , of 8U Hind III (Takara Shuzo Co., Ltd.)
Mixture containing 20 and 8 U of EcoRI (Takara Shuzo)
Digested for 1 hour at 37 ° C in μl. The digestion mixture was extracted with phenol and chloroform, the DNA was recovered by ethanol precipitation, and 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution 8
Dissolved in μl.

[0125] Similarly, Hind III
And digested with EcoRI, extracted with phenol and chloroform, recovered by ethanol precipitation, and BAP-treated with alkaline phosphatase (E. coli C75, Takara Shuzo). The reaction solution was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation, then 10 mM Tris-HCl (pH 7.4),
It was dissolved in 10 μl of a 1 mM EDTA solution.

1 μl of BAP-treated plasmid pUC19 and 4 μl of the above PCR product were ligated using DNA Ligation Kit Ver. 2 (Takara Shuzo) and transformed into E. coli JM109 competent cells (Nippon Gene) in the same manner as described above. This was spread on 2 × YT agar medium containing 50 μg / ml ampicillin and cultured at 37 ° C. overnight. The obtained transformant was cultured overnight at 37 ° C. in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin. QIAprep Sp from cell fraction
The plasmid was purified using the in Plasmid Kit (QIAGEN). After determining the nucleotide sequence, the plasmid having the correct nucleotide sequence was designated as CHL / pUC19.

Each 1 μg of each of the plasmids Cλ / pUC19 and Cκ / pUC19 was added to 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ ,
20 μl of reaction mixture containing 1 mM DTT, 100 mM KCl, 8 U Hind III (Takara Shuzo) and 2 U BlnI (Takara Shuzo)
For 1 hour at 37 ° C. The digestion mixture was extracted with phenol and chloroform, DNA was recovered by ethanol precipitation, and BAP treatment was performed at 37 ° C. for 30 minutes. The reaction solution was extracted with phenol and chloroform, the DNA was recovered by ethanol precipitation, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA
Dissolved in 10 μl of solution. 8 μg of the plasmid CHL / pUC19 containing the # 23-57-137-1 L chain V region was similarly digested with HindIII and BlnI. The obtained 409 bp DNA fragment was electrophoresed on a 3% low melting point agarose gel, and then GENECLEANII Ki
Recover from the gel using t (BIO101), purify, 10 mM Tris
-HCl (pH 7.4) was dissolved in 10 μl of 1 mM EDTA solution.

4 μl of this L chain V region DNA was BAP-treated and the plasmid Cλ / pUC19 or Cκ / pUC19, 1 μl each
And transformed into E. coli JM109 competent cells. The cells were cultured overnight in 3 ml of 2 × YT medium containing 50 μg / ml ampicillin, and QIAprep
The plasmid was purified using the Spin Plasmid Kit (QIAGEN). These are called plasmid MBC1L (λ) / pUC19,
MBC1L (κ) / pUC19. Plasmid MBC1L (λ) / pUC19
And MBC1L (κ) / pUC19 were digested with EcoRI.
After electrophoresis on a low-melting point agarose gel, the 743 bp DN
The A fragment was recovered and purified from the gel using the GENECLEANII Kit (BIO101), and 10 mM Tris-HCl (pH 7.4), 1 mM
l.

As an expression vector, plasmid HEF-PM1k-g
2.7 μg of k was digested with EcoRI, extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation. After BAP treatment of the recovered DNA fragment, it was electrophoresed on a 1% low melting point agarose gel, and the 6561 bp DNA fragment was subjected to GENECLEANII K
It was recovered from the gel using it (BIO101), purified, and 10 mM Tris
-HCl (pH 7.4) was dissolved in 10 μl of 1 mM EDTA solution. BA
2 μl of the P-treated HEF vector was added to the above plasmid MBC1.
L (λ) or MBC1L (κ) Ligation with each EcoRI fragment 3μl,
E. coli JM109 competent cells were transformed. 50
After culturing in 2 ml of 2 × YT medium containing μg / ml ampicillin, a QIAprep Spin Plasmid Kit (QIAGEN)
Was used to purify the plasmid.

[0130] The purified plasmid was added to 20 mM Tris-HCl (p
H8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, 8 U Hi
Digestion was performed at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing ndIII (Takara Shuzo) and 2 U of PvuI (Takara Shuzo). If the fragment is inserted in the correct direction, 5104/2195 bp, if inserted in the reverse direction, a digested fragment of 4378/2926 bp will be generated, so that the plasmid inserted in the correct direction is MBC1L (λ) / neo, MBC1L (κ) / neo.

(4) Transfection of COS-7 cells In order to evaluate the antigen binding activity and the neutralizing activity of the chimeric antibody, the expression plasmid was transiently expressed in COS-7 cells. That is, the transient expression of the chimeric antibody is determined by using a combination of a plasmid MBC1HcDNA / pCOS1 and MBC1L (λ) / neo or a combination of MBC1HcDNA / pCOS1 and MBC1L (κ) / neo with a GenePulser device (BioRad).
Was used to co-transduce COS-7 cells by electroporation. COS-7 cells suspended at a cell concentration of 1 × 10 ⁷ cells / ml in PBS (−)
To 8 ml, 10 μg of each plasmid DNA was added, and 1,50
A pulse was applied with a capacitance of 0 V and 25 μF. After a 10 minute recovery period at room temperature, the electroporated cells were harvested with 2% Ultra Low IgG fetal calf serum (G
The suspension was suspended in a DMEM medium (GIBCO) containing IBCO) and cultured in a CO ₂ incubator using a 10 cm culture dish. After 72 hours of culture, the culture supernatant was collected,
Cell debris was removed by centrifugation and used for ELISA samples. Purification of the chimeric antibody from the culture supernatant of COS-7 cells was performed using AffiGel Protein A
The test was carried out using a MAPSII kit (BioRad) according to the instructions attached to the kit.

(5) ELISA (i) Measurement of Antibody Concentration An ELISA plate for antibody concentration measurement was prepared as follows. 96-well plate for ELISA (Maxi
sorp, NUNC) in a solid phase buffer (0.1 M
NaHCO ₃ , 0.02% NaN ₃ ) was immobilized with 100 μl of a goat anti-human IgG antibody (TAGO) adjusted to a concentration of 1 μg / ml, and 200 μl of a dilution buffer (50 mM Tris-HC)
l, 1 mM MgCl ₂ , 0.1 M NaCl, 0.05% Tween20, 0.02
% NaN ₃ , 1% bovine serum albumin (BSA), pH 7.2
After blocking in step ()), the CO
S cell culture supernatant or purified chimeric antibody was serially diluted and added to each well. Incubate for 1 hour at room temperature
After washing with S-Tween 20, 100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added. Incubate for 1 hour at room temperature, PBS-Tw
After washing with een20, a 1 mg / ml substrate solution (Si
gma104, p-nitrophenylphosphate, SIGM
A) was added and then the absorbance at 405 nm was measured on a microplate reader (BioRad). As a standard for concentration measurement, Hu IgG1λ Purifi
ed (The Binding Site) was used.

(Ii) Measurement of antigen binding ability An ElISA plate for antigen binding measurement was prepared as follows. 100 μl of human PTHrP (1-34) (Peptide Research Laboratories) in which each well of an ELISA 96-well plate was adjusted to a concentration of 1 μg / ml with a solid phase buffer
and immobilized. After blocking with 200 μl of the dilution buffer, the culture supernatant of the COS cells expressing the chimeric antibody or the purified chimeric antibody was serially diluted and added to each well. Incubate at room temperature with PBS-Tween20
After washing with alkaline phosphatase-conjugated goat anti-human I
100 μl of gG antibody (TAGO) was added. After incubating at room temperature and washing with PBS-Tween 20, 1
mg / ml substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, then 405 nm
Absorbance measured by microplate reader (BioRa)
Measured in d). As a result, the chimeric antibody is human PTH
It had the ability to bind to rP (1-34) and was shown to have the correct structure of the cloned mouse antibody V region (FIG. 5). Also, in the chimeric antibody, the light chain C
Whether the region is a λ chain or a κ chain, the PT
Since the binding ability to HrP (1-34) does not change, the L chain C region of the humanized antibody is
Constructed using chains.

(6) Establishment of Stable CHO Producing Cell Line In order to establish a stable chimeric antibody producing cell line, the above expression plasmid was introduced into CHO cells (DXB11). That is, the establishment of a cell line for stably producing a chimeric antibody is performed by using the expression plasmids MBC1HcDNA / pCHO1 for CHO cells and MBC1L.
(Λ) / neo or MBC1H cDNA / pCHO1 and MBC
GenePuls in combination of 1L (κ) / neo
CHO cells were co-transduced by electroporation using an er device (BioRad). Each expression vector was cleaved with a restriction enzyme PvuI to obtain linear DNA, extracted with phenol and chloroform, recovered by ethanol precipitation, and used for electroporation. Each plasmid DNA was added to 0.8 ml of CHO cells suspended at a cell concentration of 1 × 10 ⁷ cells / ml in PBS (−).
10 μg was added, and a pulse was applied with a capacitance of 1,500 V and 25 μF. After a 10 minute recovery period at room temperature, the electroporated cells were transferred to MEM-α medium (GIBC) supplemented with 10% fetal calf serum (GIBCO).
O) and three 96-well plates (Falcon)
And cultured in a CO ₂ incubator. The day after the start of culture, 10% fetal bovine serum (GIBCO) and 50%
0 mg / ml GENETICIN (G418Sulf
ate, GIBCO), and a MEM-α medium without ribonucleoside and deoxyribonucleoside (GIBC
The selection medium in O) was replaced, and cells into which the antibody gene had been introduced were selected. After exchanging the selective medium, the cells were observed under a microscope about two weeks, and after successful cell growth was observed, the antibody production was measured by the antibody concentration measurement ELISA, and cells having a large antibody production were selected. .

[0135] The culture of the established antibody-producing stable cell line was expanded, and 2% Ultra Low IgG fetal bovine serum was added using a roller bottle, and a large-scale culture was performed using a MEM medium free of ribonucleoside and deoxyribonucleoside. Was. On the third or fourth day of the culture, the culture supernatant was collected, and cell debris was removed with a 0.2 μm filter (Millipore). CHO
Purification of the chimeric antibody from the cell culture supernatant was performed using a POROS Protein A column (PerSeptive Biosys).
tems) using ConSep LC100 (Mill
ipore) according to the attached prescription, and subjected to the measurement of neutralizing activity and the efficacy test in a hypercalcemia model animal. The concentration and antigen-binding activity of the obtained purified chimeric antibody were measured by the above-mentioned ELISA system.

[Reference Example 4] Construction of humanized antibody (1) Construction of humanized antibody H chain (i) Construction of humanized H chain V region Humanized # 23-57-137-1 antibody H The chain is used for CDR by PCR.
-Made by grafting. Human antibody S31679 (N
BRF-PDB, Cuisinier A. M. E
ur. J. Immunol. , 23, 110-118,
Six PCR primers were used to generate a humanized # 23-57-137-1 antibody H chain (version "a") with an FR from 1993). CDR-grafting primers MBC1HGP1 (SEQ ID NO: 23) and MBC1HG
P3 (SEQ ID NO: 24) has a sense DNA sequence, and CDR grafting primers MBC1HGP2 (SEQ ID NO: 25) and MBC1HGP4 (SEQ ID NO: 26) have an antisense DNA sequence, and 15 to 21 bp at each end of the primer. Has a complementary sequence of External primers MBC1HVS1 (SEQ ID NO: 27) and M
BC1HVR1 (SEQ ID NO: 28) has homology to CDR grafting primers MBC1HGP1 and MBC1HGP4.

CDR-grafting primer MBC
1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4 were separated using a urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Ma
nual, Sambrook et al., Cold Spring Harbor Laboratory Pr
ess, 1989), extraction from gel
ak method (Molecular Cloning: A Laboratory Manual, Sa
mbrook et al., Cold Spring Harbor Laboratory Press, 198
9).

That is, each 1 nmole CDR
The grafting primers are separated on a 6% denaturing polyacrylamide gel and the DNA fragments of the desired size are identified by irradiating them with ultraviolet light on a silica gel thin plate,
The gel was collected from the gel by the soak method and
0 mM Tris-HCl (pH 7.4), 1 mM ED
Dissolved in TA solution. PCR was performed using TaKaRa Ex T
Using aq (Takara Shuzo), the CDR-grafting primer MB prepared as described above was added to 100 μl of the reaction mixture.
1 μl each of C1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4, 0.25 mM
Of dNTP and 2.5 U of TaKaRaEx Taq for 55 minutes at 94 ° C. for 1 minute using the attached buffer.
5 minutes with a temperature cycle of 1 minute at 72 ° C. and 1 minute at 72 ° C., and 50 pmole of external primer MBC1
HVS1 and MBC1 HVR1 were added and the same temperature cycle was performed 30 times. 4% DNA fragment amplified by PCR
Nu Sieve GTG agarose (FMC Bio.
Products) on agarose gel electrophoresis.

A piece of agarose containing a 421 bp DNA fragment was cut out, and the GENECLEANII Kit (B
The DNA fragment was purified using IO101) according to the instructions attached to the kit. After the purified DNA was precipitated with ethanol, 10 mM Tris-HCl (pH 7.4), 1 m
Dissolved in 20 μl of M EDTA solution. The resulting PCR reaction mixture was subcloned into pUC19 prepared by digestion with BamHI and HindIII,
The nucleotide sequence was determined. The plasmid having the correct sequence was named hMBCHv / pUC19.

(Ii) H chain V for humanized H chain cDNA
Construction of Region In order to link to the cDNA of human H chain C region Cγ1, the humanized H chain V region constructed as described above was modified by PCR. The rear primer MBC1HVS2 hybridizes with the sequence encoding the 5'-side of the V region leader sequence, and has a Kozak consensus sequence (Kozak).
k, M, et al. Mol. Biol. 196,947-
950, 1987), HindIII and EcoRI.
It was designed to have a recognition sequence. The forward primer MBC1HVR2 for the H chain V region hybridizes to the DNA sequence encoding the 3′-side of the J region and 5 ′ of the C region.
The negative sequence was designed to encode the ApaI and SmaI recognition sequences.

PCR was performed using TaKaRa Ex Taq (Takara Shuzo) and 0.4 μg of hMBCHv as a template DNA.
/ PUC19 and MBC1HVS as primer
2 and MBC1HVR2 were each 50 pmole,
Performed 30 times at a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute using the attached buffer under conditions containing 2.5 U of TaKaRa ExTaq and 0.25 mM dNTP. Was. The DNA fragment amplified by the PCR method was subjected to 3% NuSieve GTG agarose (FMCBio.
Products) on agarose gel electrophoresis.

A piece of agarose containing a 456 bp DNA fragment was cut out, and the GENECLEANII Kit (B
The DNA fragment was purified using IO101) according to the instructions attached to the kit. After the purified DNA was precipitated with ethanol, 10 mM Tris-HCl (pH 7.4), 1 m
Dissolved in 20 μl of M EDTA solution. The obtained PCR reaction mixture was subcloned into pUC19 prepared by digesting with EcoRI and SmaI, and the nucleotide sequence was determined. Hybridoma # 23-57-137 thus obtained
-1 containing a gene encoding a mouse H chain V region, an EcoRI and HindIII recognition sequence and a Kozak sequence on the 5′-side, and ApaI and S on the 3′-side.
Plasmid having the maI recognition sequence was designated as hMBC1Hv / p
It was named UC19.

(2) Construction of Humanized Antibody H Chain Expression Vector Plasmid RVh Containing the hPM1 Antibody H Chain cDNA Sequence
HMBC prepared by digesting PM1f-cDNA with ApaI and BamHI, collecting a DNA fragment containing the H chain C region, and digesting with ApaI and BamHI.
Introduced into 1Hv / pUC19. The plasmid thus prepared was designated as hMBC1HcDNA / pUC19.
This plasmid contains the H chain V region of the humanized # 23-57-137-1 antibody and the human H chain C region Cγ1 at the 5′-end.
RI and HindIII recognition sequences, Bam at 3'-end
It has an HI recognition sequence. Plasmid hMBC1HcDNA /
SEQ ID NO: 58 shows the nucleotide sequence and the corresponding amino acid sequence of humanized H chain version "a" contained in pUC19. In addition, the amino acid sequence of version a was replaced with SEQ ID NO: 56
Shown in

The hMBC1H cDNA / pUC19 was
After digestion with RI and BamHI, the obtained DNA fragment containing the H chain sequence was introduced into an expression plasmid pCOS1 prepared by digestion with EcoRI and BamHI. The expression plasmid for the humanized antibody thus obtained was transformed into hMBC1
It was named HcDNA / pCOS1. HMBC1Hc to prepare a plasmid for use in expression in CHO cells.
DNA / pUC19 was digested with EcoRI and BamHI, and the obtained DNA fragment containing the H chain sequence was introduced into an expression plasmid pCHO1 prepared by digesting with EcoRI and BamHI. The expression plasmid for the humanized antibody thus obtained was named hMBC1HcDNA / pCHO1.

(3) Construction of L chain hybrid variable region (i) Preparation of FR1, 2 / FR3, 4 hybrid antibody An L chain gene in which the humanized antibody and the FR region of a mouse (chimeric) antibody were recombined was constructed. Each region for humanization was evaluated. By utilizing the restriction enzyme AflII cleavage site in CDR2, hybrid antibodies were prepared in which FR1 and FR2 were derived from human antibodies and FR3 and FR4 were derived from mouse antibodies. Plasmid MBC1L (λ) / n
10 μg each of eo and hMBC1L (λ) / neo
mM Tris-HCl (pH 7.5), 10 mM MgC
l ₂ , 1 mM DTT, 50 mM NaCl, 0.01% (w
/ V) Digestion was performed at 37 ° C. for 1 hour in 100 μl of a reaction mixture containing 10 U of BSA and AflII (Takara Shuzo).
The reaction solution was subjected to electrophoresis on a 2% low-melting point agarose gel, and a fragment (hereinafter referred to as c1) of 6282 bp and a fragment (hereinafter referred to as c2) of plasmid MBC1L (λ) / neo and plasmid hMBC1L (λ) / neo of 622 bp were obtained.
The 82 bp fragment (h1) and the 1022 bp fragment (h2) were combined with the GENECLEANII Kit.
It was recovered and purified from the gel using (BIO101).

BAP treatment was performed on 1 μg of each of the collected c1 and h1 fragments. After extracting DNA with phenol and chloroform and recovering it by ethanol precipitation, 10 mM
Tris-HCl (pH 7.4) was dissolved in 10 μl of a 1 mM EDTA solution. 1 μl of the BAP-treated c1 and h1 fragments were ligated to 4 μl of the h2 and c2 fragments, respectively (4
E. coli JM109 competent cells overnight. 2 × containing 50 μg / ml ampicillin
The cells were cultured in 2 ml of YT medium, and QIAprep
Plasmids were purified using SpinPlasmidKit (QIAGEN).

The purified plasmid was added to 10 mM Tris
-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DT
The digestion was performed at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing 2 U of T, ApaLI (Takara Shuzo), 8 U of BamHI (Takara Shuzo) and 8 U of HindIII (Takara Shuzo). c1
If -h2 is correctly linked, 556 in ApaLI
0/1246/498 bp, BamHI / HindII
The plasmid was confirmed by generating a digested fragment of 7134/269 bp with I.

This was converted to human FR1,2 / mouse FR3,4
The expression vector encoding the hybrid antibody L chain
mMBC1L (λ) / neo. On the other hand, h1-c2
Since no clone was obtained, it was cloned into the HEF vector after recombination on the pUC vector. At this time, plasmids hMBC1Laλ / pUC19 containing a humanized antibody L chain V region without amino acid substitution, and FR3
At position 91 (amino acid number 87 as defined by Kabat)
HMBC1Ldλ / pUC containing a humanized antibody L chain V region in which tyrosine at position 2) is replaced with isoleucine
19 was used as a template.

Plasmid MBC1L (λ) / pUC1
9, hMBC1Laλ / pUC19 and hMBC1Ld
Each 10 μg of λ / pUC19 was added to 10 mM Tris-HC.
1 (pH 7.5), 10 mM MgCl ₂ , 1 mM DT
T, 50 mM NaCl, 0.01% (w / v) BSA,
Digestion was performed at 37 ° C. for 1 hour in 30 μl of a reaction mixture containing 16 U of HindIII and 4 U of AflII. The reaction solution was electrophoresed on a 2% low melting point agarose gel, and the plasmid M
BC1L (λ) / pUC19 to 215 bp (c
2 ′), 3218 from plasmids hMBC1Laλ / pUC19 and hMBC1Ldλ / pUC19, respectively.
bp (ha1 ', hd1') DNA fragment
The gel was recovered and purified using ANII Kit (BIO101).

The ha1 'and hd1' fragments were each ligated to the c2 'fragment and transformed into E. coli JM109 competent cells. 2 × containing 50 μg / ml ampicillin
The cells were cultured in 2 ml of YT medium, and QIAprep
Plasmids were purified using SpinPlasmidKit (QIAGEN). These were isolated from the plasmids m / hMBC1Laλ / pUC19 and m / hMBC1L, respectively.
dλ / pUC19. Obtained plasmid m / hM
BC1Laλ / pUC19, m / hMBC1Ldλ / p
UC19 was digested with EcoRI. 743 bp each
Was electrophoresed on a 2% low-melting-point agarose gel, then recovered and purified from the gel using GENECLEANII Kit (BIO101), and purified using 10 mM Tris-H.
Cl (pH 7.4) was dissolved in 20 μl of a 1 mM EDTA solution.

4 μl of each DNA fragment was ligated to 1 μl of the above-mentioned BAP-treated HEF vector, and transformed into E. coli JM109 competent cells. The cells were cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and QIAprepSpinPlasmidKit (Q
The plasmid was purified using IAGEN). Each purified plasmid was used in 20 mM Tris-HCl (pH 8.
5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KC
1, 20 μl of a reaction mixture containing 8 U of HindIII (Takara Shuzo) and 2 U of PvuI (Takara Shuzo) was digested at 37 ° C. for 1 hour. 5104/2195 bp if the fragment is inserted in the right direction, 4 if the fragment is inserted in the opposite direction.
The plasmid was confirmed by the generation of a digested fragment of 378/2926 bp. These were used for mouse FR
The expression vector encoding the 1,2 / human FR3,4 hybrid antibody L chain was constructed using m / hMBC1Laλ / neo, m
/ HMBC1Ldλ / neo.

(Ii) Preparation of FR1 / FR2 Hybrid Antibody A hybrid antibody of FR1 and FR2 was prepared in the same manner by utilizing the SnaBI cleavage site in CDR1. Plasmid MBC1L (λ) / neo and h /
10 μg of each of mMBC1L (λ) / neo was added to 10 mM
Tris-HCl (pH 7.9), 10 mM MgCl ₂ , 1
mM DTT, 50 mM NaCl, 0.01% (w /
v) Digestion was performed at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing 6 U of BSA and SnaBI (Takara Shuzo). Then 2
0 mM Tris-HCl (pH 8.5), 10 mM MgC
l ₂ , 1 mM DTT, 100 mM KCl, 0.01%
(W / v) Digestion was performed at 37 ° C. for 1 hour in 50 μl of a reaction mixture containing 6 U of BSA and PvuI.

After electrophoresis of the reaction mixture on a 1.5% low-melting point agarose gel, plasmid MBC1L (λ) / neo
From 4955 bp (m1) and 2349 bp (m
2), 4955 bp (hm1) and 2349 bp (hm2) from plasmid h / mMBC1L (λ) / neo
Of each DNA fragment of GENECLEANII Kit (BI
Recovered from the gel using O101) and purified to 10 mM
Tris-HCl (pH 7.4) was dissolved in 40 μl of a 1 mM EDTA solution.

1 µl of the m1 and hm1 fragments
2. Ligation was performed with 4 μl of the m2 fragment, and transformed into E. coli JM109 competent cells. The cells were cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and QIAprepSpinPlasmidKit (QIprep
(AGEN) to purify the plasmid. Each of the purified plasmids was added to 10 mM Tris-HCl (pH 7.
5) Digestion was performed at 37 ° C. for 1 hour in 20 μl of a reaction mixture containing 10 mM MgCl ₂ , 1 mM DTT, 8 U of ApaI (Takara Shuzo) or 2 U of ApaLI (Takara Shuzo).

If each fragment is correctly ligated, Apa
7304 bp for I and 5560/1246 / for ApaLI
498 bp (m1-hm2), 6538/7 with ApaI
66bp, 3535/2025/1246 with ApaLI
The plasmid was confirmed by the generation of a digested fragment of / 498 bp (hm1-m2). These were isolated from the human FR1 / mouse FR2,3,4 hybrid antibody L, respectively.
HmmMBC1L (λ)
/ Neo, mouse FR1 / human FR2 / mouse FR3
Expression vector encoding the four-hybrid antibody L chain
hmmBC1L (λ) / neo.

(4) Construction of humanized antibody L chain The humanized # 23-57-137-1 antibody L chain was subjected to CDR by PCR.
-Made by grafting. Human antibody HSU03
868 (GEN-BANK, Defos M et al., Sc
and. J. Immunol. , 39, 95-103,
1994) and FR2 from human antibody S25755 (NBRF-PDB)
Six PCR primers were used to generate a humanized # 23-57-137-1 antibody light chain with version 4 (version "a").

CDR-grafting primer MBC
1LGP1 (SEQ ID NO: 29) and MBC1LGP3 (SEQ ID NO: 30) have a sense DNA sequence and the CDR grafting primer MBC1LGP2 (SEQ ID NO: 3)
1) and MBC1LGP4 (SEQ ID NO: 32) have an antisense DNA sequence and each has a complementary sequence of 15 to 21 bp at each end of the primer. External primers MBC1LVS1 (SEQ ID NO: 33) and MBC1L
VR1 (SEQ ID NO: 34) has homology to CDR grafting primers MBC1LGP1 and MBC1LGP4.

CDR-grafting primer MBC
1LGP1, MBC1LGP2, MBC1LGP3 and MBC1LGP4 were separated using a urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Ma
nual, Sambrook et al., Cold Spring Harbor Laboratory Pr
ess, 1989), extraction from gel
ak method (Molecular Cloning: A Laboratory Manual, Sa
mbrook et al., Cold Spring Harbor Laboratory Press, 198
9). That is, each 1 nmole CD
The R-grafting primer was separated on a 6% denaturing polyacrylamide gel, and the DNA fragment of the desired size was identified by irradiating ultraviolet rays on a silica gel thin plate.
20 μl of 10 mM Tris-HCl (pH 7.4), 1 mM, collected from the gel by the hand and soak method.
Dissolved in DTA solution.

The PCR was carried out using TaKaRa Ex Taq (Takara Shuzo), and the CDR-grafting primer MBC1LG prepared as described above was added to 100 μl of the reaction mixture.
P1, MBC1LGP2, MBC1LGP3 and MB
1 μl each of C1LGP4 and 0.25 mM dNT
P, 5 times with a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute using the attached buffer under conditions including 2.5 U of TaKaRa Ex Taq,
50 pmole of external primer MB was added to this reaction mixture.
C1LVS1 and MBC1LVR1 were added, and further reacted 30 times in the same temperature cycle. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio. Products).

A piece of agarose containing a 421 bp DNA fragment was cut out, and the GENECLEANII Kit (B
The DNA fragment was purified using IO101) according to the instructions attached to the kit. PUC prepared by digesting the resulting PCR reaction mixture with BamHI and HindIII
19 and the nucleotide sequence was determined. The thus obtained plasmid was designated as hMBCL / pUC19. However, position 104 of CDR4 (Kabat
Since the amino acid at amino acid No. 96 according to the above-mentioned rules was arginine, a modified primer MBC1LGP10R (SEQ ID NO.
5) was designed and synthesized. PCR is TaKaRa Taq
(Takara Shuzo) and add template DNA to 100 μl of the reaction mixture.
0.6 μg of plasmid hMBCL / pUC1
9. MBC1LVS1 and MBC1L as primers
GP10R was 50 pmole and 2.5 U of Ta, respectively.
KaRa Ex Taq (Takara Shuzo) 0.25 mM dN
50 μl of mineral oil was layered on the upper layer under the conditions containing TP using the attached buffer solution at 94 ° C. for 1 minute, 55 ° C. for 1 minute, 7
The test was performed 30 times at a temperature of 2 ° C. for 1 minute. 3% Nu Sieve GT
Separation was performed by agarose gel electrophoresis using G agarose (FMC Bio. Products). 421
A piece of agarose containing a DNA fragment of bp length was cut out and G
The DNA fragment was purified using ENECLEANII Kit (BIO101) according to the instructions attached to the kit. The resulting PCR reaction mixture was digested with BamHI and HindIII.
And subcloned into pUC19 prepared by digestion.

The nucleotide sequence was determined using the M13 Primer M4 primer and the M13 Primer RV primer. As a result, the correct sequence was obtained. This plasmid was digested with HindIII and BlnI, and the 416 bp fragment was digested with 1% agarose gel. Separated by electrophoresis. GENECLEANII Kit
DNA using (BIO101) according to the instructions attached to the kit
The fragment was purified. The obtained PCR reaction mixture was used for HindI.
II and BlnI, and introduced into plasmid Cλ / pUC19.
It was named 1Laλ / pUC19. This plasmid is
After digestion with coRI, a sequence containing a sequence encoding the humanized L chain was introduced into plasmid pCOS1, so that the initiation codon of the humanized L chain was located downstream of the EF1α promoter. The thus obtained plasmid was used for hMBC1Laλ.
/ PCOS1. SEQ ID NO: 66 shows the nucleotide sequence of the humanized L chain version "a" (including the corresponding amino acid). The amino acid sequence of version a is shown in SEQ ID NO: 47.

[0162] Version "b" was prepared using mutagenesis by the PCR method. 43rd in version "b" (Kaba
It was designed so that glycine at amino acid number 43 according to t is changed to proline and lysine at position 49 (amino acid number 49 according to Kabat) to aspartic acid. PCR was performed using the mutagenic primer MBC1LGP5R (SEQ ID NO: 36) and the primer MBC1LVS1 with the plasmid hMBC1Laλ / pUC19 as a template, and the obtained DNA fragment was analyzed using BamHI and HindII
I, digested with BamHI, HindIII of pUC19
The site was subcloned. After determining the nucleotide sequence, digestion with restriction enzymes HindIII and AflII,
HMBC1Laλ / digested with III and AflII
Ligation with pUC19. The plasmid thus obtained was designated as hMBC1Lbλ / pUC19, this plasmid was digested with EcoRI, a fragment containing DNA encoding the humanized L chain was introduced into plasmid pCOS1, and the start of the humanized L chain was placed downstream of the EF1α promoter. Codons are located. The thus obtained plasmid was designated as hMBC1L.
It was named bλ / pCOS1.

The version "c" was prepared by introducing a mutation by the PCR method. 84th in version "c" (Kaba
Serine at amino acid number 80 (defined by t) was designed to be changed to proline. Mutagenic primer MBC
1LGP6S (SEQ ID NO: 37) and primer M13P
plasmid hMBC1Laλ /
PCR was performed using pUC19 as a template, and the obtained DNA fragment was digested with BamHI and HindIII, and BamHI was digested.
And p prepared by digestion with HindIII
It was subcloned into UC19.

After the nucleotide sequence was determined, digestion was performed with the restriction enzymes BstPI and Aor51HI, and BstPI and Aor5HI were digested.
Ligation with hMBC1Laλ / pUC19 digested with 1HI. The thus obtained plasmid was designated as hMBC1Lcλ.
/ PUC19, and this plasmid is replaced with the restriction enzyme EcoR.
The sequence containing the sequence encoding the humanized L chain after digestion with I was introduced into the EcoRI site of plasmid pCOS1, and EF1α
The start codon of the humanized light chain was located downstream of the promoter. The plasmid thus obtained was transformed into hMB
It was named C1Lcλ / pCOS1.

Versions "d", "e" and "f" were prepared by using mutagenesis by the PCR method. For each version, the "a", "b", and "c" versions are ranked 91st (Kaba
The tyrosine at amino acid number 87 according to the definition of t) was designed to be changed to isoleucine. Mutagenic primer MBC1LGP11R (SEQ ID NO: 38) and primer M
HMBC1La by -S1 (SEQ ID NO: 44)
λ / pCOS1, hMBC1Lb λ / pCOS1, hMBC1Lc
PCR was performed using λ / pCOS1 as a template, and the obtained DNA fragment was digested with BamHI and HindIII, and BamHI was digested.
And p prepared by digestion with HindIII
It was subcloned into UC19. After determining the nucleotide sequence,
Digest with indIII and BlnI, HindIII
And Cλ / pU prepared by digestion with BlnI
Connected to C19.

The plasmids thus obtained were sequentially transformed into hMB
C1Ldλ / pUC19, hMBC1Leλ / pUC1
9, hMBC1Lfλ / pUC19. These plasmids were digested with EcoRI, and a sequence containing a sequence encoding the humanized L chain was introduced into the EcoRI site of plasmid pCOS1, so that the start codon of the humanized L chain was located downstream of the EF1α promoter. The plasmids thus obtained were respectively sequenced into hMBC1Ldλ / pCOS1,
Named hMBC1Leλ / pCOS1, hMBC1Lfλ / pCOS1.

Versions "g" and "h" were prepared using mutagenesis by the PCR method. "A" for each version in order
The "d" version was designed to change histidine at position 36 (amino acid number 36 as defined by Kabat) to tyrosine. Mutagenic primer MBC1LGP9
R (SEQ ID NO: 39) and M13 Primer RV
HMBC1Laλ / pUC using as primer
19 was used as a template to perform PCR.
Further PCR was performed using Primer M4 as a primer and plasmid hMBC1Laλ / pUC19 as a template. The obtained DNA fragment was digested with HindIII and BlnI, and subcloned into plasmid Cλ / pUC19 prepared by digestion with HindIII and BlnI. Using this plasmid as a template,
Primers MBC1LGP13R (SEQ ID NO: 40) and M
PCR was performed using BC1LVS1 as a primer. The resulting PCR fragment was digested with ApaI and HindII and the plasmids hMBC1Laλ / pUC19 and hMBC1Ld digested with ApaI and HindIII.
λ / pUC19. The nucleotide sequence was determined, and plasmids containing the correct sequence were sequentially identified as hMBC1Lgλ / pUC.
19 and hMBC1Lhλ / pUC19, and these plasmids were digested with EcoRI to obtain humanized L.
The sequence containing the sequence coding for the chain is replaced by the plasmid pCOS1 E
A coRI site was introduced so that the start codon of the humanized light chain was located downstream of the EF1α promoter. The plasmids obtained in this manner were each sequentially sequenced with hMBC1Lg.
λ / pCOS1 and hMBC1Lhλ / pCOS1.

Versions "i", "j", "k", "l"
, "M", "n" and "o" were made using mutagenesis by PCR. Mutagenic primer MBC1LGP14
S (SEQ ID NO: 41) and the primer VIRV (λ) (SEQ ID NO: 43) to give the plasmid hMBC1Laλ / pUC
19 was used as a template, and the obtained DNA fragment was
Plasmid hMBC1Lg prepared by digestion with ApaI and BlnI and digestion with ApaI and BlnI
It was subcloned into λ / pUC19. The nucleotide sequence was determined, and clones in which the mutation corresponding to each version was introduced were selected. The plasmid thus obtained was transformed into hMBC1Lxλ / pUC19 (x = i, j, k,
l, m, n, o), this plasmid was digested with EcoRI, a sequence containing the sequence encoding the humanized L chain was introduced into the EcoRI site of plasmid pCOS1, and the humanized L chain was downstream of the EF1α promoter. The start codon was located. The thus obtained plasmid was designated as hMBC1.
Lxλ / pCOS1 (x = i, j, k, l, m, n, o). Versions "j", "l", "m" and "o"
Are shown in SEQ ID NOs: 67, 68, 69, and 70, respectively (including the corresponding amino acids). The amino acid sequences of these versions are shown in SEQ ID NOs: 48, 49, 50 and 51, respectively.

Versions "p", "q", "r", "s" and "t" are versions "i", "j", "m" and "l".
Or, a version in which the tyrosine at position 87 in the amino acid sequence of "o" is replaced with isoleucine. Using the restriction enzyme Aor51MI cleavage site in FR3, version "h" is replaced with version "i", "j". , "M", "l" or "o". That is, the expression plasmid hMBC1Lxλ / pCOS
1 (x = i, j, m, l, o), CDR3 and FR
Aor51HI fragment 514b containing a part of FR3 and FR4
except for the expression plasmid hMBC1Lhλ / pC
In OS1, a 514 bp Aor51HI fragment containing CDR3 and part of FR3 and FR4 was linked to 91
The tyrosine at position (amino acid position 87 according to Kabat's definition) was changed to isoleucine. A nucleotide sequence was determined, and a clone in which the tyrosine at position 91 (amino acid number 87 according to Kabat's definition) of each version "i", "j", "m", "l" and "o" was replaced with isoleucine was obtained. Select the corresponding version "p", "" respectively
q "," s "," r "and" t ", and the obtained plasmid was designated as hMBC1Lxλ / pCOS1 (x = p, q, s,
r, t). The nucleotide sequences of the versions "q", "r", "s" and "t" (including the corresponding amino acids) are shown in SEQ ID NOs: 71, 72, 73 and 74, respectively. In addition, the amino acid sequences of each of these versions are shown in SEQ ID NOs: 52 and 5, respectively.
3, 54 and 55 are shown.

The plasmid hMBC1Lqλ / pCOS1 was replaced with H
Digest with indIII and EcoRI, HindII
I and EcoRI were subcloned into plasmid pUC19 and the plasmid hMBC1Lqλ / p
It was named UC19. Table 2 shows the position of the substituted amino acid in each version of the humanized L chain.

[0171]

[Table 2]

In the table, Y indicates tyrosine, P indicates proline, K indicates lysine, V indicates valine, D indicates aspartic acid, and I indicates isoleucine. The plasmid hMBC1HcDN
Escherichia coli having A / pUC19 and hMBC1Lqλ / pUC19 was obtained from Escherichia coli J
M109 (hMBC1HcDNA / pUC19) and
Escherichia coli JM109 (hMB
(C1Lqλ / pUC19), Institute of Biotechnology, Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki)
On August 15, 1996, Escherichia
coli JM109 (hMBC1HcDNA / pUC1
For 9), refer to FERM BP-5629, Esche
richia coli JM109 (hMBC1Lq
λ / pUC19) for FERM BP-5630
International deposit under the Budapest Treaty.

(5) Transfection into COS-7 cells In order to evaluate the antigen binding activity and neutralizing activity of the hybrid antibody and the humanized # 23-57-137-1 antibody, the expression plasmid was transformed into COS-7 cells. For transient expression. That is, in the transient expression of the L-chain hybrid antibody, the plasmids hMBC1HcDNA / pCOS1 and h / mMBC1L (λ) / ne
o, hMBC1HcDNA / pCOS1 and m / hMBC1Laλ / ne
o, hMBC1HcDNA / pCOS1 and m / hMBC1Ldλ / ne
o, hMBC1HcDNA / pCOS1 and hmmMBC1L (λ) / n
eo or hMBC1HcDNA / pCOS1 and mhmmBC1L
(Λ) / neo is combined with GenePulse
COS-7 cells were co-transduced by electroporation using an r apparatus (BioRad). PBS
Each plasmid DNA was added to 0.8 ml of COS-7 cells suspended at a cell concentration of 1 × 10 ⁷ cells / ml in (−).
0 μg was added, and a pulse was applied with a capacitance of 1,500 V and 25 μF. After a recovery period of 10 minutes at room temperature, the electroporated cells were treated with 2% Ultra Lo.
w DMEM containing IgG fetal bovine serum (GIBCO)
The cells were suspended in a culture solution (GIBCO) and cultured in a CO ₂ incubator using a 10 cm culture dish. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and used for an ELISA sample.

For the transient expression of the humanized # 23-57-137-1 antibody, plasmids hMBC1HcDNA / pCOS1 and hMBC1Lxλ
/ PCOS1 (x = a to t)
Using a nePulser device (BioRad), COS-7 was obtained in the same manner as in the case of the hybrid antibody.
Cells were transfected and the resulting culture supernatant was
It was subjected to LISA. Purification of a hybrid antibody or a humanized antibody from the culture supernatant of COS-7 cells is performed by A
The test was performed using the fifiGel Protein A MAPSII kit (BioRad) according to the instructions attached to the kit.

(6) ELISA (i) Measurement of Antibody Concentration An ELISA plate for antibody concentration measurement was prepared as follows. 96-well plate for ELISA (Maxi
sorp, NUNC) into the solid phase buffer (0.
1 μg with 1M NaHCO ₃ , 0.02% NaN ₃ )
Goat anti-human IgG antibody (TAG
O) Immobilize with 100 μl, and add 200 μl of dilution buffer (50 mM Tris-HCl, 1 mM MgC
l ₂ , 0.1 M NaCl, 0.05% Tween2
After blocking with 0, 0.02% NaN ₃ , 1% bovine serum albumin (BSA), pH 7.2), COS-7 expressing hybrid antibody or humanized antibody
Cell culture supernatant or purified hybrid antibody or humanized antibody was serially diluted and added to each well. After incubating for 1 hour at room temperature and washing with PBS-Tween 20,
100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added. After incubating for 1 hour at room temperature and washing with PBS-Tween 20, 1 m
g / ml substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, then absorbance at 405 nm was measured using a microplate reader (BioRad).
Was measured. Hu I as a standard for concentration measurement
gG1λ Purified (TheBinding
Site).

(Ii) Measurement of antigen binding ability An ELISA plate for antigen binding measurement was prepared as follows. Each well of the 96-well ELISA plate was immobilized with 100 µl of human PTHrP (1-34) prepared at a concentration of 1 µg / ml with a solid phase buffer. 20
After blocking with 0 μl of the dilution buffer, the culture supernatant of COS-7 cells expressing the hybrid antibody or humanized antibody or the purified hybrid antibody or humanized antibody was serially diluted and added to each well. After incubation at room temperature and washing with PBS-Tween 20, alkaline phosphatase-conjugated goat anti-human IgG antibody (TAG
O) 100 μl was added. Incubate at room temperature
After washing with BS-Tween 20, 1 mg / ml substrate solution (Sigma 104, p-nitrophenyl phosphate,
SIGMA) and then the absorbance at 405 nm was measured on a microplate reader (BioRad).

(7) Activity confirmation (i) Evaluation of humanized H chain An antibody obtained by combining the humanized H chain version "a" and the chimeric L chain had the same PTHrP binding ability as the chimeric antibody (FIG. 6). This result indicates that version "a" is sufficient for humanization of the H chain V region. Hereinafter, the humanized H chain version "a" was used as the H chain of the humanized antibody.

(Ii) Activity of hybrid antibody (ii-a) When the FR1, 2 / FR3, and 4 hybrid antibody L chains were h / mMBC1L (λ), no activity was observed, but m / hMBC1Laλ or m / hMBC1Laλ. / H
In the case of MBC1Ldλ, all exhibited the same binding activity as the chimeric # 23-57-137-1 antibody (FIG. 7). These results indicate that although FR3 and FR4 have no problem as a humanized antibody,
It suggests that there is an amino acid residue to be substituted in 1,2. (ii-b) FR1 / FR2 hybrid antibody No activity was observed when the L chain was mhmMBC1L (λ), but when hmmMBC1L (λ), the binding was equivalent to that of the chimeric # 23-57-137-1 antibody. It showed activity (FIG. 8). These results suggest that although FR1 among FR1 and FR2 is not a problem as a humanized antibody, there is an amino acid residue to be substituted in FR2.

(Iii) Activity of Humanized Antibody The antigen-binding activity of a humanized antibody using one of each of the versions "a" to "t" as an L chain was measured. As a result, the light chain versions "j", "l", "m", "o"
, "Q", "r", "s", and "t" exhibited PTHrP binding ability equivalent to that of the chimeric antibody (FIG. 9).
~ 12).

(8) Establishment of a Stable CHO Producing Cell Line In order to establish a stable humanized antibody producing cell line, the above expression plasmid was introduced into CHO cells (DXB11).
That is, the establishment of a cell line for stably producing a humanized antibody is performed by CHO
Expression plasmid for cells hMBC1HcDNA / pCHO1 and hM
BC1Lmλ / pCOS1 or hMBC1H cDNA / pCHO1
And hMBC1Lqλ / pCOS1 or hMBC1H cDNA
/ PCHO1 and hMBC1Lrλ / pCOS1 combine G
CHO cells were co-transduced by electroporation using an enePulser device (BioRad). Each expression vector was cleaved with a restriction enzyme PvuI to obtain linear DNA, extracted with phenol and chloroform, recovered by ethanol precipitation, and used for electroporation. 1 × 10 ⁷ cells / ml in PBS (-)
0.8 ml of CHO cells suspended at a cell concentration of
10 μg of each plasmid DNA was added, and 1,500 V, 25
A pulse was applied with a capacitance of μF. After a 10 minute recovery period at room temperature, the electroporated cells were added to 10% fetal calf serum (GIBCO), MEM-
Suspended in α medium (GIBCO), 96-well plate (Fa
1con) in a CO ₂ incubator. The day after the start of culture, 10% fetal bovine serum (GIBC
O) and 500 mg / ml GENETICIN (G
418 Sulfate, GIBCO), MEM-α without ribonucleosides and deoxyribonucleosides
The medium was replaced with a selection medium (GIBCO), and cells into which the antibody gene had been introduced were selected. The cells were observed under a microscope about two weeks after the selection medium was exchanged, and after successful cell growth was observed, the amount of antibody production was measured by the antibody concentration measurement ELISA, and cells having high antibody production ability were selected. .

The culture of the established antibody-producing stable cell line was expanded, and 2% Ultra Low IgG fetal bovine serum was added using a roller bottle, and mass culture was performed using MEM-α medium free of ribonucleoside and deoxyribonucleoside. went. On the third or fourth day of the culture, the culture supernatant was collected, and cell debris was removed with a 0.2 μm filter (Millipore).
Purification of the humanized antibody from the culture supernatant of CHO cells
ROS protein A column (PerSeptive Bio
systems) using ConSepLC100
(Millipore) according to the attached prescription and used for measurement of neutralizing activity and drug efficacy test in a hypercalcemia model animal. The concentration and antigen-binding activity of the obtained purified humanized antibody were measured by the above-mentioned ELISA system.

Reference Example 5 Measurement of Neutralizing Activity The neutralizing activity of a mouse antibody, a chimeric antibody and a humanized antibody was measured using a rat osteosarcoma cell line ROS17 / 2.8-5.
This was performed using cells. That is, ROS17 / 2.8-
5 cells were treated with Ha containing 10% fetal calf serum (GIBCO).
The cells were cultured in m'SF-12 medium (GIBCO) in a CO ₂ incubator. ROS17 / 2.8-5 cells were seeded on a 96-well plate at 10 ⁴ cells / 100 μl / well, cultured for 1 day, and replaced with Ham'SF-12 medium (GIBCO) containing 4 mM hydrocortisone and 10% fetal bovine serum. I do. After further culturing for 3-4 days,
Washed with Ham'SF-12 medium (GIBCO)
mM isobutyl-1-methylxanthine (IBMX,
SIGMA) and 10% fetal calf serum and 10 mM H
Add 80 μl of Ham's F-12 containing EPES,
Incubated for 30 minutes at 37 ° C.

A mouse antibody, a chimeric antibody or a humanized antibody whose neutralizing activity is to be measured was previously prepared at 10 μg / ml.
3.3 μg / ml, 1.1 μg / ml and 0.37 μ
g / ml group, 10 μg / ml, 2 μg / ml, 0.5
μg / ml and 0.01 μg / ml groups, or 10
μg / ml, 5 μg / ml, 1.25 μg / ml, 0.
PTHrP (1-34) serially diluted to a group of 63 μg / ml and 0.31 μg / ml and adjusted to 4 ng / ml
And a mixture of each antibody and PTHrP (1-34) (80 μl) was added to each well. The final concentration of each antibody is one-fourth of the above antibody concentration and PTHrP (1-34)
Will be 1 ng / ml. After treatment at room temperature for 10 minutes, the culture supernatant is discarded, and the cells are washed three times with PBS. Then, intracellular cAMP is extracted with 100 μl of 0.3% hydrochloric acid and 95% ethanol. Evaporate the ethanol from hydrochloric acid with a water aspirator and use the cAMP EIA kit
(CAYMANCHEMICAL'S) After adding 120 μl of the attached EIA buffer to extract cAMP, cAM
P EIA kit (CAYMANCHEMICAL '
S) cAMP was measured according to the attached prescription. As a result, a humanized antibody having versions “q”, “r”, “s”, and “t” in which the tyrosine at position 91 was replaced with isoleucine in the L chain version having the same antigen binding as the chimeric antibody was obtained. The neutralization ability was close to that of the chimeric antibody, and among them, version "q" showed the strongest neutralization ability (Figs. 13 to 1).
5).

[0184]

Industrial Applicability According to the present invention, there is provided a therapeutic agent for cachexia, which comprises, as an active ingredient, a substance that inhibits the binding between a parathyroid hormone-related peptide and its receptor. The above substance is useful as a therapeutic agent for cachexia, since it suppresses weight loss compared to a control in a drug efficacy test in a cachexia model animal and also has an effect of extending the survival period.

[0185]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: AAATAGCCCT TGACCAGGCA 20

SEQ ID NO: 2 Sequence length: 38 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Sequence: CTGGTTCGGC CCACCTCTGA AGGTTCCAGA ATCGATAG 38

SEQ ID NO: 3 Sequence length: 28 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGATCCCGGG CCAGTGGATA GACAGATG 28

SEQ ID NO: 4 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGATCCCGGG TCAGRGGAAG GTGGRAACA 29

SEQ ID NO: 5 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTTTTCCCAG TCACGAC 17

SEQ ID NO: 6 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CAGGAAACAG CTATGAC 17

SEQ ID NO: 7 Sequence length: 31 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTAAGCTT CCACCATGAA ACTTCGGGCT C 31

SEQ ID NO: 8 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGGATCC CTGCAGAGAC AGTGACCAGA 30

SEQ ID NO: 9 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTGAATTC AAGCTTCCAC CATGGGGTTT GGGCTG 36

SEQ ID NO: 10 Sequence length: 41 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TTTCCCGGGC CCTTGGTGGA GGCTGAGGAG ACGGTGACCA G 41

SEQ ID NO: 11 Sequence length: 109 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTGAATTC AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA CCCCACGGTC ACCCTGTTCC 60 CGCCCTCCTC TGAGGAGCTC CAAGCCAACA AGGCCACACT AGTGTGTCT 109

SEQ ID NO: 12 Sequence length: 110 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGTTTGGTGG TCTCCACTCC CGCCTTGACG GGGCTGCCAT CTGCCTTCCA GGCCACTGTC 60 ACAGCTCCCG GGTAGAAGTC ACTGATCAGA CACACTAGTG TGGCCTTGTT 110

SEQ ID NO: 13 Sequence length: 98 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGAGTGGAGA CCACCAAACC CTCCAAACAG AGCAACAACA AGTACGCGGC CAGCAGCTAC 60 CTGAGCCTGA CGCCCGAGCA GTGGAAGTCC CACAGAAG 98

SEQ ID NO: 14 Sequence length: 106 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGAATTC TTACTATGAA CATTCTGTAG GGGCCACTGT CTTCTCCACG GTGCTCCCTT 60 CATGCGTGAC CTGGCAGCTG TAGCTTCTGT GGGACTTCCA CTGCTC 106

SEQ ID NO: 15 Sequence length: 43 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTGAATTC AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA CCC 43

SEQ ID NO: 16 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGAATTC TTACTATGAA 20

SEQ ID NO: 17 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CAACAAGTAC GCGGCCAGCA GCTACCTGAG CCTGACGCC 39

SEQ ID NO: 18 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTAGCTGCTG GCCGCGTACT TGTTGTTGCT CTGTTTGGA 39

SEQ ID NO: 19 Sequence length: 46 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTGAATTC AAGCTTAGTC CTAGGTCGAA CTGTGGCTGC ACCATC 46

SEQ ID NO: 20 Sequence length: 34 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGAATTC TTACTAACAC TCTCCCCTGT TGAA 34

SEQ ID NO: 21 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTAAGCTT CCACCATGGC CTGGACTCCT CTCTT 35

SEQ ID NO: 22 Sequence length: 48 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGAATTC AGATCTAACT ACTTACCTAG GACAGTGACC TTGGTCCC 48

SEQ ID NO: 23 Sequence length: 128 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTAAGCTT CCACCATGGG GTTTGGGCTG AGCTGGGTTT TCCTCGTTGC TCTTTTAAGA 60 GGTGTCCAGT GTCAGGTGCA GCTGGTGGAG TCTGGGGGAG GCGTGGTCCA GCCTGGGAGG 120 TCCCTGAG 128

SEQ ID NO: 24 Sequence length: 125 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACCATTAGTA GTGGTGGTAG TTACACCTAC TATCCAGACA GTGTGAAGGG GCGATTCACC 60 ATCTCCAGAG ACAATTCCAA GAACACGCTG TATCTGCAAA TGAACAGCCT GAGAGCTGAG 120 GACAC 125

SEQ ID NO: 25 Sequence length: 132 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTACCACCAC TACTAATGGT TGCCACCCAC TCCAGCCCCT TGCCTGGAGC CTGGCGGACC60 CAAGACATGC CATAGCTACT GAAGGTGAAT CCAGAGGCTG CACAGGAGAG TCTCAGGGAC 120 CTCCCAGGCT GG 132

SEQ ID NO: 26 Sequence length: 110 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGGATCC CTGAGGAGAC GGTGACCAGG GTTCCCTGGC CCCAGTAAGC AAAGTAAGTC60 ATAGTAGTCT GTCTCGCACA GTAATACACA GCCGTGTCCT CAGCTCTCAG 110

SEQ ID NO: 27 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCTAAGCTT CCACCATGGG GTTTGGGCTG 30

SEQ ID NO: 28 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TGTTGGATCC CTGAGGAGAC GGTGACCAGG 30

SEQ ID NO: 29 Sequence length: 133 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACAAAGCTTC CACCATGGCC TGGACTCCTC TCTTCTTCTT CTTTGTTCTT CATTGCTCAG 60 GTTCTTTCTC CCAGCTTGTG CTGACTCAAT CGCCCTCTGC CTCTGCCTCC CTGGGAGCCT 120 CGGTCAAGCT CAC 133

SEQ ID NO: 30 Sequence length: 118 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Sequence: AGCAAGATGG AAGCCACAGC ACAGGTGATG GGATTCCTGA TCGCTTCTCA GGCTCCAGCT 60 CTGGGGCTGA GCGCTACCTC ACCATCTCCA GCCTCCAGTC TGAGGATGAG GCTGACTA 118

SEQ ID NO: 31 Sequence length: 128 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTGTGGCTTC CATCTTGCTT AAGTTTCATC AAGTACCGAG GGCCCTTCTC TGGCTGCTGC 60 TGATGCCATT CAATGGTGTA CGTACTGTGC TGACTACTCA AGGTGCAGGT GAGCTTGACC 120 GAGGCTCC 128

SEQ ID NO: 32 Sequence length: 114 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CCCTCACAAA 60 TTGTTCCTTA ATTGTATCAC CCACACCACA GTAATAGTCA GCCTCATCCT CAGA 114

SEQ ID NO: 33 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACAAAGCTTC CACCATG 17

SEQ ID NO: 34 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTGGATCCG GGCTGACCT 19

SEQ ID NO: 35 Sequence length: 75 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CGTACACAAA 60 TTGTTCCTTA ATTGT 75

SEQ ID NO: 36 Sequence length: 43 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: AAAGGATCCT TAAGATCCAT CAAGTACCGA GGGGGCTTCT CTG 43

SEQ ID NO: 37 Sequence length: 46 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACAAAGCTTA GCGCTACCTC ACCATCTCCA GCCTCCAGCC TGAGGA 46

SEQ ID NO: 38 Sequence length: 111 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CGTACACAAA 60 TTGTTCCTTA ATTGTATCAC CCACACCACA GATATAGTCA GCCTCATCCT C 111

SEQ ID NO: 39 Sequence length: 42 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTCTCTGGC TGCTGCTGAT ACCATTCAAT GGTGTACGTA CT 42

SEQ ID NO: 40 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CGAGGGCCCT TCTCTGGCTG CTGCTG 26

SEQ ID NO: 41 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GAGAAGGGCC CTARGTACST GATGRAWCTT AAGCA 35

SEQ ID NO: 42 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CACGAATTCA CTATCGATTC TGGAACCTTC AGAGG 35

SEQ ID NO: 43 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGCTTGGAGC TCCTCAGA 18

SEQ ID NO: 44 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GACAGTGGTT CAAAGTTTTT 20

SEQ ID NO: 45 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser Phe Ser Leu Gly 1 5 10 15 Ala Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro Lys 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg 65 70 75 Tyr Leu Ser Ile Ser Asn Ile Gln Pro Glu Asp Glu Ala Met Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 46 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly 1 5 10 15 Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met Ser Trp Ile Arg Gln Thr Pro Asp Lys Arg Leu 35 40 45 Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr 50 55 60 Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp 80 85 90 Thr Ala Met Phe Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe 95 100 105 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 110 115

SEQ ID NO: 47 Sequence length: 116 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 110 115

SEQ ID NO: 48 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 49 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 50 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 51 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 52 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 53 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 54 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 55 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Leu Val Leu Thr Gln Ser Pro Ser
Ala Ser Ala Ser Leu Gly 1 5 10 15 Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30 Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg 35 40 45 Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg 65 70 75 Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90 Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 110 115

SEQ ID NO: 56 Sequence length: 118 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly 1 5 10 15 Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr 50 55 60 Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe 95 100 105 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115

SEQ ID NO: 57 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG AAC TTC GGG CTC AGC TTG ATT TTC CTT GCC CTC ATT TTA AAA 45 Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Ala Leu Ile Leu Lys -15 -10 -5 GGT GTC CAG TGT GAG GTG CAA CTG GTG GAG TCT GGG GGA GAC TTA 90 Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu 1 5 10 GTG AAG CCT GGA GGG TCC CTG AAA CTC TCC TGT GCA GCC TCT GGA 135 Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly 15 20 25 TTC ACT TTC AGT AGC TAT GGC ATG TCT TGG ATT CGC CAG ACT CCA 180 Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Ile Arg Gln Thr Pro 30 35 40 GAC AAG AGG CTG GAG TGG GTC GCA ACC ATT AGT AGT GGT GGT AGT 225 Asp Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser 45 50 55 TAC ACC TAC TAT CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC 270 Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 60 65 70 AGA GAC A AT GCC AAG AAC ACC CTA TAC CTG CAA ATG AGC AGT CTG 315 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu 75 80 85 AAG TCT GAG GAC ACA GCC ATG TTT TAC TGT GCA AGA CAG ACT ACT 360 Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys Ala Arg Gln Thr Thr 90 95 100 ATG ACT TAC TTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC 405 Met Thr Tyr Pyr Ahe Tla Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 105 110 115 TCT GCA 411 Ser Ala

SEQ ID NO: 58 Sequence length: 411 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GGG TTT GGG CTG AGC TGG GTT TTC CTC GTT GCT CTT TTA AGA 45 Met Gly Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg -15 -10 -5 GGT GTC CAG TGT CAG GTG CAG CTG GTG GAG TCT GGG GGA GGC GTG 90 Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val 1 5 10 GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA 135 Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 15 20 25 TTC ACC TTC AGT AGC TAT GGC ATG TCT TGG GTC CGC CAG GCT CCA 180 Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro 30 35 40 GGC AAG GGG CTG GAG TGG GTG GCA ACC ATT AGT AGT GGT GGT AGT 225 Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser 45 50 55 TAC ACC TAC TAT CCA GAC AGT GTG AAG GGG CGA TTC ACC ATC TCC 270 Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 60 65 70 AGA GAC A AT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG 315 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 75 80 85 AGA GCT GAG GAC ACG GCT GTG TAT TAC TGT GCG AGA CAG ACT ACT 360 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Thr Thr 90 95 100 ATG ACT TAC TTT GCT TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC 405 Met Thr Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 105 110 115 TCC TCA 411 Ser Ser

SEQ ID NO: 59 Sequence length: 11 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 60 Sequence length: 7 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 61 Sequence length: 9 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 62 Sequence length: 5 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 63 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Ser Ile Phe Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys Phe Lys Gly 1 5 10 15

SEQ ID NO: 64 Sequence length: 11 Sequence type: amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 65 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAA CTT GTG CTC ACT CAG TCA TCT TCA GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser 1 5 10 TTC TCC CTG GGA GCC TCA GCA AAA CTC ACG TGC ACC TTG AGT AGT 135 Phe Ser Leu Gly Ala Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAA CAG CCA CTC 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu 30 35 40 AAG CCT CCT AAG TAT GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Pro Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCT GGA TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGT GCT G AT CGC TAC CTT AGC ATT TCC AAC ATC CAG CCA GAA GAT 315 Gly Ala Asp Arg Tyr Leu Ser Ile Ser Asn Ile Gln Pro Glu Asp 75 80 85 GAA GCA ATG TAC ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Met Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAT GTT TTC GGC GGT GGG ACC AAG GTC ACT GTC CTA GGT 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 66 Sequence length: 405 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG CAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp His Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT CGG TAC TTG ATG AAA CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Leu Met Lys Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGT 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115

SEQ ID NO: 67 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AAG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 68 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AAG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 69 Sequence length: 411 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 70 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT TAC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 71 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AAG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 72 Sequence length: 411 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC CTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 73 Sequence length: 411 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AAG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Lys Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 74 Sequence length: 411 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence: ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT GTT CTT CAT TGC TCA 45 Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT 90 Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACC TTG AGT AGT 135 Ala Ser Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser 15 20 25 CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 180 Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu 30 35 40 AAG GGC CCT AGG TAC GTG ATG GAT CTT AAG CAA GAT GGA AGC CAC 225 Lys Gly Pro Arg Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CGC TTC TCA GGC TCC AGC TCT 270 Ser Thr Gly Asp Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser 60 65 70 GGG GCT G AG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT 315 Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 75 80 85 GAG GCT GAC TAT ATC TGT GGT GTG GGT GAT ACA ATT AAG GAA CAA 360 Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln 90 95 100 TTT GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTC CTA GGC 405 Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115 CAG CCC 411 Gln Pro

SEQ ID NO: 75 Sequence length: 34 Sequence type: amino acid Topology: Linear Sequence type: Peptide Sequence: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile 1 5 10 15 Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu 20 25 30 Ile His Thr Ala

[Brief description of the drawings]

FIG. 1 is a view showing the therapeutic effect of anti-PTHrP antibody on cachexia.

FIG. 2 shows the therapeutic effect of anti-PTHrP antibody on cachexia.

FIG. 3 shows the therapeutic effect of anti-PTHrP antibody on cachexia.

FIG. 4 is a graph showing the therapeutic effect of anti-PTHrP antibody on cachexia.

FIG. 5 is a view showing the measurement results of antibody binding activity.

FIG. 6 is a view showing the measurement results of antibody binding activity.

FIG. 7 shows the results of measuring antibody binding activity.

FIG. 8 is a view showing the measurement results of antibody binding activity.

FIG. 9 shows the results of measuring antibody binding activity.

FIG. 10 shows the results of measuring antibody binding activity.

FIG. 11 is a view showing the measurement results of antibody binding activity.

FIG. 12 shows the results of measuring antibody binding activity.

FIG. 13 shows the neutralizing activity of a humanized antibody.

FIG. 14 shows the neutralizing activity of a humanized antibody.

FIG. 15 shows the neutralizing activity of a humanized antibody.

FIG. 16 shows the therapeutic effect of humanized antibodies on cachexia.

FIG. 17 shows the therapeutic effect of humanized antibodies on cachexia.

FIG. 18 shows the therapeutic effect of humanized antibodies on cachexia.

FIG. 19 shows the therapeutic effect of humanized antibodies on cachexia.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12P 21/08 C12R 1:91)

Claims (8)

[Claims] 1. A cachexia therapeutic agent comprising as an active ingredient a substance that inhibits the binding of a parathyroid hormone-related peptide to its receptor. 2. The therapeutic agent for cachexia according to claim 1, wherein the substance is an antagonist to a parathyroid hormone-related peptide receptor. 3. The agent for treating cachexia according to claim 1, wherein the substance is an anti-parathyroid hormone-related peptide antibody. 4. The therapeutic agent for cachexia according to claim 1, wherein the substance is an anti-parathyroid hormone-related peptide antibody fragment and / or a modified product thereof. 5. The therapeutic agent for cachexia according to claim 3, wherein the antibody is humanized or chimerized. 6. The therapeutic agent for cachexia according to claim 5, wherein the humanized antibody is a humanized # 23-57-137-1 antibody. 7. The therapeutic agent for cachexia according to claim 3, wherein the antibody is a monoclonal antibody. 8. The cachexia according to claim 1, wherein the cachexia is derived from a cancer.
8. The therapeutic agent for cachexia according to any one of 7.