JP2006306895A - Therapeutic agent for cachexia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent for cachexia comprising, as an active ingredient, a substance capable of inhibiting the binding between a parathyroid hormone relating protein (PTHrP) and a receptor thereof. <P>SOLUTION: The therapeutic agent for cachexia includes an antibody against the PTHrP as an active ingredient. Further, the therapeutic agent for cachexia includes a humanized or chimerized antibody against the PTHrP. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cachexia therapeutic agent containing, as an active ingredient, a substance that inhibits the binding between a parathyroid hormone related peptide (PTHrP) and its receptor. Specifically, the present invention relates to a cachexia therapeutic agent containing an antibody against a parathyroid hormone-related peptide as an active ingredient.

  Cachexia in patients with end-stage cancer is one of the accompanying syndromes of malignant tumors and causes poor general condition mainly due to anorexia, weight loss, anemia, abnormalities of water electrolytes, immune abnormalities, etc. . For cancer patients, the development of cachexia not only results in life-threatening end-stage symptoms, but also significantly impairs the patient's quality of life (QOL). Physical and social impact.

  Recently, it has become clear that cachectin, which was thought to be a causative agent of cancer cachexia, is the same factor as tumor necrosis factor (TNF). Later, similar effects on cytokines such as interleukin 1 (IL-1), IL-6, LIF, and IFN were revealed, and cancer cachexia is a pathological condition that acts in combination with multiple factors. Has become clear.

  The human oral floor cancer-derived OCC-1 cell line is known to produce various humoral factors related to cancer cachexia, and when OCC-1 cells are transplanted into nude mice, Symptoms such as quality (Kajimura N. et al., Cancer Chemother. Pharmacol., 1996, 38 Suppl. PS48-52, Tanaka R. et al., Jpn. J. Clin. Oncology 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 proliferate. It is thought to act and cause the above symptoms.

  Thus, the symptoms of nude mice transplanted with the OCC-1 cell line are considered to be very common with the symptoms of human terminal cancer patients. However, until now, there are no known reports on drugs for such cachexia.

  An object of the present invention is to provide a therapeutic agent for cachexia.

  As a result of intensive studies to provide such therapeutic agents, the present inventors have found that the object can be achieved by a substance that inhibits the binding between a parathyroid hormone-related peptide and its receptor. It came to be completed. That is, the present invention is a cachexia therapeutic agent containing, 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.

  That is, the present invention relates to a cachexia therapeutic agent comprising an antibody against a parathyroid hormone-related peptide as an active ingredient.

  The antibody against the parathyroid hormone-related peptide is preferably humanized or chimerized.

For example, the humanized antibody may comprise at least one of the following (a) to (c):
(A) an L chain V region having any one of the amino acid sequences represented by SEQ ID NOs: 48 to 51,
(B) an L chain V region having any one of the amino acid sequences represented by SEQ ID NOs: 52 to 55,
(C) an H chain V region having the amino acid sequence shown in SEQ ID NO: 56;

  The humanized antibody includes, for example, an L chain V region having any one of the amino acid sequences shown in SEQ ID NOs: 48 to 55 and an H chain V region having the amino acid sequence shown in SEQ ID NO: 56.

  The humanized antibody may further contain an L chain C region and / or an H chain C region of a human antibody. The humanized antibody is preferably a humanized # 23-57-137-1 antibody.

  The antibody against the parathyroid hormone-related peptide may be, for example, an antibody fragment and / or a modified product thereof. The antibody against the parathyroid hormone-related peptide may be a monoclonal antibody.

The antibody against the parathyroid hormone related peptide is, for example,
(a) effective in the treatment of cachexia,
(b) Those that do not significantly affect the growth of human oral floor cancer OCC-1 cells can also be used.
The cachexia that is the target of the cachexia therapeutic agent can be derived from, for example, cancer.

  INDUSTRIAL APPLICABILITY According to the present invention, a cachexia therapeutic agent containing a substance that inhibits binding between a parathyroid hormone-related peptide and its receptor as an active ingredient is provided. The above-mentioned substance is useful as a cachexia therapeutic agent because it suppresses body weight loss in comparison with a control in a medicinal effect test in a cachexia model animal and also has an effect of extending the survival time.

  The present invention is a cachexia therapeutic agent comprising as an active ingredient a substance that inhibits the binding between a parathyroid hormone related peptide (PTHrP) and its receptor (PTHrP receptor).

  In the present specification, “PTHrP receptor” refers to a receptor that binds to PTHrP described in, for example, JP-T-6-506598, and is present on a target organ (for example, bone or kidney). It doesn't matter whether the body.

  In addition, “substances that inhibit the binding of PTHrP and PTHrP receptor” are substances that inhibit PTHrP binding to PTHrP receptor by binding to PTHrP (for example, anti-PTHrP antibody), and PTHrP receptor. A substance that inhibits PTHrP from binding to the PTHrP receptor by binding to (for example, an antagonist of the PTHrP receptor (also referred to as a PTHrP antagonist), specifically, at least one amino acid of the PTHrP peptide is substituted or deleted. Or a partial sequence of a PTHrP peptide).

Examples of the anti-PTHrP antibody include known antibodies such as humanized antibodies, human antibodies (WO96 / 33735) or chimeric antibodies (JP-A-4-28089), as well as antibodies (# 23-57) of the present invention. -137-1 antibody). The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody. Examples of PTHrP antagonists include polypeptides and small molecules, for example, substances that bind to PTHrP receptor antagonistically with respect to PTHrP, for example, JP-A-7-165790, Peptides (UNITED STATES) 1995, 16 ( 6) 1031-1037, Biochemistry (UNITED STATES) Apr. 28 1992, 31 (16) 4026-4033, and a polypeptide having PTHrP antagonist activity described in JP-T-5-509098. Among the above exemplified polypeptides, those 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 is not limited in its origin, type (monoclonal, polyclonal) and shape as long as it has a cachexia therapeutic effect.

  The anti-PTHrP antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means. As the anti-PTHrP antibody used in the present invention, 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 binds to PTHrP, thereby inhibiting PTHrP from binding to the PTH / PTHrP receptor, blocking PTHrP signaling, and inhibiting the biological activity of PTHrP.

  Examples of such an antibody include # 23-57-137-1 antibody produced by hybridoma clone # 23-57-137-1.

  Hybridoma clone # 23-57-137-1 is designated as mouse-mouse hybridoma # 23-57-137-1, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1, Higashi 1-chome, Tsukuba, Ibaraki) In the middle 6), on August 15, 1996, FERM BP-5631 was deposited internationally under the Budapest Treaty.

2. Antibody-producing hybridomas Monoclonal antibody-producing hybridomas can basically be prepared as follows using known techniques. That is, using PTHrP as a sensitizing antigen, this is immunized according to a normal immunization method, the obtained immune cells are fused with a known parent cell by a normal cell fusion method, and monoclonal antibodies are obtained by a normal screening method. It can be produced by screening antibody-producing cells.

Specifically, the monoclonal antibody can be produced as follows.
First, human PTHrP used as a sensitizing antigen for antibody acquisition is obtained by expressing the PTHrP gene / amino acid sequence disclosed in Suva, LJ et al., Science (1987) 237, 893. That is, a gene sequence encoding PTHrP is inserted into a known expression vector system to transform an appropriate host cell, and then the target PTHrP protein is purified from the host cell or culture supernatant by a known method. .

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

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters, rabbits, monkeys and the like are used.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and the suspension is mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. Administer to mammals several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen.

  After immunizing in this way and confirming that the desired antibody level rises in the serum, immune cells are collected from the mammal and subjected to cell fusion. Preferred immune cells include spleen cells. It is done.

  Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells. This myeloma cell is known in various known cell lines such as P3 (P3x63Ag8.653) (J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1 -7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. DHet al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, SF et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133) are preferably used. The

  The cell fusion between the immune cells and myeloma cells is basically performed by a known method such as the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). It can carry out according to etc.

  More specifically, the cell fusion is performed in a normal nutrient culture medium in the presence of a cell fusion promoter, for example. As the fusion accelerator, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired.

  The use ratio of immune cells and myeloma cells can be arbitrarily set. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture can be used. Serum supplements such as fetal calf serum (FCS) can be used in combination.

  In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preliminarily heated to about 37 ° C. is usually 30-60% ( The target fusion cell (hybridoma) is formed by adding at a concentration of w / v) and mixing. Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.

  The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to die. Subsequently, a normal limiting dilution method is performed, and screening and single cloning of a hybridoma that produces the target antibody are performed.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes are sensitized to PTHrP in vitro, and the sensitized lymphocytes are fused with human-derived myeloma cells having a permanent division ability. A desired human antibody having binding activity to PTHrP can also be obtained (see Japanese Patent Publication No. 1-59878). Furthermore, PTHrP as an antigen may be administered to a transgenic animal having all repertoires of human antibody genes to obtain anti-PTHrP antibody-producing cells, and human antibodies against PTHrP may be obtained from the immortalized cells. (See International Patent Application Publication No. WO 94/25585, WO 93/12227, WO 92/03918, WO 94/02602).

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

  In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible therewith to proliferate, and its ascites The method obtained as follows is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

3. Recombinant antibody In the present invention, as a monoclonal antibody, an antibody gene is cloned from a hybridoma, incorporated into an appropriate vector, introduced into a host, and a recombinant antibody produced using gene recombination technology. (See, 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. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) and the like to prepare total RNA, and the target mRNA is prepared using mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

  The cDNA of the antibody V region is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis is performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation). For cDNA synthesis and amplification, 5'-AmpliFINDER RACE Kit (manufactured by Clontech) and 5'-RACE method using PCR (Frohman, MAet al., Proc. Natl. Acad. Sci. USA ( 1988) 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) and the like.

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

  After obtaining DNA encoding the V region of the desired anti-PTHrP antibody, this is incorporated into an expression vector containing DNA encoding the desired antibody constant region (C region).

  In order to produce the anti-PTHrP antibody used in the present invention, an antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody.

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

  In addition to the above host cells, transgenic animals can be used for the production of recombinant antibodies. For example, an antibody gene is inserted in the middle of a gene encoding a protein (such as goat β casein) inherently produced in milk to prepare a fusion gene. A DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its offspring. In addition, hormones may be used in transgenic goats as appropriate to increase the amount of milk containing the desired antibody produced from the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).

4). Modified antibody In the present invention, in addition to the above-mentioned antibody, a genetically modified antibody artificially modified for the purpose of reducing heteroantigenicity to humans, for example, a chimeric antibody or a humanized antibody is used. it can. These modified antibodies can be produced using known methods.

  A chimeric antibody is obtained by ligating the DNA encoding the antibody V region obtained as described above with DNA encoding the human antibody C region, incorporating it into an expression vector, introducing it into a host, and producing it. Using this known method, a chimeric antibody useful in the present invention can be obtained.

  A humanized antibody is also called a reshaped human antibody, which is a complementarity determining region (CDR) of a mammal other than a human, eg, a mouse antibody, to the complementarity determining region of a human antibody. It is transplanted and its general gene recombination technique is also known (see European Patent Application Publication No. EP 125023, WO 96/02576).

  Specifically, a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody has a portion that overlaps both terminal regions of CDR and FR. The prepared oligonucleotides are used as primers and synthesized by PCR. The obtained DNA is linked to 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).

  As the framework region of the human antibody to be ligated via CDR, a region in which the complementarity determining region forms a favorable antigen binding site is selected. If necessary, framework region amino acids 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., CancerRes. (1993) 53, 851-856).

  For the C region of the chimeric antibody and humanized antibody, human antibodies are used. For example, Cγ1, Cγ2, Cγ3, and Cγ4 can be used for the H chain, and Cκ and Cλ can be used for the L chain. In addition, the human antibody C region may be modified to improve the stability of the antibody or its production.

  A chimeric antibody consists of a variable region of a non-human mammal-derived antibody and a constant region derived from a human antibody. On the other hand, a humanized antibody consists of a complementarity determining region of a non-human mammal-derived antibody, and a framework region and C region derived from a human antibody. Since humanized antibodies have reduced antigenicity in the human body, they are useful as active ingredients of the therapeutic agent of the present invention.

  Examples of humanized antibodies that can be used in the present invention include humanized # 23-57-137-1 antibodies. The humanized # 23-57-137-1 antibody shows 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) for the L chain. J. Immunol., 39, 95-103, 1994) derived from three FR fragments (FR1, FR2 and FR3) and a human antibody S25755 (NBRF-PDB) derived FR fragment (FR4), The H chain is linked to the framework region of human antibody S31679 (NBRF-PDB, Cuisinier AM et al., Eur. J. Immunol., 23, 110-118, 1993), and the framework region has an antigen binding activity. The amino acid residue is partially substituted.

  In addition, Escherichia coli having a plasmid containing DNA encoding the L chain or H chain of the humanized # 23-57-137-1 antibody is the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (East 1 in Tsukuba City, Ibaraki Prefecture). On the 15th day of August 1996, Escherichia coli JM109 (hMBC1HcDNA / pUC19), which has a plasmid containing DNA encoding the H chain, was designated as FERM BP-5629. Escherichia coli JM109 (hMBC1Lqλ / pUC19), which is an Escherichia coli having a plasmid containing a DNA encoding a chain, has been internationally deposited under the Budapest Treaty as FERM BP-5630.

5. Modified Antibody The antibody used in the present invention may be a fragment of an antibody 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 (ab ′) ₂ , Fv, or single chain Fv (scFv) in which Hv or L chain Fvs are linked by an appropriate linker. Specifically, the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, followed by appropriate host cells. (Eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc. Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc., Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669, Bird, RE et al., TIBTECH (1991) 9, 132-137).

  scFv is 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 the 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). The H chain V region and L chain V region in scFv may be derived from any of those described as antibodies herein. As the peptide linker that links the V regions, for example, any single chain peptide consisting of amino acid residues 12-19 is used.

  The scFv-encoding DNA is a DNA encoding the H chain or H chain V region of the antibody, and a DNA encoding the L chain or L chain V region. Amplify by PCR using a DNA pair that encodes as a template and a primer pair that defines both ends, and then further encode the DNA that encodes the peptide linker part, and both ends are linked to the H and L chains, respectively. Obtained by combining and amplifying the pair of rimers defined in 1.

  In addition, once a DNA encoding scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method. ScFv can be obtained according to the method.

  These antibody fragments can be produced by the host by obtaining and expressing the gene in the same manner as described above. The “antibody” in the present invention includes fragments of these antibodies.

  As a modified antibody, an anti-PTHrP antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used. The “antibody” in the present invention includes these modified antibodies. Such a modified antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have 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. In the case of mammalian cells, it can be expressed by functionally binding a useful promoter commonly used, an antibody gene to be expressed, and a poly A signal downstream of the 3 ′ side thereof. For example, examples of the promoter / enhancer include human cytomegalovirus immediate early promoter / enhancer.

  In addition, other promoters / enhancers that can be used for expression of antibodies used in the present invention include viral promoters / enhancers such as retrovirus, polyoma virus, adenovirus, simian virus 40 (SV 40), or human elongation factor 1α ( HEF1α) and other promoters / enhancers derived from mammalian cells.

  When using the SV 40 promoter / enhancer, the method of Mulligan et al. (Nature (1979) 277, 108), and when using the HEF1α promoter / enhancer, the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322), gene expression can be easily performed.

  In the case of Escherichia coli, the gene can be expressed by functionally combining a commonly used useful promoter, a signal sequence for antibody secretion, and an antibody gene to be expressed. Examples of the promoter include lacz promoter and araB promoter. When using the lacz promoter, the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), or when using the araB promoter, the method of Better et al. (Science (1988) 240, 1041-1043).

  As a signal sequence for antibody secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the periplasm of E. coli is produced. Then, after separating the antibody produced in the periplasm, the structure of the antibody is appropriately refolded and used.

  As the origin of replication, those derived from SV 40, polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can be used. Furthermore, the expression vector is a selection marker for gene copy number amplification in the host cell system. As an aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene and the like.

  Any expression system, such as a eukaryotic or prokaryotic cell system, can be used for the production of the antibodies used in the present invention. Examples of eukaryotic cells include established mammalian cell systems, insect cell systems, filamentous fungal cells, and animal cells such as yeast cells. Examples of prokaryotic cells include bacterial cells such as E. coli cells.

  Preferably, the antibodies used in the present invention are expressed in mammalian cells such as CHO, COS, myeloma, BHK, Vero, HeLa cells.

  Next, the transformed host cell is cultured in vitro or in vivo to produce the desired antibody. Host cells are cultured according to a known method. For example, DMEM, MEM, RPMI1640, and IMDM can be used as the culture medium, and serum supplements such as fetal calf serum (FCS) can be used in combination.

7). Separation and Purification of Antibody 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, Hyper D, POROS, Sepharose FF (Pharmacia) etc. are mentioned as a column using a protein A column. In addition, it is only necessary to use a separation and purification method used in ordinary proteins, and the method is not limited at all. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns other than the affinity column, filters, 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 antibodies used in the present invention (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988), ligand receptor binding inhibitory activity (Harada, A. et al., For the measurement of International Immunology (1993) 5, 681-690), known means can be used.

  ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method is used as a method for measuring the antigen binding activity of the anti-PTHrP antibody used in the present invention. be able to. For example, when an enzyme immunoassay is used, a sample containing an anti-PTHrP antibody, for example, a culture supernatant of an anti-PTHrP antibody-producing cell or a purified antibody is added to a plate coated with PTHrP (1-34). It is possible to evaluate the antigen binding activity by adding a secondary antibody labeled with an enzyme such as alkaline phosphatase, incubating the plate, washing, adding an enzyme substrate such as p-nitrophenyl phosphate and measuring the absorbance. it can. In order 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 purpose of treating or improving cachexia. It does not matter whether the cachexia is derived from cancer or not. For example, those derived from cancer include 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. Pancreatol. (UNITED STATES) Aug-Nov 1990, 7 (1-3) p141-150, J. Natl. Cancer Inst. (UNITEDSTATES) Dec 19 , 1990, 82 (24) p1922-1926, and the like.

  Moreover, JPEN J. Parenter. Enteral Nutr. (UNITEDSTATES) Nov-Dec 1990, 14 (6) p605-609, Chest (UNITED STATES) Nov 1990, 98 (5) p1091-1094, Bone Marrow Examples include cachexia described in Transplant. (ENGLAND) 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 orally or parenterally, but is preferably parenterally administered. Specifically, it is a transpulmonary dosage form (for example, nephriser etc. Pulmonary administration using a device), nasal administration type, transdermal administration type (for example, ointment, cream), injection type and the like. As an example of the injection type, it can be administered systemically or locally by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. The administration method can be appropriately selected depending on the age and symptoms of the patient. Effective doses are selected in the range of 0.001 mg to 1000 mg per kg body weight per dose. Alternatively, a dose of 0.01 to 100,000 mg / body can be selected per patient. However, the therapeutic agent containing the anti-PTHrP antibody of the present invention is not limited to these doses.

  In addition, the administration time may be administered before or after the occurrence of cachexia, or may be administered when 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 Science, latest edition, Mark Publishing Company, Easton, USA), and a pharmaceutically acceptable carrier And may contain both additives.

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

  The actual additive is selected from the above alone or in appropriate combination depending on the dosage form of the therapeutic agent of the present invention, but is of course not limited thereto. For example, when used as an injectable preparation, the purified anti-PTHrP antibody is dissolved in a solvent, such as physiological saline, buffer solution, glucose solution, etc., and an adsorption inhibitor such as Tween 80, Tween 20, gelatin, human serum albumin The thing which added etc. can be used. Alternatively, it may be lyophilized to obtain a dosage form that is reconstituted before use, and as an excipient for lyophilization, for example, sugar alcohols or saccharides such as mannitol and glucose are used. be able to.

  Hereinafter, the present invention will be described more specifically with reference examples and examples. However, the technical scope of the present invention is not limited to these examples.

Drug efficacy test in cachexia model animal Using a human tumor-nude mouse transplant cachexia model animal, the therapeutic effect of mouse monoclonal antibody against PTHrP on cachexia was examined.

  As a model animal, a nude mouse transplanted with human oral floor cancer OCC-1 (purchased from Central Laboratory for Experimental Animals) was used. Nude mice transplanted with human oral floor cancer OCC-1 increase in blood calcium concentration as tumors increase, and develop cachexia symptoms such as weight loss and decreased exercise. The cachexia symptom caused by human oral floor cancer OCC-1 was evaluated by the improvement of mouse monoclonal antibody using blood calcium concentration, body weight and life-prolonging effect as indicators.

  The passage of human oral floor cancer OCC-1 was performed in vivo using BALB / c-nu / nu nude mice (Claire Japan). For evaluation of drug efficacy, 6-week-old male BALB / c-nu / nu nude mice (Claire Japan) were purchased, and after acclimatization for 1 week, 7-week-old animals were used.

  Preparation and grouping of cachexia model animals were performed as follows. That is, the human oral floor cancer OCC-1 that had been passaged was excised, and a tumor mass finely chopped into 3 mm square blocks was transplanted one by one subcutaneously into the flank of the mouse. After confirming that the tumor volume became sufficiently large on the 10th day after transplantation of the tumor mass, grouped so that each index would be averaged using blood calcium concentration, body weight and tumor volume as indices, and cachexia A model animal was used.

The therapeutic effect on cachexia was examined as follows.
(1) Observation of survival period In the examination of the life-prolonging effect, the mouse monoclonal antibody was administered twice a week, and the survival period was observed. In addition, pamidronate (Aredia), which has already been prescribed as a therapeutic agent for hypercalcemia, was administered once into the tail vein at a dose of 15 mg / Kg. As a control, phosphate buffered saline (PBS) was administered at 0.2 ml / mouse into the tail vein twice a week. The result is shown in FIG.
(2) Observation of blood calcium concentration The mouse monoclonal antibodies against 10 μg or 100 μg of PTHrP per mouse were administered into the tail vein twice every two days to the cachexia model animals prepared and grouped as described above. In addition, pamidronate (Aredia), which has already been prescribed as a therapeutic agent for hypercalcemia, was administered once into the tail vein at a dose of 15 mg / Kg. As a control, phosphate buffered saline (PBS) was administered twice every 2 days into the tail vein at 0.2 ml / mouse.
(3) Measurement of blood calcium Blood calcium concentration was measured on the 1st and 4th days after administration of the mouse monoclonal antibody, and the efficacy of each antibody was evaluated. The blood calcium concentration was collected as a whole blood ionized calcium concentration using a 643 automatic Ca / pH analyzer (CIBA-CORNING) by collecting blood from the orbit using a hematocrit tube. Body weight was measured daily until the fourth day after antibody administration. The results are shown in FIG. 2 and FIG.
(4) Measurement tumor volume Tumor weight is calculated 4 days after antibody administration, the tumor major axis (a mm) and minor axis (b mm) were measured, as tumor volume by equation ab ^2/2 of Galant did. The result is shown in FIG.

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

Drug efficacy test in hypercalcemia / cachexia model animal Using human tumor-nude mouse transplant system cachexia model animal, the therapeutic effect of humanized antibody version q against PTHrP on cachexia was examined.

  As a model animal, a nude mouse transplanted with human oral floor cancer OCC-1 (purchased from Central Laboratory for Experimental Animals) was used. Nude mice transplanted with human oral floor cancer OCC-1 increase in blood calcium concentration as tumors increase, and develop cachexia symptoms such as weight loss and decreased exercise. The cachexia symptom caused by human oral floor cancer OCC-1 was evaluated by the improvement of humanized antibody version q using blood calcium concentration, body weight and life-prolonging effect as indicators.

  The passage of human oral floor cancer OCC-1 was performed in vivo using BALB / c-nu / nu nude mice (Claire Japan). For evaluation of drug efficacy, 6-week-old male BALB / c-nu / nu nude mice (Claire Japan) were purchased, and after acclimatization for 1 week, 7-week-old animals were used.

  Preparation and grouping of cachexia model animals were performed as follows. That is, the human oral floor cancer OCC-1 that had been passaged was excised, and a tumor mass finely chopped into 3 mm square blocks was transplanted one by one subcutaneously into the flank of the mouse. After confirming that the tumor volume became sufficiently large on the 10th day after transplantation of the tumor mass, grouping was performed so that each index was averaged using the tumor volume, blood calcium concentration and body weight as indices, and cachexia A model animal was used.

The therapeutic effect on cachexia was examined as follows.
(1) Observation of survival period In the examination of the survival effect, humanized antibody version q was administered twice a week into the tail vein, and the survival period was observed. As a control, phosphate buffered saline (PBS) was administered into the tail vein twice a week at 0.1 ml / mouse. The results are shown in FIG.
(2) Observation of blood calcium concentration 10 μg or 100 μg of humanized antibody version q per mouse was administered to the cachexia model animals prepared and divided as described above twice in the tail vein for 2 days. . As a control, phosphate buffered saline (PBS) was similarly administered at 0.1 ml / mouse.
(3) Measurement of blood calcium Blood calcium concentration was measured on the 1st and 4th day after the initial administration of humanized antibody version q, and the efficacy of the antibody was evaluated. The blood calcium concentration was measured as a whole blood ionized calcium concentration using a 643 automatic Ca / pH analyzer (CHIRON) by collecting blood from the orbit using a hematocrit tube. Body weight was measured daily until day 4. The results are shown in FIG. 17 and FIG.
(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 results are shown in FIG.

  As described above, administration of humanized antibody version q at 10 μg or 100 μg suppressed the increase in blood calcium concentration and the decrease in body weight associated with malignant tumors as compared with the control group. In addition, when 100 μg of humanized antibody version q was continuously administered twice a week, a significant increase in survival time (p = 0.0108: Log Rank test) was observed compared to the control group. The effect of the humanized antibody version q on the cachexia model animal associated with the malignant tumor was similar to the effect of the previously reported mouse monoclonal antibody. This indicated 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 # 23-57-137-1 against human PTHrP (1-34) (SEQ ID NO: 75) were designated as Mikiji Sato. (Sato, K. et al., J. Bone Miner. Res. 8, 849-860, 1993).

  For use as an immunogen, PTHrP (1-34) (manufactured by Peninsula) and thyroglobulin as a carrier protein were combined using carbodiimide (Dojinn). PTHrP (1-34) bound to thyroglobulin was dialyzed and prepared to have a protein concentration of 2 μg / ml, then mixed 1: 1 with Freund's adjuvant (Difco), and after emulsion preparation, 16 female BALBs were prepared. 100 μg per animal was immunized 11 times subcutaneously or intraperitoneally in the back of / C mice. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second and subsequent boosters.

  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, antiserum diluted with RIA buffer after serum separation and 125I-labeled PTHrP (1-34) were mixed, and the binding activity was measured. Mice with increased antibody titers were finally immunized with 50 μg of PTHrP (1-34) not conjugated with carrier protein per animal.

On the third day of the final immunization, the mice were sacrificed and the spleen was removed, and then the spleen cells and the mouse myeloma cell line P3x63Ag8U.1 were cell-fused according to a conventional method using 50% polyethylene glycol 4000. The cell-fused cells were seeded into 85 96-well plates at a cell number of 2 × 10 ⁴ / well. Hybridoma selection was performed using HAT medium.

  Hybridoma screening was carried out by measuring and selecting the presence or absence of a PTHrP-recognizing antibody in a culture supernatant in a hole in which growth was observed in a HAT medium by a solid-phased RIA method. Hybridomas are collected from the wells that have been confirmed to be capable of binding to the antibody, suspended in RPMI-1640 medium containing 15% FCS and supplemented with OPI-supplement (Sigma), and hybridomas are unified by limiting dilution. Carried out. Clones # 23-57-154 and # 23-57-137-1 having strong binding ability to PTHrP (1-34) were obtained.

  In addition, hybridoma clone # 23-57-137-1 is called mouse-mouse hybridoma # 23-57-137-1, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1, Higashi 1-chome, Tsukuba, Ibaraki) In the middle 6), on August 15, 1996, FERM BP-5631 was deposited internationally under the Budapest Treaty.

[Reference Example 2]
Cloning of DNA encoding mouse monoclonal antibody V region against human PTHrP (1-34) DNA encoding the variable region of mouse monoclonal antibody # 23-57-137-1 against human PTHrP (1-34) is as follows. And cloned.
(1) Preparation of mRNA mRNA from hybridoma # 23-57-137-1 was prepared using Quick Prep mRNA Purification Kit (Pharmacia Biotech). The cells of hybridoma # 23-57-137-1 were completely homogenized with Extraction Buffer, and mRNA was purified with oligo (dT) -Cellulose Spun Column according to the instructions attached to the kit, followed by ethanol precipitation. The mRNA precipitate was dissolved in Elution Buffer.

(2) Preparation and amplification of cDNA of gene encoding mouse H chain V region
(i) Cloning of # 23-57-137-1 antibody H chain V region cDNA Cloning of the gene encoding the H chain V region of the mouse monoclonal antibody against human PTHrP was performed by the 5'-RACE method (Frohman, MA et al. , Proc. Natl. Acad. Sci. USA, 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-2932, 1989). For the 5′-RACE method, 5′-Ampli FINDER RACE kit (CLONETECH) was used, and 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 heavy chain constant region (C region) was used. Using about 2 μg of the mRNA prepared as described above as a template, 10 pmole of MHC2 primer was added and reacted with reverse transcriptase at 52 ° C. for 30 minutes for reverse transcription to cDNA.

  After RNA was hydrolyzed with 6N NaOH (65 ° C., 30 minutes), the 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 FINDER Anchor (SEQ ID NO: 42) was ligated to the 5 ′ end of the synthesized cDNA. Anchor primers (SEQ ID NO: 2) and MHC-G1 primers (SEQ ID NO: 3) (S. T. Jones, et al., Biotechnology, 9, 88, 1991) were used as primers for amplification by PCR using this as a template.

In 50 μl of the PCR solution, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl ₂ , 2.5 units TaKaRa Taq (Takara Shuzo), 10 pmole Contains 1 μl of an anchor primer and a cDNA reaction mixture ligated with MHC-G1 primer and Ampli FINDER Anchor. This solution was overlaid with 50 μl of mineral oil. PCR was performed 30 times in a thermal cycle using Thermal Cycler Mode 1480J (Perkin Elmer) at 94 ° C. for 45 seconds, 60 ° C. for 45 seconds, and 72 ° C. for 2 minutes.

(ii) Cloning of cDNA of # 23-57-137-1 Antibody L Chain V Region Cloning of the gene encoding the L chain V region of the mouse monoclonal antibody against human PTHrP can be performed by the 5′-RACE method (Frohman, MA et al , Proc. Natl. Acad. Sci. USA 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-2932, 1989). For the 5′-RACE method, a 5′-AmpliFinder RACE Kit (Clonetech) was used, and the operation followed the attached prescription. Oligo-dT primer was used as a primer for cDNA synthesis. The oligo-dT primer was added using about 2 μg of the mRNA prepared as described above as a template and reacted with reverse transcriptase at 52 ° C. for 30 minutes for reverse transcription to cDNA. After RNA was hydrolyzed with 6N NaOH (65 ° C., 30 minutes), the cDNA was purified by ethanol precipitation. The Ampli FINDER Anchor was ligated to the 5 ′ end of the synthesized cDNA by reacting with T4 RNA ligase at 37 ° C. for 6 hours and at room temperature for 16 hours.

A PCR primer MLC (SEQ ID NO: 4) was designed from the conserved sequence of the mouse L chain λ chain constant region and synthesized using a 394 DNA / RNA synthesizer (ABI). In 100 μl of the PCR solution, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl ₂ , 2.5 units of AmpliTaq (PERKIN ELMER), Contains 50 pmole of Anchor primer (SEQ ID NO: 2), and 1 μl of a reaction mixture of cDNA linked to MLC (SEQ ID NO: 4) and Ampli FINDER Anchor. This solution was overlaid with 50 μl of mineral oil. PCR was performed 35 times using a Thermal Cycler Model 480J (Perkin Elmer) at a temperature cycle of 94 ° C. for 45 seconds, 60 ° C. for 45 seconds, and 72 ° C. for 2 minutes.

(3) Purification and fragmentation of PCR products The DNA fragments amplified by PCR as described above were separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio. Products). A piece of agarose containing a DNA fragment of about 550 bp in length as the H chain V region and about 550 bp in length as the L chain V region was excised, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. The purified DNA was precipitated with ethanol, and then dissolved in 10 mM Tris-HCl (pH 7.4) and 20 μl of 1 mM EDTA solution. 1 μl of the obtained DNA solution was digested with the restriction enzyme XmaI (New England Biolabs) at 37 ° C. for 1 hour, and then digested with the restriction enzyme EcoRI (Takara Shuzo) at 37 ° C. for 1 hour. This digestion mixture was extracted with phenol and chloroform, and DNA was collected by ethanol precipitation.

  Thus, a DNA fragment containing a gene encoding a mouse H chain V region and L chain V region having an EcoRI recognition sequence at the 5'-end and an XmaI recognition sequence at the 3'-end was obtained.

  The EcoRI-XmaI DNA fragment containing the gene encoding the mouse H chain V region and L chain V region prepared as described above and the pUC19 vector prepared by digestion with EcoRI and XmaI were used as DNA ligation kit ver.2 (Takara Shuzo) ) And reacted at 16 ° C. for 1 hour according to the attached formulation. Next, 10 μl of the above ligation mixture was added to 100 μl of E. coli JM109 competent cells (Nippon Gene), and the cells were allowed to stand for 15 minutes on ice, 1 minute at 42 ° C., and 1 minute on ice. Subsequently, 300 μl of SOC medium (Molecular Cloning: A Labgoratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) was added and incubated at 37 ° C. for 30 minutes, and then 100 μg / ml or 50 μg / ml ampicillin, This E. coli is seeded on LB agar medium or 2xYT agar medium (Molecular Cloning: A Labgoratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) containing 0.1 mM IPTG, 20 μg / ml X-gal. E. coli transformants were obtained by incubating overnight at 37 ° C.

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

(4) Determination of the base sequence of the gene encoding the mouse antibody V region The DNA sequencer 373A (Perkin-Elmer, ABI) was used to determine the base sequence of the cDNA coding region in the above plasmid using the Dye Terminator Cycle Sequencing kit (Perkin-Elmer). Determined by. Using M13 Primer M4 (Takara Shuzo) (SEQ ID NO: 5) and M13 Primer RV (Takara Shuzo) (SEQ ID NO: 6) as sequencing primers, the sequence was determined by confirming the base sequence in both directions.

  The plasmid containing the gene encoding the mouse H chain V region derived from hybridoma # 23-57-137-1 thus obtained was named MBC1H04, and the plasmid containing the gene encoding the L chain V region was named MBC1L24. The nucleotide sequences (including the corresponding amino acid sequences) of the genes encoding the H chain V region and L chain V region of the mouse # 23-57-137-1 antibody contained in the plasmids MBC1H04 and MBC1L24 are shown in SEQ ID NOs: 57 and 65, respectively. Show. The polypeptides of the H chain V region and L chain V region fragments are both started from the 58th position (encoding glutamine) of the base sequences represented by SEQ ID NOs: 57 and 65. These amino acid sequences are shown in SEQ ID NO: 46 for the H chain V region fragment and SEQ ID NO: 45 for the L chain V region fragment.

  In addition, Escherichia coli JM109 (MBC1H04) and Escherichia coli JM109 (MBC1L24) are the Escherichia coli having the plasmids MBC1H04 and MBC1L24. On August 15, 1996, Escherichia coli JM109 (MBC1H04) was internationally deposited under the Budapest Treaty as FERM BP-5628 and Escherichia coli JM109 (MBC1L24) as FERM BP-5627.

(5) CDR determination of mouse monoclonal antibody # 23-57-137-1 against human PTHrP The general structures of the H chain V region and the L chain V region are similar to each other, and each has four frameworks. The parts are linked by three hypervariable regions, the complementarity determining regions (CDRs). The amino acid sequence of the framework is relatively well conserved, while the variability of the amino acid sequence of the CDR region is extremely high (Kabat, EA et al., “Sequence of Proteins of Immunological Interest” US Dept. Health and Human Services, 1983).

 Based on these facts, the amino acid sequence of the variable region of the mouse monoclonal antibody against human PTHrP is applied to the antibody amino acid sequence database created by Kabat et al., And the CDR regions are as shown in Table 1 by examining the homology. Were determined.

  The amino acid sequences of CDR1 to CDR3 of the L chain V region are shown in SEQ ID NOs: 59 to 61, respectively, and the amino acid sequences of CDR1 to CDR3 of the H chain V region are shown in SEQ ID NOs: 62 to 64, respectively.

[Reference Example 3]
Construction of chimeric antibody
(1) Construction of chimeric antibody heavy chain
(i) Construction of H chain V region The cloned mouse H chain V region was modified by PCR in order to be ligated to an expression vector containing the genomic DNA of human H chain C region Cγ1. The back primer MBC1-S1 (SEQ ID NO: 7) hybridizes to the DNA encoding the 5′-side of the V region leader sequence and the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196). , 947-950, 1987) and the recognition sequence of restriction enzyme Hind III. The forward primer MBC1-a (SEQ ID NO: 8) was designed to hybridize to the DNA sequence encoding the 3′-side of the J region and to have a splice donor sequence and a restriction enzyme BamHI recognition sequence. For PCR, TaKaRa Ex Taq (Takara Shuzo) was used. In a 50 μl reaction mixture, 0.07 μg of plasmid MBC1H04 as template DNA, MBC1-a and MBC1-S1 as primers were 50 pmole, 2.5 U TaKaRa Ex Taq and 0.25 mM, respectively. 50 μl of mineral oil was overlaid using the attached buffer under conditions containing dNTPs, and performed 30 times with a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes. 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 437 bp long DNA fragment was excised and purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. The purified DNA was recovered by ethanol precipitation, and then dissolved in 10 mM Tris-HCl (pH 7.4) and 20 μl of 1 mM EDTA solution. 1 μl of the obtained DNA solution was digested with restriction enzymes BamHI and Hind III (Takara Shuzo) at 37 ° C. for 1 hour. This digestion mixture was extracted with phenol and chloroform, and DNA was collected by ethanol precipitation.

  The Hind III-BamHI DNA fragment containing the gene encoding the mouse H chain V region prepared as described above was subcloned into the pUC19 vector prepared by digesting with Hind III and BamHI. In order to confirm the base sequence of this plasmid, the base sequence was determined by DNA Sequencer 373A (Perkin-Elmer) using Dye Terminator Cycle Sequencing kit (Perkin-Elmer) using primers M13 Primer M4 and M13 Primer RV as primers. It contains a gene encoding the mouse H chain V region derived from hybridoma # 23-57-137-1 having the correct base sequence, and contains a Hind III recognition sequence and Kozak sequence on the 5′-side, and BamHI recognition on the 3′-side. The plasmid having the sequence was named MBC1H / pUC19.

(ii) Construction of H chain V region for preparation of cDNA type mouse-human chimeric H chain In order to link with cDNA of human H chain C region Cγ1, the mouse H chain V region constructed as described above was subjected to PCR. Modified by law. The rear primer MBC1HVS2 (SEQ ID NO: 9) for the H chain V region converts the second asparagine of the sequence encoding the beginning of the V region leader sequence to glycine, and the Kozak consensus sequence (Kozak, M. et al. , J. Mol. Biol., 196, 947-950, 1987) and designed to have HindIII and EcoRI recognition sequences. 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 to encode ApaI and SmaI. It was designed to have a recognition sequence.

  PCR was performed using TaKaRa Ex Taq (Takara Shuzo), with 50 μl of reaction mixture containing 0.6 μg of plasmid MBC1H / pUC19 as template DNA, MBC1HVS2 and MBC1HVR2 as primers, 50 pmole and TaKaRa Ex Taq of 2.5 U and 0.25 mM dNTP, respectively. And 50 μl of mineral oil was overlaid using the attached buffer at 30 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 1% Sea Kem GTG agarose (FMC Bio.Products). A piece of agarose containing a 456 bp long DNA fragment was excised, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. The purified DNA was ethanol precipitated and dissolved in 20 mM of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution.

  1 μl of the obtained DNA solution was digested with restriction enzymes EcoRI and SmaI (Takara Shuzo) at 37 ° C. for 1 hour. This digestion mixture was extracted with phenol and chloroform, and DNA was collected by ethanol precipitation. The EcoRI-SmaI DNA fragment containing the gene encoding the mouse H chain V region prepared as described above was subcloned into the pUC19 vector prepared by digesting with EcoRI and SmaI. In order to confirm the base sequence of this plasmid, the base sequence was determined by DNA Sequencer 373A (Perkin-Elmer) using Dye Terminator Cycle Sequencing kit (Perkin-Elmer) using primers M13 Primer M4 and M13 Primer RV as primers. Contains a gene encoding the mouse H chain V region derived from hybridoma # 23-57-137-1 having the correct base sequence, EcoRI and Hind III recognition sequence and Kozak sequence on the 5′-side, and 3′-side A plasmid having ApaI and SmaI recognition sequences was named MBC1Hv / pUC19.

(iii) Construction of chimeric antibody H chain expression vector A cDNA containing the human antibody H chain C region Cγ1 was prepared as follows. That is, the expression vector DHFR-ΔE-RVh encoding the genomic DNA of humanized PM1 antibody H chain V region and human antibody H chain C region IgG1 (N. Takahashi, et al., Cell 29, 671-679 1982). -PM-1-f (see WO92 / 19759) and an 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; MRNA was prepared from the CHO cells introduced with, and cDNA containing the humanized PM1 antibody H chain V region and human antibody C region Cγ1 was cloned by RT-PCR and subcloned into the Hind III and BamHI sites of pUC19. After confirming the base sequence, the plasmid having the correct sequence was named pRVh-PM1f-cDNA.

  A Hind III site between the SV40 promoter and DHFR gene on DHFR-ΔE-RVh-PM-1-f, and an EcoRI site between the EF-1α promoter and the humanized PM1 antibody H chain V region Was used to construct an expression vector for cDNA containing the humanized PM1 antibody H chain V region and human antibody C region Cγ1.

  pRVh-PM1f-cDNA was digested with BamHI, then blunted with a Klenow fragment, and further digested with Hind III to prepare a Hind III-BamHI blunted fragment. This Hind III-BamHI blunted fragment was ligated to an expression vector prepared by digesting DHFR-ΔE-RVh-PM1-f from which the Hind III and EcoRI sites were deleted with Hind III and SmaI, An expression vector RVh-PM1f-cDNA containing cDNA encoding humanized PM1 antibody H chain V region and human antibody C region Cγ1 was constructed.

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

  Plasmid MBC1HcDNA / pUC19 was digested with EcoRI and BamHI, and the resulting DNA fragment containing the nucleotide sequence encoding the H chain of the chimeric antibody was introduced into expression vector pCOS1 prepared by digesting with EcoRI and BamHI. The thus obtained chimeric antibody expression plasmid was designated as MBC1HcDNA / pCOS1. The expression vector pCOS1 was constructed by deleting the antibody gene from HEF-PMh-gγ1 (see WO92 / 19759) by digestion with EcoRI and SmaI, and ligating EcoRI-NotI-BamHI Adapter (Takara Shuzo).

  Further, in order to prepare a plasmid for use in expression in CHO cells, plasmid MBC1HcDNA / pUC19 was digested with EcoRI and BamHI, and the resulting DNA fragment containing the chimeric antibody heavy chain sequence was digested with EcoRI and BamHI. It was introduced into the prepared expression plasmid pCHO1. The chimeric antibody expression plasmid thus obtained was named MBC1HcDNA / pCHO1. The expression vector pCHO1 is obtained by deleting the antibody gene from DHFR- △ E-rvH-PM1-f (see WO92 / 19759) by digestion with EcoRI and SmaI, and ligating EcoRI-NotI-BamHI Adapter (Takara Shuzo). It was constructed.

(2) Construction of human L chain constant region
(i) Preparation of Cloning Vector In order to construct a pUC19 vector containing a human L chain constant region, a Hind III site-deleted pUC19 vector was prepared. 2 μg of pUC19 vector was digested for 1 hour at 37 ° C. in 20 μl of a reaction mixture containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, 8 U Hind III (Takara Shuzo). The digested mixture was extracted with phenol and chloroform, and DNA was recovered by ethanol precipitation. The recovered DNA was incubated at room temperature in a 50 μl reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM NaCl, 0.5 mM dNTP, 6 U Klenow fragment (GIBCO BRL). For 20 minutes to smooth the ends. The reaction mixture was extracted with phenol and chloroform, and the vector DNA was recovered by ethanol precipitation.

The recovered vector DNA contains 50 mM Tris-HCl (pH 7.6), 10 mM MgCl ₂ , 1 mM ATP, 1 mM DTT, 5% (v / v) polyethylene glycol-8000, 0.5 U T4 DNA ligase (GIBCO BRL) The reaction mixture was reacted for 2 hours at 16 ° C. in 10 μl of the reaction mixture and self-ligated. 5 μl of the reaction mixture was added to 100 μl of E. coli JM109 competent cells (Nippon Gene), allowed to stand on ice for 30 minutes, then allowed to stand at 42 ° C. for 1 minute and further on ice for 1 minute. After adding 500 μl of SOC medium and incubating at 37 ° C. for 1 hour, 2 × YT agar medium (containing 50 μg / ml ampicillin) coated with X-gal and IPTG (Molecular Cloning: A Laboratory Manual, Sambrook, et. al., Cold Spring Harbor Laboratory Press, 1989) and cultured overnight at 37 ° C. to obtain transformants.

  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 the Plasmid Mini Kit (QIAGEN) according to the attached instructions. The purified plasmid was digested with Hind III, and the plasmid that was confirmed to lack the Hind III site was named pUC19ΔHind III.

(ii) Construction of gene encoding human L chain λ chain constant region Human antibody L chain λ chain C region is Mcg + Ke + Oz −, Mcg − Ke − Oz −, Mcg − Ke − Oz +, Mcg − Ke + At least four isotypes of Oz- are known (P. Dariavach, et al., Proc. Natl. Acad. Sci. USA, 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 was searched in the EMBL database. As a result, isotype was Mcg + Ke + Oz-(accession No. X57819) (P. Dariavach, et al., Proc. Natl. Acad. Sci. USA, 84, 9074-9078, 1987), the human antibody L chain λ chain showed the highest homology, and # 23-57-137-1 The homology with the mouse L chain λ chain C region was 64.4% in terms of amino acid sequence and 73.4% in terms of base sequence.

  Therefore, the gene encoding the human antibody L chain λ chain C region was constructed 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) have a sense DNA sequence, HLAMB2 (SEQ ID NO: 12) and HLAMB4 (SEQ ID NO: 14) have an antisense DNA sequence, 20 at each end of each primer. To 23 bp of complementary sequence.

  External primers HLAMBS (SEQ ID NO: 15) and HLAMBR (SEQ ID NO: 16) have sequences homologous to HLAMB1 and HLAMB4, HLAMBS recognizes EcoRI, Hind III and BlnI, and HLAMBR recognizes EcoRI recognition sequence. Contains. In the first PCR, HLAMB1-HLAMB2 and HLAMB3-HLAMB4 were reacted. After the reaction, they were mixed in equal amounts and assembled by the second PCR. Furthermore, external primers HLAMBS and HLAMBR were added, and full length DNA was amplified by the third PCR.

  PCR was performed using TaKaRa Ex Taq (Takara Shuzo) according to the attached prescription. In the first PCR, 100 μl of reaction mixture containing 5 pmole of HLAMB1 and 0.5 pmole of HLAMB2 and 5 U of TaKaRa Ex Taq (Takara Shuzo), or 0.5 pmole of HLAMB3 and 5 pmole of HLAMB4 and 5 U of TaKaRa Ex Taq (Takara Shuzo) And 50 μl of mineral oil was used as an upper layer, and the reaction was repeated 5 times in a temperature cycle of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute.

  In the second PCR, 50 μl of the reaction solution was mixed, 50 μl of mineral oil was overlaid, and the reaction was performed three times with a temperature cycle of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute.

  In the third PCR, 50 pmole each of the external primers HLAMBS and HLAMBR was added to the reaction solution, and the reaction was performed 30 times with a temperature cycle of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute.

  The DNA fragment of the third PCR product was electrophoresed on a 3% low melting point agarose gel (NuSieve GTG Agarose, FMC), and then collected and purified from the gel using GENECLEAN II Kit (BIO101) according to the attached recipe.

The obtained DNA fragment was digested for 1 hour at 37 ° C. in a 20 μl reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM NaCl, 8 U EcoRI (Takara Shuzo). . The digested mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation, and then dissolved in 8 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution.

0.8 μg of plasmid pUC19ΔHind III was similarly digested with EcoRI, extracted with phenol and chloroform, and recovered by ethanol precipitation. Dephosphorylated by reacting digested plasmid pUC19ΔHind III in 50 μl of reaction mixture containing 50 mM Tris-HCl (pH 9.0), 1 mM MgCl ₂ , alkaline phosphatase (E. coli C75, Takara Shuzo) at 37 ° C. for 30 minutes. Treated (BAP treatment). The reaction solution was extracted with phenol and chloroform, and DNA was recovered by ethanol precipitation, and then dissolved in 10 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution.

  The above-mentioned BAP-treated plasmid pUC19 ΔHind III (1 μl) and the previous PCR product (4 μl) were ligated using DNA Ligation Kit Ver.2 (Takara Shuzo) and transformed into E. coli JM109 competent cells. The obtained transformant was cultured overnight in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN).

  For the above plasmid, the base sequence of the cloned DNA was confirmed. A 373A DNA sequencer (ABI) was used to determine the base sequence, 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 inside the cloned DNA. The plasmid containing this DNA was named CλΔ / pUC19. Therefore, primers HCLMS (SEQ ID NO: 17) and HCLMR (SEQ ID NO: 18) were newly synthesized to compensate for that portion, and correct DNA was constructed again by PCR.

  In the first PCR, the plasmid CλΔ / pUC19 containing the deleted DNA was used as a template, and the reaction was performed with primers HLAMBS and HCLMR, HCLMS and HLAMB4. Each PCR product was purified and assembled in a second PCR. Further, external primers HLAMBS and HLAMB4 were added, and full length DNA was amplified by the third PCR.

  In the first PCR, CλΔ / pUC19 0.1 μg as a template, primers HLAMBS and HCLMR 50 pmole each, or HCLMS and HLAMB4 50 pmole each, 5 μ TaKaRa Ex Taq (Takara Shuzo), 100 μl reaction mixture, 50 μl mineral oil And then 30 times with a temperature cycle of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute.

  The PCR products HLAMBS-HCLMR (236 bp) and HCLMS-HLAMB4 (147 bp) were each electrophoresed on a 3% low melting point agarose gel, and then recovered and purified from the gel using GENECLEAN II Kit (BIO101). In the second PCR, 20 ng of the reaction mixture containing 40 ng of each purified DNA fragment and 1 U TaKaRa Ex Taq (Takara Shuzo) was used, and 25 μl of mineral oil was overlaid at 94 ° C. for 1 minute, at 60 ° C. for 1 minute, Five 1 minute temperature cycles at 72 ° C. were performed.

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

  The obtained DNA fragment (0.1 μg) was digested with EcoRI and then subcloned into BAP-treated plasmid pUC19ΔHind III. E. coli JM109 competent cells were transformed, cultured overnight in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN).

  The base sequence of the purified plasmid was determined with a 373A DNA sequencer (ABI) using M13 Primer M4 and M13 Primer RV (Takara Shuzo). The plasmid that was confirmed to have the correct base sequence without deletion was designated as Cλ / pUC19.

(iii) Construction of gene encoding human L chain κ chain constant region A DNA fragment encoding L chain κ chain C region was cloned from plasmid HEF-PM1k-gk (WO92 / 19759) by PCR. 394 The front primer HKAPS (SEQ ID NO: 19) synthesized using a DNA / RNA synthesizer (ABI) has EcoRI, Hind III and BlnI recognition sequences, and the rear primer HKAPA (SEQ ID NO: 20) has EcoRI recognition sequences. Designed.

  Plasmid HEF-PM1k-gk 0.1 μg used as a template, primers HKAPS and HKAPA 50 pmole each, 5 μl of TaKaRa Ex Taq (Takara Shuzo) were used, and 100 μl of a reaction mixture was used to overlay 50 μl of mineral oil. 30 cycles of reaction at 94 ° C. for 1 minute, 60 ° C. for 1 minute and 72 ° C. for 1 minute were performed. A 360 bp PCR product was electrophoresed on a 3% low melting point agarose gel, and then recovered from the gel and purified using GENECLEAN II Kit (BIO101).

  The obtained DNA fragment was digested with EcoRI and then cloned into BAP-treated plasmid pUC19ΔHindIII. E. coli JM109 competent cells were transformed, cultured overnight in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN). The base sequence of the purified plasmid was determined with a 373A DNA sequencer (ABI) using M13 Primer M4 and M13 Primer RV (Takara Shuzo). The plasmid confirmed to have the correct base 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. By linking a gene encoding the # 23-57-137-1 L chain V region to the Hind III and Bln sites immediately before the human antibody constant regions of plasmids Cλ / pUC19 and Cκ / pUC19, respectively, chimera # 23- The pUC19 vector encoding the 57-137-1 antibody L chain V region and the L chain λ chain or L chain κ chain constant region was constructed. The chimeric antibody L chain gene was excised by EcoRI digestion and subcloned into a HEF expression vector.

  That is, the # 23-57-137-1 antibody L chain V region was cloned from the plasmid MBC1L24 using the PCR method. Each primer was synthesized using a 394 DNA / RNA synthesizer (ABI). The rear primer MBCCHL1 (SEQ ID NO: 21) is a Hind III recognition sequence and Kozak sequence (Kozak, M. et al., J. Mol. Biol. 196, 947-950, 1987), the front primer MBCCHL3 (SEQ ID NO: 22) is BglII, It was designed to have an EcoRI recognition sequence.

PCR was performed using 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.2 mM dNTP, 0.1 μg MBC1L24, MBCCHL1 and MBCCHL3 as primers, 50 μmole each, 100 μl containing 1 μl AmpliTaq (PERKIN ELMER). The reaction mixture was used, and 50 μl of mineral oil was overlayered and carried out 30 times at a temperature cycle of 94 ° C. for 45 seconds, 60 ° C. for 45 seconds, and 72 ° C. for 2 minutes.

A 444 bp PCR product was electrophoresed on a 3% low-melting point agarose gel, recovered from the gel using GENECLEAN II kit (BIO101), purified, and dissolved in 10 mM Tris-HCl (pH 7.4) and 20 μl of 1 mM EDTA solution. . 1 μl of the PCR product is 37 ° C. in 20 μl of a reaction mixture containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, 50 mM NaCl, 8 U Hind III (Takara Shuzo) and 8 U EcoRI (Takara Shuzo), respectively. Digested for 1 hour. The digested mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation and dissolved in 8 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution.

  Similarly, 191 μg of plasmid pUC was digested with Hind III and 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 DNA was collected by ethanol precipitation, and then dissolved in 10 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution.

  1 μl of BAP-treated plasmid pUC19 and 4 μl of the previous PCR product were ligated using DNA LigationKit Ver.2 (Takara Shuzo), and transformed into E. coli JM109 competent cells (Nippon Gene) as described above. This was seeded on a 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. The plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN). After determining the base sequence, the plasmid having the correct base sequence was designated as CHL / pUC19.

1 μg each of plasmid Cλ / pUC19 and Cκ / pUC19 containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, 8 U Hind III (Takara Shuzo) and 2 U BlnI (Takara Shuzo) Digestion was performed in 20 μl of the mixture at 37 ° C. for 1 hour. The digested mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation, followed by BAP treatment at 37 ° C. for 30 minutes. The reaction solution was extracted with phenol and chloroform, and DNA was collected by ethanol precipitation and dissolved in 10 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution.

  8 μg of the plasmid CHL / pUC19 containing # 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, then recovered and purified from the gel using GENECLEAN II Kit (BIO101), and 10 mL of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution was used. Dissolved.

  4 μl of this L chain V region DNA was subcloned into 1 μl each of BAP-treated plasmid Cλ / pUC19 or Cκ / pUC19 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 the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN). These were designated as plasmid MBC1L (λ) / pUC19 and MBC1L (κ) / pUC19, respectively.

  Plasmids MBC1L (λ) / pUC19 and MBC1L (κ) / pUC19 were each digested with EcoRI, electrophoresed on a 3% low melting point agarose gel, and then a 743 bp DNA fragment was recovered from the gel using GENECLEANII Kit (BIO101). Purified and dissolved in 10 μl of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution.

  As an expression vector, 2.7 μg of plasmid HEF-PM1k-gk was digested with EcoRI, extracted with phenol and chloroform, and DNA was collected by ethanol precipitation. The recovered DNA fragment was subjected to BAP treatment and then electrophoresed on a 1% low melting point agarose gel. The 6561 bp DNA fragment was recovered and purified from the gel using GENECLEAN II Kit (BIO101), and 10 mM Tris-HCl (pH 7.4). Dissolved in 10 μl of 1 mM EDTA solution.

  2 μl of BAP-treated HEF vector was ligated with 3 μl each of the above plasmid MBC1L (λ) or MBC1L (κ) EcoRI 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 the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN).

The purified plasmid was obtained at 37 ° C. in 20 μl of a reaction mixture containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, 8 U Hind III (Takara Shuzo) and 2 U PvuI (Takara Shuzo). Digested for 1 hour. If the fragment is inserted in the correct direction, 5104/2195 bp, and if it is inserted in the reverse direction, a digested fragment of 4378/2926 bp is 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 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 carried out by electroporation using the GenePulser apparatus (BioRad) with the combination of plasmid MBC1HcDNA / pCOS1 and MBC1L (λ) / neo or MBC1HcDNA / pCOS1 and MBC1L (κ) / neo by electroporation. Seven cells were co-transduced. 10 μg of 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 PBS (−), and pulsed at a capacitance of 1,500 V and 25 μF. . After a 10 minute recovery period at room temperature, the electroporated cells are suspended in DMEM medium (GIBCO) containing 2% Ultra Low IgG fetal calf serum (GIBCO) and CO is used in a 10 cm culture dish. _The cells were cultured in ₂ incubators. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and the sample was subjected to ELISA. Further, purification of the chimeric antibody from the culture supernatant of COS-7 cells was performed using an AffiGel Protein 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. Each well of a 96-well plate for ELISA (Maxisorp, NUNC) was solid-phased with 100 μl of goat anti-human IgG antibody (TAGO) prepared to a concentration of 1 μg / ml with an immobilization buffer (0.1 M NaHCO ₃ , 0.02% NaN ₃ ) After blocking with 200 μl of dilution buffer (50 mM Tris-HCl, 1 mM MgCl ₂ , 0.1 M NaCl, 0.05% Tween 20, 0.02% NaN ₃ , 1% bovine serum albumin (BSA), pH 7.2) The culture supernatant of the expressed COS cells or purified chimeric antibody was serially diluted and added to each well. After incubating at room temperature for 1 hour and washing with PBS-Tween 20, 100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added. After incubating at room temperature for 1 hour and washing with PBS-Tween 20, 1 mg / ml of a substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, and then the absorbance at 405 nm was measured with a microplate reader (BioRad). It was measured. Hu IgG1λ Purified (The Binding Site) was used as a standard for concentration measurement.

(ii) Measurement of antigen binding ability An ElISA plate for antigen binding measurement was prepared as follows. Each well of the 96-well plate for ELISA was solid-phased with 100 μl of human PTHrP (1-34) (Peptide Institute) adjusted to a concentration of 1 μg / ml with a solid-phase buffer. After blocking with 200 μl of dilution buffer, the culture supernatant of COS cells expressing the chimeric antibody or purified chimeric antibody was serially diluted and added to each well. After incubating at room temperature and washing with PBS-Tween 20, 100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added. After incubation at room temperature and washing with PBS-Tween 20, 1 mg / ml substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, and then the absorbance at 405 nm was measured with a microplate reader (BioRad). . As a result, it was shown that the chimeric antibody has a binding ability to human PTHrP (1-34) and has the correct structure of the cloned mouse antibody V region (FIG. 5). Further, since the binding ability of the antibody to PTHrP (1-34) does not change even if the L chain C region is a λ chain or κ chain in the chimeric antibody, the L chain C region of the humanized antibody is human. It was constructed using a typed antibody L chain λ chain.

(6) Establishment of stable production cell line of CHO In order to establish a stable production cell line of the chimeric antibody, the expression plasmid was introduced into CHO cells (DXB11).

That is, a stable production cell line of a chimeric antibody is established by using a GenePulser device (BioRad) with a combination of expression plasmids for CHO cells MBC1HcDNA / pCHO1 and MBC1L (λ) / neo or MBC1HcDNA / pCHO1 and MBC1L (κ) / neo. CHO cells were co-transduced by poration. Each expression vector was cleaved with the restriction enzyme PvuI to obtain linear DNA, extracted with phenol and chloroform, and then recovered by ethanol precipitation and used for electroporation. 10 μg of 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 (−), and pulsed at a capacitance of 1,500 V and 25 μF. After a recovery period of 10 minutes at room temperature, the electroporated cells were suspended in MEM-α medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO) and three 96-well plates (Falcon) And cultured in a CO ₂ incubator. On the day after the start of culture, 10% fetal bovine serum (GIBCO) and 500 mg / ml GENETICIN (G418 Sulfate, GIBCO) were added, and the ribonucleoside and deoxyribonucleoside-free MEM-α medium (GIBCO) selection medium was replaced, and the antibody gene Introduced cells were selected. After exchanging the selective medium, the cells were observed under a microscope around two weeks, and after smooth cell growth was observed, the antibody production amount was measured by the antibody concentration measurement ELISA, and cells with a large antibody production amount were selected. .

  The culture of the established stable antibody-producing cell line was expanded, and mass culture was carried out using a roller bottle with 2% Ultra Low IgG fetal bovine serum added, and a ribonucleoside and deoxyribonucleoside-free MEM medium. On the 3rd to 4th day of culture, the culture supernatant was collected, and cell debris was removed with a 0.2 μm filter (Millipore).

  Purification of the chimeric antibody from the culture supernatant of CHO cells was performed using a POROS protein A column (PerSeptive Biosystems) with ConSep LC100 (Millipore) according to the attached recipe, and measurement of neutralizing activity and hypercalcemia model It was subjected to a medicinal effect test in animals. The concentration and antigen binding activity of the obtained purified chimeric antibody were measured by the above ELISA system.

[Reference Example 4]
Construction of humanized antibody
(1) Construction of humanized antibody heavy chain
(i) Construction of humanized H chain V region Humanized # 23-57-137-1 antibody H chain was prepared by CDR-grafting by PCR. Humanized # 23-57-137-1 antibody heavy chain with FR derived from human antibody S31679 (NBRF-PDB, Cuisinier AM et al., Eur. J. Immunol., 23, 110-118, 1993) Six PCR primers were used for the production of version "a"). CDR-grafting primers MBC1HGP1 (SEQ ID NO: 23) and MBC1HGP3 (SEQ ID NO: 24) have 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 has a complementary sequence of 15 to 21 bp on each end of the primer. External primers MBC1HVS1 (SEQ ID NO: 27) and MBC1HVR1 (SEQ ID NO: 28) have homology with CDR grafting primers MBC1HGP1 and MBC1HGP4.

  CDR-grafting primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4 were separated using urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989), and extraction from the gel was crushed and soaked. (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).

  That is, each 1 nmole of CDR-grafting primer was separated on a 6% denaturing polyacrylamide gel, and a DNA fragment of the desired size was identified by irradiating with ultraviolet rays on a silica gel thin layer plate, and then by the crash and soak method. It was recovered from the gel and dissolved in 20 μl of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution. For PCR, TaKaRa Ex Taq (Takara Shuzo) was used, and 100 μl of the reaction mixture was mixed with CDR-grafting primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4 prepared as described above in 1 μl, 0.25 mM dNTP and 2.5 U, respectively. 5 times in 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 TaKaRaEx Taq, and 50 pmole of external primers MBC1HVS1 and MBC1HVR1 In addition, the same temperature cycle was performed 30 times. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 4% Nu Sieve GTG agarose (FMC Bio. Products).

  A piece of agarose containing a 421 bp long DNA fragment was excised, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. The purified DNA was precipitated with ethanol, and then dissolved in 20 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution. The obtained PCR reaction mixture was subcloned into pUC19 prepared by digesting with BamHI and HindIII, and the nucleotide sequence was determined. The plasmid with the correct sequence was named hMBCHv / pUC19.

(Ii) Construction of H chain V region for humanized H chain cDNA In order to link with the cDNA of human H chain C region Cγ1, the humanized H chain V region constructed as described above was obtained by PCR. Qualified. The back primer MBC1HVS2 hybridizes with the sequence encoding the 5′-side of the leader region of the V region, and the Kozak consensus sequence (Kozak, M, et al., J. Mol. Biol. 196, 947-950, 1987), HindIII And designed to have an EcoRI recognition sequence. The forward primer MBC1HVR2 for the H chain V region is designed to hybridize to the DNA sequence encoding the 3′-side of the J region and to encode the sequence 5′-side of the C region and to have ApaI and SmaI recognition sequences did.

  PCR uses TaKaRa Ex Taq (Takara Shuzo), 0.4 μg hMBCHv / pUC19 as template DNA, MBC1HVS2 and MBC1HVR2 as primers, 50 pmole, 2.5 U TaKaRa ExTaq, 0.25 mM dNTP, respectively. Using a buffer solution, it was performed 30 times in a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 3% NuSieve GTG agarose (FMCBio. Products).

  A piece of agarose containing a 456 bp long DNA fragment was excised and purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. The purified DNA was precipitated with ethanol, and then dissolved in 20 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution. The obtained PCR reaction mixture was subcloned into pUC19 prepared by digesting with EcoRI and SmaI, and the nucleotide sequence was determined. A gene encoding the mouse H chain V region derived from the hybridoma # 23-57-137-1 thus obtained was contained, EcoRI and HindIII recognition sequence and Kozak sequence on the 5′-side, ApaI on the 3′-side and The plasmid having the SmaI recognition sequence was named hMBC1Hv / pUC19.

(2) Construction of humanized antibody H chain expression vector Plasmid RVh-PM1f-cDNA containing the hPM1 antibody H chain cDNA sequence was digested with ApaI and BamHI, and a DNA fragment containing the H chain C region was recovered. It was introduced into hMBC1Hv / pUC19 prepared by digestion with ApaI and BamHI. The thus prepared plasmid was designated as hMBC1HcDNA / pUC19. This plasmid contains the H chain V region and human H chain C region Cγ1 of the humanized # 23-57-137-1 antibody, and contains an EcoRI and HindIII recognition sequence at the 5′-end and a BamHI recognition sequence at the 3′-end. Have. The nucleotide sequence of the humanized H chain version “a” contained in the plasmid hMBC1HcDNA / pUC19 and the corresponding amino acid sequence are shown in SEQ ID NO: 58. The amino acid sequence of version a is shown in SEQ ID NO: 56.

  hMBC1HcDNA / pUC19 was digested with EcoRI and BamHI, and the resulting DNA fragment containing the heavy chain sequence was introduced into the expression plasmid pCOS1 prepared by digesting with EcoRI and BamHI. The thus obtained humanized antibody expression plasmid was designated as hMBC1HcDNA / pCOS1.

  Furthermore, in order to prepare a plasmid for use in expression in CHO cells, hMBC1HcDNA / pUC19 was digested with EcoRI and BamHI, and the resulting DNA fragment containing the heavy chain sequence was digested with EcoRI and BamHI. Introduced. The thus obtained humanized antibody expression plasmid was designated as hMBC1HcDNA / pCHO1.

(3) Construction of L chain hybrid variable region
(i) Preparation of FR1,2 / FR3,4 hybrid antibody An L chain gene was constructed by recombining the FR region of humanized antibody and mouse (chimeric) antibody, and each region for humanization was evaluated. . By using the restriction enzyme AflII cleavage site in CDR2, a hybrid antibody was prepared in which FR1 and 2 were derived from a human antibody and FR3 and 4 were derived from a mouse antibody.

10 μg each of plasmid MBC1L (λ) / neo and hMBC1L (λ) / neo were added to 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, 50 mM NaCl, 0.01% (w / v) BSA, AflII (Takara Shuzo) 10U Was digested at 37 ° C. for 1 hour in 100 μl of the reaction mixture. The reaction solution was electrophoresed on a 2% low-melting point agarose gel, and a 6282 bp fragment (referred to as c1) and a 1022 bp fragment (referred to as c2) from the plasmid MBC1L (λ) / neo, and a plasmid hMBC1L (λ) / neo to 6282 bp A fragment (designated h1) and a 1022 bp fragment (designated h2) were recovered from the gel and purified using GENECLEAN II Kit (BIO101).

  BAP treatment was performed on 1 μg of each of the collected c1 and h1 fragments. DNA was extracted with phenol and chloroform and recovered by ethanol precipitation, and then dissolved in 10 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution. 1 μl of c1 and h1 fragments treated with BAP were ligated to 4 μl of h2 and c2 fragments, respectively (4 ° C., overnight), and transformed into E. coli JM109 competent cells. The mixture was cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN).

The purified plasmid was treated at 37 ° C. in 20 μl of a reaction mixture containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, ApaLI (Takara Shuzo) 2 U, or BamHI (Takara Shuzo) 8 U, HindIII (Takara Shuzo) 1 Digested for hours. If c1-h2 was correctly ligated, a digested fragment of 5560/1246/498 bp with ApaLI and 7134/269 bp with BamHI / HindIII was generated, thereby confirming the plasmid.

  The expression vector encoding the human FR1,2 / mouse FR3,4 hybrid antibody L chain was designated as h / mMBC1L (λ) / neo. On the other hand, since a clone of h1-c2 was not obtained, it was cloned on the HEF vector after recombination on the pUC vector. In that case, humanized antibody L in which the tyrosine at position 91 (amino acid number 87 as defined by Kabat) in FR3 is substituted with isoleucine in plasmid hMBC1Laλ / pUC19 containing humanized antibody L chain V region without amino acid substitution. Plasmid hMBC1Ldλ / pUC19 containing the chain V region was used as a template.

10 μg each of plasmid MBC1L (λ) / pUC19, hMBC1Laλ / pUC19 and hMBC1Ldλ / pUC19 were added to 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, 50 mM NaCl, 0.01% (w / v) BSA, HindIII16U, AflII4U Digestion was carried out in 30 μl of the contained reaction mixture at 37 ° C. for 1 hour. The reaction solution was electrophoresed on a 2% low melting point agarose gel, and DNA fragments of plasmid MBC1L (λ) / pUC19 to 215 bp (c2 ′), plasmid hMBC1Laλ / pUC19 and hMBC1Ldλ / pUC19 were respectively 3218 bp (ha1 ′, hd1 ′). It was recovered from the gel and purified using GENECLEAN II Kit (BIO101).

  The ha1 ′ and hd1 ′ fragments were each ligated to the c2 ′ fragment and transformed into E. coli JM109 competent cells. The mixture was cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN). These were designated as plasmids m / hMBC1Laλ / pUC19 and m / hMBC1Ldλ / pUC19, respectively.

  The obtained plasmids m / hMBC1Laλ / pUC19 and m / hMBC1Ldλ / pUC19 were digested with EcoRI. Each 743 bp DNA fragment was electrophoresed on a 2% low-melting point agarose gel, then recovered and purified from the gel using GENECLEAN II Kit (BIO101), and dissolved in 20 μl of a 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution.

  4 μl of each DNA fragment was ligated to 1 μl of the aforementioned BAP-treated HEF vector and transformed into E. coli JM109 competent cells. The mixture was cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN).

Each purified plasmid was digested for 1 hour at 37 ° C. in 20 μl of a reaction mixture containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, HindIII (Takara Shuzo) 8 U, PvuI (Takara Shuzo) 2 U did. The plasmid was confirmed by generating a digested fragment of 5104/2195 bp if the fragment was inserted in the correct direction and 4378/2926 bp if it was inserted in the reverse direction. The expression vectors encoding the mouse FR1,2 / human FR3,4 hybrid antibody L chain were m / hMBC1Laλ / neo and m / hMBC1Ldλ / neo, respectively.

(ii) Preparation of FR1 / FR2 hybrid antibody A hybrid antibody of FR1 and FR2 was similarly prepared by using the SnaBI cleavage site in CDR1.

10 μg each of plasmid MBC1L (λ) / neo and h / mMBC1L (λ) / neo were added to 10 mM Tris-HCl (pH 7.9), 10 mM MgCl ₂ , 1 mM DTT, 50 mM NaCl, 0.01% (w / v) BSA , SnaBI (Takara Shuzo) digested in 20 μl of a reaction mixture containing 6 U at 37 ° C. for 1 hour. Next, it was digested at 37 ° C. for 1 hour in 50 μl of a reaction mixture containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, 0.01% (w / v) BSA, PvuI6U.

  After the reaction solution was electrophoresed on a 1.5% low melting point agarose gel, plasmids MBC1L (λ) / neo to 4955 bp (m1) and 2349 bp (m2), plasmids h / mMBC1L (λ) / neo to 4955 bp (hm1) and Each DNA fragment of 2349 bp (hm2) was recovered from the gel using GENECLEAN II Kit (BIO101), purified, and dissolved in 40 μl of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution.

  1 μl of m1 and hm1 fragments were ligated to 4 μl of hm2 and m2 fragments, respectively, and transformed into E. coli JM109 competent cells. The mixture was cultured in 2 ml of 2 × YT medium containing 50 μg / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN).

Each purified plasmid was digested for 1 hour at 37 ° C. in 20 μl of a reaction mixture containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT, ApaI (Takara Shuzo) 8U, or ApaLI (Takara Shuzo) 2U. .

  If each fragment is correctly ligated, digested fragment of 7304 bp with ApaI, 5560/1246/498 bp (m1-hm2) with ApaLI, 6538/766 bp with ApaI, 3535/2025/1246/498 bp (hm1-m2) with ApaLI As a result, the plasmid was confirmed. These are expressed as hmmMBC1L (λ) / neo, an expression vector encoding human FR1 / mouse FR2,3,4 hybrid antibody L chain, and hmHMBC1L, an expression vector encoding mouse FR1 / human FR2 / mouse FR3,4 hybrid antibody L chain, respectively. (Λ) / neo.

(4) Construction of humanized antibody L chain Humanized # 23-57-137-1 antibody L chain was prepared by CDR-grafting by PCR. Humans with FR1, FR2 and FR3 from human antibody HSU03868 (GEN-BANK, Deftos M et al., Scand. J. Immunol., 39, 95-103, 1994) and FR4 from human antibody S25755 (NBRF-PDB) Six PCR primers were used for the preparation of the typed # 23-57-137-1 antibody light chain (version "a").

  CDR-grafting primers MBC1LGP1 (SEQ ID NO: 29) and MBC1LGP3 (SEQ ID NO: 30) have a sense DNA sequence, and CDR grafting primers MBC1LGP2 (SEQ ID NO: 31) and MBC1LGP4 (SEQ ID NO: 32) have an antisense DNA sequence. And has a complementary sequence of 15 to 21 bp on each end of the primer. External primers MBC1LVS1 (SEQ ID NO: 33) and MBC1LVR1 (SEQ ID NO: 34) have homology with CDR grafting primers MBC1LGP1 and MBC1LGP4.

  CDR-grafting primers MBC1LGP1, MBC1LGP2, MBC1LGP3 and MBC1LGP4 were separated using urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989), and extraction from the gel was crushed and soaked. (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).

  That is, each 1 nmole of CDR-grafting primer was separated on a 6% denaturing polyacrylamide gel, and a DNA fragment of the desired size was identified by irradiating with ultraviolet rays on a silica gel thin layer plate, and then by the crash and soak method. It was recovered from the gel and dissolved in 20 μl of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA solution.

  For PCR, TaKaRa Ex Taq (Takara Shuzo) was used, and 100 μl of the reaction mixture was mixed with CDR-grafting primers MBC1LGP1, MBC1LGP2, MBC1LGP3 and MBC1LGP4 prepared as described above in 1 μl, 0.25 mM dNTP and 2.5 U, respectively. 5 times in 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 TaKaRa Ex Taq. Primers MBC1LVS1 and MBC1LVR1 were added and further reacted 30 times with 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 long DNA fragment was excised, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. The obtained PCR reaction mixture was subcloned into pUC19 prepared by digesting with BamHI and HindIII, and the nucleotide sequence was determined. The plasmid thus obtained was named hMBCL / pUC19. However, since the amino acid at position 104 (amino acid number 96 as defined by Kabat) of CDR4 was arginine, a modified primer MBC1LGP10R (SEQ ID NO: 35) for correcting this to tyrosine was designed and synthesized. PCR uses TaKaRa Taq (Takara Shuzo). In 100 μl of the reaction mixture, 0.6 μg of plasmid hMBCL / pUC19 as template DNA, MBC1LVS1 and MBC1LGP10R as primers are 50 pmole and 2.5 U of TaKaRa Ex Taq (Takara Shuzo) 0.25 mM, respectively. 50 μl of mineral oil was overlayered using the attached buffer under the conditions containing 2 dNTPs, and was performed 30 times in a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute. 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 long DNA fragment was excised, and the DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. The resulting PCR reaction mixture was subcloned into pUC19 prepared by digestion with BamHI and HindIII.

  As a result of determining the nucleotide sequence using the M13 Primer M4 primer and M13 Primer RV primer, the correct sequence was obtained. This plasmid was digested with HindIII and BlnI, and the 416 bp fragment was analyzed by 1% agarose gel electrophoresis. separated. The DNA fragment was purified using GENECLEAN II Kit (BIO101) according to the instructions attached to the kit. The obtained PCR reaction mixture was introduced into plasmid Cλ / pUC19 prepared by digesting with HindIII and BlnI, and named plasmid hMBC1Laλ / pUC19. This plasmid was digested with EcoRI, and a sequence containing a sequence encoding the humanized L chain was introduced into the plasmid pCOS1, so that the start codon of the humanized L chain was located downstream of the EF1α promoter. The plasmid thus obtained was designated as hMBC1Laλ / pCOS1. The nucleotide sequence (including the corresponding amino acid) of the humanized L chain version “a” is shown in SEQ ID NO: 66. The amino acid sequence of version a is shown in SEQ ID NO: 47.

  Version “b” was prepared using mutagenesis by PCR. Version “b” was designed so that glycine at position 43 (amino acid number 43 according to Kabat) was changed to proline and lysine at position 49 (amino acid position 49 according to Kabat) was changed to aspartic acid. PCR was performed using mutagen primer MBC1LGP5R (SEQ ID NO: 36) and primer MBC1LVS1 using plasmid hMBC1Laλ / pUC19 as a template, the resulting DNA fragment was digested with BamHI and HindIII, and subcloned into the BamHI and HindIII sites of pUC19. After determination of the nucleotide sequence, it was digested with restriction enzymes HindIII and AflII and ligated with hMBC1Laλ / pUC19 digested with HindIII and AflII.

  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 the plasmid pCOS1, and the start of the humanized L chain was downstream of the EF1α promoter. The codon was positioned. The plasmid thus obtained was designated as hMBC1Lbλ / pCOS1.

  Version “c” was prepared using mutagenesis by PCR. In version “c”, the serine at position 84 (amino acid number 80 according to the Kabat rule) was changed to proline. PCR was performed using mutagen primer MBC1LGP6S (SEQ ID NO: 37) and primer M13 Primer RV with plasmid hMBC1Laλ / pUC19 as a template, and the resulting DNA fragment was digested with BamHI and HindIII, and pUC19 prepared by digesting with BamHI and HindIII. Subcloned into

  After determination of the base sequence, it was digested with restriction enzymes BstPI and Aor51HI and ligated with hMBC1Laλ / pUC19 digested with BstPI and Aor51HI. The plasmid thus obtained was designated as hMBC1Lcλ / pUC19, this plasmid was digested with the restriction enzyme EcoRI, a sequence containing a sequence encoding the humanized L chain was introduced into the EcoRI site of the plasmid pCOS1, and humanized into the downstream of the EF1α promoter. The start codon of the L chain was located. The plasmid thus obtained was designated as hMBC1Lcλ / pCOS1.

  Versions “d”, “e” and “f” were prepared using PCR for mutagenesis. Each version was designed so that the tyrosine at position 91 (amino acid number 87 according to the Kabat rule) of the “a”, “b”, and “c” versions was changed to isoleucine in order. PCR was performed using the mutagens primer MBC1LGP11R (SEQ ID NO: 38) and primer M-S1 (SEQ ID NO: 44) using hMBC1Laλ / pCOS1, hMBC1Lbλ / pCOS1, and hMBC1Lcλ / pCOS1 as templates, and the resulting DNA fragment was digested with BamHI and HindIII. And subcloned into pUC19 prepared by digestion with BamHI and HindIII. After the nucleotide sequence was determined, it was digested with HindIII and BlnI, and ligated with Cλ / pUC19 prepared by digestion with HindIII and BlnI.

  The plasmids thus obtained were named hMBC1Ldλ / pUC19, hMBC1Leλ / pUC19, and hMBC1Lfλ / pUC19 in this order. These plasmids were digested with EcoRI, and a sequence containing a sequence encoding the humanized L chain was introduced into the EcoRI site of the plasmid pCOS1, so that the start codon of the humanized L chain was located downstream of the EF1α promoter. The plasmids thus obtained were named hMBC1Ldλ / pCOS1, hMBC1Leλ / pCOS1, and hMBC1Lfλ / pCOS1, respectively.

  Versions “g” and “h” were prepared using mutagenesis by PCR. Each version was designed so that the histidine at position 36 (amino acid position 36 as defined by Kabat) of the “a” and “d” versions was changed to tyrosine in order. PCR was performed using mutagen primers MBC1LGP9R (SEQ ID NO: 39) and M13 Primer RV as primers and hMBC1Laλ / pUC19 as a template, and the resulting PCR product and M13 Primer M4 as primers, and plasmid hMBC1Laλ / pUC19 as a template. Further PCR was performed. 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, PCR was performed using primers MBC1LGP13R (SEQ ID NO: 40) and MBC1LVS1 as primers. The obtained PCR fragments were digested with ApaI and HindII and introduced into plasmids hMBC1Laλ / pUC19 and hMBC1Ldλ / pUC19 digested with ApaI and HindIII. The nucleotide sequence was determined, and the plasmids containing the correct sequence were sequentially designated as hMBC1Lgλ / pUC19 and hMBC1Lhλ / pUC19. These plasmids were digested with restriction enzyme EcoRI, and the sequence containing the sequence encoding the humanized L chain was converted into the EcoRI site of plasmid pCOS1. The start codon of the humanized L chain was located downstream of the EF1α promoter. The plasmids thus obtained were named hMBC1Lgλ / pCOS1 and hMBC1Lhλ / pCOS1, respectively.

  Versions “i”, “j”, “k”, “l”, “m”, “n” and “o” were prepared using mutagenesis by PCR. PCR was performed using mutagen primer MBC1LGP14S (SEQ ID NO: 41) and primer VlRV (λ) (SEQ ID NO: 43) using plasmid hMBC1Laλ / pUC19 as a template, and the resulting DNA fragment was digested with ApaI and BlnI, and digested with ApaI and BlnI. Was subcloned into the plasmid hMBC1Lgλ / pUC19 prepared as described above. The nucleotide sequence was determined, and clones into which mutations corresponding to each version were introduced were selected. The plasmid thus obtained was designated as hMBC1Lxλ / pUC19 (x = i, j, k, 1, m, n, o), this plasmid was digested with EcoRI, and a sequence containing a sequence encoding the humanized L chain was expressed as plasmid pCOS1. The start codon of the humanized L chain was located downstream of the EF1α promoter. The plasmid thus obtained was designated as hMBC1Lxλ / pCOS1 (x = i, j, k, l, m, n, o). The nucleotide sequences (including the corresponding amino acids) of versions “j”, “l”, “m” and “o” are shown in SEQ ID NOs: 67, 68, 69 and 70, respectively. 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 the tyrosine at position 87 of the amino acid sequence of version “i”, “j”, “m”, “l” or “o”. Is replaced with isoleucine, and version “h” is replaced with version “i”, “j”, “m”, “l”, or “o” using the restriction enzyme Aor51MI cleavage site in FR3. It was produced by changing the connection. That is, in the expression plasmid hMBC1Lxλ / pCOS1 (x = i, j, m, l, o), CDR3 and a part of FR3 and Aor51HI fragment 514 bp containing FR4 were excluded, and in this expression plasmid hMBC1Lhλ / pCOS1, CDR3 and FR3 A tyrosine at position 91 (amino acid number 87 according to the Kabat rule) was made isoleucine by linking 514 bp of an Aor51HI fragment containing FR4 and a part of the fragment. The clones in which the tyrosine at position 91 (amino acid number 87 as defined by Kabat) in each version “i”, “j”, “m”, “l” and “o” was replaced with isoleucine Select the corresponding versions as “p”, “q”, “s”, “r” and “t”, respectively, and the resulting plasmid is hMBC1Lxλ / pCOS1 (x = p, q, s, r, t) Named. The nucleotide sequences (including corresponding amino acids) of versions “q”, “r”, “s” and “t” are shown in SEQ ID NOs: 71, 72, 73 and 74, respectively. The amino acid sequences of these versions are shown in SEQ ID NOs: 52, 53, 54, and 55, respectively.

  Plasmid hMBC1Lqλ / pCOS1 was digested with HindIII and EcoRI, subcloned into plasmid pUC19 digested with HindIII and EcoRI, and designated plasmid hMBC1Lqλ / pUC19. Table 2 shows the positions of the substituted amino acids in each version of the humanized L chain.

  In the table, Y is tyrosine, P is proline, K is lysine, V is valine, D is aspartic acid, and I is isoleucine.

  Escherichia coli having the plasmids hMBC1HcDNA / pUC19 and hMBC1Lqλ / pUC19 is Escherichia coli JM109 (hMBC1HcDNA / pUC19) and Escherichia coli JM109 (hMBC1Lqλ / pUC19). 1-chome, 1-address 1 center 6), on August 15, 1996, for Escherichia coli JM109 (hMBC1HcDNA / pUC19), FERM BP-5629, Escherichia coli JM109 (hMBC1Lqλ / pUC19) for FER30 Deposited internationally 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 humanized # 23-57-137-1 antibody, the expression plasmid was transiently expressed in COS-7 cells. It was expressed in sex. That is, in transient expression of the L chain hybrid antibody, plasmids hMBC1HcDNA / pCOS1 and h / mMBC1L (λ) / neo, hMBC1HcDNA / pCOS1 and m / hMBC1Laλ / neo, hMBC1HcDNA / pCOS1 and m / hMBC1Ldλ / neo, hMBC1HcDNA COS-7 cells were co-transfected by electroporation with hmmMBC1L (λ) / neo or a combination of hMBC1HcDNA / pCOS1 and mhmmBC1L (λ) / neo using a GenePulser device (BioRad). 10 μg of 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 PBS (−), and pulsed at a capacitance of 1,500 V and 25 μF. Gave. After a recovery period of 10 minutes at room temperature, the electroporated cells were suspended in DMEM culture medium (GIBCO) containing 2% Ultra Low IgG fetal calf serum (GIBCO) and used in a 10 cm culture dish. The cells were cultured in a CO ₂ incubator. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and the sample was subjected to ELISA.

  In the transient expression of the humanized # 23-57-137-1 antibody, any combination of the plasmid hMBC1HcDNA / pCOS1 and hMBC1Lxλ / pCOS1 (x = at) was used using the GenePulser apparatus (BioRad), COS-7 cells were transfected by the same method as for the hybrid antibody, and the resulting culture supernatant was subjected to ELISA.

  In addition, purification of the hybrid antibody or humanized antibody from the culture supernatant of COS-7 cells was performed using an AffiGel 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. 100 μl of goat anti-human IgG antibody (TAGO) in which each well of a 96-well plate for ELISA (Maxisorp, NUNC) was prepared to a concentration of 1 μg / ml with an immobilization buffer (0.1 M NaHCO ₃ , 0.02% NaN ₃ ) 200 μl of dilution buffer (50 mM Tris-HCl, 1 mM MgCl ₂ , 0.1 M NaCl, 0.05% Tween 20, 0.02% NaN ₃ , 1% bovine serum albumin (BSA), pH 7.2) After blocking, the culture supernatant of COS-7 cells expressing hybrid antibody or humanized antibody or purified hybrid antibody or humanized antibody was serially diluted and added to each well. After incubating at room temperature for 1 hour and washing with PBS-Tween 20, 100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added. After incubating at room temperature for 1 hour and washing with PBS-Tween 20, 1 mg / ml of a substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, and then the absorbance at 405 nm was measured with a microplate reader (BioRad). It was measured. Hu IgG1λ Purified (The Binding Site) was used as a standard for concentration measurement.

(ii) Measurement of antigen binding ability An ELISA plate for measuring antigen binding was prepared as follows. Each well of the 96-well plate for ELISA was immobilized with 100 μl of human PTHrP (1-34) prepared at a concentration of 1 μg / ml with a solid phase buffer. After blocking with 200 μl of dilution buffer, the culture supernatant of COS-7 cells expressing hybrid antibody or humanized antibody or purified hybrid antibody or humanized antibody was serially diluted and added to each well. After incubating at room temperature and washing with PBS-Tween 20, 100 μl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO) was added. After incubation at room temperature and washing with PBS-Tween 20, 1 mg / ml substrate solution (Sigma 104, p-nitrophenyl phosphate, SIGMA) was added, and then the absorbance at 405 nm was measured with a microplate reader (BioRad). .

(7) Activity confirmation
(i) Evaluation of humanized H chain The antibody combining the humanized H chain version “a” and the chimeric L chain was equivalent in PTHrP binding ability to the chimeric antibody (FIG. 6). This result shows 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) FR1,2 / FR3,4 hybrid antibody No activity was observed when the L chain was h / mMBC1L (λ). However, in the case of m / hMBC1Laλ or m / hMBC1Ldλ, the chimera # 23 The binding activity was equivalent to that of the -57-137-1 antibody (FIG. 7). These results suggest that FR3 and 4 have no problem as humanized antibodies, but there are amino acid residues to be substituted in FR1 and FR2.

(ii-b) FR1 / FR2 hybrid antibody No activity was observed when the L chain was mhmmBC1L (λ), but in the case of hmmMBC1L (λ), binding equivalent to that of the chimeric # 23-57-137-1 antibody Activity was shown (FIG. 8). These results suggest that FR1 of FR1 and FR2 has no problem as a humanized antibody, but there is an amino acid residue to be substituted in FR2.

(iii) Activity of humanized antibody Antigen binding activity was measured for a humanized antibody using each one of versions "a" to "t" as the L chain. As a result, the humanized antibody having the L chain versions “j”, “l”, “m”, “o”, “q”, “r”, “s”, “t” is equivalent to the chimeric antibody PTHrP. The binding ability was shown (FIGS. 9-12).

(8) Establishment of stable production cell line of CHO In order to establish a stable production cell line of humanized antibody, the expression plasmid was introduced into CHO cells (DXB11).

That is, the stable production of a humanized antibody cell line was established by combining the expression plasmids hMBC1HcDNA / pCHO1 and hMBC1Lmλ / pCOS1 or hMBC1HcDNA / pCHO1 and hMBC1Lqλ / pCOS1 or hMBC1HcDNA / pCHO1 and hMBC1Lrλs / pCnePerul with the CHO cell expression plasmid, Were cotransduced into CHO cells by electroporation. Each expression vector was cleaved with a restriction enzyme PvuI to obtain linear DNA, and after phenol and chloroform extraction, the DNA was recovered by ethanol precipitation and used for electroporation. 10 μg of 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 (−), and pulsed at a capacitance of 1,500 V and 25 μF. . After a recovery period of 10 minutes at room temperature, the electroporated cells were suspended in MEM-α medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO), and CO using a 96-well plate (Falcon). _The cells were cultured in ₂ incubators. The day after the start of culture, 10% fetal bovine serum (GIBCO) and 500 mg / ml GENETICIN (G418 Sulfate, GIBCO) were added, and the ribonucleoside and deoxyribonucleoside-free MEM-α medium (GIBCO) was replaced with a selective medium. Introduced cells were selected. After exchanging the selective medium, the cells were observed under a microscope around 2 weeks, and after normal cell growth was observed, the amount of antibody produced was measured by the antibody concentration measurement ELISA, and cells with high antibody producing ability were selected. .

  The culture of the established stable antibody-producing cell line was expanded, and mass culture was carried out using a roller bottle with 2% Ultra Low IgG fetal bovine serum added and ribonucleoside and deoxyribonucleoside-free MEM-α medium. On the 3rd to 4th day of 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 is performed using a POROS protein A column (PerSeptive Biosystems) with ConSepLC100 (Millipore) according to the attached instructions, measuring neutralizing activity and hypercalcemia It used for the drug efficacy test with a model animal. The concentration and antigen binding activity of the purified humanized antibody obtained were measured by the above ELISA system.

[Reference Example 5]
Measurement of neutralizing activity The neutralizing activity of mouse antibody, chimeric antibody and humanized antibody was measured using rat osteosarcoma cell line ROS17 / 2.8-5 cells. That is, ROS17 / 2.8-5 cells were cultured in a Ham 2 SF-12 medium (GIBCO) containing 10% fetal calf serum (GIBCO) in a CO ₂ incubator. ROS17 / 2.8-5 cells were seeded in 96-well plates at 10 ⁴ cells / 100 μl / well and cultured for 1 day, and replaced with Ham'SF-12 medium (GIBCO) containing 4 mM hydrocortisone and 10% fetal calf serum. To do. After further culturing for 3 to 4 days, the cells were washed with 260 μl of Ham'SF-12 medium (GIBCO), and 1 mM isobutyl-1-methylxanthine (IBMX, SIGMA), 10% fetal bovine serum and 10 mM HEPES were added. 80 μl of Ham'sF-12 containing was added and incubated for 30 minutes at 37 ° C.

  Mouse antibody, chimeric antibody or humanized antibody to be measured for neutralizing activity was previously added in groups of 10 μg / ml, 3.3 μg / ml, 1.1 μg / ml and 0.37 μg / ml, 10 μg / ml, 2 μg / ml. , 0.5 μg / ml and 0.01 μg / ml, or 10 μg / ml, 5 μg / ml, 1.25 μg / ml, 0.63 μg / ml and 0.31 μg / ml An equal amount of PTHrP (1-34) prepared in ml was mixed, and 80 μl of a mixed solution of each antibody and PTHrP (1-34) was added to each well. The final concentration of each antibody is one fourth of the antibody concentration, and the concentration of PTHrP (1-34) is 1 ng / ml. After treating at room temperature for 10 minutes, the culture supernatant is discarded, washed with PBS three times, and intracellular cAMP is extracted with 100 μl of 0.3% hydrochloric acid 95% ethanol. Ethanol hydrochloride was evaporated with a water aspirator, 120 μl of EIA buffer attached to cAMP EIA kit (CAYMANCHEMICAL'S) was added to extract cAMP, and cAMP was measured according to the prescription attached to cAMP EIA kit (CAYMANCHEMICAL'S). As a result, humanized antibodies having versions “q”, “r”, “s” and “t” in which the 91-position tyrosine is replaced with isoleucine among the L chain versions having the same antigen binding as the chimeric antibody are obtained. The neutralizing ability close to that of the chimeric antibody was exhibited, and among them, version “q” showed the strongest neutralizing ability (FIGS. 13 to 15).

  INDUSTRIAL APPLICABILITY According to the present invention, a cachexia therapeutic agent containing a substance that inhibits binding between a parathyroid hormone-related peptide and its receptor as an active ingredient is provided.

  The above-mentioned substance is useful as a cachexia therapeutic agent because it suppresses body weight loss in comparison with a control in a medicinal effect test in a cachexia model animal and also has an effect of extending the survival time.

It is a figure which shows the therapeutic effect with respect to the cachexia of an anti- PTHrP antibody. It is a figure which shows the therapeutic effect with respect to the cachexia of an anti- PTHrP antibody. It is a figure which shows the therapeutic effect with respect to the cachexia of an anti- PTHrP antibody. It is a figure which shows the therapeutic effect with respect to the cachexia of an anti- PTHrP antibody. It is a figure which shows the measurement result of antibody binding activity. It is a figure which shows the measurement result of antibody binding activity. It is a figure which shows the measurement result of antibody binding activity. It is a figure which shows the measurement result of antibody binding activity. It is a figure which shows the measurement result of antibody binding activity. It is a figure which shows the measurement result of antibody binding activity. It is a figure which shows the measurement result of antibody binding activity. It is a figure which shows the measurement result of antibody binding activity. It is a figure which shows the neutralization activity of a humanized antibody. It is a figure which shows the neutralization activity of a humanized antibody. It is a figure which shows the neutralization activity of a humanized antibody. It is a figure which shows the therapeutic effect with respect to the cachexia of a humanized antibody. It is a figure which shows the therapeutic effect with respect to the cachexia of a humanized antibody. It is a figure which shows the therapeutic effect with respect to the cachexia of a humanized antibody. It is a figure which shows the therapeutic effect with respect to the cachexia of a humanized antibody.

  SEQ ID NOs: 1-44: Synthetic DNA

Claims (13)

  A cachexia treatment agent comprising an antibody against a parathyroid hormone-related peptide as an active ingredient.   The cachexia therapeutic agent according to claim 1, wherein the antibody against the parathyroid hormone-related peptide is humanized or chimerized. The cachexia therapeutic agent according to claim 2, wherein the humanized antibody comprises at least one of the following (a) to (c).
(A) an L chain V region having any one of the amino acid sequences represented by SEQ ID NOs: 48 to 51,
(B) an L chain V region having any one of the amino acid sequences represented by SEQ ID NOs: 52 to 55,
(C) H chain V region having the amino acid sequence shown in SEQ ID NO: 56   The cachexia therapeutic agent according to claim 2 or 3, wherein the humanized antibody comprises an L chain V region having any one of the amino acid sequences shown in SEQ ID NOs: 48 to 51.   The cachexia therapeutic agent according to claim 2 or 3, wherein the humanized antibody comprises an L chain V region having any one of the amino acid sequences shown in SEQ ID NOs: 52 to 55.   The cachexia therapeutic agent according to claim 2 or 3, wherein the humanized antibody comprises an H chain V region having the amino acid sequence represented by SEQ ID NO: 56.   The humanized antibody comprises an L chain V region having any one of the amino acid sequences shown in SEQ ID NOs: 48 to 55 and an H chain V region having the amino acid sequence shown in SEQ ID NO: 56. The cachexia therapeutic agent of any one of -6.   The cachexia therapeutic agent according to any one of claims 2 to 7, wherein the humanized antibody further comprises an L chain C region and / or an H chain C region of a human antibody.   The cachexia therapeutic agent according to any one of claims 2 to 8, wherein the humanized antibody is a humanized # 23-57-137-1 antibody.   The cachexia therapeutic agent of any one of Claims 1-9 whose antibody with respect to a parathyroid hormone related peptide is an antibody fragment and / or its modification.   The cachexia therapeutic agent of any one of Claims 1-10 whose antibody with respect to a parathyroid hormone related peptide is a monoclonal antibody. Antibodies against parathyroid hormone related peptides
(a) effective in the treatment of cachexia,
(b) The cachexia treatment agent according to any one of claims 1 to 11, which does not significantly affect the growth of human oral floor cancer OCC-1 cells.   The cachexia therapeutic agent according to any one of claims 1 to 12, wherein the cachexia is derived from cancer.

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