JPH1192399A - Myeloma medicine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine capable of exhibiting an excellent cell-damaging activity against myeloma cells by including an antibody specifically acting on a protein having a specific amino acid sequence. SOLUTION: This myeloma medicine contains as an active ingredient an antibody (preferably a monoclonal antibody) that specifically binds to (A1 ) a protein having (A') an amino acid sequence of the formula or (A2 ) a protein comprising the protein A1 in which one or more (e.g. one to several) amino acids are substituted, deleted or inserted and which has a pre cell B proliferation-supporting ability, and further that has an activity to damage cells such as antibody-dependent cellular cells. The medicine is preferably administered at a dose of 0.01-100 mg/kg body as the active ingredient by a parenteral administration method such as an intravenous, intramuscular, intraabdominal or hypodermic injection method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a therapeutic agent for myeloma comprising, as an active ingredient, an antibody that specifically binds to a BST-2 protein having the amino acid sequence shown in SEQ ID NO: 1.

[0002]

2. Description of the Related Art A living body has a complex mechanism for protecting an individual from exogenous microorganisms or foreign substances, that is, an immune mechanism. B cells play a major role in the immune system.Hematopoietic stem cells differentiate into mature B cells via pro-B cells and pre-B cells by stimulation of various cytokines in the bone marrow, and are activated by antigen stimulation. And
The end result is plasma cells that produce and secrete antibodies. It is known that stromal cells (supporting cells) in the bone marrow are deeply involved in the differentiation and proliferation of B cells. That is, direct adhesion between B cells and bone marrow stromal cells
It has been reported that it is deeply involved in cell differentiation and proliferation (Miyake, K. et al., J. Exp. Med., (1991) 173,
599-607).

[0003] Myeloma is a neoplastic disease in which plasma cells (myeloma cells) that produce and secrete immunoglobulin are mainly increased in the bone marrow in a monoclonal manner. Monoclonal immunoglobulin or its component is present in serum or urine. L chain, H chain, etc. are detected (Masaaki Kosaka et al.,
Japanese clinical practice (1995) 53, 91-99). Myeloma is a disease in which plasma cells, which are the final stage of B cell differentiation, proliferate monoclonally.Therefore, similar to normal B cells, proliferation of myeloma cells involves interaction with bone marrow stromal cells. Conceivable. It is thought that adhesion factors such as CD21, VLA-5 and MPC-1 are involved in the adhesion between myeloma cells and bone marrow stromal cells (Michio Kono, Clinical Science (1996) 32, 703-710). ) And other factors are still being studied.

On the other hand, Ishikawa et al. Have reported monoclonal antibodies (RS38, RS38) obtained by immunizing mice with rheumatoid patient synovial cells.
38-E) (Ishikawa, J. et al., GENOMIC
S (1995) 26, 527-534). Ishikawa et al. Used this antibody to isolate and identify the antigen BST-2 recognized by this antibody (Ishikaw
a, J. et al., GENOMICS (1995) 26, 527-534). In addition, Ishikawa and colleagues reported in this paper that BST-2 was expressed on bone marrow stromal cells and that BST-2
States that it may be involved in B cell proliferation.

[0005]

In recent years, as a result of the use of various chemotherapy and interferon therapy for the treatment of myeloma, the temporary antitumor effect has been considerably increased. There has been little progress in the survival of tumor patients in recent decades, at 30-40 months. Accordingly, there is a need for innovative therapeutic agents or treatments that lead to remission of myeloma and prolong the survival of patients. An object of the present invention is to provide a new therapeutic agent for myeloma.

[0006]

Means for Solving the Problems In order to achieve the intended purpose, the present inventors have developed Northern blot analysis using a BST-2 probe or FCM (flow cyto memory) using an anti-BST-2 antibody. As a result of repeated in vitro studies such as analysis and measurement of cytotoxic activity such as CDC activity, it was found that BST-2 is strongly expressed at the gene and protein levels in myeloma cells. The inventors have found that the BST-2 antibody has cytotoxic activity against myeloma cells, and have completed the present invention.

Accordingly, the present invention relates to an antibody which specifically binds to the amino acid sequence shown in SEQ ID NO: 1 or a protein having an amino acid sequence modified with respect to this amino acid sequence and preferably has a cytotoxic activity as an active ingredient. And a therapeutic agent for myeloma. Said antibody is preferably a monoclonal antibody. The cytotoxic activity includes ADCC activity or CDC activity. The modified amino acid sequence refers to the amino acid sequence of SEQ ID NO: 1 which has been modified by substitution, deletion and / or insertion of one or more, for example, one to several amino acids, and It means an amino acid sequence capable of supporting growth.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION

1. Preparation of antibody 1-1. Preparation of hybridoma The hybridoma producing the antibody used in the present invention is:
Basically, it can be manufactured as follows using a known technique. That is, BST-2 protein or a cell expressing BST-2 is used as a sensitizing antigen, and immunized according to a usual immunization method, and the obtained immunocytes are compared with known parent cells by a normal cell fusion method. It can be produced by fusing and screening monoclonal antibody-producing cells by a conventional screening method.

Specifically, a monoclonal antibody may be prepared as follows. For example, BST-2 expressing cells which are sensitizing antigens for obtaining antibodies include SynSV6-14 derived from synovial cells of rheumatic patients (Ishikawa, J. et al., GEN.
OMICS (1995) 26, 527-534) can be used. Further, a protein having the amino acid sequence shown in SEQ ID NO: 1 or a peptide or polypeptide containing an epitope recognized by an anti-BST-2 antibody can be used as the sensitizing antigen.

[0010] The cDNA of the protein having the amino acid sequence shown in SEQ ID NO: 1 used as the sensitizing antigen is pUC19.
The plasmid is inserted between the XbaI cleavage site of the vector and the plasmid
It has been prepared as pRS38-pUC19. This plasmid pR
E. coli containing S38-pUC19 was introduced in 1993 (1993).
Year) On October 5th, the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Escherichia coli DH5a (pRS38-pUC19))
It has been deposited internationally under accession number FERM BP-4434 under the Budapest Treaty (see Japanese Patent Application Laid-Open No. Hei 7-96694). Using the cDNA fragment contained in the plasmid pRS38-pUC19, a peptide or polypeptide containing an epitope recognized by the anti-BST-2 antibody can be prepared by a genetic engineering technique.

As a mammal immunized with a sensitizing antigen,
Although not particularly limited, selection is preferably made in consideration of compatibility with a parent cell used for cell fusion, and rodent animals such as mice, rats, and hamsters are generally used. Is done. Immunization of an animal with a sensitizing antigen is performed according to a known method. For example, as a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and then mixed with an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. Preferably, it is administered to the mammal several times every 4-21 days. In addition, a suitable carrier can be used at the time of immunization of the sensitizing antigen.

After immunization in this way and confirming that the level of the desired antibody in the serum increases, immune cells are removed from the mammal and subjected to cell fusion. Preferred immune cells to be subjected to cell fusion particularly include spleen cells. Mammalian myeloma cells as the other parent cells to be fused with the immune cells are already known in various cell lines, for example, P3X63Ag8.653 (J. Immnol. (1979) 123: 1).
548-1550), P3X63Ag8U.1 (Current Topics in Microb
iology and Immunology (1978) 81: 1-7), NS-1 (Kohl
er.G. and Milstein, C. Eur. J. Immunol. (1976) 6:
511-519), MPC-11 (Margulies. DH et al., Cell
(1976) 8: 405-415), SP2 / 0 (Shulman, M. et al.,
Nature (1978) 276: 269-270), FO (de St. Groth, S.
F. et al., J. Immunol.Methods (1980) 35: 1-21
), S194 (Trowbridge, ISJ Exp. Med. (1978) 1
48: 313-323), R210 (Galfre, G. et al., Nature (197
9) 277: 131-133) etc. are used as appropriate.

The cell fusion of the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymo).
l. (1981) 73: 3-46). More specifically, the cell fusion is carried out, for example, in a normal nutrient culture in the presence of a cell fusion promoter. Examples of the fusion promoter include polyethylene glycol (PEG),
Sendai virus (HVJ) or the like is used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency.

The ratio of the use of the immune cells and the myeloma cells is preferably, for example, 1 to 10 times the number of the immune cells to the myeloma cells. As a culture solution used for the cell fusion, for example, RP suitable for growth of the myeloma cell line
MI1640 culture solution, MEM culture solution, and other normal culture solutions used for this type of cell culture can be used.
A serum replacement solution such as fetal calf serum (FCS) can also be used in combination. For cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution preliminarily heated to about 37 ° C., for example, a PEG solution having an average molecular weight of about 1,000 to 6,000 is usually used. At a concentration of 30-60% (w / v),
The desired fused cells (hybridomas) are formed by mixing. Subsequently, by repeatedly adding an appropriate culture solution and centrifuging to remove the supernatant, a cell fusion agent or the like unfavorable for hybridoma growth can be removed.

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

In addition to obtaining the above-mentioned hybridomas by immunizing non-human animals with an antigen, human lymphocytes are
Sensitized with BST-2 or BST-2 expressing cells, and sensitized lymphocytes are fused with human myeloma cells, for example, U266, and BST-2
Alternatively, a desired human antibody having a binding activity to BST-2 expressing cells can be obtained (see Japanese Patent Publication No. 1-59878). Furthermore, BST-2 or BST-2 expressing cells serving as an antigen may be administered to a transgenic animal having the entire repertoire of human antibody genes to obtain a desired human antibody according to the method described above (International Patent Application Publication Number WO 93/12227, WO
92/03918, WO94 / 02602, WO 94/25585, WO 96/34096
, WO 96/33735).

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

Specifically, the production of hybridomas producing anti-BST-2 antibody was carried out by the method of Ishikawa, J. et al.
GENOMICS (1995) 26, 527-534). A method for purifying an anti-BST-2 antibody from the ascites fluid by injecting the anti-BST-2 antibody-producing hybridoma into the intraperitoneal cavity of a BALB / c mouse (manufactured by CLEA Japan) and a method for purifying the hybridoma from a suitable medium, for example, , 10% fetal bovine serum, 5%
RPMI16 containing BM-Condimed H1 (Boehringer Mannheim)
40 medium, hybridoma SFM medium (GIBCO-BRL), PFH
Culture can be performed in an M-II medium (manufactured by GIBCO-BRL) or the like, and the anti-BST-2 antibody can be purified from the culture supernatant.

1-2. Recombinant Antibody In the present invention, as a monoclonal antibody, an antibody gene is cloned from a hybridoma, inserted into an appropriate vector, introduced into a host, and produced using a gene recombination technique. Recombinant antibodies can be used (eg, Carl, AK Borrebaeck, James, W. Larric
k, TERAPEUTIC MONOCLONAL ANTIBODIES, Published in
the United Kingdom by MACMILLAN PUBLISHERS LTD, 19
90).

Specifically, from the hybridoma producing the desired antibody, m encoding the variable (V) region of the antibody is obtained.
Isolate the RNA. mRNA isolation can be performed by known methods, for example,
Guanidine ultracentrifugation (Chirgwin, JM et al., Biochemist
ry (1979) 18, 5294-5299), the AGPC method (Chmczynski, P. et al.
And (1987) 162, 156-159).
Using mRNA Purification Kit (Pharmacia) etc.
Prepare mRNA. Also, QuickPrep mRNA Purification
MRNA can be directly prepared by using Kit (Pharmacia).

An antibody is obtained from the obtained mRNA by using a reverse transcriptase.
Synthesize V region cDNA. cDNA synthesis is performed using AMV Reverse
Transcriptase First-strand cDNA Synthesis Kit can be used. To synthesize and amplify cDNA, 5'-Ampli FINDERRACE Kit (Clontech) and 5'-RACE method using PCR (Frohman, MA et al., Pr.
oc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Be
lyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919
-2932) can be used. A desired DNA fragment is purified from the obtained PCR product and ligated to a vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. The base sequence of the target DNA can be determined by a known method,
For example, it is confirmed by the deoxy method.

DNA encoding the V region of the desired antibody
Is obtained, this is ligated to DNA encoding the desired antibody constant region (C region), and this is inserted into an expression vector. Alternatively, the DNA encoding the V region of the antibody is
It may be incorporated into an expression vector containing the DNA of the region. In order to produce the 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, for example, an enhancer or a promoter, as described below. Next, host cells can be transformed with the expression vector to express the antibody.

1-3. Expression and Production The antibody gene constructed as described above can be expressed and obtained by a known method. For mammalian cells,
It can be expressed by a useful useful promoter commonly used, an antibody gene to be expressed, a DNA having a polyA signal operably linked to its 3 ′ downstream, or a vector containing the DNA. For example, as the promoter / enhancer, human cytomegalovirus immediate early pr
omoter / enhancer).

Other promoters / enhancers that can be used for the expression of antibodies used in the present invention include retroviruses, polyomaviruses, adenoviruses, and the like.
Viral promoter / enhancer such as Simian virus 40 (SV40) and human elongation factor 1
A promoter / enhancer derived from a mammalian cell such as α (HEF1α) may be used. For example, when using the SV40 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) can be easily implemented.

In the case of Escherichia coli, expression can be carried out by operably linking a commonly used useful promoter, a signal sequence for antibody secretion, and an antibody gene to be expressed.
For example, as the promoter, lacz promoter, araB
Promoters can be mentioned. When using the lacz promoter, the method of Ward et al. (Nature (1098) 341,54
4-546; FASEB J. (1992) 6, 2422-2427), when using the araB promoter, the method of Better et al. (Science (198
8) 240, 1041-1043). As a signal sequence for antibody secretion, a pelB signal sequence (Lei, SP et al J. Ba
cteriol. (1987) 169, 4379). After separating the antibody produced in the periplasm, the structure of the antibody is appropriately refolded and used (for example,
See WO96 / 30394).

The origin of replication includes SV40, polyoma virus, adenovirus, bovine papilloma virus (BP
V) and the like, and the expression vector can be selected as an aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine as a selection marker for gene copy number amplification in a host cell system. Phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr)
It can contain genes and the like. For the production of the antibodies used in the present invention, any production system can be used. Production systems for antibody production are in vitro and in vivo.
There is a production system for o. Examples of the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells.

When eukaryotic cells are used, there are production systems using animal cells, plant cells, and fungal cells. Animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, (2)
Amphibian cells, for example, Xenopus oocytes, or (3) insect cells, for example, sf9, sf21, Tn5 and the like are known. Nicotiana (Nico
genus, for example, Nicotian Tabacum
a tabacum) -derived cells are known and may be callus cultured. As fungal cells, yeasts such as Saccharomyces, for example, Saccharomyces cerevisiae, and filamentous fungi, for example, Aspergillus, for example, Aspergillus niger are known.

When a prokaryotic cell is used, there is a production system using a bacterial cell. Bacterial cells include E. coli
), Bacillus subtilis are known. An antibody can be obtained by introducing a desired antibody gene into these cells by transformation and culturing the transformed cells in vitro. Culture is performed according to a known method. For example, DMEM, MEM, RPMI1640, IMDM can be used as a culture solution, and a serum supplement such as fetal calf serum (FCS) can be used in combination. In addition, antibodies may be produced in vivo by transferring the cells into which the antibody gene has been introduced into the peritoneal cavity or the like of the animal.

On the other hand, examples of the in vivo production system include a production system using animals and a production system using plants.
When using animals, there are production systems using mammals and insects. Goats can be used as mammals. Silkworms can be used as insects. When using a plant, for example, tobacco can be used.

The antibody gene is introduced into these animals or plants, and the antibodies are produced in the animals or plants and collected. For example, the antibody gene is prepared as a fusion gene by inserting the antibody gene into a gene such as goat β casein, which encodes a protein uniquely produced in milk. A DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat. A desired antibody is obtained from milk produced by a transgenic goat born from a goat that has received an embryo or a progeny thereof. Hormones may optionally be used in the transgenic goat 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).

When using silkworms, the silkworms are infected with a baculovirus into which the desired antibody gene has been inserted,
The desired antibody is obtained from the body fluid of the silkworm (Susumu, Me.
t al., Nature (1985) 315, 592-594). Furthermore, when tobacco is used, the desired antibody gene is inserted into a plant expression vector, for example, pMON530, and this vector is
Introduce into bacteria such as erium tumefaciens. The bacteria are infected with tobacco, for example, Nicotiana tabacum, and the desired antibody is obtained from the leaves of this tobacco (Julian,
K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-1
38).

As described above, the term "host" in the present invention includes cells in these in vitro production systems, and animals and plants in in vivo production systems. When producing antibodies in an in vitro or in vivo production system, it is possible to co-transform the host by separately incorporating the DNAs encoding the antibody heavy chain (H chain) or light chain (L chain) into an expression vector. Alternatively, the host may be transformed by incorporating the DNAs encoding the H and L chains into a single expression vector (see International Patent Application Publication No. WO 94-11523).

The antibody obtained as described above can be bound to various molecules such as polyethylene glycol (PEG) and used as a modified antibody. The “antibody” referred to in the claims of the present application also includes these modified antibodies. Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.

2. Separation and Purification of Antibodies 2-1. Separation and Purification of Antibodies Antibodies produced and expressed as described above can be separated from inside and outside cells and from hosts and purified to homogeneity. The antibody used in the present invention can be separated and purified by affinity chromatography. Columns used for affinity chromatography include, for example, a protein A column, a protein G column, and an antibody binding column. Carriers used for the protein A column include, for example, Hyper D, POROS, Sepharose FF and the like. In addition, the separation and purification methods used for ordinary proteins may be used, and are not limited at all. For example, the antibody used in the present invention can be separated and purified by appropriately selecting and combining chromatography, filters, ultrafiltration, salting out, dialysis, etc. other than the above-mentioned affinity chromatography. Examples of the chromatography include ion exchange chromatography, hydrophobic chromatography, and gel filtration.

The antibody of the present invention thus obtained is a protein having the amino acid sequence shown in SEQ ID NO: 1 or one or more, usually one or several, amino acids in the amino acid sequence having substitutions, deletions or insertions. Specifically binds to a protein that has the ability to support pre-B cell growth. In the present invention, an antibody having cytotoxic activity is preferably used.

2-2. Measurement of Antibody Concentration The concentration of the antibody obtained in 2-1 was measured by measuring absorbance or EL.
This can be done by ISA, etc. That is, when the absorbance is measured, the antibody used in the present invention or a sample containing the antibody is appropriately diluted with PBS (−), and then the absorbance at 280 nm is measured, and 1 mg / ml is set to 1.35 OD. calculate.
In addition, in the case of using ELISA, it can be measured as follows. That is, 1M in 0.1M bicarbonate buffer (pH 9.6)
Goat anti-human IgG (BIO SOURCE) 10 diluted to μg / ml
Add 0 μl to a 96-well plate (manufactured by Nunc) and incubate overnight at 4 ° C to solidify the antibody.

After blocking, 100 μl of an appropriately diluted antibody used in the present invention or a sample containing the antibody, or 100 μl of human IgG (manufactured by CAPPEL) as a standard is added, and the mixture is incubated at room temperature for 1 hour. After washing, alkaline phosphatase-labeled anti-human IgG (BIO
SOURCE) (100 μl) and incubate at room temperature for 1 hour. After washing, a substrate solution is added, and after incubation, absorbance at 405 nm is measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate the concentration of the target antibody.

3. FCM Analysis The reactivity of myeloma cells with the antibody used in the present invention was determined by FC
It can be performed by M (flow cytometry) analysis.
As the cells, established cell lines or freshly isolated cells can be used. For example, as established cell lines, multiple myeloma-derived KPMM2 (JP-A-7-236475), multiple myeloma-derived RP
MI8226 (ATCC CCL-155), U266B1 (AT
CC TIB-196), HS-Sultan from IgG multiple myeloma (AT
CC CRL-1484), ARH-77 derived from plasmacytoma (ATCC CRL-162)
1), K562 derived from chronic myeloid leukemia (ATCC CCL-243) and the like can be used.

After washing the cells with PBS (-), the cells were washed with FACS buffer (containing 2% fetal bovine serum and 0.05% sodium azide).
Add 100 μl of antibody or control antibody diluted to 25 μg / ml with PBS (-), and incubate on ice for 30 minutes. After washing with FACS buffer, 100 μl of 25 μg / ml FITC-labeled goat anti-mouse antibody (GAM, manufactured by Becton Dickinson)
And incubate on ice for 30 minutes. After washing with FACS buffer, suspend in 600 μl FACS buffer and
Scan (Becton Dickinson) may be used to measure the fluorescence intensity of each cell.

4. Northern Blot The level of BST-2 mRNA expression in myeloma cells can be measured by Northern blot analysis. In practice, this can be done as follows. That is, as a myeloma cell line, for example, KPMM2 derived from multiple myeloma (JP-A-7-23
6475), RPMI8226 from multiple myeloma (ATCC CCL-155),
U266B1 derived from IgE myeloma (ATCC TIB-196), HS-Sultan derived from IgG multiple myeloma (ATCC CRL-1484), ARH-77 derived from plasmacytoma (ATCC CRL-1621), K562 derived from chronic myelogenous leukemia (ATCC CCL 243) 1.0x10 ⁷ were washed with PBS (-), and 1 ml of TRIzol (manufactured by GIBCO-BRL) was added and dissolved. 200 μl of chloroform was added to the mixture, and after stirring, the mixture was cooled to 14000 rpm using a high-speed centrifuge (MRX-152, manufactured by Tommy Seiko).
And centrifuge for 15 minutes to collect the upper layer.

To this was added 500 μl of isopropanol,
After gentle stirring, centrifuge at 15,000 rpm for 10 minutes, and wash the precipitate with 70% ethanol. After drying under reduced pressure, dissolve in DEPC-treated water to obtain total RNA. The RNA concentration can be calculated by diluting a part of the RNA with DEPC-treated water and measuring the absorbance at a wavelength of 260 nm using a spectrophotometer DU 650 (manufactured by BECKMAN). All RNs obtained in this way
A is applied to a 1% agarose gel containing formaldehyde at 20 μg / lane, and electrophoresed at 100V. After the electrophoresis, the RNA is transferred from the agarose gel to nylon membrane Hybond N (manufactured by Amersham) by capillary transfer.

After the transfer, the nylon membrane was subjected to Rapid Hybr
After prehybridization with an idization buffer (manufactured by Amersham) at 68 ° C. for 15 minutes, a HindIII fragment (31) of plasmid pUC-HM (315) in which BST-2 antigen cDNA was cloned was used.
5bp) or a random prime DNA labeling system (Amersham) using EcoR I-Not I fragment (about 900bp) as a template.
Then, probe DNA labeled with ³² P is added, and the mixture is hybridized at 68 ° C. for 2 hours. Use a nylon membrane.
After washing twice with 2% SSC solution containing 1% SDS and 0.2x SSC solution containing 0.1% SDS, BST-2 mRNA can be detected using BAS-5000 (manufactured by Fuji Film).

5. Cytotoxic Activity 5-1. Measurement of CDC Activity The antibodies used in the present invention have cytotoxic activities such as complement-dependent cytotoxicity.
oxicity (CDC). The CDC activity of the therapeutic agent for myeloma of the present invention on myeloma cells can be measured as follows. First, target cells are cultured in an appropriate medium such as RPMI containing 10% fetal bovine serum (GIBCO-BRL).
Prepare a 1640 medium (GIBCO-BRL) at 4 x 10 ⁵ cells / ml. Target cells include multiple myeloma-derived KP
MM2 (JP-A-7-236475), RPMI8226 derived from multiple myeloma
(ATCC CCL-155), U266B1 derived from IgE myeloma (ATCC TIB
-196), HS-Sultan derived from IgG multiple myeloma (ATCC CRL
-1484), plasma cell tumor-derived ARH-77 (ATCC CRL-1621),
K562 derived from chronic myeloid leukemia (ATCC CCL 243) and the like can be used.

These cells were placed in a 96-well flat bottom plate (FALCON
And culture overnight at 37 ° C in a CO ₂ incubator. Next, an antibody for measuring CDC activity was added, and 60
After incubating for 2 minutes, an appropriately diluted complement such as Baby Rabbit Complement (CEDARLANE) is added, and
Incubate for hours. Alamar Bule (BIO SO
URCE (10 μl) was added to each well and incubated for 4 hours. After that, the fluorescence intensity (excitation wavelength 530 nm, detection wavelength 590
nm) fluorescence measurement system CytoFluor 2350 (MILLIPORE
Manufactured). Cytotoxic activity (%) is (AC) /
It can be calculated by (BC) x 100. Here, A is the fluorescence intensity when incubated in the presence of the antibody, B is the fluorescence intensity when incubated only with the culture solution without containing the antibody, and C is the fluorescence intensity in the well containing no cells.

5-2. Measurement of ADCC Activity The antibody used in the present invention has a cytotoxic activity, for example, antibody-dependent cellular cytotoxicity (antibody-dependent cytotoxicity).
ell-mediated cytotoxicity (ADCC). AD for myeloma cells of the therapeutic agent for myeloma of the present invention
CC activity can be measured as follows. First, mononuclear cells are separated from human peripheral blood and bone marrow by specific gravity centrifugation to prepare effector cells. In addition, as target cells, multiple myeloma-derived KPMM2 (JP-A-7-236475),
Multiple myeloma-derived RPMI8226 (ATCC CCL-155), IgE-type myeloma-derived U266B1 (ATCC TIB-196), IgG-type multiple myeloma-derived HS-Sultan (ATCC CRL-1484), Plasmacytoma-derived AR
H-77 (ATCC CRL-1621) or the like is labeled with ⁵¹ Cr to prepare target cells. The labeled target cells are then subjected to AD
An antibody for measuring CC activity is added and incubated, and then an appropriate ratio of effector cells to target cells is added and incubated.

After the incubation, the supernatant is taken and the radioactivity is measured with a gamma counter. In this case, 1% of NP-40 can be used for measuring the maximum free radioactivity. The cytotoxic activity (%) can be calculated by (AC) / (BC) × 100. A is the radioactivity released in the presence of the antibody (cpm), B is the radioactivity released by NP-40 (cpm), and C is the radioactivity released in the culture medium alone without the antibody (cpm). It is.

5-3. Enhancement of cytotoxic activity In order to exhibit cytotoxic activities such as ADCC activity and CDC activity, in humans, antibody constant region (C region) is used as C region.
It is preferred to use γ, especially Cγ1 or Cγ3.
Furthermore, stronger ADCC activity or CDC activity can be induced by partially adding, modifying, or modifying amino acids in the antibody C region. For example, IgM-like polymerization of IgG by amino acid substitution (Smith, RIF & Morris
on, SLBIO / TECHNOLOGY (1994) 12, 683-688), IgM-like polymerization of IgG by amino acid addition (Smith, RI
F. et al., J. Immunology (1995) 154, 2226-2236
), Expression of the gene encoding the L chain in tandem ligation (Shu
ford, W. et al., Science (1991) 252, 724-727),
Dimerization of IgG by amino acid substitution (Caron, PC et
al., J. Exp.Med. (1992) 176, 1191-1195, Shopes,
B., J. Immunology (1992) 148, 2918-2922), IgG dimerization by chemical modification (Wolff, EA et al., Canc.
er Res. (1993) 53, 2560-2565) and the introduction of effector functions by amino acid modification of the antibody hinge region (No.
rderhaug, L. et al., Eur. J. Immunol. (1991) 21, 2
379-2384). These can be achieved by using an oligomer site-directed mutagenesis method using a primer, adding a base sequence using a restriction enzyme cleavage site, and using a chemical modifier that causes a covalent bond.

6. Administration Route and Preparation The therapeutic agent for myeloma of the present invention can be parenterally administered systemically or locally. For example, intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection such as infusion can be selected, and the administration method can be appropriately selected according to the age and symptoms of the patient. Effective dose is 1 kg body weight at a time
The range is from 0.01 mg to 100 mg. Alternatively, a dose of 1-1000 mg, preferably 5-50 mg, per patient can be chosen.

The therapeutic agent for myeloma of the present invention may contain pharmaceutically acceptable carriers and additives depending on the administration route. Examples of such carriers and additives include:
Water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, Xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose,
Surfactants and the like that are acceptable as pharmaceutical additives. The additives to be used are appropriately or in combination selected from the above depending on the dosage form, but are not limited thereto. The disease to be treated according to the present invention is myeloma in which BST-2 is present on a target tumor cell. The therapeutic agent of the present invention is useful as a therapeutic agent for myeloma.

[0050]

EXAMPLES Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Example 1. Preparation of RS38 (IgM-type anti-BST-2 antibody) 1. Preparation of mouse ascites containing RS38 RS38-producing hybridoma was prepared by the method of Ishikawa et al.
J. et al., GENOMICS (1995) 26, 527-534).

2,6,10,14-tetramethylpentadecane (manufactured by Wako Pure Chemical Industries, Ltd.) was added 50 and 14 days in advance, respectively.
5 × 10 ⁶ hybridomas were intraperitoneally injected into BALB / c mice (manufactured by CLEA Japan) administered intraperitoneally at 0 μl each. From day 9 after injection of the hybridoma, ascites collected in the abdominal cavity of the mouse was collected with a 19 gauge indwelling needle Happy Cas (made by Medikit). The collected ascites is a low-speed centrifuge
Rotation speed 1000 and 3000 using RLX-131 (Tomy Seiko)
The mixture was centrifuged twice at rpm to remove foreign matter such as hybridomas and blood cells.

2. Purification of RS38 from mouse ascites Purification of RS38 from the mouse ascites was performed by the following method. An equal volume of PBS (-) was added to mouse ascites, filtered using a hollow fiber filter media prep (MILLIPORE), and then purified using a high-speed antibody purification device ConSep LC100 (MILLIPORE).
) And an anti-mouse IgG + IgM antibody binding column (column volume: 20 ml, NGK) based on the attached instructions, 0.02 M phosphate buffer (pH 7.6) as the adsorption buffer, and Wash buffer as the washing solution. A (manufactured by NGK) and affinity purification was performed using 0.2 M glycine-HCl buffer (pH 2.5) as an elution buffer.

The eluted fraction was immediately purified with 1 M Tris-HCl (pH 8.0)
And adjust the pH to around 7.4.
Concentrate using ntriprep 10 and replace the buffer with PBS (-). MILLEX-G membrane filter with a pore size of 0.22 μm
The solution was sterilized by filtration with V (manufactured by Millipore) to obtain purified RS38. 3. Measurement of Antibody Concentration The concentration of the purified antibody was measured by measuring the absorbance. That is, after the purified antibody was diluted with PBS (-), the absorbance at 280 nm was measured, and 1 mg / ml was calculated as 1.35 OD.

Example 2 RS38-E (IgG type anti-BST-2 antibody)
1. Preparation of mouse ascites containing RS38-E RS38-E-producing hybridoma was prepared by the method of Ishikawa et al.
a, J. et al., GENOMICS (1995) 26, 527-534). According to the above Example 1-1, mouse ascites containing RS38-E was obtained.

2. Purification of RS38-E from mouse ascites Purification of RS38-E from the above mouse ascites was performed by the following method. An equal volume of PBS (-) was added to mouse ascites, filtered using a hollow fiber filter media prep (MILLIPORE), and then purified using a high-speed antibody purification device ConSep LC100 (MILLIPORE).
Manufactured) and a HyperD Protein A column (column volume 20 m)
l, NGK) and 1.5 M glycine + 3 M sodium chloride buffer (pH 8.9) as the adsorption buffer and 0.1 M sodium citrate buffer (pH 6) as the elution buffer based on the attached instructions. For affinity purification.

The eluted fraction was immediately mixed with 1 M Tris-HCl (pH 8.0)
And adjust the pH to around 7.4.
Concentrate using ntriprep 10 and replace the buffer with PBS (-). MILLEX-G membrane filter with a pore size of 0.22 μm
The solution was sterilized by filtration with V (manufactured by Millipore) to obtain purified RS38-E.
The concentration of the purified antibody was measured in accordance with Example 1-3 described above.

Example 3 Anti-BST-2 Antibody against Myeloma Cells
Preparation and purification of reactive Study 1. control mouse IgG1 to (MOPC-31C) Preparation and Purification control mouse IgG1 (MOPC-31C) was performed by the following method. 1-a.Production of mouse ascites containing MOPC-31C MOPC-31C-producing hybridoma (ATCC CCL 130) was injected intraperitoneally into BALB / c mice according to Example 1-1 above, and MOPC-3
Mouse ascites containing 1C was obtained. 1-b. Purification of MOPC-31C from mouse ascites Purification of MOPC-31C from the mouse ascites was performed according to Example 2-2 described above.
Followed. The concentration measurement of the purified antibody was performed according to Example 1-3 above.

2. FCM Analysis The reactivity of anti-BST-2 antibody against myeloma cells
M (flow cytometry) analysis was performed. KPMM2 derived from multiple myeloma (JP-A-7-236475), ARH-77 derived from IgG-type plasmacytoma (ATCC CRL-1621), RPMI82 derived from multiple myeloma
26 (ATCC CCL-155), U266B1 derived from IgE myeloma (ATCC T
IB-196), HS-Sultan derived from IgG multiple myeloma (ATCC C
RL-1484), K562 derived from chronic myeloid leukemia (ATCC CCL 24
3) After washing with PBS (-), 25µl of FACS buffer (PBS (-) containing 2% fetal bovine serum and 0.05% sodium azide)
100 μl of RS38-E or MOPC-31C diluted to g / ml was added, and the mixture was incubated on ice for 30 minutes.

After washing with FACS buffer, 25 μg / ml FI
100 μl of TC-labeled goat anti-mouse antibody (GAM) was added, and the mixture was incubated on ice for 30 minutes. After washing with FACS buffer, the cells were suspended in 600 μl of FACS buffer and FACScan (Bect
on Deckinson) was used to measure the fluorescence intensity of each cell. As a result, as shown in FIG.
It reacted strongly with 38-E, confirming that BST-2 was highly expressed on the cells. On the other hand, K562, a non-myeloma cell line
It hardly reacted with 38-E and appeared to have no or very weak expression of BST-2 on the cell surface.

Example 4 Northern Blot Analysis Northern blot analysis for examining BST-2 mRNA expression in cells was performed as follows. 1. Preparation of Total RNA KPMM2 derived from multiple myeloma (JP-A-7-236475), RPMI8226 derived from multiple myeloma (ATCC CCL-155), IgE type myeloma
U266B1 (ATCC TIB-196), K562 from chronic myeloid leukemia
(ATCC CCL 243) Each 1.0 × 10 ⁷ cells were washed with PBS (−), and 1 ml of TRIzol (manufactured by GIBCO-BRL) was added and dissolved. Add 200 μl of chloroform to this, stir and cool
The mixture was centrifuged at 14000 rpm for 15 minutes using MRX-152 (manufactured by Tommy Seiko) to recover the upper layer.

To this was added 500 μl of isopropanol,
After gently stirring and centrifuging at 15,000 rpm for 10 minutes, the precipitate was washed with 70% ethanol. After drying under reduced pressure, the residue was dissolved in DEPC-treated water to obtain total RNA. About the RNA concentration, a part was further diluted with DEPC-treated water, and the absorbance at a wavelength of 260 nm was measured using a spectrophotometer DU 650 (manufactured by BECKMAN) to calculate the RNA concentration.

2. Northern hybridization The total RNA obtained in the above 1 was applied to a 1% agarose gel containing formaldehyde at a concentration of 20 μg / lane.
Electrophoresis was performed at 0V. After electrophoresis, transfer RNA from agarose gel to nylon membrane Hybond N (Amersh
am) by capillary transfer.
After transfer, the nylon membrane is placed in Rapid Hybridization bu
After prehybridization at 68 ° C. for 15 minutes with ffer (manufactured by Amersham), the BST-2 cDNA cloned plasmid pUC-HM (315) Hind III fragment (315 bp) or Ec
Using the oR I-Not I fragment (about 900 bp) as a template, random pri
Probe DNA labeled with ³² P was added using a meDNA labeling system (manufactured by Amersham), and hybridization was performed at 68 ° C. for 2 hours.

2 × S containing 0.1% SDS with nylon membrane
After washing twice with SC solution and 0.2x SSC solution containing 0.1% SDS twice, respectively, BST-2 with BAS-5000 (manufactured by Fuji Film)
mRNA was detected. As a result, as shown in FIG.
BST-2 mRNA expression is weaker compared to KPMM2 and RP
In both MI8226 and U266B1, the band of BST-2 mRNA was strong, and it was revealed that BST-2 mRNA was strongly expressed in these myeloma cell lines.

Example 5 Measurement of CDC Activity The CDC activity of the anti-BST-2 antibody on myeloma cells was measured as follows. 1. Preparation of target cells As a target cell, multiple myeloma-derived RPMI8226 (ATCC CCL-1
55), U266B1 derived from IgE myeloma (ATCC TIB-196), IgG
Medium-type multiple myeloma-derived HS-Sultan (ATCC CRL-1484) and chronic myelogenous leukemia-derived K562 (ATCC CCL 243) in RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum (GIBCO)
Was adjusted to 4 × 10 ⁵ cells / ml. Add 50 μl of these cell suspensions to a 96-well flat bottom plate (FALCON), and add
The cells were cultured overnight in a 5% CO ₂ high humidity incubator (manufactured by TABAI).

2. Preparation of RS38 Purified RS38 obtained in Example 1 was used in an RPMI1640 medium (manufactured by GIBCO-BRL) containing 10% fetal bovine serum (manufactured by GIBCO-BRL).
Adjusted to 0, 0.2, 2, and 20 μg / ml and prepared in 1 above
50 μl was added to each well of a 96-well flat bottom plate. 37 ℃, 5
After incubating in a% CO ₂ high humidity incubator (TABAI) for 60 minutes, the mixture was centrifuged at 1000 rpm for 5 minutes using a low-speed centrifuge 05PR-22 (Hitachi) to remove 50 μl of the supernatant. 3. Preparation of complement Dissolve Baby Rabbit Complement (manufactured by CEDARLANE) with 1 ml of purified water per vial, and add RPMI 164 without FCS.
0 Diluted with 5 ml of medium (GIBCO). This is the above 2
Add 50 μl to each well of a flat-bottomed plate at 37 ° C, 5% CO ₂
Incubated for 2 hours in a high humidity incubator (TABAI).

4. Measurement of CDC Activity After the incubation, place each well of the 96-well flat-bottom plate
Add 10 μl of Alamar Bule (BIO SOURCE) and add 37
℃, 5% CO _{2 in a} high humidity incubator (TABAI) 4
After incubation for an hour, the fluorescence intensity (excitation wavelength)
530 nm, detection wavelength 590 nm) using the fluorescence measurement system CytoFluo
r2350 (manufactured by Millipore). The cytotoxic activity (%) was calculated by (AC) / (BC) × 100. Here, A is the fluorescence intensity when incubated in the presence of the antibody, B is the fluorescence intensity when incubated only with the culture solution without containing the antibody, and C is the fluorescence intensity in the well containing no cells.

As a result, as shown in FIG.
The reaction with RS38-E is weak, and even in Northern blot analysis
In K562, where BST-2 mRNA expression was relatively weak, cell damage did not occur even when RS38 was added, whereas FCM analysis strongly reacted with RS38-E, and BST-2 mRNA expression was also reduced. Strong RPMI
For 8226, U266B1, and HS-Sultan, RS38
Cytotoxicity was observed in a concentration-dependent manner. From this, the anti
The BST-2 antibody was found to show CDC activity against myeloma cells that highly express BST-2 on the cell surface.

[0068]

EFFECT OF THE INVENTION CDC-induced cytotoxicity was observed in myeloma cells by treatment with the anti-BST-2 antibody. This indicates that the anti-BST-2 antibody has an antitumor effect on myeloma cells.

[0069]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1013 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA sequence 0 GAATTCGGCA CGAGGGATCT GG ATG GCA TCT ACT TCG TAT GAC TAT TGC AGA 52 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg 1 5 10 GTG CCC ATG GAA GAC GGG GAT AAG CGC TGT AAG CTT CTG CTG GGG ATA 100 Val Pro Met Glu Asp Gly Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile 15 20 25 GGA ATT CTG GTG CTC CTG ATC ATC GTG ATT CTG GGG GTG CCC TTG ATT 148 Gly Ile Leu Val Leu Leu Ile Ile Val Ile Leu Gly Val Pro Leu Ile 30 35 40 ATC TTC ACC ATC AAG GCC AAC AGC GAG GCC TGC CGG GAC GGC CTT CGG 196 Ile Phe Thr Ile Lys Ala Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg 45 50 55 GCA GTG ATG GAG TGT CGC AAT GTC ACC CAT CTC CTG CAA CAA GAG CTG 244 Ala Val Met Glu Cys Arg Asn Val Thr His Leu Leu Gln Gln Glu Leu 60 65 70 ACC GAG GCC CAG AAG GGC TTT CAG GAT GTG GAG GCC CAG GCC GCC ACC 292 Thr Glu Ala Gln Lys Gly Phe Gln Asp Val Glu Ala Gln Ala Ala Thr 75 80 85 90 TGC AAC CAC ACT GTG ATG GCC CTA AT G GCT TCC CTG GAT GCA GAG AAG 340 Cys Asn His Thr Val Met Ala Leu Met Ala Ser Leu Asp Ala Glu Lys 95 100 105 GCC CAA GGA CAA AAG AAA GTG GAG GAG CTT GAG GGA GAG ATC ACT ACA 388 Ala Gln Gly Gln Lys Lys Val Glu Glu Leu Glu Gly Glu Ile Thr Thr 110 115 120 TTA AAC CAT AAG CTT CAG GAC GCG TCT GCA GAG GTG GAG CGA CTG AGA 436 Leu Asn His Lys Leu Gln Asp Ala Ser Ala Glu Val Glu Arg Leu Arg 125 130 135 AGA GAA AAC CAG GTC TTA AGC GTG AGA ATC GCG GAC AAG AAG TAC TAC 484 Arg Glu Asn Gln Val Leu Ser Val Arg Ile Ala Asp Lys Lys Tyr Tyr 140 145 150 CCC AGC TCC CAG GAC TCC AGC TCC GCT GCG GCG CCC CAG CTG CTG ATT 532 Pro Ser Ser Gln Asp Ser Ser Ser Ala Ala Ala Pro Gln Leu Leu Ile 155 160 165 170 GTG CTG CTG GGC CTC AGC GCT CTG CTG CAG TGAGATCCCA GGAAGCTGGC 582 Val Leu Leu Gly Leu Ser Ala Leu Leu Gln 175 180 ACATCTTGGA TGCTCGGCTT TTCGCTTGAA CATTCCCTTG ATCTCATCAG 642 TTCTGAGCGG GTCATGGGGC AACACGGTTA GCGGGGAGAG CACGGGGTAG CCGGAGAAGG 702 GCCTCTGGAG CAGGTCTGGA GGGGCCATGG GGCAGTCCTG GGTGTGGGGA CA CAGTCGGG 762 TTGACCCAGG GCTGTCTCCC TCCAGAGCCT CCCTCCGGAC AATGAGTCCC CCCTCTTGTC 822 TCCCACCCTG AGATTGGGCA TGGGGTGCGG TGTGGGGGGC ATGTGCTGCC TGTTGTTATG 882 GGTTTTTTTT GCGGGGGGGG TTGCTTTTTT CTGGGGTCTT TGAGCTCCAA AAAAATAAAC 942 ACTTCCTTTG AGGGAGAGCA CACCTTAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAATTC 1002 GGGCGGCCGC C 1013

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows RS38-E and control mouse IgG1.
(MOPC-31C) shows the histogram when KPMM2 was subjected to FCM analysis by the indirect method.

FIG. 2 shows RS38-E and control mouse IgG1.
(MOPC-31C) shows a histogram obtained by FCM analysis of ARH-77 by the indirect method.

FIG. 3 shows RS38-E and control mouse IgG1.
(MOPC-31C) shows a histogram obtained by FCM analysis of RPMI8226 by the indirect method.

FIG. 4 shows RS38-E and control mouse IgG1.
(MOPC-31C) shows a histogram when U266B1 was subjected to FCM analysis by the indirect method.

FIG. 5 shows RS38-E and control mouse IgG1.
(MOPC-31C) shows a histogram obtained by FCM analysis of HS-Sultan by the indirect method.

FIG. 6 shows RS38-E and control mouse IgG1.
(MOPC-31C) shows a histogram when K562 was subjected to FCM analysis by the indirect method.

FIG. 7 shows autoradiography when Northern blot was performed using a probe against BST-2. In K562, BST-2 mRNA expression was weak, whereas KPMM
2. RPMI8226 and U266B1 all show strong BST-2 mRNA expression.

FIG. 8. RS38 shows no cytotoxic activity against K562, whereas myeloma cell lines RPMI8226, U266
It is a graph which shows that B1 and HS-Sultan cause cell damage by CDC activity in a concentration-dependent manner.

Claims (5)

[Claims] 1. A therapeutic agent for myeloma comprising, as an active ingredient, an antibody that specifically binds to a protein having the amino acid sequence shown in SEQ ID NO: 1. 2. A therapeutic agent for myeloma comprising, as an active ingredient, an antibody that specifically binds to a protein having the amino acid sequence shown in SEQ ID NO: 1 and has cytotoxic activity. 3. The therapeutic agent for myeloma according to claim 1, wherein the antibody is a monoclonal antibody. 4. The therapeutic agent for myeloma according to claim 2, wherein the cytotoxic activity is ADCC activity or CDC activity. 5. An amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have an amino acid sequence in which substitution, deletion and / or insertion has been performed, and a protein and pre-B
The therapeutic agent for myeloma according to any one of claims 1 to 4, wherein an antibody that specifically binds to a protein capable of supporting cell growth is used as an active ingredient.