JPH05276981A - Monoclonal antibody, polypeptide and its production - Google Patents

Abstract

PURPOSE:To provide a monoclonal antibody and polypeptide useful for metastasis inhibition. CONSTITUTION:The first objective anticancer antibody inhibiting the activity of cancer cells; the second objective hybridoma capable of producing said antibody; the third objective recognition antigen polypeptide for said antibody; the fourth objective DNA coding said polypeptide and the fifth objective transformant containing the DNA; and the sixth objective production method for said polypeptide by culture of said transformant. The above monoclonal antibody can be used for inhibiting metastasis (e.g. for lung cancer, glioblastoma). And, the polypeptide of the present invention and the fragments thereof can also be used for metastasis inhibition against carcinoma.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a monoclonal antibody having a cancer metastasis inhibitory action. The antibody of the present invention exhibits a metastasis-suppressing action on human cancer such as lung cancer and glioblastoma, and can be used for the prevention and treatment of cancer. The present invention also relates to polypeptides and methods for making the same.
More specifically, the present invention relates to a polypeptide involved in the motility of cancer cells, wherein the polypeptide is
It can be used to suppress metastasis of cancer cells, especially lung cancer, glioblastoma and other metastatic cancers.

[0002]

2. Description of the Related Art In recent years, deaths due to cancer have been increasing year by year. On the other hand, the development of therapeutic methods for cancer has been rapidly progressing, and some therapeutic methods are being established. In particular, many anti-cancer antibodies have been developed since the announcement of Koehler and Milstein's monoclonal antibody production [Koehler, G. and Milstein, C., Nature, 256, 495 (1975)], and they are used as cancer therapeutics and diagnostics. It is applied as a medicine. In the treatment of cancer, these antibodies are used for so-called antibody missile therapy, which specifically binds to cancer cells without damaging normal tissues and eliminates or damages them, and in blood-related cancers such as leukemia and lymphoma. Has achieved some results.

However, at present, almost no effective preventive and therapeutic method for cancer metastasis has been found. Cell surface molecules, cytoskeletons, proteases, etc. are assumed as factors involved in metastasis, and some of them have been clarified in detail. However, metastasis is established through a very complicated process, and it is far from clearing the whole picture. At present, it is considered most efficient to use an in vitro experimental model system of cancer metastasis to develop a drug that suppresses metastasis. As such a system, using a chemotaxis chamber, the pore size of 5
8μm filter with uniform holes (Nucleopore filter
er; nucleopore membrane filter, etc.) is widely used. As a factor involved in the metastasis of cancer cells found using this system, for example, its promotion, autocrine motility factor (AMF)
[Procedures of National Academy
Science USA (Proc. Natl. Acad. Sci.
USA), 83 , 3302-3306 (198).
6)] and chemotactic factor (chemotacti
c factor) [The History of Medicine, 150 , 805-806 (1989)] has been reported. If a drug that suppresses the motility of cancer cells could be created in such an experimental system, it would be extremely useful as a drug for suppressing cancer metastasis.

[0004]

In the treatment of cancer, some effective treatments for specific cancers have been established. However, it is difficult to prevent cancer metastasis, and it is hoped that an effective preventive and therapeutic method will be established, but there is currently no such method.

[0005]

Based on this technical background, the present inventors have found that among the monoclonal antibodies that specifically bind to cancer cells, the antigen is involved in the motility of cancer cells. Assuming that it contains an antibody that suppresses cancer motility by binding to it, a monoclonal antibody is screened using the chemotaxis chamber described above in a system that examines the motility of cancer cells. , A monoclonal antibody that suppresses the motility of cancer cells has been found. The antibody specifically binds to a protein on the surface of cancer cells and suppresses the motility of cancer cells. Therefore, it is presumed that the protein recognized by this antibody plays a very important role in the motility of cancer cells. It is considered that such a protein or a peptide fragment thereof is extremely useful as a drug for suppressing cancer metastasis, and the present inventors have utilized genetic engineering techniques to determine the amino acid sequence of the peptide recognized by the present monoclonal antibody. Is isolated, and a plasmid containing the cDNA is constructed, a transformant transformed with the plasmid is prepared, and the desired peptide is obtained from the transformant culture. Thus, the present invention has been completed. That is, the present application includes the following inventions (1) to (7). (1) An anti-human cancer monoclonal antibody that suppresses the motility of human cancer cells. Specifically, the antibody of the present invention uses a protein involved in the motility of human cancer cells as a recognition antigen, and as human cancer cells, a monoclonal IgG having specificity for human lung cancer cells and human glioblastoma, preferably I
The gG ₁ antibody. (2) A polypeptide containing the amino acid sequence of SEQ ID NO: 1 [hereinafter sometimes abbreviated as M], and this polypeptide can be recognized by the antibody of (1) above. (3) A polypeptide that has a partial amino acid sequence of the amino acid sequence of SEQ ID NO: 1 and suppresses the motility of cancer cells, and this polypeptide has a partial amino acid sequence of the amino acid sequence of SEQ ID NO: 1 Any substance may be used as long as it exhibits the motor activity suppressing action. For example, amino acid residue numbers 35 to 60, 113 to 142, 131
To 166, 163 to 191 and the like. The polypeptide may be selected from a site having a receptor function, a site interacting with other proteins on the cell membrane and extracellularly. (4) A recombinant DNA containing a nucleotide sequence encoding M. (5) A vector containing the DNA of (4) above. (6) A transformant transformed with the vector of (5) above. (7) A method for producing M, which comprises culturing a transformant transformed with the vector containing the DNA of (4) above, causing M to be produced and accumulated in the culture, and collecting the M. The above-mentioned recombinant DNA may be any one as long as it contains the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, but for example, DNA containing the nucleotide sequence of SEQ ID NO: 2 is preferable.

The antibody of the present invention can be used in the treatment of cancer, particularly in the prevention of metastasis of cancer, in combination with a metastasis suppressor having a different mechanism of action such as the present antibody alone or a substance having an inhibitory effect on adhesion of cancer cells. it can. Further, substances having appropriate antitumor activity to the antibody of the present invention, for example, anticancer chemotherapeutic agents such as methotrexate, daunomycin, vincristine and cisplatin, or Pseudomonas aeruginosa exotoxin A, ricin, abrin, diphtheria toxin, neocarzino It can be used for treating cancer as a so-called missile therapeutic agent by binding to a toxin protein such as statin, and a cyclotoxin such as tumor necrosis factor, lymphotoxin and interferon. In this case, by using an antitumor active substance that does not suppress the action of the antibody of the present invention, the antibody of the present invention suppresses the metastasis of cancer while specifically killing cells It is advantageous in treatment.

[0007] The antibody of the present invention can be obtained by screening an anti-human cancer antibody obtained from an anti-human cancer antibody-producing hybridoma obtained by a method known per se, using the activity of suppressing cancer cell motility as an index. Such an anti-human cancer antibody-producing hybridoma may be any hybridoma that produces an antibody capable of specifically binding to a human cancer cell, and for example, an anti-human transferrin receptor (hereinafter abbreviated as hTfR). MoAb producing mouse hybridoma 22C6 [IFO 50172,
FERM BP-2054] [see JP-A-2-79970] or anti-human renal cancer MoAb producing mouse hybridoma RCS-1 [IFO 50184, FERM
BP-2333] [see WO 91-09134 publication] and the like.

In the production of these antibody-producing hybridomas, usual hybridoma production methods are used [G. Koehler et al .: Nature, 256 , 49.
5 (1975)]. For example, a method of immunizing an animal with cancer cells according to a conventional method and fusing the obtained antibody-producing cells with myeloma cells or the like is used. Examples of cancer cells used for immunization include renal cancer (AM-RC-3, AM-RC-6, AM-RC-7, SK-RC-
1, SK-RC-9, SK-RC-18), bladder cancer (T-24, KK-47, MGH-U)
-1), Prostate cancer (DU-145), Gastric cancer (NUGC-2, NUGC-3, NUGC)
-4, MKN-28, KATO-III, MRK-1), bowel cancer (SW-403, SW-62)
0, SW-1116, SW-1222, CaOV-4, HT-29), cervical cancer (ME-
180), melanoma (SK-MEL-33, SK-MEL-37), breast cancer (MCF
-7), glioma (MG-178), lung cancer (Luci-10, Calu-6, P
C-10, ADLC-DA, SBC-3, SCLC-SA, Luci-6, CADO-LC3, O
KADA, QG-56), T cell leukemia (HUT-78, CCRF-CEM, HPB-
ALL, HSB-2, HUT-102, RPMI-8402, P12 / Ichikawa, MT-
1, MT-2), B cell leukemia (Raji, Daudi, BALL-1, RPMI-17
88, Ly-16), Null cell leukemia (NALL-1, NALM-6, NALM-1
8, KOPN-K, P30 / Ohkubo), myeloma leukemia (HL-60), etc.

It is also possible to use cancer cells established from the cancer tissue of an actual cancer patient, such as a lung cancer patient, such as MAC8 cells. Further, by using a cell surface antigen recognized by the antibody of the present invention as an immunizing antigen, improvement in the production amount of the antibody of the present invention can be expected. In order to identify such cancer cell surface antigens, in addition to the conventional fluorescent antibody method, FACS (fluorescence activated cell selection device) which has been recently developed has been developed.
It has become common to use. When the cancer cells used are adherent cells, a cell suspension is usually prepared using PBS containing 5 mM EDTA. Depending on the purpose, trypsin, collagenase or the like may be used. These methods can be carried out, for example, by using the method described in the experimental manual [Zokusei Kagaku Kenkyu Koza 5 Immunobiochemistry Research Method, p. 122 Tokyo Kagaku Dojin].

Examples of immunized animals include mammals (eg,
Humans, mice, rats, guinea pigs, cats, dogs, monkeys, rabbits, sheep, goats, hamsters, etc.), birds (eg, chickens, geese, ducks, etc.), among others, preferred are mice, rats, rabbits, etc. ,
Mice are particularly preferred for the production of MoAbs. The antibody-producing cells that are induced in the immunized animal may be derived from any of spleens, lymph nodes, peripheral blood lymphocytes, etc., as long as they produce antibodies, but spleen cells are particularly preferably used. In the case of these antibody-producing cells such as mice, 4-
10-week-old mice are immunized with cancer cells to prepare splenocytes. The immunization method is usually a phosphate buffer (hereinafter, PB
A small amount of live cells washed with
Suspended in BS, 10 ⁶ to 10 ⁸ cells / 0.1 ml, preferably 10 ⁷ cells / 0.1 ml, are subcutaneously or intraperitoneally administered to the back or abdomen of a mouse. Thereafter, the cells are administered every 2 weeks in the same manner 2 to 5 times. For final immunity,
After intravenous administration, splenocytes can be prepared from mice 3 to 4 days after administration according to a usual method.

This antibody-producing cell can stably produce an antibody by fusing with a proliferative cell to form a hybridoma. The fusion partner cell is particularly a myeloma cell (eg, NS). -1, P3-X63-AgU
I, X45, SP2, X68-Ag8, etc.) are conveniently used. Among them, especially MOPC21 (BALB /
(c mouse) -derived myeloma cells, X68-Ag8 and improved strains thereof are preferably used. When any of these improved strains or other myeloma-derived cell lines are selected and used for producing a monoclonal antibody, this type of cell line is resistant to 8-azaguanine, and hypoxanthine / guanine phosphoribosyl transferase is obtained. Therefore, it cannot grow in HAT medium (medium containing hypoxanthine-aminopterin-thymidine), which is advantageous for selection of hybridoma after fusion.

Cell fusion is carried out according to known methods,
As a fusing agent, for example, polyethylene glycol (hereinafter,
(Sometimes abbreviated as PEG), Sendai virus, etc. are particularly preferably used. As PEG, polyethylene glycol having an average molecular weight of 1000 to 6000 is preferable, PEG 4000 is particularly preferably used, and the concentration is 10 to 80%, preferably 40 to 50%. Cell fusion involves RP of antibody-producing cells and myeloma cells
The cells are washed with MI1640 medium, usually mixed at a ratio of 2: 1 to 10: 1, and centrifuged at room temperature at 700 × g for 5 minutes to obtain a cell pellet. The cell pellet is loosened while warming it in a constant temperature bath at 37 ° C., and the pre-warmed PEG solution is gradually added and mixed well. Usually, 1 ml is added per 10 ⁸ cells, but it may be increased or decreased in each case.
Then, pre-warmed medium is gradually added dropwise and mixed,
Decrease PEG concentration. It usually takes about 10 minutes
Add 30 ml of medium. Collect the cells by centrifugation at room temperature,
Splenocytes are dispensed in a medium containing 10% fetal calf serum (hereinafter sometimes abbreviated as FCS) at a cell concentration of 1 to 2 × 10 ⁶ / ml into a 96-well plate by 100 μl each.
After standing overnight, 100 μl of a medium containing HAT and 10% FCS (HAT medium) is added. In order to omit this operation, cells may be suspended in HAT medium after cell fusion. The medium is changed several times in a few weeks. As a method for replacing the medium, the medium 100 is usually fed from each hole of the plate.
˜200 μl to remove fresh HAT medium 100-200
Add μl. During this period, if the appearance of hybridomas is observed, an appropriate screening system is used as soon as possible to detect the growing wells of the hybridomas producing the target antibody. The obtained hybridoma is immediately cloned by an appropriate method.

Various known methods can be used for screening such anti-cancer cell antibody-producing hybridomas. For example, mixed hemagglutination reaction (hereinafter abbreviated as MHA) as seen by adsorption of indicator red blood cells to cancer cells. , An immunoadhesive reaction that observes the phenomenon in which red blood cells adhere to the antibody bound to cancer cells via complement, and a fluorescently labeled second antibody (usually
(Anti-immunoglobulin antibody) immunofluorescence method (hereinafter sometimes abbreviated as IF) in which cancer cells are stained by fluorescence analysis, or cancer cells adhered to a microplate, for example, lung cancer cell lines for screening lung cancer-specific monoclonal antibodies,
For example, A549 (ATCC CCL 185) or M
Cell binding to AC8 (lung cancer cell line) etc. seen by enzyme-linked immunosorbent assay (hereinafter sometimes abbreviated as ELISA)
-ELISA method etc. are mentioned. Hybridomas positive for antibody activity are immediately subjected to cloning, which is usually easily performed by the limiting dilution method or the like. Regarding the culture supernatant of the cloned hybridoma, its antibody titer is measured by the above method, a hybridoma that stably produces an antibody with high titer is selected, and an antibody that suppresses the motility of cancer cells is further selected. Screening of modified Boyd using the nucleopore membrane filter described above.
en chamber assay [J. Immunol. Methods, 33,239-247 (1
980)] or using a commercially available Transwell plate (Costar). The pore size of the filter used for this screening is, for example, 5 μm or 8 μm depending on the experimental conditions.
You can choose one. Although melanoma cells and glioma cells are often used as the cancer cells to be used, suitable cells may be screened from other cancer cell lines and used.
For example, MAC10 cells screened from lung cancer cells can be used. By selecting a hybridoma that produces an antibody having a strong inhibitory effect on cancer cells by this method, the target monoclonal hybridoma can be obtained.

The production and accumulation of the anti-human cancer cell monoclonal antibody is carried out by culturing the hybridoma of the present invention in a liquid medium, preferably a serum-free medium or intraperitoneally in a warm-blooded animal other than human (usually mouse). However, it is desirable to use a serum-free medium, especially when high-purity antibodies are required. Examples of those cultures are given below.

As a liquid medium, for example, fetal bovine serum or the like is added to a basal medium for animal cell culture [eg, an equal volume mixed medium (I / H medium) such as Iscove's medium and Ham's F12 medium or RPM1-1640 medium]. Or GIT medium (Wako Pure Chemical Industries, Ltd.) (JP-A-60-14508)
No. 8) and the like. Culture is usually about 3-6
0 days, preferably about 5-10 days, about 30-38 ° C,
It is preferably carried out at about 37 ° C. For intraperitoneal transplantation into a mouse, about 2 × 10 ^{5 to} 5 × 10 ⁷ , preferably about 1 to 5 × 10 ⁶ , antibody-producing hybridomas per mouse are intraperitoneally transferred for about 10 to 35 days. 3-10 ml of a sufficient amount of antibody-containing ascites fluid can be obtained by breeding.

The antibody in the liquid medium or ascites fluid can be purified by combining known biochemical techniques. For example, after centrifuging the antibody-containing liquid,
Salt out the supernatant (usually using ammonium sulfate or sodium sulfate). The obtained protein precipitate was dissolved in an appropriate buffer solution, dialyzed, and subjected to column chromatography (DE
AE ion exchange column, hydroxyapatite column,
The target antibody can be separated and purified by subjecting it to gel filtration column, protein A column, protein G column, etc.) or immunoaffinity chromatography. By the above-mentioned separation and purification operation, for example, about 5 to 30 mg of anti-cancer cell MoAb having a protein weight ratio of 90% or more can be obtained from 500 ml of liquid medium. The same antibody was found to be 10-2 in 50 ml of ascites fluid.
00 mg is obtained. In particular, chromatography using a protein A or G column, gel filtration column carrier, D
By combining chromatography using an EAE ion exchange column carrier, hydroxyapatite and the like, a higher purity antibody can be obtained. In these purified antibody preparations, the content of heterologous protein is about 0.1%.
The following is advantageous when administered to humans as a medicine.

The MoAb obtained as described above is treated with proteolytic enzymes (papain, pepsin, etc.) and a reducing agent while Fab, Fab ′ or F (ab ′) ₂ while maintaining the ability to bind to cancer cells. Fragments can be obtained and serve the same purpose as the MoAbs of the invention. When these hybridomas produce mouse IgG MoAb, DNA encoding the variable region containing the antigen recognition site of the anti-cancer cell MoAb is obtained, and the DNA is engineered by a gene manipulation technique [Steplewski, Z. et al .: Proceedings]. ·of·
National Academy Sciences, USA (Proc. Natl. Acad. Sci. USA), 85 , 4852 (19)
88)] can be used to bind a gene encoding the constant region of human IgG to prepare a mouse-human chimeric antibody. Such a chimeric antibody is advantageously used when administered to humans because it has low antigenicity. The anti-cancer cell MoAb produced by the method of the present invention is mixed with itself or with an appropriate pharmacologically acceptable carrier, excipient, diluent or the like after filtering and sterilizing operation with a membrane filter, It can be formulated as an injection or the like. Such a MoAb preparation is expected not only as an anti-cancer metastasis-suppressing effect itself, but also as a so-called missile therapeutic agent in which an appropriate anti-cancer agent is bound, in particular. In addition, like other anti-human cancer antibodies, it can be used for detecting human cancer cells, particularly human lung cancer cells and human glioblastoma. Furthermore, it is useful in the screening, purification, etc. of M of the present invention involved in the motility of human cancer cells.
The M of the present invention can be obtained by a combination of known genetic engineering techniques as described below.

That is, the expression vector containing the DNA having the nucleotide sequence encoding M in the present invention is
For example, (i) a phage containing the target DNA is isolated by a protoblot immunoscreening system using an appropriate cDNA library, (ii) the target cloned DNA is excised from the phage, and (i)
ii) It can be produced by ligating the cloned DNA downstream of the promoter in the vehicle. As a vector, a plasmid derived from Escherichia coli (eg, pBR3
22, pBR325, pUC12, pUC13), and a Bacillus subtilis-derived plasmid (eg, pUB110, pTB5, pC)
194), a yeast-derived plasmid (eg, pSH19, pS
H15) or bacteriophages such as phages and animal viruses such as retroviruses and vaccinia viruses. The gene may have ATG as a translation initiation codon at its 5'end and TAA, TGA or TAG as a translation stop codon at its 3'end. Furthermore, in order to express the gene, a promoter is connected upstream thereof. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.

For example, when the host for transformation is Escherichia coli, T7 promoter, trp promoter, la
c promoter, rec promoter, λP _L promoter, lpp promoter, etc., when the host is Bacillus subtilis, SP01 promoter, SP02 promoter, pe
If the host is yeast, such as the nP promoter, PH
O5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. In particular, the host is E. coli and the promoter is the trp promoter or
Is preferably the λP _L promoter. When the host is an animal cell, a promoter derived from SV40,
Examples thereof include retrovirus promoters, and SV40-derived promoters are particularly preferable.

A transformant is produced using the vector containing the DNA thus produced. As a host, prokaryotic organisms such as Escherichia coli, Bacillus subtilis, actinomycetes, yeast,
Examples include eukaryotes such as mold and animal cells. Specific examples of the above-mentioned Escherichia coli and Bacillus subtilis include, for example, Escherichia
co li K12DH1 [Proceedings of National Academy of Sciences US
A. (Proc. Natl. Acad. Sci. USA), 60 , 160
(1968)], JM103 [Nucl. Acids, Res.], 9 , 309 (19).
81)], JA221 [Journal of Molecular Biology (J. Mol. Biol.), 120 , 517].
(1978)], HB101 [J. Mol. Biol., 41 ,
459 (1969)] and C600 [Genetics, 39 , 440 (1954)] and the like. Examples of the Bacillus subtilis include Bacillus su
btilis MI114 [Gene, 24 , 255
(1983)], 207-21 [Journal of Biochemistry, J. Biochem., 95 , 87 (19).
84)] and the like. Specific examples of the above yeast include Saccharomyces cerevi siae AH22,
AH22R ^-, NA87-11A, and the like DKD-5D. Specific examples of the above animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, mouse L cells and the like. To transform the above E. coli, for example, Proc. Natl. Aca
d. Sci. USA, 6 9 , 2110 (1972) and Gene, 1
7 , 107 (1982) and the like. Transformations of Bacillus subtilis include, for example, Molecular and General Genetics (Molec. Ge.
n. Genet.), 168 , 111 (1979) and the like. For transforming yeast, for example, Proc. Natl. Acad. Sci. USA, 75 , 1929.
It is performed according to the method described in (1978). For transforming animal cells, for example, Virology is used.
y), 52 , 456 (1973). Thus, a transformant transformed with the vector containing the DNA encoding M is obtained.

The host is Escherichia coli, Bacillus subtilis, actinomycete, yeast or
Is used for culturing fungal transformants.
A suitable liquid medium is a liquid medium in which the form
Carbon source, nitrogen source, inorganic substances, etc. necessary for the growth of the transformant
Is included. As a carbon source, for example, gluco
Such as glucose, dextrin, soluble starch or sucrose
As the source, for example, ammonium salts, nitrates,
Corn steep liquor, peptone, casein, meat d
Inorganic such as kiss, soybean meal or potato extract or
Calcium chloride and dihydrogen phosphate as organic and inorganic substances
Such as sodium or magnesium chloride
It The pH of the medium is preferably about 5-8. Host is E. coli
In this case, the medium used is, for example, glucose or casamino acid.
M9 medium containing [Miller, J., Experiments in
 Molecular Genetics (Exp. Mol. Genet.,
p.431), Cold Spring Harbor Laboratory, New Yor
k, 1972] is preferred. Culturing is usually about 14-43 ℃
For about 3 to 24 hours, add aeration and agitation if necessary.
You can also do it. When the host is Bacillus subtilis, the culture is usually about 3
Perform for about 6 to 24 hours at 0 to 40 ° C, aeration or stirring if necessary.
Stirring can be added. Transformant whose host is yeast
When culturing cells, for example, Burkholder's medium
Small medium [Bostian, K. L. et al., Proc. Natl. Acad. Sci.
 USA,77, 4505 (1980)]. Cultivation
The pH of the ground is preferably adjusted to about 5-8. Culture
Always perform at about 20-35 ° C for about 24-72 hours, if necessary
Aeration and stirring. Transformation in which the host is an animal cell
When culturing the body, as a medium, for example, about 5-20%
MEM medium containing FBS [Science,
122, 501 (1952)], DMEM medium [Virol
ogy,8, 396 (1959)], RPMI 1640
Place [Journal of the American Medical
Association (J. Am. Med. Assoc.),199, 5
19 (1967)], 199 medium [Proceedings]
 Of The Society For Experimental
Biology and Medicine (Proc. Soc. Exp.
Biol. Med.),73, 1 (1950)] and the like.
It The pH is preferably about 6-8. Culture is usually about
Perform for about 15 to 60 hours at 30 ℃ to 40 ℃, and if necessary
Add aeration and agitation.

Although the M protein is produced and accumulated in the culture by the production method of the present invention, it may be collected intracellularly or extracellularly. The whole molecular form M is mainly produced in cells, but can be secreted as a fusion protein by a conventional method, for example, with an appropriate leader sequence. When the intracellular M is extracted from the culture, for example, after culturing the cells inside the cells, the cells are collected by a known method, and a buffer containing a protein denaturing agent such as guanidine hydrochloride or urea or Triint X-10 is used.
The cells are suspended in a buffer solution containing a surfactant such as 0, and then centrifuged to obtain a supernatant containing M, or the cells are sonicated, treated with an enzyme such as lysozyme, or frozen and thawed. After the disruption, a method of obtaining a supernatant containing M by centrifugation may be appropriately used. In order to separate and purify these supernatants and M produced and accumulated outside the cells, separation and purification methods known per se may be combined appropriately. As these known separation and purification methods, methods such as salting out and solvent precipitation methods that utilize the difference in solubility, dialysis method, ultrafiltration method, gel filtration method and SDS-polyacrylamide gel electrophoresis method are mainly used. How to take advantage of the difference between
Methods using specific affinity such as affinity chromatography, methods utilizing difference in hydrophobicity such as reversed-phase high performance liquid chromatography, methods utilizing difference in isoelectric points such as isoelectric focusing Is mentioned. In particular, M can be recognized by the monoclonal antibody of the present invention, for example, M31-15 obtained in the Examples described below, and thus immunoaffinity chromatography using the monoclonal antibody is extremely effective for purifying the protein.

On the other hand, M and its partial peptide of the present invention can be chemically synthesized, for example, by an automatic peptide synthesizer. This basic synthesis process is described in RBMerrifield [Advances in Enzymology 32 , 221-296 (19
69)]. In this method, a carboxyl-terminal amino acid is covalently bound to a resin carrier, and α-
The principle is to obtain a peptide resin having a desired amino acid sequence by sequentially repeating the removal of the amino-protecting group and the condensation of the protected amino acid to extend the peptide chain toward the amino terminus. Condensation of each amino acid and removal of the protecting group for the α-amino group are carried out under substantially the same conditions, and no intermediates are purified, so generally high skill is not required in the synthesis. Moreover, this method is rapid and is a very convenient method for synthesizing various peptides. The protected peptide resin thus obtained is reacted with, for example, anhydrous hydrogen fluoride, trifluoromethanesulfonic acid or trifluoroacetic acid in the presence of various additives to remove the peptide from the resin and remove all protecting groups. Can be done in one step. The obtained crude peptide product can be purified by a known means for purifying a peptide or protein. Examples thereof include gel filtration, ion exchange chromatography using a cation exchange resin or an anion exchange resin, column chromatography by various principles such as hydrophobic chromatography, partition adsorption chromatography and high performance liquid chromatography.
The polypeptide of the present invention thus obtained can be obtained in the form of various salts. Examples of the salt include inorganic acids, salts with organic acids such as formic acid, acetic acid, tartaric acid, and citric acid, or inorganic bases such as sodium and ammonia, and salts with organic bases such as triethylamine, ethylamine, and methylamine. Be done. The polypeptide of the present invention thus obtained can be used as a therapeutic agent for cancer and the like. To use the polypeptide of the present invention as a medicine,
As a powder as it is, or together with other pharmacologically acceptable carriers, excipients or diluents, the pharmaceutical composition (eg,
Can be safely administered parenterally or orally to warm-blooded animals (eg, humans, mice, rats, hamsters, rabbits, dogs, cats) as injections, tablets, capsules, solutions, ointments) it can. The injection is formulated according to a conventional method using, for example, physiological saline or an aqueous solution containing glucose and other auxiliary agents. Pharmaceutical compositions such as tablets and capsules can also be prepared according to a conventional method. When the polypeptide of the present invention is used as the above-mentioned drug, for example, the above-mentioned warm-blooded animal is administered in an appropriate dose selected from the daily dose of about 1 μg to 1 mg / kg, in consideration of the administration route, symptoms and the like. To be done.

In the present specification and drawings, when abbreviations are used for bases, amino acids, etc., IUPAC-IUB Commi
sion on Biochemical Nomenclature or abbreviations commonly used in this field, examples of which are given below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. Unless otherwise specified, the left-to-right direction of the sequence is the direction from the N-terminus to the C-terminus in the amino acid sequence,
In the base sequence, the direction from the 5'end to the 3'end is shown. In the M of the present invention, a part (for example, up to about 5%) of the amino acid sequence may be modified (addition, removal, substitution with other amino acid, etc.). DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: 2'-deoxyadenylic acid residue T: thymidylate residue G: 2'-deoxyguanidylate residue C: 2'-deoxycytidylic acid residue RNA : Ribonucleic acid mRNA: messenger RNA dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate dCTP: deoxycytidine triphosphate ATP: adenosine triphosphate EDTA: ethylenediaminetetraacetic acid SDS: dodecylsulfate Sodium FBS: Fetal bovine serum Gly or G: Glycine Ala or A: Alanine Val or V: Valine Leu or L: Leucine Ile or I: Isoleucine Ser or S: Serine Thr or T: Threonine Cys or C: Cysteine Met or M: Methionine Glu or E: Glutamic acid Asp or D: Aspartic acid Lys or K: Lysine Arg or R: Arginine His or H: Histidine Phe or F: Phenylalanine Tyr or Y: Tyrosine Trp or W : Tryptophan Pro or P: Proline Asn or N: Asparagine Gln or Q: Glutamine

[0025]

EXAMPLES The present invention will be specifically described below with reference to Reference Examples, Examples and Test Examples, but it goes without saying that these do not limit the scope of the present invention. The hybridomas and transformants disclosed in Reference Examples and Examples are as follows: Fermentation Research Institute (IFO;
2-1785, 13-Honcho, Yodogawa-ku, Osaka) and the Institute of Microbial Technology, Ministry of International Trade and Industry (FRI; 1-3-1, Higashi, Tsukuba, Ibaraki) based on the Budapest Treaty. ───────────────────────────────── IFO FRI Name (IFO No.) (FERM BP No.) ── ─────────────────────────────── Mouse hybridoma 50324 3340 M31-15 (1991.4.2) (1991.4.9) Escherichia coli 15165 3357 DH5αF'IQ / pTB1352 (1991.4.9) (1991.4.15) Hamster fibroblast 50365 3746 CHO / M-1 (1992.1.31) (1992.2.13) Escherichia coli 15259 3745 DH1 / pTB1442 (1992.2.5) (1992.2.5) (1992.2.10) ────────────────────────────────── In the table, () indicates the date of deposit.

Reference Example 1 Establishment of MAC8 cells Cancer tissue pieces excised from a human lung cancer patient were treated with phosphate buffer (P
BS) (137 mM NaCl, 2.7 mM KCl, 8.1 m
M Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ ; pH 7.4)
After washing with, cut into 1-2 mm squares with scissors. The obtained strip was subcutaneously transplanted into a nude mouse, and 1-2 weeks later, the tumor was excised from a nude mouse in which the tumor had grown.
After washing with PBS, it was cut into 1-2 mm squares with scissors. Then, it was digested with PBS containing 0.25% trypsin for 10 minutes. Discard the first digestive fluid, collect the second and third digestive fluids, and centrifuge (700 × g, 5 minutes) to give 10% F cells.
After washing BS with RPMI1640 medium, the cells were cultured in the same medium to establish human lung cancer cell line MAC8. The established cell line was then subjected to cloning by the limiting dilution method.

Reference Example 2 Anticancer Antibody ELISA The lung cancer cell line MAC8 established in Reference Example 1 was suspended in RPMI1640-10% FBS medium at a concentration of 4 × 10 ⁵ cells / ml, and 100 μl of each suspension was spread on a 96-well microplate. The cells were cultured overnight in a CO ₂ incubator at 37 ° C.
Next day, plate the plate with 50 mM Tris buffer (pH 7.
After washing 3 times in 4), test culture supernatant 1 in each hole of the plate.
00 μl was added and the mixture was heated at 37 ° C. for 1 hour. Plate 4
After washing twice, it was reacted with biotinylated goat anti-mouse immunoglobulin for 1 hour. After washing the plate 4 times, avidin biotinylated peroxidase (Vectastatin) was added and reacted for 1 hour. After washing the plate 4 times, ortho-phenylenediamine solution (1 mg / ml, 0.5 μl hydrogen peroxide solution, 5.1 mg citric acid, 18.4 mg Na ₂
50 μl of HPO ₄ .12H ₂ O / ml) was added and color was developed for 10 minutes. 100 μl of 2N H ₂ SO ₄ was added, and the absorbance at 450 nm was measured using an absorptiometer for 96-well microplate.

Example 1 Production of Monoclonal Antibody (1) Preparation of Mouse Spleen Cells MAC8 cell suspension obtained in Reference Example 1 in PBS (1 ×
Balb / c mice (8-week-old female) were intraperitoneally administered at 10 ⁷ cells / 0.2 ml / mouse, and booster was given after 2, 3, 4 and 7 weeks. Immunization after 7 weeks was administered intravenously. The spleen was taken out from the mouse 3 days after the final immunization, and a splenocyte suspension was prepared aseptically. (2) Cell fusion The mouse splenocyte suspension (2 × 10 ⁸ cells) obtained in (1) was mixed with the mouse myeloma cell line SP2 suspension (5 × 10 ⁷ cells) and centrifuged at 700 × g for 5 minutes. A cell pellet was obtained. 1 ml of 50% polyethylene glycol 4 on cell pellet
000 solution (Sigma Chemical Island, NY) was gradually added dropwise with mixing for 2 minutes. Subsequently, at intervals of 2 minutes, 1,1.5,2,3,4,5 ml of RPMI
1640 medium was added sequentially to gradually reduce the polyethylene glycol concentration. Collect the cells by centrifugation at 700 xg for 5 minutes, wash the cells with RPMI1640 medium, and then
Suspend (10 ⁷ cells / ml) in RPMI1640 medium supplemented with 0% FBS / HAT, and add 1 to each well of a 96-well plate.
00 μl was sprinkled on each side. After 2, 5 and 10 days, half of the medium was replaced with fresh HAT medium, and on the 21st day the culture supernatant 100
μl was taken and the antibody was screened by the ELISA described in Reference Example 2. (3) Limiting dilution method The hybridomas screened in (2) above were HA
The cells were cultured in T medium, diluted to a cell number of 5 cells / ml, and each 100 μl was added to a 96-well tissue culture plate and cultured.
The antibody-producing ability of the culture solution of the well in which the growth of a single colony was observed was examined according to the ELISA described in Reference Example 2 to obtain a single clone cell. (4) Screening for Cancer Cell Motility Suppressing Antibody 0.6 ml of the culture supernatant of the hybridoma obtained in (3) above was added to the lower chamber of the Transwell plate, and the lung cancer cell line was placed on the nucleopore membrane filter (pore size 5 μm). MA
C10 suspension (5 × 10 ⁵ / ml; RPMI1640
-10% FBS) (0.1 ml) was added and the mixture was cultured in a CO ₂ incubator at 37 ° C for 16 hours. The number of cells passing through the Nucleopore membrane filter and sinking to the bottom of the plate was counted using a phase contrast microscope. The 135 hybridomas obtained in (3) above were screened and a mouse hybridoma (M31-15) having a strong inhibitory activity was selected. (5) Preparation of antibody 0.5 BALB / c mice (female, 10 weeks old) were injected into the abdominal cavity of 0.5 mice.
1 ml of pristane (mineral oil; Wako Pure Chemical Industries, Ltd.) was administered, and after 2 weeks, 0.5 ml of a hybridoma M31-15 suspension (2 × 10 ⁷ cells / ml) was administered to the abdominal cavity of each mouse. Ascites was collected from the mice 14 days later, and
Centrifuge at 0 rpm for 10 minutes to remove cells, and add 1800
Centrifuge at 0 rpm for 60 minutes, then glass filter (Whatman GF / C) and Millex GV (0.22μ
m, Millipore) and sterilized by filtration to obtain 28.5 ml of ascites. The MAPSII kit (Protein A column; manufactured by Bio-Rad) was used for purification of IgG antibody from ascites. 0.5 ml of ascites binding buffer
5 ml was added, 0.5 ml of the mixed solution was adsorbed on a Protein A column (Affiprep; 30 × 4.6 mm), and elution was carried out according to the following elution program using high performance liquid chromatography manufactured by Varian. Elution solvent A: binding buffer; Elution solvent B: PBS
[8.1 mM Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ ,
137 mM NaCl, 2.7 mM KCl (pH 7.
4)]; Elution solvent C: elution buffer; Elution program: 0 minutes 100% A; 3 minutes 100% A; 3.01
Min 100% B; 15 min 100% B; 15.01
Min 100% C; 20 min 100% C; 20.01
Min 100% B; elution rate: 0.8 ml / min; detection wavelength: 280 nm; loop: 1 ml IgG antibody eluted from the column was immediately dialyzed against PBS, and then sterilized by filtration through a Millex GV filter (Millipore). Purified mouse anti-human cancer antibody M31-15 (Ig
G ₁ ) 0.4 mg was obtained.

Example 2 Specificity of Monoclonal Antibody The binding of M31-15 antibody to cancer cells was FACSt.
The measurement was performed using ar (Becton Dickinson). The adherent cell line was PBS / 0.02 containing 0.125% trypsin.
With 10% EDTA solution RPMI1640-10% FB
After washing with S medium, the cells were incubated at a concentration of 1 × 10 ⁶ cells / ml at 0 ° C. for 1 hour. Purified M31-15 antibody (final concentration 1 μg / ml) was added and reacted at 0 ° C. for 1 hour. The cells were washed 3 times with PBS-0.1% NaN ₃ and then FITC-.
Labeled anti-mouse IgG (H + L) antibody (20-fold dilution) 10
The reaction was performed with 0 μl at 0 ° C. for 30 minutes. PBS-0.1%
After washing with NaN ₃ , cells were washed with 2 ml of PBS-0.1% Na.
Suspended in N ₃ and analyzed using FACStar. M3
The 1-15 antibody bound to most cancer cells except liver cancer cells and some lymphoid cancer cells [Table 1].

[Table 1] Cell line Staining example Positive ────────────────────────── Lung cancer 4 4 Gastric cancer 2 2 Pancreatic cancer 2 2 Breast cancer 2 2 Liver cancer 20 Colon cancer 1 1 Cervical cancer 1 1 Epidermoid cancer 1 1 Melanoma 1 1 Glioma 1 1 Lymphoid cancer cells 9 4 Fibroblasts 4 2 ──────────────────── ───────

Example 3 Insolubilization of M31-15 antibody 0.43 mg of the purified monoclonal antibody obtained according to the method described in Example 1- (5) was added to 1 L of 0.1 M NaHCO _3.
It was dialyzed against (pH 8.4) overnight. AffiGel
After washing 2 ml of 10 (Bio-Rad) according to the instructions for use and inactivating the M31-15 antibody by reacting with the antibody at 4 ° C. for 24 hours, the gel was washed with 0.1 M Tris buffer (p.
H 8.1) Wash with 10 ml and then in the same buffer at 4 ℃ for 3
After leaving for one day to block the remaining active groups, the gel was washed with PBS. As a control gel, a gel treated in the same manner using a buffer containing no antibody was prepared.

Example 4 Confirmation of antigen binding activity of insolubilized M31-15 antibody MIA PaCa-2 cells were treated with RPMI1640-10%.
Roller bottle using FCS medium (Corning 2524
It was cultured in 0). Remove cells with scraper 0.1M
1% after washing twice with Tris buffer (pH 8.1)
Triton X114 / 0.1M Tris / 10mM ED
TA / 0.2 mM p-APMSF ((p-amidinophenyl) me
thanesulfonyl fluoride hydrochloride)
(2U / ml) Trasylol (pH 8.1) 2ml
Was solubilized in ice for 10 minutes. Centrifugal supernatant (150
(00 rpm, 10 minutes) was heated at 37 ° C. for 10 minutes and centrifuged at room temperature at 3000 rpm for 10 minutes. Discard the upper layer and add 2 ml of 0.1 M Tris buffer (pH 8.1) to the lower layer.
It was centrifuged at room temperature for 10 minutes. 1% CHAPS (3-
[[3-cholamidopropyl] dimethylammonio] -1-propans
ulfonat; Boehringer Mannheim) /0.1M Tris (p
H 8.1) 0.5 ml was added at 15000 rpm at 4 ° C.
After centrifugation for 30 minutes, about 0.9 ml of supernatant was obtained. 5 μl of the supernatant was transferred to an Eppendorf tube and PBS 15 μl, M31-15 antibody-bound AffiGel obtained in Example 3 was used.
About 10 μl of 10 was added and the mixture was reacted at room temperature for 1 hour. Thirty
After centrifugation at 00 rpm for 10 minutes, the supernatant was collected with a microsyringe and subjected to SDS-PAGE and Western blotting. Antigen bound to gel was 10% SDS 5 μl, 1
% DTT 5 μl Electrophoresis buffer 10 μl was added and the mixture was heated in boiling water for 5 minutes to elute. Although the antigen was not bound to the gel in the control gel and was recovered in the supernatant, the M31-15
In the case of the antibody-bonded gel, all of it remained in the supernatant and was adsorbed on the gel [Fig. 1].

Test Example 1 Monoclonal antibody M31-1
Suppression of in vivo metastasis by 5 MAC8 cells cultured in a flask were detached with trypsin, and then once with 10% FBS / RPMI1640 medium,
Then wash with RPMI 1640 twice and then 2 or 6
× 10 ⁵ cells were iv administered to BALB / c nu / nu mice via the tail vein. In order to see the metastasis-suppressing effect of the monoclonal antibody M31-15 obtained in Reference Example 1, cancer cell administration 3
After 30 hours, M31-15 (50 μg) was administered iv through the tail vein. After 3 months, the number of colonies of cancer cells metastasized to the lung was counted. The results are shown in [Table 2]. A marked decrease in the number of metastatic colonies was observed by antibody administration.

[Table 2] ──────────────────────────── Metastatic positive cases / number Average colonies ───────── ──────────────────── Antibody non-administration group 6/7 54.4 Antibody administration group 3/4 1.8 ──────────── ─────────────────

Test Example 2 Suppression of Motility by Purified M31-15 Antibody (1) Suppression of Motility of MAC10 Cells MAC10 cells were cultured in a culture flask at 0.01% EDTA.
With PBS containing, peel off RPMI1640-10% FCS
After washing with the medium, the cells were suspended at a concentration of 5 × 10 ⁵ / ml. T
RPMI1640- in the lower chamber of the raswell plate (Costar)
10% FCS medium and purified M31-15 antibody (0-
10 μg) 0.6 ml was added, and 0.1 ml of MA was added to the upper chamber.
C10 cell suspension was added, and the cells were cultured at 37 ° C. for 16 hours.
The cells that had passed through the membrane and migrated into the medium in the lower chamber were collected, concentrated by centrifugation, transferred to a 96-well microplate, and the number of cells was counted by a phase contrast microscope. As shown in FIG. 2, 50% inhibition of motility was observed at an antibody concentration of 0.1 μg / ml. (2) Inhibition of T98G cell motility T98G cells were cultured in a 75 cm flask (Corning 25110) using MEM-10% FCS (containing 1% non-essential amino acid and 1% sodium pyruvate) medium. After the cells formed a dense monolayer, the medium was discarded, washed twice with the above serum-free medium, 3 ml of the medium was added, and the mixture was cultured at 37 ° C. for 24 hours. Collect the culture supernatant and
The cells were removed by centrifugation at 0 rpm for 5 minutes to obtain a chemotactic factor. In the lower chamber of the Microchemotaxis chamber (Academia), crude chemotactic factor and purified antibody (0-1 μg /
Put mixture 30μl of ml), and 4 hours at 37 ° C. was added a T98G cell suspension 2.5 × 10 ^4/50 [mu] l in the upper chamber carrying a pore size 8μm nucleosomal pore membrane (PVPF). The cells on the upper surface of the membrane are removed, and cells passing through the membrane and adhering to the lower surface of the membrane are diff-
Stained with Quick (green cross). The number of cells in two fields of view of 0.25 mm square was counted using a 40 × objective lens. The results were as shown in [Table 3]. This antibody has a motility of T98G cells of 50 at a concentration of 0.1 μg / ml.
% Inhibition.

Table 3 Chemotactic factor M31-15 antibody Number of migrating cells (μg / ml) − − 0 + − 128 + 0.1 42 + 0.5 6 6 + 1.0 4 ─────────────────────────────── ────

Test Example 3 Absorption of chemotactic factor by insolubilized M31-15 antibody About 20 μl of the gel obtained in Example 3 was patted with an Eppendorf tube (3).
810), 0.4 ml of the crude chemotactic factor obtained in Test Example 2-2) was added, and the mixture was reacted at room temperature for 1 hour. After centrifugation at 3000 rpm for 10 minutes, the suppressive activity of the supernatant was measured according to the method described in Test Example 2-2). However, all concentrations of chemotactic factor were used at 2-fold dilution. As a result, the chemotactic factor was not absorbed [Table 4].

[Table 4] Chemotactic Factor Antibody-A ffiGel10 * Number of migrating cells --0 + -194 + Incubation with MEM 152 + + 52 + Control gel 95 *: Antibody-AffiGel 10 (or control gel) was chemotactic factor. (Or MEM) was used for 1 hour at room temperature.

Example 5 Identification of antigen recognized by M31-15 antibody MIA PaCa-2 (pancreatic cancer cell), HEL (erythroleukemia cell), ZR-75-30 (breast cancer cell), MAC10
(Lung cancer cells) and AZ521 (gastric cancer cells) were monolayer-cultured on a Falcon dish (3003), and the cells were adjusted to 0.
After washing twice with 1 M Tris (pH 8.1), the cells were collected with a scraper (Costar, 3010), washed by centrifugation, and the cells were washed with 1% CHAPS / 0.1 M Tris / 10 mM E.
DTA / 0.2 mM p-APMSF / Tracylol
(2 U / ml) 100 μl was added and solubilized at 0 ° C. for 2 hours. 20 μl of centrifugation supernatant at 15,000 rpm for 30 minutes
Laemmli SDS-PAGE (gel concentration 12%) [N
Nature Lond., 227, 680-685 (1970)], and the antigen was detected by Western blotting. As shown in [FIG. 3], a major band having a molecular weight of 25,000 and a minor band having a molecular weight of 28,000 were observed in all cancer cells.

Example 6 Cloning of cDNA encoding M M cDNA library of λgt11 (Human Breast Carcinom)
a cDNA library, CLONTECH Lab. Inc.) was infected with E. coli Y1090 attached to the library kit, and then spread on a soft agar plate of L broth. IPTG (Isopropylthiogalactoside) when plaques start to appear
Place the nitrocellulose membrane containing the above on the plate, and 3.
Incubated for 5 hours. Next, remove the nitrocellulose membrane, and remove the TBST buffer solution (10 mM Tris, 15 mM).
0 mM NaCl, 0.05% Tween 20, pH
8.0) and then 30% in 20% FBS solution.
I put it on for minutes. The nitrocellulose membrane treated in this way is
The cells were immersed in TBST containing the 31-15 antibody (2 μg / ml) and E. coli extract (1 mg / ml) and reacted at room temperature for 50 minutes. After washing the membrane 3 times with TBST buffer, alkaline phosphatase-labeled anti-mouse IgG antibody (50
After reacting with (00-fold dilution) for 30 minutes and washing 3 times with TBST, the substrate (Nitro-bluetetrazalium and 5-bromo-4) was washed.
Color was developed using chloro-3-indoyl phosphate). 1
1 out of a x10 ⁶ pfu (plaque forming unit) charge
Four positive clones were selected. As a result of rescreening these 14 clones according to the above method,
One clone was positive again and was selected as positive.

Example 7 Preparation of transformant containing plasmid pTB1352 After infecting Escherichia coli Y1090 with the selected positive clone, it was spread on soft agar medium of L broth and cultured at 37 ° C. for 16 hours, and then about 10 ⁹ from soft agar. The pfu phage was extracted. From the phage thus obtained, QIAGEN> lambda <
DNA was purified using a kit (DIAGEN GmbH). DN obtained by digesting the purified DNA with the restriction enzyme EcoRI
The A fragment was transformed into plasmid pU using T4 DNA ligase.
It was inserted into the EcoRI site of C118 (Takara Shuzo) to construct the plasmid pTB1352. The resulting pTB1352
E. coli DH5arufaF'IQ ^TM in transfected transformant Escherichia coli DH5αF'IQ ^TM / pTB
1352 was obtained.

Example 8 Nucleotide Sequence Determination of DNA The transformant obtained in Example 7 was transformed with helper phage KO.
・ After infecting 7 with 2 × YT medium (1.6% Bacto-tr
yptone, 1% Yeast extract, 0.5% NaCl) 3
After culturing at 7 ° C for 16 hours, PEG # 60 was obtained from the culture supernatant.
00 single-stranded DNA was purified by the ethanol precipitation method. The base sequence of the purified DNA is α-
³² P-dCTP (-800 Ci / mmole, Amersham), Sequenase Ver. 2.5 kit (UBC)
Was used to determine the sequence of the cDNA portion. The determined nucleotide sequence and the amino acid sequence deduced from the sequence are shown in [FIG. 4] to [FIG. 9] and SEQ ID NO: 3.

Example 9 Preparation of Gene Map of pTB1352 The transformant obtained in Example 7 was treated with 2 × YT medium at 37 ° C. for 1 hour.
QIAGEN> plasmid <kit from transformants that had been cultured for 6 hours
Was used to purify the double-stranded DNA. The obtained DNA was digested with restriction enzymes SacI, KpnI, SmaI, BamHI, XbaI,
It was digested with SalI, PstI, SphI and HindIII and the digest was analyzed by 1% agarose gel electrophoresis. As a result, it was revealed that there were two cleavage sites for XbaI and one cleavage site for SphI. Next, XbaI-EcoRI and SphI-
Double cutting of EcoRI was performed to complete the gene map shown in FIG.

Example 10 Construction of plasmid for expressing animal cells Known plasmid pTB399 [JP-A-60-6328]
No. 2 publication Example 1 (v)] is cleaved with a restriction enzyme EcoRI,
After blunting the ends using DNA polymerase (Klenow fragment), BglII linker (CAGATCTG) having phosphorylated 5'ends was ligated according to a known method. After digestion with BglII, a 3.9 Kb DNA fragment was separated and purified by agarose gel electrophoresis, and the DNA fragment was circularized by T4 DNA ligase reaction to form a plasmid containing A-MuLV LTR region, splice region and polyA addition region. pTB1308 was constructed. This plasmid pTB1308 is then cleaved with the restriction enzyme EcoRI, while the restriction enzyme E is obtained from the plasmid pTB1352 obtained in Example 7.
The plasmid pTB1308 / M was constructed by mixing the M gene fragments (1.1 Kb) separated and prepared by agarose gel electrophoresis after cleaving with coRI and performing T4 DNA ligase reaction. Similarly, the known plasmid pTB348 [JP-A-60-63]
No. 282, Example 1 (v)] is treated with restriction enzymes SalI and Hin.
cleaved with dIII, while the plasmid pTB130 obtained above
A-MuLV L, M gene isolated from 8 / M by digestion with restriction enzymes SalI and HindIII and separated and prepared by agarose gel electrophoresis
A DNA fragment (2.7 Kb) containing a TR region, a splice region and a polyA addition region was mixed, and a M expression plasmid pTB1442 (Fig. 11) was constructed by T4 DNA ligase reaction. Transformant Escherichia in which Escherichia coli DH1 was transfected with the obtained plasmid pTB1442
coli DH1 / pTB1442 was obtained.

Example 11 Expression of pTB1442 and acquisition of transformant i) CHO cells Hamster-DHFR to CHO cells (hereinafter abbreviated as CHO cells) [Proc. Natl. Acad. Sci. USA, 77 , 4216-4220]
(1980)] was cultured in a Falcon dish (diameter 6 cm; BECTON DECKINSON, CA) using 5% FBS / Ham's F12 medium.
CHO cells were transfected with the plasmid pTB1442 (10 μg per dish) according to Graham's method [Virology, 52 , 456-467 (1973)]. After 2 days, the medium was changed to 10% dialyzed FBS / proline (35 μg / ml) / Dulbecco's modified MEM medium, and 2 weeks later, DHFR ⁺ was grown to obtain colonies. Whether or not M was expressed was confirmed according to the method described in Example 3. The results are shown in [Fig. 12]. ii) MAC10 cells
Electroporation method for transfection of MAC10 cells [EMBO J., 1 , 841 (1982)]
Was used. That is, 20 μg of plasmid pTB1442 was added to 5 × 10 ^{6 of} MAC10 cells by pRc / CMV (Invitr).
(ogen, CA) mixed with 2 μg and transfected, 10%
It was diluted to a concentration of 2.5 × 10 ⁵ / ml with FBS / RPMI1640 medium, and 100 μl of the solution was spread in each well of the 96-well plate. G418 (Sigma) as selection medium 1 mg / ml
10% FBS / RPMI1640 medium containing the above was used.
After about 2-3 weeks, a G418-resistant colony-17 strain was obtained.

Example 12 Determination of M by Dot Plot Method i) Solubilization of cells The cells obtained in Example 11 were cultured in a dish having a length of 10 cm, 2 ml of PBS and 0.1 M Tris buffer (pH).
8.1) Wash with 1 ml, 0.1 M Tris buffer 0.5 ml
And cells were collected in an Eppendorf tube 3810 using a cell scraper (Costar). The cells remaining in the dish were collected with an additional 0.5 ml of 0.1 M Tris buffer.
It was Cells were supplemented with 1% CHAPS / 0.1M Tris pH 8.
1/10 mM EDTA / 0.2 mM p-APMSF / tr
Add 50 μl of asylol (2 U / ml) at 0 ℃, 30
It was solubilized for minutes and the supernatant was used. ii) Quantification by dot plot method 1 μl of the extract obtained in the above i) or its diluted solution was spotted on a nitrocellulose membrane (BIO-RAD; Richmond, CA), and after air-drying, TBS (20 mM Tris / 500
Immersion in mM sodium chloride, pH 7.5) overnight. Then, it was reacted with 1% gelatin / TBS containing a monoclonal antibody, M31-15 (1 μg / ml), at 37 ° C. for 1 hour. The membrane was made of TBS (TT containing 20% Tween 20).
(BS) twice for 10 minutes and once with TBS, then HRP
The mixture was allowed to react with 1% gelatin / TBS containing a labeled anti-mouse antibody (diluted 1000 times) at 37 ° C for 1 hour. After the membrane was washed twice with TTBS for 10 minutes and once with TBS, the coloring solution (HR
P color development reagent (BIO-RAD) 20mg / methanol 6.6ml / hydrogen peroxide solution 20μl / TBS 3
(3.4 ml) and developed for 10 minutes. The membrane was thoroughly washed with distilled water and then air dried. 1 extract of MAC10 cells
Expressed as 0 unit (U) / ml. MA obtained by expressing the plasmid pTB1442 containing M in [Table 5]
The measurement result of the clonal extract of C10 cells is shown. BCA Protein Assay Reagent (PIERCE, Roc
kford, Illynois).

[Table 5] ────────────────────────── M (U / ml) Protein amount (mg / ml) U / mg ──── ────────────────────── MAC 10 10 10 1.0 Clone 1 60 19 3.2 Clone 4 20 19 1.1 Clone 7 10 11 0.9 Clone 9 40 17 2.4 Clone 11 10 9.6 1.0 ───────────────────────────

Example 13 Synthesis and Purification of M Fragment Peptide The synthesis of the M fragment peptide was carried out by the solid phase synthesis method for peptides [RB Merrifiel developed by Merrifield et al.
d, Advance in Enzymology
Enzymology) 32 , 221-296 (1969)] and an automated peptide synthesis group 430A (Applied Biosystems) was used. The protected peptide-resin was synthesized using a protocol designated by Applied Biosystems. Protected amino acid-p-oxymethylphenylacetamidomethyl resin (polystyrene-1% divinylbenzene) was used as a starting material, and the protected amino acid was successively condensed thereto.
A tertiary butyloxycarbonyl (BOC) group was used to protect the α-amino group of each amino acid during condensation.
The side chain functional group protection was performed as follows. The hydroxyl groups of serine and threonine are O-benzyl ethers,
The hydroxyl group of tyrosine is p-bromobenzyloxycarbonyl ester, the carboxyl group of glutamic acid and aspartic acid is benzyl ester, the imidazole nitrogen of histidine is benzyloxymethyl, and the side chain amino group of lysine is 2-chlorobenzyloxy. With carbonyl, the guanidine functional group of arginine was protected with p-toluenesulfonyl group, and the indo-luimine of tryptophan was protected with formyl group. All amino acids were obtained from Applied Biosystems Japan or Bachem Chemicals. After condensation of all amino acids on the resin, the protected peptide resin was removed from the synthesizer and dried. Peptide resin (1 g) was added to p-
Cresol (1 ml), 1,2-ethanedithiol (1 ml),
It was reacted with anhydrous hydrogen fluoride (8 ml) containing 2-mercaptopyridine (100 mg) at 0 ° C. for 2 hours. After completion of the reaction, hydrogen fluoride was distilled off and the residue was washed with diethyl ether to remove most of the mixed reagents. The peptide was extracted with trifluoroacetic acid (10 ml) and the resin was removed by filtration. After the filtrate was concentrated, ether was added, the resulting precipitate was collected by centrifugation, and the precipitate was washed with ether three more times.
The obtained powder preparation was further purified by reverse phase high performance liquid chromatography [column YMC A-303 ODS (4.6 × 250 m
m); elution solvent A, 0.1% trifluoroacetic acid-99.9
% Water; elution solvent B, 0.1% trifluoroacetic acid-9
9.9% acetonitrile; elution gradient program, 0
Min (80% A + 20% B) -30 min (50% A + 50%
B) (However, other elution programs may be used if necessary) Elution rate, 1 ml / min; detection wavelength 230 or 280 nm]. The peak fraction containing the pure target substance was collected and passed through a column of Bio-Rad AGI × 8 (acetic acid type, 1.8 × 5 cm), washings were also collected, and acetonitrile was distilled off, followed by freeze-drying. The results of amino acid analysis of the peptides thus obtained are shown in [Table 6]. In addition, the amino acid sequences of these peptides and the retention times in reversed phase high performance liquid chromatography are shown below.

[Table 6] ━━━━━━━━━━━━━━━━━━━━━━━━━ Peptide ─────────────────── 1 2 3 4 ━━━━━━━━━━━━━━━━━━━━━━━━━ Real side value (theoretical value) Asp 4.0 (4) 2.0 (2) 5.1 (5) 5.0 (5 ) Thr 2.6 (3) 0.9 (1) 1.9 (2) 2.5 (3) Ser 0.9 (1) 1.9 (2) 3.3 (4) Glu 7.1 (7) 4.2 (4) 2.1 (2) 4.0 (4) Pro 2.1 (2) Gly 3.0 (3) 1.1 (1) Ala 3.0 (3) 1.1 (1) 1/2 Cys 2.8 (3) 1.8 (2) Val 1.6 (2) 1.0 (1) 2.9 (3) 1.0 (1) Ile 0.7 (1) 2.8 (3) 1.9 (2) 0.9 (1) Leu 2.0 (2) 3.0 (3) 1.0 (1) Tyr 1.9 (2) 1.0 (1) 2.0 (2) Phe 1.0 (1) 1.0 (1 ) 2.0 (2) 3.0 (3) Lys 5.7 (6) 1.0 (1) 4.9 (5) 0.9 (1) His 0.9 (1) Arg 0.9 (1) 1.0 (1) 0.9 (1) ━━━━━━━ ━━━━━━━━━━━━━━━━━━━ It was subjected to amino acid analysis after hydrolysis with 6N hydrochloric acid in a vacuum sealed tube at 110 ° C. for 24 hours. However, Cys was quantified as cysteic acid after the oxidation of performic acid. Peptide 1: Lys-Asp-Glu-Val-Ile-Lys-Glu-Val-Gln-Gl
u-Phe-Tyr-Lys-Asp-Thr-Tyr-Asn-Lys-Leu-Lys-Thr-Lys-
Asp-Glu-Pro-Gln-Arg-Glu-Thr-Leu Retention time: 19.0 minutes (0 minutes (74% A + 26% B) -30 minutes (59% A + 41% B),
0.7 ml / min] [sequence of amino acid residue numbers 113 to 142 of SEQ ID NO: 1] Peptide 2: Gln-Arg-Glu-Thr-Leu-Lys-Ala-Ile-His-Ty
r-Ala-Leu-Asn-Cys-Cys-Gly-Leu-Ala-Gly-Gly-Val-Glu-
Gln-Phe-Ile-Ser-Asp-Ile-Cys retention time: 28.2 minutes [sequence of amino acid residue numbers 131 to 166 of SEQ ID NO: 1] peptide 3: Ser-Asp-Ile-Cys-Pro-Lys-Lys- Asp-Val-Le
u-Glu-Thr-Phe-Thr-Val-Lys-Ser-Cys-Pro-Asp-Ala-Ile-
Lys-Glu-Val-Phe-Asp-Asn-Lys- Retention time: 16.0 minutes [Sequence of amino acid residues 163-191 of SEQ ID NO: 1] Peptide 4: Arg-Phe-Asp-Ser-Gln-Thr-Lys -Ser-Ile-Ph
e-Glu-Gln-Glu-Thr-Asn-Asn-Asn-Asn-Ser-Ser-Phe-Tyr-
Thr-Gly-Val-Tyr retention time: 24.3 minutes [0.7 ml / min] [sequence of amino acid residue numbers 35 to 60 of SEQ ID NO: 1] Peptides 1 and 3 were dissolved in PBS at a concentration of 1 mg / ml 2 dissolved in 5% acetic acid, RPMI1640
It was diluted with the medium and used for the following experiments.

Test Example 4 Inhibition of motility of cancer cells by M fragment peptide MAC10 cells were cultured in a culture flask at 0.01% EDTA.
And stripped with PBS containing 0.125% trypsin,
10% FBS / RPMI1640 then RPMI16
After washing twice with 40 medium, a concentration of 5 × 10 ⁵ / ml (FBS
(Concentration 0.05%). Transwell plate (Corsta
r; Cambridge, MA) 0.1% containing peptides 2 and 3 (10-1000 ng / ml) obtained in Example 13 in the lower chamber
0.6 ml of FBS / RPMI1640 medium was added, 0.1 ml of MAC10 cell suspension was added to the upper chamber, and the mixture was cultured at 37 ° C. for 16 hours. The number of cells migrated through the membrane into the medium in the lower chamber was counted by a phase contrast microscope. As shown in [FIG. 13], inhibitory activities were observed in peptides 2 and 3 in a concentration-dependent manner.

Test Example 5 Change in motility by M expression i) CHO cell clone Regarding the CHO cell clone obtained in Example 11,
The values of locomotor activity measured by the method described in Test Example 4 are shown in FIG. In the clones in which M was expressed as compared with the parent strain (CHO), a marked decrease in motor performance was observed. ii) MAC10 cell clone For some of the MAC10 clones obtained in Example 11, motility values measured by the method described in Test Example 4 were used to determine the expression of M obtained in Example 12. The results are shown in [Table 7] together with the amounts. A decrease in motility was observed due to overexpression of M.

[Table 7] ────────────────────────────── Number of migrated cells M ───────────── ─ (U / mg) Experiment 1 Experiment 2 ────────────────────────────── MAC10 208 ± 23 (100%) 235 ± 33 (100%) 1.0 Clone 1 83 ± 10 (40%) 102 ± 23 (42%) 3.2 Clone 4 205 ± 10 (99%) 188 ± 27 (80%) 1.1 Clone 9 73 ± 23 (35%) 150 ± 18 (64%) 2.4 ────────────────────────────── n = 3 ± SD

[0046]

INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention is a cancer cell,
In particular, it has extremely excellent properties of suppressing the motility of lung cancer cells and glioblastoma cells. The polypeptide M of the present invention is present on the surface of various human cancer cells and plays an important role in the motility of cancer cells. Therefore, any of the monoclonal antibody, peptide or peptide fragment thereof of the present invention is very effective as a drug for suppressing metastasis of cancer cells.

[0047]

[Sequence listing] SEQ ID NO: 1 Sequence length (SEQUENCE LENGTH): 227 Sequence type (SEQUENCE TYPE): Amino acid Number of chains (STRANDEDNESS): single Topology- (TOPOLOGY): Linear sequence MOLECULE TYPE: protein Sequence: Pro Val Lys Gly Gly Thr Lys Cys Ile Lys Tyr Leu Leu Phe Gly Phe 1 5 10 15 Asn Phe Ile Phe Trp Leu Ala Gly Ile Ala Val Leu Ala Ile Gly Leu 20 25 30 Trp Leu Arg Phe Asp Ser Gln Thr Lys Ser Ile Phe Glu Gln Glu Thr 35 40 45 Asn Asn Asn Asn Ser Ser Phe Tyr Thr Gly Val Tyr Ile Leu Ile Gly 50 55 60 Ala Gly Ala Leu Met Met Leu Val Gly Phe Leu Gly Cys Cys Gly Ala 65 70 75 80 Val Gln Glu Ser Gln Cys Met Leu Gly Leu Phe Phe Gly Phe Leu Leu 85 90 95 Val Ile Phe Ala Ile Glu Ile Ala Ala Ala Ile Trp Gly Tyr Ser His 100 105 110 Lys Asp Glu Val Ile Lys Glu Val Gln Glu Phe Tyr Lys Asp Thr Tyr 115 120 125 Asn Lys Leu Lys Thr Lys Asp Glu Pro Gln Arg Glu Thr Leu Lys Ala 130 135 140 Ile His Tyr Ala Leu Asn Cys Cys Gly Leu Ala Gly Gly Val Glu Gln 145 150 155 160 Phe Ile Ser Asp Ile Cys Pro Lys Lys Asp Val Leu Glu Thr Phe Thr 165 170 175 Val Lys Ser Cys Pro Asp Ala Ile Lys Glu Val Phe Asp Asn Lys Phe 180 185 190 His Ile Ile Gly Ala Val Gly Ile Gly Ile Ala Val Val Met Ile Phe 195 200 205 Gly Met Ile Phe Ser Met Ile Leu Cys Cys Ala Ile Arg Arg Asn Arg 210 215 220 Glu Met Val 225 227

Sequence number (SEQ ID NO): 2 Sequence length (SEQUENCE LENGTH): 682 Sequence type (SEQUENCE TYPE): Nucleic acid Number of chains (STRANDEDNESS): Double topology- (TOPOLOGY): Linear sequence type (MOLECULE TYPE): cDNA to genomic RNA (HIPOTHTICAL): No Antisense (ANTI-SENCE): No Origin (ORIGINAL SOURCE) Organ name (ORGANISM): human cell type (CELL TYPE): breast carcinoma cell line (CELL lINE): ZR-75-1 sequence: ATGCCGGTCA AAGGAGGCAC CAAGTGCATC AAATACCTGC TGTTCGGATT TAACTTCATC 60 TTCTGGCTTG CCGGGATTGC TGTCCTTGCC ATTGGACTAT GGCTCCGATT CGACTCTCAG 120 ACCAAGAGCA TCTTCGAGCA AGAAACTAAT AATAATAATT CCAGCTTCTA CACAGGAGTC 180 TATATTCTGA TCGGAGCCGG CGCCCTCATG ATGCTGGTGG GCTTCCTGGG CTGCTGCGGG 240 GCTGTGCAGG AGTCCCAGTG CATGCTGGGA CTGTTCTTCG GCTTCCTCTT GGTGATATTC 300 GCCATTGAAA TAGCTGCGGC CATCTGGGGA TATTCCCACA AGGATGAGGT GATTAAGGAA 360 GTCCAGGAGT TTTACAAGGA CACCTACAAC AAGCTGAAAA CCAAGGATGA GCCCCAGCGG 420 GAAACGCTGA AAGCCATCCA CT ATGCGTTG AACTGCTGTG GTTTGGCTGG GGGCGTGGAA 480 CAGTTTATCT CAGACATCTG CCCCAAGAAG GACGTACTCG AAACCTTCAC CGTGAAGTCC 540 TGTCCTGATG CCATCAAAGA GGTCTTCGACATATGCGT ATCCCGTATGATCATAGTGCTC ATGACATCGT ATCCCCATCATGACTG

Sequence number (SEQ ID NO): 3 Sequence length (SEQUENCE LENGTH): 1120 Sequence type (SEQUENCE TYPE): Nucleic acid Number of chains (STRANDEDNESS): Double topology- (TOPOLOGY): Linear sequence type (MOLECULE TYPE): cDNA to genomic RNA (HIPOTHTICAL): No Antisense (ANTI-SENCE): No Origin (ORIGINAL SOURCE) Organ name (ORGANISM): human cell type (CELL TYPE): breast carcinoma cell CELL LINE: ZR-75-1 Sequence Features (FEATURE) 115..795 E Mat peptide Sequence: GACCAGCCTA CAGCCGCCTG CATCTGTATC CAGCGCCAGG TCCTGCCAGT CCCAGCTGCG 60 CGCGCCCCCC AGTCCCGCAC CCGTTCGGCC CAGGCTAAGT TAGCCCTCAC C ATG CCG 1
17
Met Pro
1 GTC AAA GGA GGC ACC AAG TGC ATC AAA
TAC CTG CTG TTC GGA TTT AAC 165 Val Lys Gly Gly Thr Lys Cys Ile Lys
Tyr Leu Leu Phe Gly Phe Asn 5 10
15 TTC ATC TTC TGG CTT GCC GGG ATT GCT
GTC CTT GCC ATT GGA CTA TGG 213 Phe Ile Phe Trp Leu Ala Gly Ile Ala
Val Leu Ala Ile Gly Leu Trp 20 25
30 CTC CGA TTC GAC TCT CAG ACC AAG AGC
ATC TTC GAG CAA GAA ACT AAT 261 Leu Arg Phe Asp Ser Gln Thr Lys Ser
Ile Phe Glu Gln Glu Thr Asn 35 40
45 AAT AAT AAT TCC AGC TTC TAC ACA GGA
GTC TAT ATT CTG ATC GGA GCC 309 Asn Asn Asn Ser Ser Phe Tyr Thr Gly
Val Tyr Ile Leu Ile Gly Ala 50 55
60 65 GGC GCC CTC ATG ATG CTG GTG GGC TTC
CTG GGC TGC TGC GGG GCT GTG 357 Gly Ala Leu Met Met Leu Val Gly Phe
Leu Gly Cys Cys Gly Ala Val 70
75 80 CAG GAG TCC CAG TGC ATG CTG GGA CTG
TTC TTC GGC TTC CTC TTG GTG 405 Gln Glu Ser Gln Cys Met Leu Gly Leu
Phe Phe Gly Phe Leu Leu Val 85 90
95 ATA TTC GCC ATT GAA ATA GCT GCG GCC
ATC TGG GGA TAT TCC CAC AAG 453 Ile Phe Ala Ile Glu Ile Ala Ala Ala
Ile Trp Gly Tyr Ser His Lys 100 105
110 GAT GAG GTG ATT AAG GAA GTC CAG GAG
TTT TAC AAG GAC ACC TAC AAC 501 Asp Glu Val Ile Lys Glu Val Gln Glu
Phe Tyr Lys Asp Thr Tyr Asn 115 120
125 AAG CTG AAA ACC AAG GAT GAG CCC CAG
CGG GAA ACG CTG AAA GCC ATC 549 Lys Leu Lys Thr Lys Asp Glu Pro Gln
Arg Glu Thr Leu Lys Ala Ile 130 135
140 145 CAC TAT GCG TTG AAC TGC TGT GGT TTG
GCT GGG GGC GTG GAA CAG TTT 597 His Tyr Ala Leu Asn Cys Cys Gly Leu
Ala Gly Gly Val Glu Gln Phe 150
155 160 ATC TCA GAC ATC TGC CCC AAG AAG GAC
GTA CTC GAA ACC TTC ACC GTG 645 Ile Ser Asp Ile Cys Pro Lys Lys Asp
Val Leu Glu Thr Phe Thr Val 165 170
175 AAG TCC TGT CCT GAT GCC ATC AAA GAG
GTC TTC GAC AAT AAA TTC CAC 693 Lys Ser Cys Pro Asp Ala Ile Lys Glu
Val Phe Asp Asn Lys Phe His 180 185
190 ATC ATC GGC GCA GTG GGC ATC GGC ATT
GCC GTG GTC ATG ATA TTT GGC 741 Ile Ile Gly Ala Val Gly Ile Gly Ile
Ala Val Val Met Ile Phe Gly 195 200
205 ATG ATC TTC AGT ATG ATC TTG TGC TGT
GCT ATC CGC AGG AAC CGC GAG 789 Met Ile Phe Ser Met Ile Leu Cys Cys
Ala Ile Arg Arg Asn Arg Glu 210 215
220 225 ATG GTC TAGAGTCAGC TTACATCCCT GAGCAG
GAAA GTTTACCCCAT GAAGATTTGGT 845 Met Val
GGGATTTTTTGTTTGTTTGTTTTTGTTTTTGT TTG
TTGTTTTG TTGTTTGTTTTTTTGCCCACT 905 AATTTTTAGTA TTCATTCTGC ATTGCTAGAT AAA
AGCTGAA GTTACTTTTAT GTTTGTCTTT 965 TAATGCTTCA TTCAATATTG ACATTTTGTAG TTG
AGCGGGGGGTTTGGTTTT GCTTTGGTTT 1025 ATATTTTTC AGTTGTTTTGTTTTGTCTGTGT TAT
ATTAAGC AGAAATCCTG CAATGAAAGG 1085 TACATATTTT GCTAGACTCT AGACAAAGATA TTG
TA 1120

[Brief description of drawings]

FIG. 1 is a diagram showing the result of Western blot analysis in Example 4.

FIG. 2 MA by the purified antibody of the present invention in Test Example 2
It is the figure which showed the suppression result of the motility of C10 cell.

FIG. 3 is a diagram showing the result of Western blot analysis in Example 5.

FIG. 4 shows the nucleotide sequence of the cDNA of the positive clone obtained in Example 2 and the amino acid sequence deduced from the sequence.

FIG. 5 shows the nucleotide sequence of the cDNA of the positive clone obtained in Example 2 and the amino acid sequence deduced from the sequence.

FIG. 6 shows the nucleotide sequence of the cDNA of the positive clone obtained in Example 2 and the amino acid sequence deduced from the sequence.

FIG. 7 shows the nucleotide sequence of the cDNA of the positive clone obtained in Example 2 and the amino acid sequence deduced from the sequence.

FIG. 8 shows the nucleotide sequence of the cDNA of the positive clone obtained in Example 2 and the amino acid sequence deduced from the sequence.

FIG. 9 shows the nucleotide sequence of the cDNA of the positive clone obtained in Example 2 and the amino acid sequence deduced from the sequence.

FIG. 10 is a gene map of pTB1352 constructed in Example 2.

FIG. 11 is a diagram showing a restriction enzyme map of the M expression plasmid pTB1442 constructed in Example 5.

FIG. 12 is a FACStar analysis diagram of CHO (dhfr) cells and their M-expressing clones in Example 6.

FIG. 13 is a diagram showing the results of suppressing the motility of MAC10 cells by the M fragment peptide in Test Example 1.

FIG. 14 is a diagram showing the motility of CHO (dhfr ~) cells and their M-expressing clones in Test Example 2.

[Explanation of symbols]

1 A control gel (supernatant) is shown in FIG. 2, and a molecular weight marker is shown in FIG. 2 In FIG. 2, a control gel (SDS eluate) is shown, and in FIG. 3, MIAPaCa-2 is shown. 3 In Figure 2, antibody-AffiGel 10 (supernatant)
Is shown in FIG. 4 In FIG. 2, antibody-AffiGel 10 (SDS
(Eluate), HEL is shown in FIG. 5 Molecular weight marker in FIG. 2, AZ5 in FIG.
21 is shown. 6 ZR-75-30

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 15/06 (C12P 21/08 C12R 1:91) (72) Inventor Masayuki Miyake Kyoto City, Kyoto Prefecture 76 Daigo Ohatacho, Fushimi-ku

Claims (12)

[Claims] 1. An anti-human cancer monoclonal antibody which suppresses the motility of human cancer cells. 2. The monoclonal antibody according to claim 1, wherein the recognition antigen is a protein involved in the motility of human cancer cells. 3. The monoclonal antibody according to claim 1, wherein the human cancer cell is a human lung cancer cell. 4. The monoclonal antibody according to claim 1, wherein the human cancer cell is human glioblastoma. 5. A polypeptide having a partial amino acid sequence of the amino acid sequence of SEQ ID NO: 1 and showing an activity of suppressing motility of cancer cells. 6. A polypeptide containing the amino acid sequence of SEQ ID NO: 1. 7. A recombinant DNA containing a base sequence encoding the amino acid sequence of SEQ ID NO: 1. 8. A vector containing the recombinant DNA according to claim 7. 9. A transformant transformed with the vector according to claim 8. 10. A transformant transformed with a vector containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 is cultured, and a polypeptide containing the amino acid sequence is produced and accumulated in the culture. A method for producing the polypeptide, which comprises: 11. A method for suppressing cancer metastasis, which comprises using the monoclonal antibody according to claim 1. 12. A method for suppressing cancer metastasis, which comprises using the polypeptide according to claim 5.