TW200914044A - Anti human Sulf1 antibody - Google Patents

Abstract

The present invention provides the antibody etc. that have the inhibiting activity of inducing to cancer of the cell and / or tumor cell proliferation. The antibody bonds specially with HSulf1, having the inhibiting activity of cell proliferation for the cell expressing the protein, and the antibody bonds with epitope which is bond with the antibody produced by the mouse hybridoma DS-87A3(FERM BP-10835), and the antibody including complementarity determining region of antibody produced from the mouse hybridoma DS-87A3(FERM BP-10835), and the medicine contains these antibodies.

Description

[Technical Field] The present invention relates to an antibody or the like having an activity of inhibiting cell canceration and/or tumor cell proliferation, and more particularly, an antibody against HSulfl or the like. A pharmaceutical composition of an antibody. [Prior Art] Sulfate esterase is a general term for an enzyme which combines hydrolysis of a sulfate ester, and is present from a microorganism to a molecule having a similar structure. The in vivo matrix hydrolyzed by sulfatase has multicolored glycosaminoglycans, sulfatides, sulphates, etc. Sulfate enzymes participate in various physiological processes including fertility 'metabolism and inflammation (Current ◦ pinion in Genetics & Development (1 997) 7, p.386-39 1). The sulfatase is oxidized by the aldehyde of the -CH2SH group which retains the cysteine to produce the methionine. It is known that this modification is incomplete, and the sulfatase enzyme becomes inert, resulting in a lytic accumulation disorder known as multiple sulfatase deficiency (Cell (2003) 133, p. 445-456). Although HSlvfl (Homo sapiens sulfatase 1) and HSulf2 (Homo sapiens sulfatase 2) (Journal of Biological Chemistry (2002) 277, p. 49 1 75 -49 1 85) have been derived from breast cancer, liver cancer, and pancreas. Cancer tissues such as visceral cancer are reported to be highly expressed, but the relationship with canceration of cells is not clear (Neoplasia (2005) 7, p.1001-1010, BMC Biochem (2006) 7, p.2-14 > Gastroenterology (2006) 1 30, p. 2130-2144, Molecular Cancer (2005) 4, p. 14 1 W02001/5541 1 > WO2002/59327, WO2005/052191). Cellular cancer or proliferation of cancer cells Although various mechanisms are known, it is known that several signaling agencies are involved in 200914044. Based on the segmental gene Wingless (Wg) of the orangutan fly embryo, the Wnt of the proto-oncogene Int-Ι of the same mouse breast cancer is the glycoprotein family, and its receptor and (Frz) are 7-membrane-through type = early stage Embryonic differentiation of cells, or adult cellostasis (Nature Reviews Neruoscience, 2000, vol., p. 113-120). It is also believed that the relationship between Wnt signaling molecules: the cause of the constant activation of cancer caused by mutations such as -catenin (Genes and Development (2005) 19, p ο and Mitogen-activated protein kinase (MAPK) Cascade in fine The central role of survival and cell differentiation, and the abnormality of MAPK can be a cause of various diseases such as cancer. In particular, the development of Raf-enzyme (MEK 1/2)-extracellular signal-regulated kinase (ERK) signaling agent is active. The abnormality of Raf-MEK-ERK and Ras, one of the cancer-producing genes, is known to be recognized by most cancers. The central gene that activates Ras has epidermal growth (EGFR), and many cancer tissues are abnormally active. Chemical and high performance are also known as positive feedback. The activation of ERK will promote EGFR expression and promote the canceration of its own secretion. Therefore, it belongs to EGFR EGF or HB-EGF which is activated by RAF-MEK-ERK (Oncogene (2007) 26, p_3291-3310) The present inventors discovered that HSulfl enhances the proliferation of these cell proliferation signal tumor cells, and uses the antibody against HSulf 1 to find a constant year in which the Wnt signal can be treated as its extracellular, 4 of the APC, , became .877-890) cell proliferation, signaling MAPK lines reported as anti-cancer Ras control factor receptors. The ligand having a high ligand is involved in canceration and participates in the cancer treatment, and finally the present invention is completed in 200914044. SUMMARY OF THE INVENTION (Problem to be Solved by the Invention) An object of the present invention is to provide an antibody against HSuifl. Another object of the present invention is to provide a pharmaceutical product having a therapeutic effect on cancer. Another object of the present invention is to provide a pharmaceutical product which inhibits cell proliferation of cells or participates in cell proliferation signals of cancer cell proliferation. (Means for Solving the Problems) The present inventors have made efforts to review the above problems, and found that HSulfl, one of the sulfatase enzymes, is specifically expressed in cancer cells, and HSulfl enhances the cell proliferation signal, and further suppresses the expression of HSulfl. Proliferation of cancer cells. Further, it has been found that administration of the antibody to HSulfl can inhibit the proliferation of cancer cells, and the present invention has been completed. That is, the present invention is (1) an antibody which specifically binds to HSulfl and has cell proliferation inhibitory activity against cells expressing the protein. (2) An antibody which specifically binds to HSulfl and which has at least one activity selected from the following (a) to (c) for cells expressing HSulfl: (a) inhibition of Wnt signaling activity (b) inhibition Activity of ERK signal (c) Activity to induce apoptosis. (3) specifically binds to a protein derived from the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing, and has an antibody against cell proliferation inhibitory activity against cells expressing the protein. (4) specifically binding to a protein obtained by the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing, and having at least one of the activities selected from the following U) to (c) for the cell expressing the protein: a) inhibiting the activity of Wnt signaling (b) inhibiting the activity of ERK signaling (c) inducing apoptosis. (5) The antibody according to any one of (1) to (4) wherein the cell is a tumor cell 〇(6)-antibody, which is a mouse fusion tumor DS-ATRL-87A3 (FERM BP-1 0835, BCRC960365 (already deposited on July 29, 2008, under the number BCRC960365, deposited in the Food Industry Development Research Institute of the Foundation), antibodies that bind to the epitopes of antibody binding. (7) The antibody according to (6), which is a field of complementarity determination of an antibody produced by a mouse fusion tumor DS-ATRL-87A3 (FERM BP-10-835). (8) The antibody according to (6), which is deleted, substituted or substituted in the amino acid sequence of the complementarity of the antibody produced by the mouse fusion tumor DS-ATRL-87A3 (FERM BP-10-835) It is prepared by adding an amino acid sequence of 1 or several amino acid sequences, and specifically binds to HSulfl, and has an activity of inhibiting cell proliferation against cells expressing the protein. (9) The antibody according to (6), which is an antibody produced by mouse fusion tumor DS-ATRL-87A3 (FERM BP-1805). (10) The antibody according to any one of (1) to (8) which is a monoclonal antibody. (11) The antibody according to any one of (1) to (8) and (10) which is artificially resistant to 200914044. (12) The antibody according to any one of (1) to (8) or (10) which is a human antibody.

(13) The antibody according to any one of (1) to (8) and (10) to (12) which is an IgG antibody. (14) The antibody according to any one of (1) to (8), which is a Fab, F(ab')2, Fv, scFV, diabody, a linear antibody, and a polyspecific antibody. (15) Mouse fusion tumor DS-ATRL-87A3 (FERM BP-1688). (16) A pharmaceutical composition comprising the antibody according to any one of (1) to (14). (17) A pharmaceutical composition for cancer treatment, which comprises the antibody according to any one of (1) to (14). (18) A pharmaceutical composition for cancer treatment, which comprises a nucleotide sequence represented by SEQ ID NO: 1 in the Sequence Listing or a partial sequence of the sequence comprising an oligonucleotide having a complementary nucleotide sequence. (19) A pharmaceutical composition for cancer treatment, which comprises an siRNA comprising a nucleotide sequence represented by SEQ ID NO: 1 in the Sequence Listing. (20) The pharmaceutical composition according to any one of (16) to (19), wherein the cancer is at least one selected from the group consisting of colon cancer, breast cancer, prostate cancer, and pancreatic cancer. (21) A method for screening an antitumor substance, which is a project comprising a substance which exhibits a contact with a test substance of HSulfl, a performance amount of HSulfl, and/or an activity of inhibition. (Effect of the Invention) According to the present invention, it has been found that HSulfl significantly increases the amount of expression in the cancerous portion, and provides an antibody against HSulfl having a cancer cell injury activity of 200914044, and a pharmaceutical composition for cancer treatment containing the antibody. [Embodiment] (Best Mode for Carrying Out the Invention) In the present specification, "cancer" and "tumor" are used in the same meaning. In the present specification, "gene" includes not only DNA but also mRNA, cDNA and cRNA thereof. Therefore, the "HSulfl gene" in the present invention contains DNA, mRNA, cDNA and cRNA of HSulfl. e: In this specification, a "polynucleotide" is used in the same sense as a nucleic acid, and includes DNA, RNA, probes, oligonucleotides, and primers. In this detail, "polypeptide" is not used separately from "protein". In the present specification, "cell" also includes cells and cultured cells in an individual animal. In the present specification, "cell canceration" refers to a cell that loses its sensitivity to contact-preventing, or exhibits an abnormal proliferation of cells such as a non-dependent proliferation of a foot, and a cell exhibiting such abnormal growth is called a "cancer cell." In the present specification, a protein having a function equivalent to the canceration activity and/or cell proliferation activity of all cells of HSulfl is also referred to as HSulfl. In this specification, 'cell damage' refers to pathological changes in any form of cells, not only direct trauma, but also the formation of DN A cuts or bases, chromosome cuts, cell division devices. Damage to the structure or function of all cells, such as damage, decreased activity of various enzymes. In the present specification, "cell disorder activity" means causing the above-mentioned cell damage. In the present specification, 'Wnt signal' means that Wnt binds to the -200914044 receptor such as Frizzled receptor, and as a result, /3 -catenin is stabilized and the transcriptional activation ability is improved. "Inhibition of Wnt signal" means inhibition of Wnt signal. Inferior cold-catenin transcriptional activation ability. In the present specification, "ERK signal" refers to a MAPK pathway that plays a central role in cell proliferation, and ERK is phosphorylated. "Inhibition of ERK signaling" means inhibition of transcriptional activation of Elkl activated by ERK. ability. In the present specification, "apoptosis" refers to active cell death characterized by reversal of cell membrane, exposure of phospholipid lysine residue, blebbing, nuclear fragmentation, serotonin aggregation, DNA decomposition, etc. Apoptosis is caused by apoptosis in the target cells. 1. HSulf 1 (1) HSulf 1 gene HSulfl (Homo sapiens sulfatase 1) gene nucleotide sequence and amino acid sequence 歹! J was registered in the gene bank with Homo sapiens sulfatase l (SULFl), mRNA (accession number: NM_015170). Further, the nucleotide sequence of the open reading frame (ORF) of HSulfl is SEQ ID NO: 1 in the Sequence Listing, and the amino acid sequence is SEQ ID NO: 2 listed in the Sequence Listing. Among the amino acid sequences of sulfatase, one or several amino acids are substituted, deleted, and added with an amino acid sequence, and proteins having the same biological activity as these enzymes are also included in the sulfatase. (2) The specificity of the cancerous part of the HSulfl gene The HSulf 1 gene is the result of immunostaining of the cancer tissue. The high expression in the cancer department was discovered by the inventors. 200914044 Cancers that are considered to have excess performance of HSulfl include, for example, colorectal cancer, breast cancer, pancreatic cancer, and prostate cancer. However, as long as the expression of HSulfl is significantly higher than that of other tissues, cancer other than this can be applied. It is also known that HSulfl is highly expressed in cancerous tissues. For example, in colorectal cancer and breast cancer, a positive signal is recognized at a high frequency in the cancer department. That is, when the amount of each cell of HSulfl and/or the amount of expression in each tissue is measured, the state of canceration and/or proliferation of cancer cells which occurs due to the excessive expression of HSulfl in the subject can be determined. Further, the substance which inhibits the amount and/or activity of HSulfl is an activity which inhibits canceration of cells caused by HSulfl and/or inhibits proliferation of cancer cells. Therefore, anti-tumor substances can be screened by expressing HSulfl cells in contact with the test substance and selecting substances that inhibit the expression and/or activity of HSulfl. The siRNA against HSulfl is an anti-tumor agent for inhibiting the expression of HSulfl. The HSulfl siRNA can be based on a partial sequence of HSulfl mRNA (sense RNA) and an RNA (antisense RNA) consisting of a base sequence complementary to the base sequence of the RNA, based on the base of HSulfl mRNA. The sequence design is carried out by a chemical synthesis method known per se, and the resulting two RNAs are hybridized to produce. The sense RNA and the antisense RNA constituting the siRNA are each preferably at the 3' end, and a nucleotide combination of a sequence of one to several bases called an overhang sequence is preferred. The overhanging partial sequence is not limited as long as the RNA is protected by the nucleic acid degrading enzyme, and it is preferably one to ten, particularly preferably one to four, and more preferably two nucleotides are available. 2. Antibody to HSulfl -12- 200914044 The antibody against HSulfl of the present invention can be obtained by immunizing an animal with any polypeptide selected from the amino acid sequence of HSulfl or HSulfl, and collecting and purifying the antibody produced in vivo. It will also be produced according to known methods (for example, Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennet, R. ed_, Monoclonal Antibody, p. 365-367, Prenum Press, N_Y. (1980)). When the antibody-producing cells of the antibody of HSulfl are fused with myeloma cells to establish a fusion tumor, a monoclonal antibody is obtained. HSulfl, which is an antigen, can be produced by producing a HSulfl gene in a host cell by genetic manipulation. Specifically, a host expressing a HSulfl gene is produced, and this gene is introduced into a host cell to express the gene, and the expressed HSulfl is purified. The following is a detailed description of the method for obtaining an antibody against HSulfl. (1) Preparation of an antigen The antigen for producing an anti-HSulfl antibody may be a polypeptide obtained by HSulfl or at least 6 consecutive partial amino acid sequences thereof, or an arbitrary amine added thereto. A base acid sequence or a derivative of a carrier. HSulfl can be used for human tumor tissue or directly purified by tumor cells, or HSulfl can be synthesized in vitro or genetically manipulated in host cells. In particular, genetic manipulation can be carried out after HSulfl is implanted in a host of possible expression, in a solution containing enzymes, matrix and energy substances necessary for transcription and translation, or by transferring other prokaryotic or eukaryotic host cells. The protein is expressed in the form of HSulfl. -13- 200914044 The HSulfl cDNA can be subjected to polymerase chain reaction (hereinafter referred to as "PCR") using a primer that expresses HSulfl as a template, for example, with reference to Saiki, RK et al., Science (1 988). 239, P.487-489) is obtained by the PCR method. The in vitro synthesis of the polypeptide may be, for example, a rapid translation system (RTS) manufactured by Roche-Diagnostics, Inc., but is not limited thereto. The host of the prokaryotic cell may be, for example, Escherichia coli or Bacillus subtilis or the like. The gene of interest is transformed into these host cells, and the host cell is transformed by a plastid host containing a replication unit suitable for the host, i.e., a replication origin and a regulatory sequence. Further, it is preferred that the host has a sequence capable of imparting selectivity to the expression form (phenotype). Host cells of eukaryotic cells include cells such as vertebrates, insects, yeast, and vertebrate cells, such as COS cells of cells such as sputum (Gluzman, Y. Cell (1981) 23, p. 1 75-1 82, ATCC CRL-1650 ), mouse fibroblast NIH3T3 (ATCC No. CRL-1658) or hamster ovary cells (CH0 cells, ATCC CCL-61) dihydrofolate reductase deficiency strain 〇^131113,0· and Chasin, LA Proc. Natl. Acad. Sci. USA (1 980) 77, p. 4126-4220), etc., but not subject to such limitations. The transformant obtained as described above can be cultured in a usual manner, and the polypeptide of interest can be produced from the culture in the cells or outside the cells. The medium to be used for the culture may be appropriately selected depending on the host cell, and if it is a coliform, for example, an antibiotic or an IPMG such as ampicillin may be added to the LB medium. -14- 200914044 The recombinant protein produced by the above culture in the cells of the transformant or outside the cell can be isolated and purified according to various known separation methods using the physical properties or chemical properties of the protein. Specifically, the method can be exemplified by treatment with a usual protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and high-speed liquid chromatography (HPLC). Various liquid chromatography, dialysis methods, combinations of these, and the like. Further, in order to express a histidine acid in which a recombinant protein is composed of 6 residues, it can be efficiently purified by a nickel affinity column. By combining the above methods, the polypeptide of interest can be easily produced in large quantities with high yield and high purity. (2) An example of an antibody that specifically binds HSulfl to an antibody that binds to HSulfl may be a monoclonal antibody that specifically binds to HSulfl, and the method for obtaining the same is as follows: When manufacturing a monoclonal antibody, it is generally required to be as follows: U) Purification of a living polymer used as an antigen, (b) Immunization of an animal after immunization, blood collection, determination of the antibody valence to determine the period of spleen extraction, preparation of antibody-producing cells, (c) bone marrow tumor cells (hereinafter referred to as "myeloma") modulation, (d) fusion of antibody-producing cells with myeloma cells, (e) selection of fusion tumors producing the antibody of interest, (divided into single cell clones (optional), (g Sometimes, cultured fusion tumors for large-scale production of monoclonal antibodies, or animals that have been transplanted with fusion tumors, 200914044 (h) review the physiological activity of the monoclonal antibodies thus produced, and their binding specificity' or assay labeling reagents Characteristics, etc. The following is a detailed description of the method for producing monoclonal antibodies, but the method for producing the antibodies is not limited thereto, and antibodies such as spleen cells can also be used to produce cells and bone marrow. (a) The purified antigen of the antigen may be HSulfl or a part thereof prepared by the method described above, and the membrane fraction prepared by HSulfl expressing recombinant cells, or HSulfl expressing the recombinant cells themselves, or a method well known to the practitioner. A chemically synthesized partial peptide of the protein of the present invention is used as an antigen. (b) Preparation of antibody-producing cells (a) A complete or incomplete adjuvant of Freund's antigen and Freund's, or an auxiliary agent such as potassium alum. The immunogen is immunized to the experimental animal. The experimental animal can use the animal used in the known fusion tumor preparation method without any hindrance. Specifically, for example, mouse, rat, goat, sheep, cow, horse, etc. can be used. The mouse or rat is preferably an immunized animal from the viewpoint of easiness of obtaining cell fusion-derived myeloma cells, etc. The system of mice and rats actually used is not limited, and in the case of mice, for example, Each system A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BR, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW 'RF, R III, SJL, SWR, WB, 129 equal In the case of rats, for example, Low, Lewis, Spraque, Daweley, ACI, BN, Fischer, etc. These mice and rats can be obtained by, for example, Japanese Kelly, Charles River, Japan, and the like - 16-14014044 experimental animal breeding and sales. Among them, 'the compatibility with the myeloma cells described later is considered to be better in mice with the BALB/c system' in the low system as the immunized animal. Considering the homology of the antigen to human and mouse, the use is reduced. Mice that are living organisms that remove their own antibodies, that is, autoimmune disease mice are also preferred. The age of these mice or rats should be 5 to 12 weeks old, preferably 6 to 8 weeks old. To immunize an animal with HSuIfl or this recombinant, for example, Weir, DM, Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1 987) 'Kabat, EA and Mayer, MM, Experimental Immunochemistry, Charles C Thomas Publisher Spigfield, Illinois (1964) and the like are well known methods. Among these immunoassays, a suitable method of the present invention is specifically exemplified as follows: First, the antigen membrane protein is divided, or the cells expressing the antigen are administered intradermally or intraperitoneally to the animal. However, in order to improve the immune efficiency, it is better to use both. The first half is administered intradermally, and the second half or only the final intraperitoneal administration can improve the immune efficiency. The antigen administration procedure varies depending on the type of the immunized animal, the individual difference, etc., but generally the antigen is administered back to the number of 3 to 6 times, and the interval between the administration and the interval is 2 to 6 weeks, and the number of administrations is 3 to 4 times. It is more preferable that the administration interval is 2 to 4 weeks. Further, the amount of the antigen to be administered varies depending on the type of the animal, the individual difference, etc., but it is usually 0.05 to 5 ml, preferably 0.1 to 0.5 ml. -17- 200914044 Additional immunization can be administered after 1~6 weeks of antigen, preferably after 2~4 weeks, more preferably after 2~3 weeks. The amount of antigen administered at the time of performing the additional immunization varies depending on the type and size of the animal. In general, for example, in the case of a mouse, it is 5 to 5 ml, preferably 0.1 to 0.5 ml, more preferably 0.1 to 0.2 ml. After 1 to 10 days from the above-mentioned additional immunization, it is preferable to take 2 to 5 days later, and more preferably 2 to 3 days, after the spleen cells or lymphocytes containing the antibody-producing cells are aseptically taken out from the immunized animal. In this case, the antibody valence is measured, and an animal having a sufficiently high antibody valence is used as a supply source of the antibody-producing cells, whereby the efficiency of subsequent operations can be improved. The assay for the antibody valency used may be, for example, the RIA method or the ELISA method, but is not limited by these methods. The measurement of the antibody valence in the present invention can be carried out, for example, according to the ELISA method. The purified or partially purified antigen is first adsorbed on a solid phase surface such as a 96-well plate for ELISA, and the surface of the solid phase not adsorbing the antigen is coated with an antigen-independent protein such as bovine serum albumin (hereinafter referred to as "BSA"). After washing the surface, the antibody is contacted with a sample (for example, mouse serum) which is diluted as a first antibody, and the antibody is bound to the antigen-binding sample. Further, the antibody against the mouse antibody, which is a second antibody and is labeled with an enzyme, binds to the mouse antibody, and after washing, the substrate of the enzyme is added, and the change in absorbance is measured based on the color of the matrix decomposition to calculate the antibody valence. The separation of antibody-producing cells from these spleen cells or lymphocytes can be carried out by a known method (e.g., Kohler et al., Nature (1 975) 256, p. 495, -18-200914044

Kohler et al., Eur. J. Immno 1 · (1 97 7) 6, p. 5 1 1, ; Milstein et al., Nature (1 977), 266, p. 550, ; Walsh, Nature, ( 1 977) 266, P.495,). For example, in the case of spleen cells, a general method in which cells are finely cleaved and filtered through a stainless steel sieve and floated in Eagle's minimal essential medium (MEM) to separate antibody-producing cells can be used. (c) Modulation of bone marrow tumor cells (hereinafter referred to as "myeloma") The myeloma cells used for cell fusion are not limited, and may be selected from known cell strains. However, in consideration of the convenience in selecting a fusion tumor from a fused cell, a HGPRT (Hipoxanthine-guanine phosphoribosyl transferase)-deficient strain established using the selection procedure is preferred. That is, the mouse originated from X63-Ag8 (X63), NS1-ANS/1 (NS1), P3X63-Ag8.Ul (P3Ul) 'X63-Ag8.653 (X63.653), SP2/0-Ag 14 (SP2/ 0), MPC1 1-45.6TG1.7 (45.6TG) > FO, S149/5XXO, BU.l, etc., the origin of the rat 210.RSY3.Ag.l.2.3 (Y3), etc., U266AR (from the origin) SKO-007), GM 1 500 · GTG-A 1 2 (GM 1 500), UC729-6 I, LICR-LOW-HMy2 (HMy2), 8226AR/NIP4-1 (NP41), and the like. These HGPRT deficiency strains can be obtained, for example, from the American Type Culture Collection (ATCC) or the like. These cell lines are in a suitable medium, for example, in 8-ostrich guanidine medium [in RPMI-1 640 medium with glutamic acid, 2-hydrothioethanol, damycin-only, and fetal serum (hereinafter referred to as "FCS"). ) medium plus 8-ostrich medium], Iscove changed Dulbecco medium (hereinafter referred to as "IMDM"), or Dulbecco changed Eagle medium (hereinafter referred to as "DMEM") subculture -19- 200914044, only for cell fusion 3 to 4 days before the subculture was carried out in a normal medium (for example, ASF104 medium (manufactured by MSG) containing 10% FCS), and the number of cells of 2x1 07 or more was ensured on the day of fusion. (d) Fusion of cell fusion antibody-producing cells and myeloma cells can be carried out according to known methods (Weir, DM, Handbook of Experimental Immunology Vol. I. II. III. . Blackwell Scientific Publications, Oxford (1 987), Kabat'EA and Mayer, Μ.M., Experimental Immunochemistry, Charles C Thomas Publisher Spigfield, Illinois (1 964), etc., is suitably carried out under conditions in which the survival rate of the cells is not extremely lowered. Such a method can be, for example, a chemical method of mixing antibody-producing cells and myeloma cells, a physical method using electrical stimulation, or the like in a high-concentration polymer solution such as polyethylene glycol. The specific chemical method is as follows: when using polyethylene glycol as a high concentration polymer solution, in a polyethylene glycol solution having a molecular weight of 1,500 to 6,000, preferably 2000 to 4000, at 30 to 40 ° C, preferably The antibody-producing cells and myeloma cells are mixed at a temperature of 35 to 38 ° C for 1 to 10 minutes, preferably 5 to 8 minutes. (e) Selection of fusion tumor group The selection method of fusion tumor obtained by the above cell fusion is not limited, and HAT (hypoxanthine/aminopurine thymidine) selection method is usually used (Kohler et al., Nature (1 97) 5) 25 6, ρ·495; Milstein at al., Nature (1 977) 266, p. 550). This method is effective when the tumor cells of the HGPRT-deficient strain that cannot survive with the amine-based sputum are -20-200914044. When the unfused cells and the fusion tumor are cultured in the HAT medium, only the fusion tumor which is resistant to the aminopurine can be selected and survived. (f) Segmentation into single cell clones (planting) The method of selecting fusion cells can be carried out by known methods such as methylcellulose method, soft agarose method, 'limit dilution method (see, for example, Barbara, BM and Stanley, MS: Selected). Methods in Cellular Immunology, WH Freeman and Company, San Francisco (1980)). Among these methods, the limiting dilution method is preferred. According to this method, the fibroblast cell strain derived from the rat fetus or the spleen cells, thymocytes, ascites cells and the like of the normal mouse are inoculated on the microplate. In other aspects, the fusion tumor is pre-diluted into 0.2~0.5/0.2ml in the medium, and the diluted suspension of the tumor is added to each well to 0.1ml, and each period (for example, every 3 days) will be about 1 / The medium of 3 can be proliferated for a period of 2 weeks, and the clone of the fusion tumor can be proliferated. In the point where the antibody price is determined, for example, 2 to 4 times of sputum is repeatedly selected by the limiting dilution method, and the antibody is determined to be an anti-HSulfl monoclonal antibody to produce a fusion tumor strain. One of the fusion tumor strains thus selected was named DS-ATRL-87A3, and was deposited in the Patent Bio-storage Center of the National Institute of Industrial and Technological Research, the National Institute of Industrial and Technological Research, and was assigned the registration number FERM BP-10835 on June 6, 2007. (g) Preparation of monoclonal antibodies by fusion tumor culture The thus selected fusion tumors can be cultured to obtain monoclonal antibodies, but it is preferred to screen for fusion tumors of the desired monoclonal antibodies before culture. -21- 200914044 This screening can be done in a manner known per se. The measurement of the antibody valence in the present invention can be carried out, for example, by the ELISA method described in the item (b) above. The fusion tumor obtained according to the above method can be stored in a frozen state in liquid nitrogen or in a freezer below -80 °C. The selected fusion tumor can be cultured by changing the medium from the HAT medium to the normal medium. A large number of cultures can be cultured in a rotary culture using a large culture flask or in a spin culture. This large-scale culture supernatant can be obtained by a method known to those skilled in the art, such as gel filtration, to obtain a monoclonal antibody specifically bound to the protein of the present invention. Further, the mice in the same system (for example, the above BALB/c), or the Nu/Nu mice are intraperitoneally injected with the fusion tumor, and when the hybridoma is propagated, a large amount of ascites containing the monoclonal antibody of the present invention can be administered into the abdominal cavity. When mineral oil such as 2,6,10,14-tetramethylpentadecane (pristane) is administered beforehand (before 3 to 7 days), more ascites can be obtained. f For example, after injecting an immunosuppressive agent into the peritoneal cavity of a mouse with the same tumor system, after inactivating T cells, after 20 days, 106 to 107 fusion tumor-clonal cells are floated in serum-free medium. (0.5 ml) was administered into the abdominal cavity, and ascites was collected from the mice when the abdomen was full and the ascites was accumulated. According to this method, a monoclonal antibody having a concentration of about 100 times or more than the culture solution is obtained. The monoclonal antibodies obtained by the above methods can be purified, for example, by the method described in Weir, D.M.: Handbook of Experimental Immunology, Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978). -22- 200914044 That is, sulfur sulphate precipitation method, gel filtration method, ion exchange chromatography method, affinity stratification method, and the like. As a simple method for purification, a commercially available monoclonal antibody purification kit (for example, MAbTrap GII kit; manufactured by Pharmacia Co., Ltd.) or the like can be used. The monoclonal antibodies thus obtained have high antigen specificity for HSulfl. (h) Verification of monoclonal antibodies The determination of the isotypes and subtypes of the thus obtained monoclonal antibodies can be carried out as follows. First, as an identification method, it can be Ouchterlony method, ELISA method or RIA method f \ % 0

The Ouchterlony method is simple, but when the concentration of the monoclonal antibody is low, it must be concentrated. In other respects, when the ELISA method or the RIA method is used, the culture supernatant is reacted with the antigen-adsorbed solid phase, and the antibody corresponding to various immunoglobulin isoforms and subtypes is used as the second antibody, and the monoclonal antibody can be identified. Shaped, subtype. Further, as a simpler method, a commercially available kit for identification (e.g., mouse Typer set; Bio-Rad) can be used. The quantification of the protein can be carried out according to the method of the Foreign row Lee method and the absorbance at 280 nm [1.4 (OD280) = immunoglobulin 1 mg/ml]. (3) Other antibodies In addition to the monoclonal antibody of HSulfl, the antibody of the present invention further contains a genetically modified antibody which is artificially modified to reduce human antigenicity and the like, for example, chimeric antibodies and humanization. (Humanized) antibody, human antibody, strand antibody (scFv), Fab, F(ab')2, Fv, diabody (-23-200914044 diabody), linear antibody, and multi-specificity of antibody fragmentation Antibodies, polyclonal antibodies, etc. Further, an antibody which binds to an epitope bound by an antibody produced by mouse fusion tumor DS-ATRL-87A3 (FERM BP-1083 5) is also included in the antibody of the present invention. An antibody further comprising a complementarity determining region of an antibody produced by a mouse fusion tumor 1^-hexa 1111^87A3 (FERM BP-1 083 5), and an amine group in the field of complementarity determination An antibody of the present invention which has one or several amino acid deletions, substitutions or addition of an amino acid sequence in an acid sequence and which specifically binds to HSulfl, and which has cell proliferation inhibitory activity on cells expressing the protein. The antibody of the present invention can be produced by a known method. The field of complementarity determination of the antibody of the present invention can be determined by a known method or can be determined as follows. A portion of the solution containing the purified monoclonal antibody was separated by SDS-PAGE. The protein in the gel was transcribed from a PVDF membrane (pore size: 0.45 μm; manufactured by InvUrogen Co., Ltd.), and washed with a washing buffer (25 mM sodium chloride, 10 mM sodium borate buffer pH 8.0). After staining with a staining solution (50% methanol, 20% acetic acid, 0.05% Kumasi bright blue) for 5 minutes, it was decolorized with 90% methanol. The portion of the band corresponding to the heavy chain (small mobility) and the light chain (large mobility band) visualized on the PVDF film was extracted with Procise (registered trademark) cLC protein sequence J [J machine 492cLC type (Applied Biosystems), Each N-terminal amino acid sequence was identified by the automated Edman method (see Edman, P_, et al. (1967) Eur. J. Biochem. 1, 80). Amino acid sequence libraries of these amino acid sequences with antibodies made by Kabat et al. (cf. Kabat, E. A. et al., (1991) in Sequences of Proteins of Immunological -24- 200914044

Interest Vol. I and II, U. S. Department of Health and Human Services) determine the subtypes of heavy and light chains. The following oligonucleotide primers of each hybrid are synthesized next to the 5'-end side of the translation domain of the antibody gene belonging to these subtypes and the stop codon (refer to the literature of Kabat et al., Matti Kartinen et aL) (1 988) 25, 859-865 and Heinrich, G. et al. (1 984) J. Exp. Med. 159, 4 1 7-435). The mRNA of the heavy chain and the light chain of the antibody is amplified by a fusion-producing tumor-producing mRNA, a heavy-chain PCR primer set or a light chain using a PCR primer set. The PCR-amplified cDNA was selected and the nucleotide sequences of the selected heavy and light chains were determined by a gene sequence analysis device. A library of amino acid sequences of heavy and light chains and amino acid sequences of antibodies according to Kabat et al. (cf. Kabat, Ε·A., et al. (1991) in Sequence of Proteins of Immunological Interest Vol. I and II": US Department of Health and Human Services) compares the review and determines the variable domains of the heavy and light chains and the amino acid sequences in the field. Chimeric antibodies can be mutually variable in the variable and stationary domains of antibodies. The antibody is, for example, a chimeric antibody that binds the variable domain of a mouse-derived antibody to a constant field of human origin (see Proc. Natl. Acad. Sci. USA (1984) 81, p. 6851-6855). The antibody can be implanted only by the antibody of the complementarity determining region (refer to Nature (1986) 32 1, ρ·522-525), according to the CDR grafting method, except for the sequence of the CDR, A part of the framework amino acid residue is also transplanted to the antibody of -25-14014044 human antibody (refer to W090/07861). Human antibody refers to a human antibody having only the gene sequence of the antibody derived from human chromosome. Anti-HSulfl Human antibodies can be produced by using human antibodies containing human chromosome fragments of the η chain and L chain genes of human antibodies (see Tomizuka, K. et al., Nature Genetics (1 997) 16, p. 1 33-143 , Kuroiwa, Y.et.al., Nuc. Acids Res. (1 998) 26, p.3447-3448; Yoshida, H.et.al., Animal Cell Technology: Basic and

Applied Aspects, 10, p.69-73 (Kitagawa, Y., Matuda, T. and r'\ u: Iijima, S. eds.), Kluwer Academic Publishers, 1 999.;

Tomizuka, K. et. al., Proc. Natl. Acad. Sci. U.S.A. (2000) 97, p. 722-727, etc.). Specifically, in such a transgenic animal, the inferior immunoglobulin heavy chain and the light chain locus of the non-human mammal are destroyed, and the Yeast artificial chromosome (YAC) host is introduced into the human immunoglobulin. Recombinant animals of the heavy and light chain loci, made by removing animals and transgenic animals, and mating these animals with each other. k may also be a gene encoding the heavy and light chain of the human antibody by gene recombination technology, preferably the eukaryotic cells are transformed from the host containing the cDNA, and the transformed cells which produce the recombinant human monoclonal antibody are cultured. This antibody was obtained from the culture supernatant. Here, the host may be, for example, a eukaryotic cell, preferably a mammalian cell such as a CHO cell, a lymphocyte or a myeloma. It is also known to use a method in which a human phage-selected phage displays a human antibody (see Wormstone, I_M.et.al, Investigative -26-200914044)

Ophthalmology & Visual Science (2002) 43(7), p.230 1 -2308 ; Carmen, S. et.al., Briefings in Functional Genomics and Proteomics (2002) 1(2), p.189-203 i Siriwardena , D. et.al., Opthalmology (2002) 1 09(3), p.427-43 1 , etc.). For example, a phage display method in which a variable region of a human antibody is expressed as a strand antibody (scFv) on a phage surface and a phage that binds to an antigen can be selected (Nature Biotechnology (2005) 23, (9), ρ·1105-1116 ). By analyzing the gene of the phage which binds to the phage of the antigen, the DNA sequence encoding the variable domain of the human antibody which binds to the antigen can be determined. When the DNA sequence of the scFv that binds to the antigen is recognized, the CDR sequence is taken out to prepare a host having the sequence, and the host is introduced into an appropriate host to obtain a human antibody (W092/01047, WO92/20791, W〇93/ 062 1 3, W093/1 1 236, WO93/1 9 1 72, W095/0 1 438, W095/15388 ' Annu. Rev. Immunol. ( 1 994) 1 2, p.433-455 ' Nature Biotechnology (2005 23(9), p.1105-1116). Once the antibody gene has been isolated and introduced into an appropriate host to produce an antibody, a combination of a suitable host and a representative host can be used. When eukaryotic cells are used as a host, animal cells, plant cells, and eukaryotic microorganisms can be used. The animal cell may be (1) a mammalian cell, such as a COS cell of a cell of sputum (Gluzman, Y. Cell (1 98 1 ) 23, p. 1 75- 1 82 'ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1 658) or dihydrofolate reductase-deficient strain of hamster ovary cells (CH0 cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci. USA - 27- 200914044 (1980) 77, ρ·4126-4220). When prokaryotic cells are used, for example, coliform, subtilis bacteria can be used. In these cells, the antibody gene of interest is introduced into a shape, and the transformed cells are cultured in vitro to obtain an antibody. The isotype of the antibody of the present invention is not limited and may be, for example, IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgAl, IgA2), IgD or IgE, etc., preferably IgG or IgM. The antibody of the present invention may also be a fragment of an antibody having an antigen-binding portion of an antibody or a modification thereof. For example, the fragment of the antibody may be a single-chain Fv (scFv), a diabody, a linear antibody, and a Fb, F(ab') 2, Fv, or a heavy chain and a light chain Fv linked by an appropriate linker. A multispecific antibody or the like formed by an antibody fragment. The antibody of the present invention may also be a multispecific antibody which is specific for at least two different antigens. Usually, the molecule is a bispecific antibody (i.e., a bispecific antibody), and the "multispecific antibody" in the present invention is an antibody containing a specificity for the above (e.g., three) antigens. The antibody of the present invention may be an antibody obtained by the full-length of a multispecific antibody, or may be a fragment of such an antibody (e.g., F(ab')2 dispecific antibody). The dual-specific antibody can also be combined with the heavy chain and the light chain (HL pair) of the two antibodies to produce a fusion tumor that can produce a different monoclonal antibody, and a dual-specific antibody can be produced to produce a fusion cell. Production (Millstein et al., Nature (1983) 305, p. 537-539). -28- 200914044 The antibody of the present invention may also be a strand antibody (also referred to as scFv). A strand antibody can be obtained by linking the heavy chain V domain of the antibody and the light chain V domain with a linker of a polypeptide (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenburg & Moore ed. 'Springer Verlag, New York, p. 269- 3 1 5( 1 994) > Nature Biotechnology (2005) 23, p. 1126-1136). Methods for making a strand of antibody are well known in the art (see, for example, U.S. Patent No. 4,946,778, U.S. Patent No. 5,260,203 U.S. Patent No. 5,091,513, U.S. Patent No. 5,455,030, etc.. In the scFv, the 'heavy chain V domain and the light chain V domain are non-binding linkers, and the polypeptide linker should be linked (Huston, JS et al. Proc. Natl. Acad. Sci. USA (1988), 85, p. 5879-5883). The scFv medium heavy chain V domain and the light chain V domain may be derived from the same antibody or may be derived from separate antibodies. The peptide linker may be any one of strands of peptides, for example, from 12 to 19 residues. The DNA encoding the scFv is a DNA encoding the heavy or heavy chain V domain of the aforementioned antibody, and encodes a light or light chain V domain. In the DNA, in order to encode these The DNA portion of all or a desired amino acid sequence is a template, and the DNA of the peptide linker is amplified by a primer which defines the two ends thereof, and the DNA of the peptide linker portion is encoded, and the two ends are defined as each weight. The primers linked by the chain and the light chain are combined to increase the amplitude. Once the DNA encoding the scFv is produced, the host containing the expression, and the host transformed by the expression host are obtained according to the usual method, and the host can be used as usual. DEC- 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 The antibody may be a mixture of a plurality of antibodies of different CDR types. The polyclonal antibody may be used to culture a mixture of cells producing a different antibody, and the antibody purified by the culture may be used (refer to WO2004/061104). No.) The antibody may also be modified by binding to various molecules such as polyethylene glycol (PEG). The antibody of the present invention may be further combined with these antibodies and other agents (Immunocon Such an antibody may be an antibody to a radioactive substance or a pharmacologically active compound (Nature Biotechnology (2005) 23, p. 1137-1146). The resulting antibody can be purified to homogeneity. The separation and purification of antibodies can be carried out by separating and purifying the usual proteins. For example, chromatography, filtration, ultrafiltration, salting out, dialysis, preparation by polyacrylamide gel electrophoresis, isoelectric point electrophoresis, etc., can be used to separate and purify antibodies (Strategies for Protein Purification and Charcterization: A Laboratoy Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1 996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but not subject to these limited. The chromatography may be affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel -30-200914044 filtration, reverse phase chromatography, adsorption chromatography, and the like. These chromatography can be carried out by liquid chromatography such as HPLC or FPLC. The column for affinity chromatography can be a protein A column or a protein G column. For example, the column using the protein column may be H y p e r D, P 0 R〇S, Sepharose F.F. (Pharmacia) or the like. The antibody can also be purified by binding to an antigen using a carrier which immobilizes the antigen. 3. Medicine containing anti-HSulfl antibody ί) The anti-HSulfl antibody obtained by the method described in the above-mentioned "2. Antibody against HSulfl" can obtain an antibody which neutralizes the biological activity of HSulfl, and specifically expresses cancer cells of HSulfl. An antibody against injury and/or an antibody that inhibits proliferation of cancer cells expressing HSulfl. These antibodies are particularly useful as cancer therapeutic agents because they inhibit the biological activity of HSulfl in vivo, that is, due to the canceration of cells and/or the proliferation of cancer cells. The biological activity neutralization activity of HSulfl against HSulfl antibody in vitro can be determined, for example, by inhibiting the canceration of cells in cells expressing HSulfl excess. For example, Hs 57 8T cells are cultured, and anti-HSulfl antibodies can be added at various concentrations in the culture system to measure the inhibitory activity against cell proliferation. The nociceptive activity of the cancer cells against the HSulfl antibody in vitro, for example, exhibits an antibody-dependent cell disorder activity against the HSulfl antibody against cells expressing HSulfl, and can be determined by complement-dependent cell disorder activity or complement-dependent cell-cell disorder activity. -31- 200914044 For example, HEK293T cells with excessive expression of HSulfl were added to the culture system at various concentrations to add anti-HSulfl antibody, and mouse spleen cells were added to measure the cell death induction rate η of the HSulfl excess cells after the appropriate time. In vivo, the therapeutic effect of the test animal against the cancer of HSulfl antibody can be determined, for example, by transplanting a HSulfl antibody to a nude mouse transplanting a cancer cell expressing HSulfl, and measuring the change of the cancer cell, or administering the same to the transgenic animal having an excessive expression of HSulfl. The HSulfl antibody is used to determine the change of the cancer cell to confirm that the anti-HSulfl antibody of the present invention specifically has the following (1) Wnt signaling inhibitory activity, (2) ERK signal inhibitory activity, (3) apoptosis-inducing activity, and (4) Cell proliferation inhibits at least any activity selected for activity. Each activity can be determined by the following method. (1) Wnt signaling inhibitory activity

Wnt signal inhibitory activity can be measured by adding Wnt3A to HEK293T cells, adding a test substance to a system capable of measuring Wnt signals, and measuring a decrease rate of Wnt signal compared with a control, and measuring according to the method described in Example 2, for example. . The anti-HSulfl antibody of the present invention inhibits the enhancement of the Wnt signal of the secondary HSulfl and inhibits the proliferation of cancer cells. (2) ERK signal inhibitory activity ERK signal inhibitory activity can be determined by adding proliferation factors bFGF, EGF, HB-EGF to HEK293T cells, adding microtuberculosis substances, and measuring the decrease rate of ERK signal compared with the control, which can be determined, for example, according to the present specification. The method described in Example 3 -32- 200914044 is intended. The anti-HSulfl antibody of the present invention inhibits the enhancement of the ERK signal of the intermediate HSulfl and inhibits the proliferation of cancer cells. (3) Apoptosis-inducing activity The apoptosis-inducing activity can be confirmed by activation of Caspase 3/7 and/or fragmentation of the core when the test substance is added. When the anti-HSulfl antibody of the present invention is administered, activation of CaspaSe3/7 and/or fragmentation of the core in cells expressing HSulfl can be confirmed, and apoptosis is caused, and (4) cell proliferation inhibits active cell proliferation inhibitory activity. The survival rate of the cells at the time of the test and/or the proliferation signal of the cells. When the anti-HSulfl antibody of the present invention is administered to a cell, the inhibition of proliferation of the cancer cell can be confirmed. For example, administration of anti-HSulfl antibodies to breast cancer cell line Hs578T cells reduces cell survival. An antibody which neutralizes the biological activity of the HSulfl thus obtained or an antibody which specifically expresses damage to cancer cells of HSulfl is useful as a pharmaceutical composition, particularly a pharmaceutical composition for the treatment of cancer, or an antibody for immunological diagnosis of such a disease. The type of cancer may be colorectal cancer or breast cancer, but is not limited thereto. The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-HSulfl antibody and a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifier, preservative, and/or adjuvant. The substance to be used in the preparation of the pharmaceutical composition of the present invention is suitably administered in an amount of -33 to 200914044 or a concentration to be administered, which is preferably non-toxic to those who administer the pharmaceutical composition. The pharmaceutical composition of the present invention may contain a preparation for changing, maintaining, and maintaining pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, dissolution rate, rate of release, absorption rate, and permeability. substance. The substance used for the preparation may be 'but not limited to these: amino acids such as glycine, alanine, glutamic acid, aspartic acid, arginine or lysine, antibacterial agents, ascorbic acid, An anti-oxidant such as sodium sulfate or sodium hydrogen sulfite, a buffer such as phosphoric acid, citric acid, boric acid buffer, sodium hydrogencarbonate or Tris-hydrochloric acid solution, a mannitol or a glycine acid, or an ethylenediaminetetraacetic acid (EDTA) and other chelating agents, caffeine, polyvinylpyrrolidone, sodium-cyclodextrin or hydroxypropyl cyclodextrin and other bulking agents, glucose, mannose or dextrin extenders, monosaccharides, disaccharides Or other hydrocarbons such as glucose, mannose or dextrin, coloring agents, flavoring agents, diluents, emulsifiers or hydrophilic polymers such as polyvinylpyrrolidone, low molecular weight polypeptides, salts to form counter ions, benzyl alcohol chloride, benzene Formic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexyxixi, sorbic acid or peroxygen gas, preservatives such as glycerol, propylene glycol or polyethylene glycol Alcohol, suspension such as mannitol or sorbitol Interface activity of polysorbate, triton, tromethamine, lecithin or cholesterol, such as PEG, sorbitan ester, polysorbate 2〇 or polysorbate 80 Female tempering enhancer such as agent, recommended sugar or sorbitol, sputum enhancer such as sodium chloride, potassium chloride or mannitol sorbitol, transport agent, diluent, excipient, and/or pharmacy Agent. The amount of the substance to be added to these preparations is preferably 0.01 to 100 times, particularly 0.1 to 10 times, to the weight of the HSulfl antibody to be added -34 to 200914044. The composition of the suitable pharmaceutical composition in the preparation may be appropriately determined by the operator according to the applicable disease, the applicable investment route, and the like. The excipient or carrier in the pharmaceutical composition may be liquid or solid. Suitable excipients or carriers may be water for injection or physiological saline, artificial cerebrospinal fluid or other substances normally used for parenteral administration. . A carrier of physiological saline containing neutral physiological saline or serum albumin may also be used. The pharmaceutical composition may also contain Tris buffer of ΡΗ7.0-8.5 or acetate buffer of pH 4.0-5.5 or these and sorbitol or other compounds. The pharmaceutical composition of the present invention is prepared as a lyophilized product or a liquid in a selected composition as an appropriate agent of the necessary purity. The pharmaceutical composition containing the anti-HSulfl antibody can also be molded with a lyophilized product of a suitable excipient such as sucrose. The pharmaceutical composition of the present invention can also be adjusted for parenteral administration, and can also be adjusted for absorption by the digestive tract. The composition and concentration of the preparation can be determined according to the administration method, and the affinity of the anti-HSulfl antibody contained in the pharmaceutical composition of the present invention to HSulfl, that is, the dissociation number (Kd値) to HSulfl, the higher the affinity (Kd値 is low) The more the drug can be administered to a person with less effect, the reason can also determine the amount of human administration of the pharmaceutical composition of the present invention based on the result. When the human anti-HSulfl antibody is administered to a human, it can be administered for about 0.1 to 100 mg/kg once a day for 30 days. The pharmaceutical composition of the present invention may be in the form of a drip injecting agent, a sitting agent -35-200914044, a nasal spray, a sublingual agent, a transdermal absorbent or the like. [Examples] The present invention will be specifically described by the following examples, but the present invention is not limited by these examples. Further, the operations of the genetic manipulations in the following examples are not specifically indicated, and are based on "Molecular Cloning" (Sambrook, J., Fritsch, EF and Maniatis, T., by Cold Spring Harbor Laboratory Press, 1989). When the method is described, or when a commercially available reagent or kit is used, it is used according to the instructions of the commercial product. Ο (Example 1) The immunostaining in the cancer is equivalent to the peptide of the partial sequence of HSulfl; Leu Asp Pro Glu Lys Pro Gly Asn Arg Phe Arg Thr Asn Lys Lys Ala (SEQ ID NO: 3 of the Sequence Listing) and bovine thyroid globulin The mixture (made by Sigma) was an antigen, mixed with an adjuvant, and immunized 8 times in rabbits every 14 days to obtain HSulfl peptide antiserum. Affinity-refined HSulfl peptide antiserum and polyclonal antibody against HSulfl peptide. Immunostaining of human cancer tissues was performed using this polyclonal antibody. As a result, a positive staining result in the cancer section was obtained. In particular, positive signals are recognized at high frequency in colorectal cancer and breast cancer (Fig. 1). In the organization matrix, the positive frequency is resolved. As a result, it was judged to be positive in colorectal cancer at 36/49 (73.5 %) and breast cancer at a frequency of 18/34 (53%). HSulfl is highly expressed in the cancer department and is known to be associated with cancer. (Example 2) Wnt signal enhancing activity as a primer for PCR amplification of HSulfl cDNA, having primer 1 (PRIMER 1) (sequence number 4 of the sequence listing) and primer 2 (PRIMER 2) (preface - 36- 200914044 list) The oligonucleotide of the sequence of SEQ ID NO: 5) was synthesized by a conventional method. The PCR reaction was carried out in accordance with the instructions attached to kod PLUS (manufactured by Toyobo Co., Ltd.). The PCR product was transformed into a coliform DH5 by a BP reaction (manufactured by Invitrogen). i_Tl=The plastid was extracted from the transformed intestinal tract, and the LR reaction was inserted into the pLNCX host inserted into the GW cassette. Construct the pLNCX-HSulf 1 host. 1.5/zl Opti-MEM (Invitrogen) and GENEμΐ FuGENE6 (manufactured by Roche) were mixed at room temperature for 5 minutes, and then TOPFlash (made by Upstate) 0.1/2 g and phRL-TK (manufactured by Promega) 0.1 were added. /zg and pLNCX-HSulf l (manufactured by Clontech) 0.2#g and placed for 15 minutes. To this solution, 1 X 104 HEK293T cells were mixed and sprinkled on a 96-well plate (manufactured by Becton-Dickinson Co., Ltd.) coated with poly-D-aspartic acid. After 24 hours, 200/z g/ml mWnt3A (manufactured by RD Systems) was added, and after 48 hours, the medium was discarded, and the cells were dissolved in 100#1 passive lysis buffer (manufactured by Promega). Among them, 10# 1 was measured by the Dual-Glo luciferase assay system (Promega) to measure the luminescence of luciferase and _Xin Xue camping enzyme, and the 値 海 萤 luciferase Correction. As a result, HSulfl enhanced the Wnt signal and enhanced the reported activity of Tcf/Lef (Fig. 2). Further, a quick-change site-directed mutagenesis kit (manufactured by Stratagene) was used to convert the cysteine of 87, 88 of HSulfl into alanine and HSulfl mut, and the same as the enzyme active. Role (Figure 2). (Example 3) ERK signal enhancing activity in Opti-MEM 50〇m 1 Adding pFA-Elk (Promega) 0.15" g, -37- 200914044 pGL4-Luc2CP (Promega) 3/zg, pGL4-hRluc-TK (Promega 0.06 / / g, pLNCX-HSulfl) l / zg, pcDNA-DEST40 (Invitrogen) 2 / / g. Others in 〇pti-MEM 500 μ1 Adding force Li Lipofectamine 2000 (Invitrogen) 40 // 1, after placing 5 minutes, mix the solution, place 15 minutes at room temperature, add 5 × 105 HEK293 cells. Among them, 100//1 was sprinkled on a 96-well plate (manufactured by Becton-Dickinson Co., Ltd.) coated with poly-D-lysine. After one night of culture, proliferation factor bFGF (manufactured by Sigma), EGF (manufactured by Sigma), and HB-EGF (manufactured by Sigma f) were added, and bFGF (after 48 hours), EGF, and HB-EGF (4 hours) at an appropriate time. After an hour, the cells were lysed with 100/z 1 passive lysis buffer (manufactured by Promega). Among them, 10/zl was used to measure the luminescence of luciferase and lentinase by the Dual-Insect luciferase reporter assay system (Promega), and the sputum was corrected by the luciferase of sea mushroom. . As a result, HSulfl was expressed to enhance the EGF signal or the ΗΒ-EGF signal, and to inhibit the bFGF signal (Fig. 3). In the enzyme inert body (HSulflmut), it also exhibits the same effect as the enzyme active substance (Fig. 4). These experimental results show that HSulfl controls the growth factor by other mechanisms because of the hydrolysis of the sulfate group in the sugar chain, not only by the action of the growth factor. (Example 4) The function of siRNA blocked the 10000 cells of the breast cancer cell line Hs578T cells which were highly expressed in HSulfl to a final concentration of 200 nM: aaggacuacu ucacagacut t (sequence number 6 of the sequence listing); and agucugugaa guaguccuut t (sequence table) SEQ ID NO: 7): HSulfl siRNA composed of -38-200914044 was introduced into the gene by DMRIE-C (Invitrogen). After 48 hours, RNA extraction was performed using RNeasy (manufactured by Qiagen) to quantify HSulfl mRNA by real-time PCR. As a result, the amount of HSulfl mRNA was reduced to about 20% in the siRNA-introduced strain (Fig. 5 (1)). On the 4th day after the introduction of the siRNA, Cell Titer Glo (Promega) was used to investigate the proliferation inhibition of the cells. As a result, introduction of HSulfl-siRNA revealed an inhibition of proliferation of about 70% (Fig. 5(b)). At the same time, Apo-ONE Homogeneous Caspase-3/7 analysis (manufactured by Promega) and Cell Death Detection ELISA (manufactured by Roche) showed Caspase 3/7 activation (Fig. 5 (c)) and nuclear fragmentation (p. 5 (d)), it is speculated that the observed proliferation inhibition causes apoptosis. (Example 5) The HSulfl recombinant protein was obtained by using the coliform of the coliform of the coulmunium when the HSulfl protein was expressed by Escherichia coli (sequence number 8 of the Sequence Listing), and inserted into the Ndel- of pET-23a (Novagen). Xhol part. The transformation is BL21 (DE3) Gold (manufactured by Stratagene), cultured according to the usual method, and the expression v is induced, and the cells are disrupted by ultrasonic waves after collection. The supernatant was recovered after centrifugation and supplied to HisTrap HP 5 ml (GE Health Care). Washing buffer 1 (20 mM phosphate buffer / 500 mM sodium chloride / 0.5% N-docosylamine sodium salt (pH 7.3), and then washing buffer 2 (20 mM phosphate buffer) /500 mM sodium chloride / 10 mM imidazole / 0.5% N-dopral myosin sodium salt (pH 7.3) washed to elution buffer (20 mM phosphate buffer / 500 mM sodium chloride / 125 mM imidazole / 0.5% N - Dodecyl sarcosine sodium salt (pH 7.3) is dissolved. Secondary dialysis buffer 1 (20 mM Tris-HCl / 500 mM sodium chloride / 0.05% N - -39 - 200914044 PH7.6)) dialyzed twice with 1000 times the amount of the sample, and further dialysis buffer 2 (20 mM Tris-HCl/400 mM sodium chloride/0.05% N-doprene myosamine sodium salt (pH 7. 6)) 2 times of dialysis to obtain HSulfl recombinant protein. (Example 6) Obtaining anti-HSulfl monoclonal antibody The HSulfl protein obtained in Example 5 was intraperitoneally administered once a week in a 6-week-old BALB/c mouse. 0/zg/shot), the first-time immunization was performed by mixing Freund's complete adjuvant (FC A) (manufactured by Sigma) and HSulfl protein, and the second and subsequent immunizations were Freund's incomplete adjuvant (FICA) and HSulfl. Protein mixed dose, total immunization 10 times. 4 by final immunization The spleen of the mouse was taken out, and it was ground with a glass piece, and the cells were fused with X-63/Ag8 cells and polyethylene glycol. The HAT selection medium (manufactured by IBL) containing 10% serum was exchanged every 3 days. Half the amount, and cultured for 7 to 10. The fusion culture supernatant was assayed by Direct ELISA, and the cells determined to be positive were selected by the limiting dilution method to establish a fusion tumor. (Example 7) Reporting the inhibitory effect of activity The HSulfl gene was introduced into HEK293T cells in the same manner as in Example 2. After 24 hours, 20 " g/ml anti-HSulfl antibody was added and cultured at 37 ° C for 1 hour. Thereafter, 200 ng / ml of mWnt3A was added, 48 hours later and Example 2 The cells were also recovered, and the reporter activity was measured. As a result, the blocking effect of the reporter activity was confirmed by a plurality of antibodies (Fig. 6), and one of the fusion tumors (#87 in the figure) which produced an antibody having a strong inhibitory effect on the activity was named as 03-Six 1^-87 A3. DS-ATRL-87 A3 was deposited with the National Patent Bio-Reservation Center of Japan on June 6, 2007, and assigned the registration number FERM BP- 1 0835. -40- 200914044 (Embodiment 8) The effect of antibodies on cell proliferation in the Baiqingdi 96-well plate ( Manufactured by Coster Co., Ltd., 50# g/ml of anti-mouse IgG goat antibody (manufactured by Sigma) 50#1, and reacted at 4 ° C for 1 night. After washing twice with PBS, an anti-HSulfl antibody 50 "1 prepared in SOvg/ml was added, and the reaction was further carried out at 4 ° C for 1 night. After washing twice with PBS, Hs578T cells were seeded at 4000 cells/well and cultured at rpmi containing 1% serum. After 4 days of culture, the cell survival rate was examined using Cell Titer Glo (Promega). IgGl was used as a negative control. As a result, the anti-HSulfl antibody ' showed a proliferation inhibition effect of up to about 40% (Fig. 7). Further, the proliferation inhibition effect was positively correlated with the reported inhibition effect (r = 0.77, p < 0.01), suggesting that the proliferation inhibition effect of the antibody is caused by the signal transmission inhibitory effect (Fig. 8). Therefore, the anti-HSulfl antibody showed an anti-tumor effect. (Industrial Applicability) The anti-HSulfl antibody of the present invention has antitumor activity, and the pharmaceutical composition containing an anti-HSulfl antibody may be an anticancer agent. [Simplified Schematic] [Fig. 1] is a graph showing high performance of HSulfl in (a) colon cancer, (b) breast cancer, (c) pancreatic cancer, and (d) prostate cancer. [Fig. 2] shows that HSulfl and enzyme inert variants enhance Wnt signaling, enhance Tcf/Lef reporter activity, and enzyme inert variants also enhance Wnt signaling. [Fig. 3] is a diagram showing HSulf 1 enhancing (a) EGF signal, (b) HB-EGF signal, and (c) bFGF signal.

[Fig. 4] shows the enhancement of (a) EGF-41-200914044 signal, (b) HB-EGF signal and (c) bFGF signal of the enzyme inert variant of HSulfl. [Fig. 5] (a) The amount of HSulfl mRNA was reduced to about 20%, (b) cell proliferation was inhibited by about 70%, (c) Caspase 3/7 was activated, and (d) nucleus was presented by introduction of siRNA against HSulfl. A fragmented picture. [Fig. 6] is a graph showing the inhibitory activity of the antibody against HSulfl. In the figure, IgG1 is a control, and #4, #23, #27, #39, #55, #82, #87, #154, #160, and #165 are monoclonal antibodies derived from different fusion tumors. [Fig. 7] is a graph showing the effect of anti-HSulfl antibody on cell proliferation inhibition. IgGl is a control antibody ##, #23, #27, #39, #5 5, #82, #87, #154, #160, #165, which are individual antibodies derived from different fusion tumors. [Fig. 8] is a graph showing a positive correlation between the cell proliferation inhibitory activity and the reported inhibitory activity. [Explanation of main component symbols] 4πτ Pick 0 - 42- 200914044 Sequence Listing <110> First Sankyo Co., Ltd. <120>anti-human sulfl antibody <130> DSPCT-FP0828 <150> JP 2007-182882 <;151> 2007-07-12 <160〉 8<170> Patent version 3.4

<210> 1 <211> 2616 <212> DNA <213>People<220>221> CDS <222>(1).. (2616) <400> 1 atg aag tat tct Tgc tgt get ctg gtt ttg get gtc ctg ggo aca gaa Met Lys Tyr Ser Cys Cys Ala Leu Val Leu Ata Val Leu Gly Thr Giu 15 10 15 48 ttg ctg gga age etc tgt teg act gtc aga tee cog agg ttc aga gga Leu Leu Gly Ser Leu Cys Ser Thr Val Arg Ser Pro Arg Phe Arg Gly 20 25 30 egg ata cag cag gaa ega aaa aac ate ega ccc aac att att ett gtg Arg lie Gin Gin 6lu Arg Lys Asn lie Arg Pro Asn lie lie Leu Val 35 40 45 96 144

Cct acc gat gat caa gat gtg gag ctg ggg tee ctg caa gtc atg aac Pro Thr Asp Asp Gin Asp Val Glu Leu Gly Ser Leu Gin Val Met Asn 50 55 60 192 aaa aeg aga aag att atg gaa cat ggg ggg gee acc ttc ate Aat gee Lys Thr Arg Lys Me Met Glu His Gly Gly Aia Thr Phe Me Asn Ala 65 70 75 80 ttt gtg act aca ccc atg tgc tgc ccg tea egg tee tee atg etc acc Phe Val Thr Thr Pro Met Cys Pro Ser Arg Ser Ser Met Leu Thr 240 288 336200914044 85 90 95 ggg aag tat gtg cac aat cac aat gtc tac acc aac aac gag aac tgc Gly Lys Tyr Val His Asn His Asn Val Tyr Thr Asn Asn Glu Asn Cys 100 105 110 tot tcc ccg teg tgg Cg geo atg cat gag cct egg act ttt get gta Ser Ser Pro Ser Trp Gin Aia Met His Glu Pro Arg Thr Phe Ala Val 115 120 125 tat ett aac aac act ggc tao aga aca gee ttt ttt gga aaa tac etc Tyr Leu Asn Asn Thr Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu 130 135 140 aat gaa tat aat ggc age tac ate ccc cct ggg tgg ega gaa tgg ett Asn Glu Tyr Asn Gly Ser Tyr lie Pro Pro Giy T卬Arg Glu Trp Leu 145 150 155 16 0 f ' gga tta ate aag aat tot ogc ttc tat aat tac act gtt tgt ego aat ^ Gly Leu I le Lys Asn Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg Asn 165 170 175 ggc ate aaa gaa aag cat gga ttt gat tat Gca aag gac tac tto aca Gly lie Lys Glu Lys His Giy Phe Asp Tyr Ala Lys Asp Tyr Phe Thr 180 185 190 gac tta ate act aac gag age att aat tac ttc aaa atg tet aag aga Asp Leu lie Thr Asn Glu Ser lie Asn Tyr Phe Lys Met Ser Lys Arg 195 200 205 atg tat ccc cat agg ccc gtt atg atg gtg ate age cac get geg ccc Met Tyr Pro His Arg Pro Val Met Met Val He Ser His Aia Ala Pro 210 215 220 cac ggc ccc gag gac Tea gee cca cag ttt tet aaa ctg tac ccc aat His Gly Pro Glu Asp Ser Ala Pro Gin Phe Ser Lys Leu Tyr Pro Asn / 225 230 235 240 get tee caa cac ata act cct agt tat aac tat gca cca aat atg gat Ala Ser Gin His I le Thr Pro Ser Tyr Asn Tyr Aia Pro Asn Met Asp 245 250 255 aaa cac tgg att atg cag tac aca gga cca atg ctg ccc ate cac atg Lys His Trp Me Met Gin Tyr Thr Sly Pro Met Leu Pro lie His Met 260 265 270 gaa tt t aca aac att eta cag ege aaa agg etc cag act ttg atg tea Glu Phe Thr Asn I le Leu Gin Arg Lys Arg Leu Gin Thr Leu Met Ser 384 432 480 528 576 624 672 720 768 816 864 912200914044 275 280 285 gtg gat gat Tot gtg gag agg ctg tat aac atg etc gtg gag aeg ggg Val Asp Asp Ser Val Giu Arg Leu Tyr Asn Met Leu Val Glu Ding hr Gly 290 295 300 gag ctg gag aat act tac ate att tac acc gee gac cat ggt tac cat Glu Leu Glu Asn Thr Tyr lie Me Tyr Thr Ala Asp His Gly Tyr His 305 310 315 320 att ggg cag ttt gga ctg gtc aag ggg aaa tco atg oca tat gac ttt lie Gly Gin Phe Gly Leu Val Lys Gly Lys Ser Met: Pro Tyr Asp Phe 325 330 335 gat att cgt gtg cct ttt ttt att cgt ggt cca agt gta gaa cca gga Asp Me Arg Val Pro Phe Phe lie Arg Gly Pro Ser Val Glu Pro Gly 340 345 350 tea ata gtc cca cag ate gtt etc aac att Gac ttg gcc ccc aeg ato Ser lie Val Pro Gin lie Val Leu Asn Me Asp Leu Ala Pro Thr lie 355 360 365 ctg gat att get ggg etc gac aca cct cct gat gtg gac ggc aag tet Leu Asp lie Ala Gly Leu Asp Thr Pro Pro Asp Val Asp Gly Lys Ser 370 375 380 gtc etc aaa ett ctg gac cca gaa aag ooa ggt aac agg ttt ega aca Val Leu Lys Leu Leu Asp Pro Glu Lys Pro Giy Asn Arg Phe Arg Thr 385 390 395 400 aac aag aag gcc aaa Att tgg cgt gat aca ttc eta gtg gaa aga ggc Asn Lys Lys Ala Lys lie Trp Arg Asp Thr Phe Leu Val Glu Arg Gly 405 410 415 aaa ttt eta cgt aag aag gaa gaa tcc age aag aat ate caa cag tea Lys Phe Leu Arg Lys Lys Glu Glu Ser Ser Lys Asn lie Gin Gin Ser 420 425 430 aat cac ttg ccc aaa tat gaa egg gto aaa gaa eta tgc cag cag gcc Asn His Leu Pro Lys Tyr Glu Arg Val Lys Glu Leu Cys Gin Gin Ala 435 440 445 Agg tac cag aca gcc tgt gaa caa cog ggg cag aag tgg caa tgc att Arg Tyr Gin Th " Ala Gys Glu Gin Pro Gly Gin Lys Trp Gin Cys lie 450 455 460 gag gat aca tet ggc aag ett ega att cac aag tgt aaa gga Ccc agt Glu Asp Thr Ser Gly Lys Leu Arg lie His Lys Cys Lys Gly Pro Ser 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488200914044 465 470 475 480 gac ctg etc aca gtc egg cag age aeg egg aac et c tac get ege ggc Asp Leu Leu Thr Val Arg Gin Ser Thr Arg Asn Leu Tyr Aia Arg Gly 485 490 495 ttc cat gac aaa gac aaa gag tgc agt tgt agg gag tet ggt tac cgt Phe His Asp Lys Asp Lys Glu Cys Ser Cys Arg Glu Ser Gly Tyr Arg 500 505 510 gee age aga age caa aga aag agt caa egg caa ttc ttg aga aac cag Ala Ser Arg Ser Gin Arg Lys Ser Gin Arg Gin Phe Leu Arg Asn Gin 515 520 525 ggg act cca aag tac aag Ccc aga ttt gtc cat act egg cag aca cgt Gly Thr Pro Lys Tyr Lys Pro Arg Phe Val His Thr Arg Gin Thr Arg 530 535 540 tee ttg tee gtc gaa ttt gaa ggt gaa ata tat gac ata aat ctg gaa Ser Leu Ser Val Glu Phe Glu Gly Glu lie Tyr Asp lie Asn Leu Glu 545 550 555 560 gaa gaa gaa gaa ttg caa gtg ttg caa cca aga aac att get aag cgt Glu Glu Glu Leu Gin Val Leu 6ln Pro Arg Αεη lie Ala Lys Arg 565 570 575 1536 1584 1632 1680 1728 cat gat gaa ggc cac aag ggg cca aga gat etc cag get tee agt ggt His Asp Glu Gly His Lys Gly Pro Arg Asp Leu Gin Ala Ser Ser Gly 580 585 590 1776 ggc aac agg ggc agg atg ctg g Ca gat age age aac gee gtg ggc cca Gly Asn Arg Gly Arg Met Leu Aia Asp Ser Ser Asn Ala Val Gly Pro 595 600 605 ect acc act gtc ega gtg aca cac aag tgt ttt att ett ccc aat gac Pro Thr Thr Val Arg Val Thr His Lys Cys Phe He Leu Pro Asn Asp 610 615 620 1824 1872 tet ate cat tgt gag aga gaa ctg tac caa teg goc ass geg tgg aag Ser He His Cys Glu Arg Glu Leu Dyr Gin Ser Ala Arg Ala Trp Lys 625 630 635 640 gac cat aag gca tac att gac aaa gag att gaa get Gtg caa gat aaa Asp His Lys Aia Tyr lie Asp Lys Glu lie Glu Ala Leu Gin Asp Lys 645 650 655 att aag aat tta aga gaa gtg aga gga cat ctg aag aga Agg aag cct Ile Lys Asn Leu Arg Glu Val Arg Gly His Leu Lys Arg Arg Lys Pro 1920 1968 2016 200914044

660 665 670 gag gaa tgt age tgc agt aaa caa ago tat tac aat aaa gag aaa ggt Giu Glu Gys Ser Cys Ser Lys Gin Ser Tyr Tyr Asn Lys Glu Lys Gly 675 680 685 gta aaa aag caa gag aaa tta aag age cat ett cac Cca ttc aag gag Vai Lys Lys Gin 6lu Lys Leu Lys Ser His Leu His Pro Phe Lys Glu 690 695 700 get get cag gaa gta gat age aaa ctg caa ott ttc aag gag aac aac Ala Ala Gin Glu Val Asp Ser Lys Leu Gin Leu Phe Lys Glu Asn Asn 705 710 715 720 cgt agg agg aag aag gag agg aag gag aag aga egg cag agg aag ggg Arg Arg Arg Lys Ly$ Glu Arg Lys Giu Lys Arg Arg Gin Arg Lys Gly 725 730 735 gaa gag tgc age ctg Cct ggc etc act tgc ttc aeg cat gac aac aac Glu Glu Cys Ser Leu Pro Gly Leu Thr Cys Phe Thr His Asp Asn Asn 740 745 750 cac tgg cag aca gee ccg ttc tgg aac ctg gga tet ttc tgt get tgc His Trp 6ln Thr Ala Pro Phe Trp Asn Leu Gly Ser Phe Cys Ala Cys 755 760 765 aeg agt tet aac aat aac acc tac tgg tgt ttg cgt aca gtt aat gag Thr Ser Ser Asn Asn Asn Thr Tyr Trp Cys Leu Arg Thr Val Asn Glu 770 775 780 Aeg Cat aat ttt ett ttc tgt gag ttt get act ggc ttt ttg gag tat Thr His Asn Phe Leu Phe Cys Glu Phe Ala Thr Gly Phe Leu Glu Tyr 785 790 795 800 ttt gat atg aat aca gat cct tat cag etc aca aat aca gtg cac Aeg Phe Asp Met Asn Thr Asp Pro Tyr Gin Leu Thr Asn Thr Val His Thr 805 810 815 gta gaa ega ggc att ttg aat Gag Gta cac gta Gaa eta atg gag etc Val Glu Arg Gly i le Leu Asn Gin Leu His Val Gin Leu Met Glu Leu 820 825 830 aga age tgt caa gga tat aag cag tgo aac oca aga cct aag aat ett Arg Ser Cys Gin Gly Tyr Lys Gin Cys Asn Pro Arg Pro Lys Asn Leu 835 840 gat gtt gga aat aaa gat gga gga age Tat gac eta cac aga gga oag Asp Val Gly Asn Lys Asp Gly 6ly Ser Tyr Asp Leu His Arg Gly Gin 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2544 2592

200914044 850 855 860 tta tgg gat gga tgg gaa ggt taa 2616

Leu Trp Asp Giy Trp Glu Gly 865 870 1—T— 7 R- L 8 p <210〉 <211〉 <212><213〉<400> 2

Met Lys Tyr Ser Cys Cys Ala Leu Val Leu Ala Val Leu Gly Thr Giu 15 10 15

Leu Leu Gly Ser Leu Cys Ser Thr Val Arg Ser Pro Arg Phe Arg Gly 20 25 30

Arg lie Gin Gin Glu Arg Lys Asn Me Arg Pro Asn Me He Leu Val 35 40 45

Pro Thr Asp Asp Gin Asp Val Glu Leu Gly Ser Leu Gin Val Met Asn 50 55 60

Lys Thr Arg Lys lie Met Glu His Gly Gly Ala Thr Phe lie Asn Ala 65 70 15 80

Phe Val Thr Thr Pro Met Gys Cys Pro Ser Arg Ser Ser Met Leu Thr 85 90 95

Gly Lys Tyr Val His Asn His Asn Val Tyr Thr Asn Asn Glu Asn Cys 100 105 110

Ser Ser Pro Ser Trp Gin Ala Met His Glu Pro Arg Thr Phe Ala Val 115 120 125

Tyr Leu Asn Asn Thr Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu 130 135 140 200914044

Asn Glu Tyr Asn Gly Ser Tyr lie Pro Pro Gly Trp Arg Glu Trp Leu 145 150 彳 55 160

Gly Leu lie Lys Asn Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg Asn 165 170 175

Gly Me Lys Glu Lys Kis Gly Phe Asp Tyr Ala Lys Asp Tyr Phe Thr 180 185 190

Asp Leu lie Thr Asn Glu Ser Me Asn Tyr Phe Lys Met Ser Lys Arg 195 200 205

Met Tyr Pro His A"g Pro Val Met Met Val lie Ser His Ala Ala Pro 210 215 220

His Gly Pro Glu Asp Ser Ala Pro Gin Phe Ser Lys Leu Tyr Pro Asn 225 230 235 240

Ala Ser Gin His lie Thr Pro Ser Tyr Asn Tyr Ala Pro Asn Met Asp 245 250 255

Lys His Trp lie Met Gin Tyr Thr Gly Pro Met Leu Pro lie His Met 260 265 270

Glu Phe Thr Asn I le Leu Gin Arg Lys Arg Leu Gin Thr Leu Met Ser 275 280 285

Vai Asp Asp Ser Vai Glu Arg Leu Tyr Asn Uet Leu Val Glu Thr Gly 290 295 300

Glu Leu Glu Asn Thr Tyr lie lie Tyr Thr Ala Asp His Gly Tyr His 305 310 315 320 I le Gly Gin Phe Gly Leu Val Lys Gly Lys Ser Met Pro Tyr Asp Phe 325 330 335 200914044

Asp lie Arg Val Pro Phe Phe Ile Arg Gly Pro Ser Val Glu Pro Gly 340 345 350

Ser Me Val Pro Gin lie Val Leu Asn lie Asp Leu Ala Pro Thr lie 355 360 365

Leu Asp i ie Ala Gly Leu Asp Thr Pro Pro Asp Vai Asp Gly Lys Ser 370 375 380

Val Leu Lys Leu Leu Asp Pro Glu Lys Pro Gly Asn Arg Phe Arg Thr 385 390 395 400

Asn Lys Lys Ala Lys lie Trp Arg Asp Thr Phe Leu Val Glu Arg Gly 405 410 415

Lys Phe Leu Arg Lys Lys Glu Glu Ser Ser Lys Asn lie Gin Gin Ser 420 425 430

Asn His Leu Pro Lys Tyr Glu Arg Val Lys Glu Leu Cys Gin Gin Ala 435 440 445

Arg Tyr Gin Thr Ala Cys Glu Gin Pro Gly Gin Lys Trp Gin Cys He 450 455 460

Giu Asp Thr Ser Gly Lys Leu Arg lie His Lys Cys Lys Gly Pro Ser 465 470 475 480

Asp Leu Leu Thr Val Arg Gin Ser Thr Arg Asn Leu Tyr Ala Arg Gly 485 490 495

Phe His Asp Lys Asp Lys Glu Cys Ser Cys Arg Glu Ser Gly Tyr Arg 500 505 510

Ala Ser Arg Ser Gin Arg Lys Ser Gin Arg Gin Phe Leu Arg Asn Gin 515 520 525 200914044

Gly Thr Pro Lys Tyr Lys Pro Arg Phe Val His Thr Arg Gin Thr Arg 530 535 540

Ser Leu Ser Val 6lu Phe Glu Gly Glu lie Tyr Asp lie Asn Leu Glu 545 550 555 560

Glu Giu Giu Giu Leu Gin Vai Leu Gin Pro Arg Asn lie Ala Lys Arg 565 570 575

His Asp Glu Gly His Lys Gly Pro Arg Asp Leu Gin Ala Ser Ser Gly 580 585 590

Gly Asn Arg Gly Arg Met Leu Ala Asp Ser Ser Asn Ala Val Gly Pro 595 600 605

Pro Thr Thr Val Arg Val Thr His Lys Cys Phe lie Leu Pro Asn Asp 610 615 620

Ser Me His Cys Glu Arg Glu Leu Tyr Gin Ser Ala Arg Ala Trp Lys 625 630 635 640

Asp His Lys Ala Tyr Me Asp Lys Glu lie Glu Ala Leu Gin Asp Lys 645 650 655

Ile Lys Asn Leu Arg Giu Val Arg Gly His Leu Lys Arg Arg Lys Pro 660 665 670

Glu Glu Cys Ser Cys Ser Lys 6ln Ser Tyr Tyr Asn Lys Glu Lys Gly / 675 680 685

Val Lys Lys Gin Glu Lys Leu Lys Ser His Leu His Pro Phe Lys Glu 690 695 700

Aia Ala Gin Glu Val Asp Ser Lys Leu Gin Leu Phe Lys Glu Asn Asn 705 710 715 720 200914044

Arg Arg Arg Lys Lys Giu Arg Lys Glu Lys Arg Arg Gin Arg Lys Gly 725 730 735

Glu Glu Cys Ser Leu Pro Gly Leu Thr Gys Phe Thr His Asp Asn Asn 740 745 750

His Trp Gin Thr Aia Pro Phe Trp Asn Leu Gly Ser Phe Cys Ala Cys 755 760 765

Thr Ser Ser Asn Asn Asn Thr Tyr Trp Cys Leu Arg Thr Val Asn Glu 770 775 780

Thr His Asn Phe Leu Phe Cys Glu Phe Ala Thr Gly Phe Leu Glu Tyr 785 790 795 800 f - K Phe Asp Met Asn Thr Asp Pro Tyr Gin Leu Thr Asn Thr Val His Thr 805 810 815

Vai Glu Arg Giy lie Leu Asn Gin Leu His Val Gin Leu Met Giu Leu 820 825 830

Arg Ser Cys Gin Gly Tyr Lys Gin Cys Asn Pro Arg Pro Lys Asn Leu 835 840 845

Asp Va! Gly Asn Lys Asp Gly Gly Ser Tyr Asp Leu His Arg Gly Gin 850 855 860

Leu Trp Asp Gly Trp Glu Gly 865 870 <210> 3 <211> 18 <212> PRT <213>People <400> 3 10-200914044

Leu Asp Pro Glu Lys Pro Gly Asn Arg Phe Arg Thr Asn Lys Lys Ala 15 10 15

Lys Me

<210> 4 <211> 61 <212> DNA <213>Labor<220><223> 丨子丨1: PCR-derived primer for Hsulfl<400> 4 ggggacaagt ttgtacaaaa aagcaggctt cgagacggag acattttgtc agttttgcaa 60 <210> 5 <211> 63 <212> _ <213>manual<220><223> 丨子丨2: PCR antisense bow of Hsulfl I <400> 5 ggggaccact ttgtacaaga aagctgggtc Ctacccttcc catccatccc ataactgtcc 60 tct 63

<210>6 <211> 21 <212> DNA/RNA <213>Manual<220><223>Hsulfl siRNA sensible stock <400> 6 21 aaggacuacu ucacagacut t -11- 200914044 <lt;;210> 7 <211> 21 <212> DNA/RNA <213>manual<220><223> Hsukfl siRNA antisense stock <400> 7 agucugugaa guaguccuut t 21 <210> 8 &lt ;211> 2616 <212> DNA <213>manual<220> f

≪ 223> Hsulfl artificial sequence < 400> 8 atgaaatatt cttgctgtgc tctggttttg gctgtcctgg gcacggaatt gctgggtagc 60 ctctgttcga ctgtccgttc cccgcgtttc cgtggtcgga ttcagcagga acgtaaaaac 120 atccgtccga acattattct tgtgcttacc gatgatcaag atgtggagct ggggtccctg 180 oaagtcatga acaaaacgcg taaaattatg gaacatgggg gggccacctt catcaatgcc 240 tttgtgacta cgccgatgtg ctgcccgtca cggtcctcca tgctcaccgg gaaatatgtg 300 cacaatcaca atgtotacac caacaacgag aactgctctt ccccgtcgtg gcaggccatg 360 catgagccgG ggacttttgc tgtatatctt aacaacactg gctaccgtac ggcctttttt 420 ggtaaatacc tcaatgaata taatggcagc tacatcccgc cggggtggog tgaatggctt 480 ggtttaatca aaaattctcg cttctataat tacactgttt ftcgcaatgg oatcaaagaa 540 aaaoatggtt ttgattatgc aaaagactac ttcaoggact taatcactaa cgagagcatt 600 aattacttca aaatgtctaa acgtatgtat ccgcatcgtc cggttatgat ggtgatcagc 660 cacgotgcgc cgcacggccc ggaggactca gccocaoagt tttGtaaact gtacccgaat 720 gcttcccaac acattaotcc gagttataac tatgcaccaa Atatggataa acactggatt 780 -12- 840 200914044 atgoagtaca cgggtccaat gctgccga tc cacatggaat ttacgaacat tttacagcgc aaacgtctcc agactttgat gtcagtggat gattctgtgg agcgtctgta taacatgctc gtggagacgg gggagctgga gaatacttac atcatttaca ccgccgacca tggttaccat attgggcagt ttggtctggt caaagggaaa tccatgccat atgactttga tattcgtgtg ccgtttltta ttcgtggtcc aagtgtagaa ccaggttcaa ttgtcccaca gatcgttctc aacattgact tggcoccgac gatcctggat attgctgggc tcgacacgcc gccggatgtg gacggcaaat ctgtcctcaa acttctggac ccagaaaaac caggtaaccg ttttcgtacg aacaaaaaag ccaaaatttg gcgtgatacg ttcttagtgg aacgtggcaa atttttacgt aaaaaagaag aatccagcaa aaatatccaa cagtcaaatc acttgccgaa atatgaacgg gtcaaagaat tatgccagca ggcccgttac cagacggcct gtgaacaacc ggggcagaaa tggcaatgca ttgaggatac gtctggcaaa cttcgtattc acaaatgtaa aggtccgagt gacctgctca cggtccggca gagcacgcgg aacctctacg ctcgcggctt ccatgacaaa gacaaagagt gcagttgtcg tgagtctggt tacGgtgcca gccgtagcca acgtaaaagt caacggcaat tcttgcgtaa ccaggggact ccaaaataca aaccgcgttt tgtccatact cggcagacgc gttccttgtc cgtcgaattt gaaggtgaaa tttatgacat taatctggaa gaagaagaag aattgcaagt gttgcaacca cgtaaca ttg ctaaacgtca tgatgaaggc cacaaagggc cacgtgatct ccaggcttcc agtggtggca accgtggccg tatgctggca gatagcagca acgccgtggg cccaccgacc actgtccgtg tgacgcacaa atgttttatt cttccgaatg actctatcca ttgtgagcgt gaactgtacc aatcggcccg tgcgtggaaa ^ gaccataaag catacattga caaagagatt gaagctctgc aagataaaat taaaaattta cgtgaagtgc gtggtcatct gaaacgtagt aaaccggagg aatgtagctg cagtaaacaa agctattaca ataaagagaa aggtgtaaaa aaacaagaga aattaaaaag ccatcttoac ccattcaaag aggctgctca ggaagtagat agcaaactgc aacttttcaa agagaacaac cgtcgtcgta aaaaagagcg taaagagaaa cgtcggcagc gtaaagggga agagtgcagc 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 -13- 200914044 ctgccgggcc tcacttgctt cacgcatgac aacaaccact ggcagacggc cccgttctgg aacctgggtt ctttctgtgc ttgcacgagt tctaacaata acacctactg gtgtttgcgt acggttaatg agacgcataa ttttcttttc tgtgagtttg ctactggctt tttggagtat tttgatatga atacggatcc gtatcagctc Acgaatacgg tgcacacggt agaacgtggc attttgaatc agttaoacgt acaattaatg gagctccgta gctgtcaa Gg ttataaacag tgcaacccac gtccgaaaaa tcttgatgtt ggtaataaag atggtggtag ctatgactta caccgtggtc agttatggga tggttgggaa ggttaa 2280 2340 2400 2460 2520 2580 2616

Claims (1)

200914044 X. Patent application scope: 1. An antibody that specifically binds to HSulfl and has cell proliferation inhibitory activity on cells expressing the protein. 2. An antibody that specifically binds to HSiilf and has antibodies to at least one of the following (a) to (c) for cells expressing HSulfl: (a) inhibition of Wnt signaling activity (b) inhibition Activity of ERK signal (c) Activity to induce apoptosis. An antibody which specifically binds to a protein derived from an amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing, and which has an antibody for inhibiting cell proliferation inhibition on cells expressing the protein. 4. An antibody which specifically binds to a protein obtained by the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing, and has at least one activity selected from the following U) to (c) for the cell expressing the protein. Antibodies: (a) inhibit the activity of Wnt signaling (b) inhibit the activity of ERK signaling i (c) induce apoptosis activity. 5. The antibody of any one of claims 1 to 4 wherein the cells are tumor cells. 6. An antibody which binds to an antigen binding site of an antibody-binding antigen produced by a mouse fusion tumor DS-ATRL-87A3 (FERM BP-1 083 5, BCRC960365). 7. The antibody of claim 6 which has the complementarity of antibodies produced by mouse fusion tumor DS-ATRL-87A3 (FERM BP-10-835) 200914044. 8. The antibody according to item 6 of the patent application, which is an amino group in the field of complementarity determined by the complementation of antibodies produced by mouse fusion tumor 03-octyl 1^-87 human 3 (1^1^8?- 1 0835) In the acid sequence, the amino acid sequence of one or several amino acid sequences is deleted, substituted or added, and specifically binds to HSulfl, and has an activity of inhibiting cell proliferation against cells expressing the protein. 9. An antibody according to claim 6 which is an antibody produced by mouse fusion tumor DS-ATRL-87A3 (FERM BP-10-8). The antibody according to any one of claims 1 to 7, which is a monoclonal antibody. 1 1. An antibody which is a humanized antibody according to any one of claims 1 to 8 and 9. 1 2. The antibody as described in any one of claims 1 to 8 and 10, which is a human antibody. 1 3. An antibody according to any one of claims 1 to 8 and 10 to 12, which is an IgG antibody. An antibody according to any one of claims 1 to 8, which comprises an antibody selected from the group consisting of Fab, F(ab')2, Fv, scFV, diabody, linear antibody, and more Specific antibody. 15·- Mouse fusion tumor DS-ATRL-87A3 (FERM BP-1688). 1 6. A pharmaceutical composition comprising an antibody selected from any one of the items 1 to 14 of the patent application. A medical composition for cancer treatment, which comprises an antibody selected from at least one of the items of the first to fourth aspects of the patent application. 200914044 1 8 _ A pharmaceutical composition for cancer treatment, which is characterized in that it comprises an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 in the sequence listing or a partial nucleotide sequence of the sequence having a complementary nucleotide sequence. 19_ A pharmaceutical composition for cancer treatment, which is characterized in that it is an siRNA having a nucleotide sequence represented by SEQ ID NO: 1 in the Sequence Listing. The pharmaceutical composition of any one of the above-mentioned claims, wherein the cancer is at least one selected from the group consisting of colorectal cancer, breast cancer, prostate cancer, and pancreatic cancer. / - 21. A screening method for an antitumor substance, which is a project for selecting a substance in which HSulfl is exposed to a substance to be tested, and selecting a substance of HSulfl and/or a substance for inhibiting activity.