JP2013013327A - Antibody binding to mansc1 protein and having anticancer activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new antibody having excellent anticancer activity, and a pharmaceutical agent containing the antibody as an active ingredient for treating or diagnosing cancer.SOLUTION: By using the SST-REX method, a monoclonal antibody binding to the MANSC1 protein and having excellent anticancer activity has been found, as the result of selecting a cDNA encoding a protein expressed on a cell surface or secreted from the cell from a cDNA library originated from a cancer cell strain, and preparing the monoclonal antibody to the protein encoded by the selected cDNA and investigating anticancer activity in vitro and in vivo. In addition, a region including an epitope of the antibody in the MANSC1 protein is identified, and the structures of the light-chain and heavy-chain variable regions of this antibody are successfully determined.

Description

  The present invention relates to an antibody having anticancer activity and use thereof.

  Cancer (tumor) is the leading cause of death in Japan. According to statistics from the National Cancer Center's Cancer Information Center, there were approximately 329 thousand people who died from cancer in 2006. By region, the lungs (23%), stomach (17%), Liver (11%), colon (7%, 11% when combined with large intestine), pancreas (6%), followed by stomach (13%), lung (13%), colon (10%) When combined with the large intestine, 14%), breast (9%), liver (8%). The number of cancer patients is increasing year by year, and the development of highly effective and safe drugs and treatment methods is strongly desired.

  Gastric cancer is one of the cancers with very high morbidity and mortality in Japan, but it is now relatively easy to cure due to advances in diagnostic methods and treatment methods such as surgical excision and chemotherapy. It is also one of them. However, regarding Skills gastric cancer, it is regarded as one of very high-grade gastric cancer that is difficult to treat. Skills gastric cancer is a cancer cell that does not appear on the mucosal surface but invades the entire stomach wall or diffuses more than half to more than 1/3, resulting in thickening and hardening of the stomach wall without forming a macroscopically obvious tumor. It has the feature that the boundary with the surrounding mucous membrane is unclear. Skills gastric cancer has a lower age of onset, progresses faster, and is difficult to diagnose than normal gastric cancer. At the time of diagnosis, 60% of the patients were already inoperable due to peritoneal dissemination / metastasis, and even if resected by surgery, the 5-year survival rate is only 15-20%.

  In recent years, the use of antibodies as anticancer agents has gained recognition as an approach in the treatment of various disease states (cancer types). For example, if an antibody is targeted to a tumor-specific antigen, the administered antibody is presumed to accumulate in the tumor, so complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) can be expected to attack cancer cells via the immune system. Further, by binding a drug such as a radionuclide or a cytotoxic substance to the antibody, the bound drug can be efficiently delivered to the tumor site. As a result, the amount of drug reaching the other tissue can be reduced, and as a result, side effects can be reduced. If the tumor-specific antigen has activity to induce cell death, administer an antibody with agonistic activity, and if the tumor-specific antigen is involved in cell growth and survival, neutralize activity By administering an antibody having a tumor, tumor growth arrest or regression can be expected due to the accumulation of antibody specific to the tumor and the activity of the antibody. From such characteristics, the antibody is considered suitable for application as an anticancer agent.

  The antibody drugs marketed so far are leukemia / lymphoma targeting CD20 targeting rituximab (trade name rituxan) and iburitumomab ozogamicin (trade name Zevailn), and CD33 targeting gemutuzumab ozogamitin (trade name Mylotarg) Etc. are being developed. For breast cancer, trastuzumab (trade name Herceptin) targeting Her2 / neu and bevacizumab (trade name Avastin) targeting VEGF have been developed for breast cancer. In addition to cancer, other human diseases such as tocilizumab (trade name Actemula), which is a human IL-6 receptor antibody, have been developed for rheumatoid arthritis and Castleman's disease.

  However, the number of antibody drugs approved by 2008 is about 20 in the United States and about 10 in Japan, and few antibody drugs are still effective particularly for solid cancers. For this reason, development of a more effective antibody drug is desired.

  By the way, “Homo sapiens MANSC domain containing 1” (hereinafter referred to as “MANSC1”) is a protein having a motif containing seven cysteine sequences that are highly conserved on the N-terminal side of the extracellular domain. (MANSC is an abbreviation for motif at N terminus with seven cysteines). The MANSC domain having this motif has been clarified to be highly conserved not only in higher vertebrates but also in multicellular organisms from molluscs and chordae by analysis using TBLASTN for EST. The MANSC domain is also present in HAI-1, an activator inhibitor of hepatocyte growth factor HGF, and LRP-11, a low-density lipoprotein receptor-related factor. It has been suggested that it may be related to the development and regeneration of tissues, neurological diseases such as Alzheimer's, tumor differentiation and metastasis (Non-patent Document 1, Non-patent Document 2, and Patent Document 1). However, these documents do not disclose the detailed behavior and function of the MANSC1 molecule itself.

  On the other hand, the behavior and function of the MANSC1 molecule has been reported as one of a comprehensive list of genes that are up-regulated in melanoma. (Table 15-21 of Patent Document 2, SEQ ID NO: 947). It has also been clarified that mutations in the gene encoding the orthologue of human MANSC1 result in a decrease in depression-like response in female (− / −) mice (paragraph 892 of the specification of Patent Document 3). However, none of these documents disclose the contribution (causal relationship) of MANSC1 to the onset of cancer.

  Therefore, at present, it has not yet been clarified whether an antibody against MANSC1 can have anticancer activity.

International Publication 2007/66124 Pamphlet JP 2008-504034 A JP 2009-527227 A

Trends in Biochemical Sciences 29 (4): 172-174 (2004) Genome Research 13 (10): 2265-2270 (2003)

  This invention is made | formed in view of such a condition, The objective is to provide the novel antibody which has the outstanding anticancer activity. A further object of the present invention is to provide an anticancer agent comprising such an antibody as an active ingredient.

  In order to solve the above-mentioned problems, the present inventors first prepared a cDNA library derived from a cancer cell line, GCIY cell, and expressed it on the cell surface or secreted from the cell by the SST-REX method. The ones that code are selected. Subsequently, monoclonal antibodies against the protein encoded by the selected cDNA were prepared, and the binding to various cancer cell lines and the anticancer activity in vitro and in vivo were examined. As a result, it was found that the “ACT35-51_1B4A7D” antibody, which is one of the obtained monoclonal antibodies, binds to the MANSC1 protein and has excellent anticancer activity in vitro and in vivo. Furthermore, the present inventor succeeded in identifying the region containing the epitope of this antibody in MANSC1 protein and determining the structure of the variable region of the light chain and heavy chain of this antibody, thereby completing the present invention. It came.

That is, the present invention relates to a monoclonal antibody that binds to MANSC1 protein and has anticancer activity, and an anticancer agent comprising the antibody as an active ingredient, more specifically,
(1) an antibody that binds to a human-derived MANSC1 protein and has anticancer activity;
(2) The antibody according to (1), which binds to an extracellular region of a human-derived MANSC1 protein,
(3) The antibody according to (1), wherein the cancer is gastric cancer or glioma,
(4) The antibody according to (1), which retains a light chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 3 to 5 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 6 to 8 ,
(5) In the antibody according to (4), one or more amino acids are substituted, deleted, added and / or inserted in at least one of the amino acid sequences set forth in SEQ ID NOs: 3 to 8, and An antibody having an activity equivalent to that of the antibody according to (4),
(6) The antibody according to (1), which retains a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12.
(7) In the antibody according to (6), one or more amino acids are substituted, deleted, added, and / or inserted in at least one of the amino acid sequences set forth in SEQ ID NOs: 10 and 12, and An antibody having an activity equivalent to that of the antibody according to (6),
(8) The signal sequence is removed from at least one of the amino acid sequences described in SEQ ID NOs: 10 and 12 in the antibody described in (6), and has the same activity as the antibody described in (6) antibody,
(9) An antibody that binds to the epitope of the antibody according to (6) in human-derived MANSC1 protein and has anticancer activity,
(10) a peptide comprising the light chain of the antibody according to (1) or the variable region thereof, comprising the amino acid sequence according to SEQ ID NO: 3 to 5,
(11) The peptide according to (10), comprising the amino acid sequence set forth in SEQ ID NO: 10 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence,
(12) A peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof, comprising the amino acid sequence according to SEQ ID NO: 6 to 8,
(13) The peptide according to (12), comprising the amino acid sequence set forth in SEQ ID NO: 12 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence,
(14) DNA encoding the antibody according to any one of (1) to (9) or the peptide according to any one of (10) to (13),
(15) A hybridoma that produces the antibody according to any one of (1) to (9) or contains the DNA according to (14),
(16) An anticancer agent comprising the antibody according to any one of (1) to (9) as an active ingredient, and (17) The anticancer agent according to (16), wherein the cancer is gastric cancer or glioma,
Is to provide.

  The present invention provides an antibody that binds to human-derived MANSC1 protein and has excellent anticancer activity in vitro and in vivo. Use of the antibody of the present invention makes it possible to treat or prevent cancer. The antibody of the present invention is particularly effective in suppressing the growth of gastric cancer cells or gliomas.

It is a figure which shows the reactivity of ACT35-51_1B4A7D antibody and Ba / F3 cell which expresses a MANSC1 gene. Analysis of antibody responses to transfectant Ba / F3 cells (A) expressing the full-length MANSC1 gene (A) and control Ba / F3 cells (B) not expressing the MANSC1 gene using a flow cytometer did. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. It is a figure which shows the result of having analyzed the reactivity of ACT35-51_1B4A7D antibody and a gastric cancer cell line (GCIY) with the flow cytometer. The filled histogram portion of each flow cytometer data shows its reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) used as a negative control. It is a microscope picture which shows the result of having analyzed the reactivity of ACT35-51_1B4A7D antibody and the surface of various cultured cancer cells by cell staining. Cancer cell lines include bladder cancer cell line (T24), gastric prostate cancer cell line (PC3, Du145), pancreatic cancer cell line (BxPC3, AsPC1), glioma cell line (U251, U87MG, T98G), gastric cancer cell line (MKN1) , GCIY). The left figure shows a nuclear stained image using Hoechst33342, the figure shows an antibody-stained image, and the right figure shows an image obtained by superimposing a nuclear stained image using Hoechst33342 and an antibody-stained image. It is a microscope picture which shows the result of having analyzed the reactivity of ACT35-51_1B4A7D antibody and various cultured cancer cells which performed the cell fixation and the membrane permeation process by the cell staining. Cancer cell lines include bladder cancer cell line (T24), gastric prostate cancer cell line (PC3, Du145), pancreatic cancer cell line (BxPC3, AsPC1), glioma cell line (U251, U87MG, T98G), gastric cancer cell line (MKN1) , GCIY). The left figure shows a nuclear stained image using Hoechst33342, the figure shows an antibody-stained image, and the right figure shows an image obtained by superimposing a nuclear stained image using Hoechst33342 and an antibody-stained image. It is an electrophoresis photograph showing the result of immunoprecipitation using the ACT35-51_1B4A7D antibody on the culture supernatant of 293T cells expressing the MANSC1 gene. As negative control cells, 293T cells (mock) into which only the vector was introduced were used. Moreover, mouse IgG2a was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_1B4A7D monoclonal antibody has on the proliferation of cancer cells. The vertical axis represents the O.D. (O.D.450 nm-O.D.650 nm) value 3 hours after addition of WST-1. Bladder cancer cell lines (T24) and prostate cancer cell lines (PC3, Du145) were used as the target cancer cell lines. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_1B4A7D monoclonal antibody has on the proliferation of cancer cells. The vertical axis represents the O.D. (O.D.450 nm-O.D.650 nm) value 3 hours after addition of WST-1. Pancreatic cancer cell lines (BxPC3, AsPC1) and gastric cancer cell lines (GCIY) were used as target cancer cell lines. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_1B4A7D monoclonal antibody has on the proliferation of cancer cells. The vertical axis represents the O.D. (O.D.450 nm-O.D.650 nm) value 3 hours after addition of WST-1. Glioma cell lines (U251, U87MG, T98G) were used as the target cancer cell lines. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. It is a figure which shows the tumor volume transition in the mouse | mouth cancer bearing model which administered ACT35-51_1B4A7D antibody. Saline was used as a control and Taxotere was used as a positive control. It is a figure which shows the excised tumor weight in the mouse | mouth cancer bearing model 3 weeks after administration of ACT35-51_1B4A7D antibody. Saline was used as a control and Taxotere was used as a positive control. It is a figure which shows the body weight transition in the mouse | mouth cancer bearing model which administered ACT35-51_1B4A7D antibody. Saline was used as a control and Taxotere was used as a positive control. It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_1B4A7D antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. It is a figure which shows the reactivity of the ACT35-51_1B4A7D antibody and the transfectant Ba / F3 cell which expresses MANSC1 gene of various length. ACT35-51_1B4A7D antibody against Ba / F3 cells expressing genes corresponding to 60, 153, 186, 219, 250, 280, and 385 amino acids from the N-terminus of the antigen MANSC1 molecule and as a control The reactivity of the MPL antibody was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the antibody against each MANSC1 molecule, and the white histogram portion shows the response of mouse IgG2a (Beckman Coulter, # 731589) used as a control.

  The present invention provides an antibody that binds to a human-derived MANSC1 protein and has anticancer activity. The “antibody” in the present invention includes all classes and subclasses of immunoglobulins. “Antibody” includes polyclonal antibodies and monoclonal antibodies, and also includes forms of functional fragments of antibodies. “Polyclonal antibodies” are antibody preparations comprising different antibodies directed against different epitopes. The “monoclonal antibody” means an antibody (including an antibody fragment) obtained from a substantially homogeneous antibody population. In contrast to polyclonal antibodies, monoclonal antibodies are those that recognize a single determinant on an antigen. The antibody of the present invention is preferably a monoclonal antibody. The antibodies of the invention are antibodies that have been separated and / or recovered (ie, isolated) from components of the natural environment.

  The “human-derived MANSC1 protein (NCBI Reference Sequence: NM_018050.2)” to which the antibody of the present invention binds is present in the cell membrane and contains seven highly conserved cysteine sequences on the N-terminal side of the extracellular domain. A protein having a motif. The human-derived MANSC1 protein is a protein consisting of a 431 amino acid sequence, of which the 26 amino acid portion from the N-terminal is a signal sequence, the 27th to 385th extracellular region, the 386th to 408th is a transmembrane region, It is presumed to be a transmembrane protein having the 409th and subsequent regions in the intramembrane region. The amino acid sequence of a typical human-derived MANSC1 protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the MANSC1 gene is shown in SEQ ID NO: 1. A human-derived MANSC1 protein may naturally have a mutated amino acid in addition to such a typical amino acid sequence. Therefore, the “human-derived MANSC1 protein” in the present invention is preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, but in addition to that, in the amino acid sequence represented by SEQ ID NO: 2, Or what consists of an amino acid sequence by which several amino acid was substituted, deleted, inserted, or added is also contained. The substitution, deletion, insertion or addition of the amino acid sequence is generally within 10 amino acids (eg, within 5 amino acids, within 3 amino acids, 1 amino acid).

  In the present invention, the “anticancer activity” means an activity of suppressing the growth of cancer cells in vitro and / or in vivo. The anticancer activity can be evaluated, for example, by analysis using the MTT assay described in Example 8 or the cancer-bearing model described in Example 9. A preferred embodiment of the antibody of the present invention shows that when the MTT assay described in Example 8 is performed, the growth of a gastric cancer cell line (eg, GCIY) is 50% compared to the control after 72 hours of antibody addition. It is an antibody that suppresses the above (for example, 60% or more, 70% or more). Another preferred embodiment of the antibody of the present invention is that when the MTT assay described in Example 8 is performed, the proliferation of a glioma cell line (for example, T98G) is 72 hours after the addition of the antibody compared to the control. It is an antibody that suppresses 50% or more (for example, 60% or more, 70% or more, 80% or more, 90% or more). In another preferred embodiment of the antibody of the present invention, the analysis using the cancer-bearing model described in Example 9 shows that the tumor volume is 30% or more (for example, 35 % Or more, 40% or more, 45% or more, 50% or more, 55% or more). In another preferred embodiment of the antibody of the present invention, an analysis using the cancer-bearing model described in Example 9 shows that the weight of the excised tumor is 20% or more (for example, 25% or more, 30% or more, 35% or more). When used as an anticancer agent, these antibodies preferably further have the property of not reducing the body weight of the administration subject. It is particularly preferred that the antibody of the present invention has a plurality of the above activities.

  Other preferred embodiments of the antibody of the present invention include a light chain variable region comprising light chains CDR1 to CDR3 (amino acid sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 5) and heavy chain CDR1 to CDR3 (SEQ ID NO: 6). (Amino acid sequence described in SEQ ID NO: 8). For example, the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 10 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO: 12 (or An amino acid sequence in which a signal sequence is removed from the amino acid sequence).

  Once an antibody having the light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 10 and the heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 12 is obtained, those skilled in the art will recognize that antibody. Peptide regions (epitope) on the human-derived MANSC1 protein recognized by can be identified, and various antibodies that bind to the region and show anticancer activity can be prepared. The epitope of the antibody can be determined by a well-known method such as examining binding to an overlapping synthetic oligopeptide obtained from the amino acid sequence of MANSC1 protein derived from human (for example, Ed Harlow and D. Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, US Pat. No. 4,088,871). A peptide library by phage display can also be used for epitope mapping. Whether two antibodies bind to the same or sterically overlapping epitopes can be determined by competition assays. The peptide region on the MANSC1 protein recognized by the antibody of the present invention is preferably an extracellular region of the MANSC1 protein. The extracellular region of the MANSC1 protein recognized by the antibody of the present invention is preferably a region within the range of positions 61 to 153 of the amino acid sequence of the MANSC1 protein.

  The antibodies of the present invention include mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies, and functional fragments of these antibodies. When the antibody of the present invention is administered to a human as a pharmaceutical, a chimeric antibody, a humanized antibody, or a human antibody is desirable from the viewpoint of reducing side effects.

  In the present invention, the “chimeric antibody” is an antibody in which the variable region of a certain antibody is linked to the constant region of a heterologous antibody. A chimeric antibody, for example, immunizes an antigen to a mouse, cuts out an antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, and binds to an antibody constant region (constant region) gene derived from human bone marrow. Can be obtained by incorporating it into an expression vector and introducing it into a host for production (for example, Japanese Patent Application Laid-Open No. 8-280387, US Pat. No. 4816397, US Pat. No. 4,816,567, US Pat. No. 5807715). In the present invention, the “humanized antibody” is an antibody obtained by transplanting the gene sequence of the antigen-binding site (CDR) of a non-human-derived antibody to a human antibody gene (CDR grafting), and its production method is publicly known. (See, for example, EP239400, EP125023, WO90 / 07861, WO96 / 02576). In the present invention, a “human antibody” is an antibody derived from all regions. In the production of human antibodies, a method for screening production of active antibodies from human B cells, a phage display method, and a transgenic animal (for example, a mouse) capable of producing a repertoire of human antibodies by immunization. It can be used. Methods for producing human antibodies are known (for example, Nature, 362: 255-258 (1993), Intern. Rev. Immunol, 13: 65-93 (1995), J. Mol. Biol, 222: 581-597. (1991), Nature Genetics, 15: 146-156 (1997), Proc. Natl. Acad. Sci. USA, 97: 722-727 (2000), JP 10-146194 A, JP 10-155492 A No. 2938569, JP-A-11-206387, JP-A-8-509612, JP-A-11-505107).

  In the present invention, the “functional fragment” of an antibody means a part (partial fragment) of an antibody that specifically recognizes a human-derived MANSC1 protein. Specifically, Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.

  Here, “Fab” means a monovalent antigen-binding fragment of an immunoglobulin composed of one light chain and part of a heavy chain. It can be obtained by papain digestion of antibodies and by recombinant methods. “Fab ′” differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the hinge region of the antibody. "F (ab ') 2" means a divalent antigen-binding fragment of an immunoglobulin that consists of both light chains and parts of both heavy chains.

  A “variable region fragment (Fv)” is the smallest antibody fragment that has a complete antigen recognition and binding site. Fv is a dimer in which a heavy chain variable region and a light chain variable region are strongly linked by a non-covalent bond. “Single-chain Fv (scFv)” comprises the heavy and light chain variable regions of an antibody, and these regions are present in a single polypeptide chain. “Sc (Fv) 2” is a chain formed by joining two heavy chain variable regions and two light chain variable regions with a linker or the like. A “diabody” is a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable region bound to a light chain variable region in the same polypeptide chain, each region comprising a separate It forms a pair with the complementary region of the strand. A “multispecific antibody” is a monoclonal antibody that has binding specificities for at least two different antigens. For example, it can be prepared by co-expression of two immunoglobulin heavy / light chain pairs where the two heavy chains have different specificities.

  The present invention provides a peptide consisting of the light chain or heavy chain of an antibody comprising the CDR identified in the present invention, or a variable region thereof. A preferred peptide is a peptide consisting of the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 3 to 5 or a variable region thereof, and particularly preferably the amino acid sequence set forth in SEQ ID NO: 10 or the amino acid sequence. A peptide comprising an amino acid sequence with the signal sequence removed from the sequence. Another preferred peptide is a peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 6 to 8 or its variable region, and particularly preferably the amino acid sequence set forth in SEQ ID NO: 12 or A peptide comprising an amino acid sequence from which the signal sequence has been removed from the amino acid sequence. A functional antibody can be produced by linking these peptides with, for example, a linker.

  The antibodies of the present invention include antibodies that have been modified in their amino acid sequence without reducing the desired activity (antigen binding activity, anticancer activity, and / or other biological properties). An amino acid sequence variant of the antibody of the present invention can be prepared by introducing a mutation into DNA encoding the antibody chain of the present invention or by peptide synthesis. Such modifications include, for example, residue substitutions, deletions, additions and / or insertions within the amino acid sequences of the antibodies of the invention. The site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has an activity equivalent to that of the antibody before modification, and the variable region (framework region and CDR). Modification of amino acids other than CDR is considered to have a relatively small effect on the binding affinity with the antigen. Currently, however, the amino acid of the CDR is modified to screen for antibodies with increased affinity for the antigen. The technique is known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), International Publication No. 2002/051870, J. Biol. Chem., 280: 24880-24887 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008)).

  The number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (eg, within 2 amino acids, 1 amino acid). The amino acid modification is preferably a conservative substitution. In the present invention, “conservative substitution” means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy groups Amino acids with amino acids (serine / threonine), amino acids with sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / (Tryptophan). Further, “having equivalent activity” means that the antigen binding activity or anticancer activity is equivalent to the target antibody (typically, ACT35-51_1B4A7D antibody) (eg, 70% or more, preferably 80% or more, More preferably 90% or more). The binding activity to the antigen can be evaluated, for example, by preparing Ba / F3 cells expressing the antigen and analyzing the reactivity with the antibody sample with a flow cytometer (Examples 4 and 11). Further, as described above, the anticancer activity can be evaluated by, for example, analysis using the MTT assay described in Example 8 or the cancer-bearing model described in Example 9.

  The modification of the antibody of the present invention may also be modification of a post-translational process of the antibody, for example, changing the number or position of glycosylation sites. Thereby, for example, the ADCC activity of the antibody can be improved. Antibody glycosylation is typically N-linked or O-linked. Antibody glycosylation is highly dependent on the host cell used to express the antibody. The glycosylation pattern can be modified by a known method such as introduction or deletion of a specific enzyme involved in sugar production (JP 2008-113663, US Pat. No. 5,473,335, US Pat. No. 5,510,261, US Pat. No. 5278299, International Publication No. 99/54342). Furthermore, in the present invention, deamidation is suppressed by substituting an amino acid adjacent to the amino acid deamidated or deamidated with another amino acid for the purpose of increasing the stability of the antibody. May be. Alternatively, glutamic acid can be substituted with other amino acids to increase antibody stability. The present invention also provides the antibody thus stabilized.

  If the antibody of the present invention is a polyclonal antibody, an immunized animal is immunized with an antigen (such as a human-derived MANSC1 protein, a partial peptide thereof, or a cell expressing these), and the conventional means (for example, Salting out, centrifugation, dialysis, column chromatography, etc.). Monoclonal antibodies can be prepared by a hybridoma method or a recombinant DNA method.

  Typically, the hybridoma method includes the Kohler and Milstein method (Kohler & Milstein, Nature, 256: 495 (1975)). The antibody-producing cells used in the cell fusion step in this method are animals (eg, mice, rats, hamsters, rabbits) immunized with antigens (human-derived MANSC1 protein, partial peptides thereof, or cells expressing these). Monkeys, goats) spleen cells, lymph node cells, peripheral blood leukocytes and the like. It is also possible to use antibody-producing cells obtained by allowing an antigen to act on the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal in a medium. As the myeloma cells, various known cell lines can be used. The antibody-producing cells and myeloma cells may be of different animal species as long as they can be fused, but are preferably of the same animal species. Hybridomas are produced, for example, by cell fusion between spleen cells obtained from mice immunized with antigen and mouse myeloma cells, and subsequent screening produces monoclonal antibodies specific for human-derived MANSC1 protein. Hybridomas can be obtained. A monoclonal antibody against a human-derived MANSC1 protein can be obtained by culturing a hybridoma or from the condensate of a mammal to which the hybridoma has been administered.

  In the recombinant DNA method, a DNA encoding the antibody or peptide of the present invention is cloned from a hybridoma, a B cell or the like and incorporated into an appropriate vector, which is then introduced into a host cell (eg, a mammalian cell line, E. coli, yeast cell, insect). A method of introducing the antibody of the present invention into a recombinant antibody (for example, PJDelves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000). OXFORD UNIVERSITY PRESS, Vandamme AM et al., Eur. J. Biochem. 192: 767-775 (1990)). In the expression of the DNA encoding the antibody of the present invention, DNA encoding the heavy chain or the light chain may be separately incorporated into an expression vector to transform the host cell. Host cells may be transformed into a single expression vector (see WO94 / 11523). The antibody of the present invention can be obtained in a substantially pure and uniform form by culturing the above host cell, separating and purifying it from the host cell or culture medium. For the separation and purification of the antibody, the methods used in the usual purification of polypeptides can be used. If transgenic animals (such as cows, goats, sheep or pigs) in which an antibody gene is incorporated are produced using transgenic animal production technology, a large amount of monoclonal antibody derived from the antibody gene is produced from the milk of the transgenic animal. It is also possible to obtain.

  The present invention provides a DNA encoding the antibody or peptide of the present invention, a vector containing the DNA, a host cell holding the DNA, and a method for producing the antibody comprising culturing the host cell and recovering the antibody Is also provided.

  Since the antibody of the present invention has anticancer activity, it can be used for the treatment or prevention of cancer. Therefore, the present invention is a cancer treatment comprising the steps of administering an anticancer agent comprising the antibody of the present invention as an active ingredient and a therapeutically or prophylactically effective amount of the antibody of the present invention to mammals including humans. It also provides a preventive method. The treatment or prevention method of the present invention can be applied to various mammals including dogs, cats, cows, horses, sheep, pigs, goats, rabbits and the like, in addition to humans.

  In this example, the antibody of the present invention is particularly effective in the treatment or prevention of gastric cancer (for example, Skills gastric cancer) and glioma because it particularly strongly suppresses the growth of gastric cancer cells and glioma cells among cancers.

  The anticancer agent comprising the antibody of the present invention as an active ingredient can be used in the form of a composition containing the antibody of the present invention and an optional component such as physiological saline, a sucrose aqueous solution, or a phosphate buffer. The anticancer agent of the present invention may be formed into a liquid or lyophilized form as necessary, and optionally a pharmaceutically acceptable carrier or medium, such as a stabilizer, preservative, isotonic agent, etc. Can also be included.

  Examples of the pharmaceutically acceptable carrier include mannitol, lactose, saccharose, human albumin and the like in the case of a lyophilized preparation. In the case of a liquid preparation, physiological saline, water for injection, phosphoric acid, etc. Examples thereof include, but are not limited to, a salt buffer and aluminum hydroxide.

  The administration method of the anticancer agent varies depending on the age, weight, sex, health status, etc. of the administration subject, but it should be administered by any of the administration routes of oral administration or parenteral administration (eg, intravenous administration, arterial administration, local administration). Can do. A preferred method of administration is parenteral administration. The dose of an anticancer drug may vary depending on the patient's age, weight, sex, health status, degree of cancer progression, and the components of the anticancer drug to be administered. The daily dosage is 0.1 to 1000 mg, preferably 1 to 100 mg.

  The antibody of the present invention can be applied not only to cancer treatment and prevention but also to cancer diagnosis. When the antibody of the present invention is used for diagnosis of cancer or used for detection of a tumor site in cancer treatment, the antibody of the present invention may be labeled. As the label, for example, a radioactive substance, a fluorescent dye, a chemiluminescent substance, an enzyme, and a coenzyme can be used. Specifically, radioisotope, fluorescein, rhodamine, dansyl chloride, luciferase, peroxidase, alkaline phosphatase, Examples include lysozyme and biotin / avidin. In order to prepare the antibody of the present invention as a diagnostic agent, it can be obtained in any dosage form by employing any suitable means. For example, the antibody titer of a purified antibody can be measured and appropriately diluted with PBS (Phosphate buffer saline, phosphate buffer containing physiological saline) or the like, and then 0.1% sodium azide or the like can be added as a preservative. . In addition, for example, the antibody titer of a substance obtained by adsorbing the antibody of the present invention on latex or the like can be obtained, diluted appropriately, and added with a preservative.

  In the present invention, since it has been found that an antibody against MANSC1 protein has anticancer activity, MANSC1 protein or a partial peptide thereof can be administered as a cancer vaccine to mammals including humans (for example, (See JP 2007-277251, JP 2006-052216). The present invention also provides a cancer vaccine composition containing MANSC1 protein or a partial peptide thereof used for such cancer vaccine applications. In the case of formulating, a pharmaceutically acceptable carrier or medium such as a stabilizer, preservative, isotonic agent and the like can be contained in the same manner as the anticancer agent of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.

[Example 1] Implementation of SST-REX SST-REX was performed to comprehensively obtain information on membrane or secretory genes expressed on the cell surface of Skills gastric cancer cell line GCIY cells.

(1) Preparation of cDNA
2 × 10 ⁷ GCIY cells were suspended in 1 ml of Trizol (invitrogen, # 15596-026), allowed to stand for 5 minutes, 200 μl of chloroform was added and suspended for 15 seconds, and then centrifuged at 12,000 × g for 15 minutes. The supernatant after centrifugation and 500 μl of isopropanol were mixed, and then centrifuged at 12,000 × g for 10 minutes. The obtained pellet was washed with 80% ethanol to obtain 200 μg or more of total RNA, and used for the subsequent experiments.

All of the obtained total RNA was dissolved in 100 μl of water, and 3 μg of mRNA was obtained using FastTrack 2.0 mRNA Isolation kit (invitrogen, # K1593-02). Using the SuperScript ^™ Choice System (invitorgen, # 18090-019), double-stranded cDNA was prepared from the obtained mRNA.

  GCIY cells are a gastric cancer cell line established from Borrman IV gastric cancer and peritoneal disseminated metastasis of ascites from a woman who suffered from peritoneal disseminated metastasis, and expressed multidrug resistance gene (mdr-1). , And poorly differentiated adenocarcinoma cells in which secretion of CEA, CA19-9, and αFP is observed.

(2) Integration of cDNA sequence into pMX-SST vector (chimerization)
In order to incorporate the obtained cDNA into the retroviral vector pMX-SST, 5 μg of pMX-SST vector (Nature Biotechnology 17: 487-490 (1999)) was added at 4 ° C. in a 100 μl reaction system at 45 ° C. with the restriction enzyme Bst XI. Time cut processing. All the reaction solutions were electrophoresed on a 1% agarose gel, and a DNA fragment having a length of about 5000 bases corresponding to the vector site was excised. Further ^{Wizard (R) SV Gel and PCR} Clean-Up System (promega, # A9282) was used to purify the DNA fragment having a length of about 5000 bases. The DNA fragment thus obtained was treated with a restriction enzyme treatment of pMX-SST vector with Bst XI, and prepared to be an aqueous solution containing 50 nanogram per 1 μl.

The previously prepared double-stranded cDNA has blunt ends and cannot be directly ligated with pMX-SST treated with Bst XI. Therefore, an operation was carried out to provide both ends of the double-stranded cDNA with a DNA sequence after cleavage of the Bst XI restriction enzyme. Double-stranded cDNA was dissolved in an aqueous solution of Bst XI Adapter in which 9 μg of Bst XI Adapter (invitorgen, # N408-18) was dissolved in 10 μl of water. 5 μl of LigationHigh (TOYOBO # LGK-201) was added thereto, suspended, and reacted at 16 ° C. for 16 hours to bind the Bst XI Adapter and the double-stranded cDNA. Thereafter, using a size fractionation column attached to SuperScript ^™ Choice System (invitorgen, # 18090-019), a DNA fragment having a chain length of about 400 bases or less was removed. Thereafter, 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added and mixed by inversion, followed by centrifugation at 20,400 × g for 30 minutes. The precipitate obtained by removing the supernatant after centrifugation was dissolved in 15 μl of water and electrophoresed on a 1.5% agarose gel. Thereafter, a gel containing a conjugate of a double-stranded cDNA fragment having a length of about 500 to about 4000 bases and a Bst XI Adapter was cut out, and the Wizard ^(R) SV Gel and PCR Clean-Up System (promega # A9282) was used to purify the conjugate of double-stranded cDNA and Bst XI Adapter.

Bst XI restriction enzyme treated with pMX-SST vector 50 ng, obtained double-stranded cDNA and conjugates of the total amount of the Bst XI Adapter, and T4 DNA ligase, and 3 hours at room temperature 20μl of the reaction system, pMX -SST vector treated with restriction enzyme with Bst XI was combined with the above conjugate. The composition of the reaction solution was adjusted according to the booklet.

(3) Amplification of cDNA library
A cDNA library constructed using the pMX-SST vector was introduced into E. coli and amplified. To the cDNA library, 5 μg of tRNA, 12.5 μl of 7.5 M sodium acetate, and 70 μl of ethanol were added and mixed by inversion, followed by centrifugation at 20,400 × g for 30 minutes, and the supernatant was discarded to obtain a precipitate. To the resulting precipitate, 500 μl of 70% ethanol was added, centrifuged at 20,400 × g for 5 minutes, the supernatant was discarded, and the resulting precipitate was dissolved in 10 μl of water. To amplify cDNA in E. coli, mix 2 μl of this with 23 μl of competent cells (invitrogen, # 18920-015), perform electroporation at 1.8 kV, and suspend the entire volume in 1 ml of SOC medium. did. This operation was performed twice, and the SOC medium in which Escherichia coli was suspended was cultured at 37 ° C. for 90 minutes with shaking. Thereafter, the entire amount of the culture solution was put into 500 ml of LB medium containing 100 μg of ampicillin per ml of the medium, and cultured with shaking at 37 ° C. for 16 hours.

  In order to confirm the number of cDNA libraries introduced into Escherichia coli and the chain length of cDNA bound to the pMX-SST vector, 5 μl of the culture solution was taken out and plated on LB agar medium containing 50 μg / ml ampicillin.

As a result, 150 colonies grew on the LB agar medium plated with 5 μl. Thus, it was considered that there were 1.5 × 10 ⁷ independent cDNA libraries in 500 ml of the culture solution. In addition, a plasmid was extracted from any 16 colonies, treated with the restriction enzyme Bst XI, electrophoresed on a 1% agarose gel, and the length of the cDNA on the pMX-SST vector was determined. Measured. As a result, the average value was about 1000 bases.

Harvested from the remaining ^{culture, 10 NucleoBond (R) AX 500} columns ( Japan Genetics, # 740574) plasmid was purified using established a amplified cDNA library system.

(4) Packaging of cDNA library and implementation of SST-REX method
Virus packaging cell Plat-E (Gene Ther. 7 (12): 1063-6 (2000) Jun) 2 × to produce a retrovirus containing the pMX-SST retroviral vector RNA incorporating the cDNA library-derived gene 10 ⁶ were suspended in a 6 cm dish containing 4 ml of DMEM medium (Wako, # 044-29765) and cultured at 37 ° C. under 5% CO ₂ conditions for 24 hours. On the other hand, 100 μl of opti-MEM (GIBCO, # 31985070) and 9 μl of Fugene (Roche, # 1814443) were mixed and allowed to stand for 5 minutes at room temperature. Then, 3 μg of cDNA library was added, and the mixture was allowed to stand for 15 minutes at room temperature. The solution containing the cDNA library was dropped onto the cultured Plat-E cells, and the supernatant was replaced 24 hours later and the culture was continued under the same conditions. Further, the supernatant after 24 hours was filtered through a 0.45 μm filter.

0.5 ml of the obtained filtration supernatant was added to a 10 cm dish containing 9.5 ml of RPMI-1640 (Kohjin Bio) medium containing 4 × 10 ⁶ Ba / F3 cells.

  Furthermore, 10 μl of polybrene (CHEMICON, # TR-1003-G) and 10 ng of IL-3 were added and cultured for 24 hours. After that, the cells are washed 3 times with RPMI-1640 medium, suspended in 200 ml of fresh RPMI-1640 medium, and evenly spread onto 20 96-well plates, and selection based on the autonomous growth ability of Ba / F3 cells and Attempted cloning. Cells that grew after 10 to 20 days were selected based on SST-REX, and the culture was further continued until the cells grew to full wells.

(5) Analysis of gene product obtained by SST-REX Half the amount of cells obtained from each well was expanded and used as a cell stock. In addition, transfectant Ba / F3 cells that express the peptide molecules derived from the incorporated cDNA outside the cells are cultured as cells that are the source of antibodies and screened. Used as cells. From the remaining half of the cells obtained from each well, the genome was extracted and sequenced, and the introduced cDNA-derived gene was analyzed. In the sequence, PCR was performed on the obtained genome using LA taq DNA polymerase (Takara, # RR002) or PrimeSTAR MAX DNA polymerase (TaKaRa, # R045A). The following sequences were used as PCR primers.

SST3 'side-T7 5'-TAATACGACTCACTATAGGGCGCGCAGCTGTAAACGGTAG-3' (SEQ ID NO: 13)
SST5'-T3 5'-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3 '(SEQ ID NO: 14)

The PCR product ^{Wizard (R) SV Gel and PCR} Clean-Up System (promega, # A9282) was purified by using a. Thereafter, the purified PCR product was sequenced using BigDye Terminator v3.1 Cycle sequencing (ABI, # 4337456) and DNA sequencer ABI3100XL. The following primers were used as sequence primers.

SST5 'side -T3 5'-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3' (SEQ ID NO: 15)

The obtained sequence data is BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) and SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/). Analyzed by using.

  As described above, the SST-REX method was performed using cells as a material. As a result, in the first implementation, cDNAs from 87 transfectant Ba / F3 cells were sequenced and 40 different genes were used. I was able to get. In the second implementation, we sequenced cDNA-derived genes from 176 transfectant Ba / F3 cells and obtained 56 different genes. In addition, there were 15 types of duplicated genes in the first and second rounds, and a total of 81 types of cDNA-derived genes could be obtained in the second round. The transfectant Ba / F3 cell line used for gene analysis was confirmed to contain only one type of cDNA-derived gene, and was used in the subsequent experiments (hereinafter derived from the cDNA thus obtained). Cells containing the gene are referred to as “SST clonal cells”).

[Example 2] Cloning of MANSC1 full-length gene and establishment of Ba / F3 cell line that expresses it In addition, the MANSC1 gene included in the cDNA-derived gene list obtained in Example 1 includes the full-length gene. Cloning was performed to obtain SST clonal cells.

Design primers based on the information of NM_018050.2 on the nucleotide search site of NCBI (http://www.ncbi.nlm.nih.gov/nucleotide) using 30 ng of cDNA prepared by SST-REX of GCIY cells as a template PCR reaction was performed using PrimeSTAR MAX DNA polymerase (TaKaRa, # R045A).

Forward primer: cgggaattcatccttgacctttgaagacc (SEQ ID NO: 16)
Reverse primer: ttttccttttgcggccgcgatgtccacatagatcccat (SEQ ID NO: 17)

The obtained PCR product was electrophoresed on a 1% agarose gel, and a DNA fragment of the desired length was extracted from the gel. The extracted DNA fragments were treated with restriction enzymes with EcoRI (TaKaRa # 1040A) and NotI (TaKaRa, # 1166A). At the same time, the pMX-SST vector was treated with restriction enzymes with EcoRI and NotI. 100 ng of the restriction enzyme-treated DNA fragment and 40 ng of the pMX-SST vector were ligated over 2 hours using LigationHigh (TOYOBO, # LGK-201).

  100 μl of E. coli competent cells for heat shock were added to the total amount of the bound ones, left on ice for 30 minutes, and then incubated at 42 ° C. for 90 seconds. Thereafter, 1 ml of LB medium was added and incubated at 37 ° C. for 1 hour. Thereafter, centrifugation was performed at 15,000 × g for 1 minute, the supernatant was removed, and the E. coli pellet was suspended with the remaining solution. Apply this whole amount to LB agar medium containing 50 μg / mL of ampicillin, incubate overnight at 37 ° C, and use the obtained colonies to perform PCR and sequence analysis in the same manner as the sequence analysis of Example 1 (4). Went. For clones in which a DNA fragment of the target length was confirmed, the PCR product was sequenced to confirm that the target sequence was inserted. In addition, PrimeSTAR MAX DNA polymerase was used as a polymerase for PCR in sequence analysis.

  Thereafter, the colony into which the target sequence was inserted was inoculated into 3 ml of LB liquid medium, and cultured at 37 ° C. overnight. The whole culture was centrifuged at 3,000 × g for 15 minutes, the supernatant was removed, and purified using QuickLyse Miniprep Kit (QIAGEN, # 27406) to obtain a plasmid containing the full length of the MANSC1 gene.

  Thereafter, using the obtained plasmid, a retrovirus containing a vector was prepared by the same operation as that after packaging of the cDNA library shown in Example 1 (4) and later. Next, a Ba / F3 cell line expressing the full-length MANSC1 gene was established and used for the subsequent experiments.

[Example 3] Production of MANSC1 monoclonal antibody The mouse immunized animal uses mouse Balb / c. First, TiterMax Gold (Alexis Biochemicals, ALX-510-002-L010) was mixed with an equal amount of PBS as an immunostimulator. 50 μl of the emulsified product was administered. On the next day, 5 × 10 ⁶ SST clonal cells having the MANSC1 gene were administered as immunogen cells, and the immunogen cells were injected four times every two days. About 2 weeks after the first immunization, the excised secondary lymphoid tissue was ground to obtain a cell population containing antibody-producing cells. These cells and fusion partner cells were mixed and a hybridoma] was prepared by cell fusion using polyethylene glycol (MERCK, 1.09727.0100). As a fusion partner cell, mouse myeloma cell P3U1 (P3-X63-Ag8.U1) was used.

  Hybridomas include HAT (SIGMA, H0262), 5% BM-condimed (Roche, 663573), 15% FBS, 1% penicillin / streptomycin solution (GIBCO, 15140-122, Penicillin-streptomycin liquid, hereinafter “P / Cultured for 10-14 days in RPMI1640 (Wako) selective medium containing “S”. Next, hybridomas were selected by flow cytometry shown in Example 4 that reacted with immunogen cells and did not react with SST clonal cells (negative control cells) that did not contain the antigen gene in the immunogen cells. Monocloning was performed by limiting dilution to obtain a hybridoma clone producing the anti-MANSC1 antibody ACT35-51_1A4B7D (FIG. 1).

  The resulting hybridoma was maintained using RPMI-1640 medium containing the required amount of HT (SIGMA, HT media supplement (50X) Hybri-Max (Sigma-Aldrich H0137), 15% FBS, 1% P / S solution). The isotype of the produced antibody was determined using an isostrip kit (Roche, 1493027), resulting in IgG2a / κ.

  Acquisition of the purified ACT35-51_1B4A7D antibody from the monocloned hybridoma was performed as follows. The hybridoma was acclimated to a serum-free medium (Hybridoma-SFM: GIBCO, 12045-076) and expanded, and then cultured for a certain period to obtain a culture supernatant. Next, the IgG fraction contained in this culture supernatant was purified with Protein A Sepharose (GE Healthcare, 17-1279-03), MAPS-II binding buffer (BIO-RAD, 153-6161), MAPS-II elution buffer (BIO -RAD, 153-6162). The eluted IgG was dialyzed against PBS to obtain a purified antibody fraction.

[Example 4] Antibody screening using flow cytometry
The reactivity of ACT35-51_1B4A7D antibody with various cells (Ba / F3 cells expressing target genes, Ba / F3 cells not expressing target genes, various cancer cells, etc.) was analyzed using flow cytometry. .

In this example, PBS containing 0.5% BSA and 2 mM EDTA was used as the cell suspension buffer and the subsequent washing buffer. Various cells to be reacted with the antibody (target cells) were adjusted and dispensed in a 96-well plate (BD Falcon, 353911) so that the cell suspension contained 5 × 10 ⁴ cells per well to 100 μl. .

  Further, when the cells to be stained were cancer cell lines, they were detached from the culture plate and collected using Cell Dissociation Buffer (GIBCO, 13151-014) when they became 80% confluent.

  To each sample of the cell suspension, 50 μl of hybridoma culture supernatant or 2 μg / ml purified antibody (hereinafter referred to as “antibody solution”) was added to react the antibody with the cells. As an isotype control antibody of the antibody solution, a washing buffer containing 2 μg / ml each of mouse IgG1 (BioLegend, 400412), mouse IgG2a (BioLegend, 400224), and mouse IgG2b (BioLegend, 400324) was used. After reacting the hybridoma culture supernatant and target cells for 30 minutes at room temperature, centrifuge at 700 xg for 2 minutes to remove the culture supernatant, add 100 μl of washing buffer, and again centrifuge at 700 xg for 2 minutes. To remove the supernatant and wash the cells.

  Next, 50 μl of Goat anti-mouse IgG, F (ab ′) 2-PE (Beckman Coulter, IM0855) diluted 200-fold with washing buffer is added to the washed cell pellet as a secondary antibody for detection. And allowed to react for 30 minutes at room temperature in the dark. After the reaction, the supernatant was removed by centrifugation at 700 × g for 2 minutes, 100 μl of washing buffer was further added, and the supernatant was removed again by centrifugation at 700 × g for 2 minutes to wash the cells. Thereafter, the cells were suspended in an appropriate amount of washing buffer, and the reactivity between the antibody and the cells was analyzed by a flow cytometer (Beckman Coulter, FC500MPL).

  In the measurement of reactivity, a gate was set to select live cells from the measured values of forward scatter and side scatter. Measure the fluorescence intensity of PE based on the reactivity with the antibody against the selected live cells, and the reactivity intensity of the isotype control is used as a reference. Hybridoma cells that produced culture supernatants that were not reactive with the bacterium were selected as candidate clones (FIG. 1).

  In the analysis of the reactivity between the antibody and various cells, an antibody with a significant reactivity was selected based on the reaction intensity between the antibody and the isotype control antibody.

[Example 5] Flow cytometry using cancer cells When the cancer cells to be stained become 80% confluent, they are peeled off from the culture plate using Cell Dissociation Buffer (GIBCO, 13151-014) and collected. 1 × 10 ⁵ cells were suspended in 100 μl each in 0.5% BSA, 2 mM EDTA / PBS (washing buffer shown in Example 4), and dispensed into a 96-well plate (BD Falcon, 353911). Thereafter, the reactivity between cancer cells and antibodies was analyzed using a flow cytometer in the same manner as in Example 4.

  GCIY, a gastric cancer cell line, was used as a cancer cell for analyzing the reactivity with the ACT35-51_1B4A7D antibody. As a result, the ACT36-27_5D1 antibody reacted significantly with GCIY, but the ACT35-51_1B4A7D antibody could not detect significant reactivity with GCIY (FIG. 2).

[Example 6] Cell staining of cancer cells
The reactivity of ACT35-51_1B4A7D antibody with various cancer cells was analyzed by cell staining. In a black 96-well plate (BD Falcon, 353219), 1 × 10 ⁴ cancer cells to be stained were suspended and seeded in 100 μl of medium, and cultured for 24 hours. As a medium for various cancer cells, DMEM medium (SIGMA) containing 10% FBS (Equitech) and 1% P / S solution which had been inactivated was used. In this example, a buffer containing 25 mM HEPES (pH 7.4), 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO ₄ , and 1.3 mM CaCl ₂ was used as the washing buffer.

  When staining only the cell surface, the washing buffer in which the hybridoma culture supernatant or purified antibody is dissolved at 2 μg / ml is removed from the cells obtained by removing the culture supernatant by centrifugation at 700 × g for 2 minutes. 50 μl of each was added. As a negative control for ACT35-51_1B4A7D antibody, mouse IgG1 (BioLegend, 400412), mouse IgG2a (BioLegend, 400224), mouse IgG2b (BioLegend, 400324) were dissolved in washing buffer at a concentration of 2 μg / ml, and 50 μl added solution Was used. After reacting each antibody at room temperature for 30 minutes, centrifuge at 700 xg for 2 minutes to remove the supernatant, add 100 μl of washing buffer, and centrifuge again at 700 xg for 2 minutes to remove the supernatant. The cells were then washed.

  After washing, Goat anti-mouse IgG, F (ab ') 2-PE (Beckman Coulter, IM0855) was diluted 200-fold with washing buffer, and 10 mg / ml Hoechst33342 (Invitrogen, H1399) was diluted 2,000-fold. As a “secondary antibody for detection / nuclear staining reagent”, 50 μl was added to the cells and reacted at room temperature for 30 minutes in the dark. Thereafter, the same washing as described above was performed twice, 100 μl of washing buffer was added, and then cell staining was observed using In Cell Analyzer 1000 (GE Healthcare).

  When staining the cell surface and the cell interior, the cell culture supernatant was removed by centrifugation, and the resulting cells were washed once with 100 μl of washing buffer in the same manner as described above. Thereafter, 50 μl of 4% paraformaldehyde / phosphate buffer (Wako, 161-20141) was added, and the cells were fixed by reacting at room temperature for 10 minutes. Then, it was washed twice with 100 μl of washing buffer. Next, 100 μl of a washing buffer containing 0.1% Triton X-100 was added and reacted at room temperature for 10 minutes to increase the permeability of the cell membrane, and then washed twice with 100 μl of washing buffer. Thereafter, staining and analysis were performed in the same manner as the method of staining only the cell surface. The cells were examined for the presence or absence of antibody staining by measuring the fluorescence of PE using the nucleus stained with Hoechst33342 as the cell position reference.

  When various cancer cell lines were stained with ACT35-51_1B4A7D antibody, MANSC1 could not be detected under staining conditions on the cell surface only (FIG. 3A). However, reactivity was observed between GCIY and U87MG by subjecting cancer cells to fixation and membrane permeabilization (FIG. 3B). From this, it was considered that in GCIY and U87MG, MANSC1 was expressed in an amount exceeding the detection limit, and the ACT35-51_1B4A7D antibody was reacting with it. The ACT35-51_1B4A7D antibody was not reactive in GCIY and U87MG unless membrane permeabilization was performed, so the epitope to which this antibody reacts does not exist on the cell membrane surface, but is cleaved and secreted. It was speculated.

[Example 7] Immunoprecipitation using MANSC1 transfectant culture supernatant
Since it was suggested that the epitope of ACT35-51_1B4A7D antibody was cleaved and secreted outside the cell, immunoprecipitation was performed using the culture supernatant of MANSC1-expressing cells. Since cancer cells are difficult to culture in a serum-free medium, the MANSC1 gene was transiently expressed in 293T cells, the medium was replaced with a serum-free medium, and immunoprecipitated using the concentrated sample.

  293T cells were seeded in 10 10 cm culture dishes, and when they became 80% confluent, gene transfer was performed using Fugene6 (Roche, # 1814443). For gene transfer, 600 μl of Opti-MEM (GIBCO, # 31985070) and 18 μl of Fugene6 were mixed per dish, left for 5 minutes at room temperature, 6 μg of MANSC1 DNA construct was added, and left for 15 minutes at room temperature. The solution was added to the 293T culture solution, washed 24 times later with 4 ml of DMEM without serum, and then 20 ml of FreeStyle 293 (GIBCO, # 12338-018) was added, followed by culturing for 5 days. As a control, 293T cells into which only the vector was similarly introduced were also prepared.

  After culturing for 5 days, the culture supernatant was collected, filtered through 0.22 μm, and concentrated 100-fold using Amicon Ultra (fractionated molecular weight 3000, Millipore, # UFC9 003 96).

  Add 800 μl of PBS to 200 μl of concentrated culture supernatant, add 20 μl of Protein A Sepharose (GE Healthcare, 17-1279-03), and stir at 4 ° C for 30 minutes using a rotator (TAITEC, # RT-5) The protein that non-specifically binds to Protein A Sepharose was removed. After the reaction, the mixture was centrifuged at 15000 × g and 4 ° C. for 5 minutes to collect the culture supernatant, and 20 μl of Protein A Sepharose was added again to remove non-specifically reacting proteins by the same method.

  5 μg of ACT35-51_1B4A7D antibody or mouse IgG2a antibody was added to the culture supernatant from which the protein non-specifically reacting with Protein A Sepharose was removed, and the mixture was reacted by stirring at 4 ° C. for 60 minutes. After the reaction of the antibody, 20 μl of Protein A Sepharose was added, and the mixture was stirred at 4 ° C. for 30 minutes to react and immunoprecipitate. After immunoprecipitation, centrifugation was performed at 15000 × g, 4 ° C. for 5 minutes, and the supernatant was removed to recover Protein A Sepharose. The collected protein A sepharose was mixed with 500 μl of PBS and washed by stirring at 4 ° C. for 5 minutes. After washing, centrifugation was performed at 15000 × g, 4 ° C. for 5 minutes, and PBS was removed to recover Protein A Sepharose. This washing operation was repeated three times.

  After washing, 20 μl of SDS-PAGE sample buffer was added to Protein A Sepharose and boiled at 100 ° C. for 5 minutes. Sample buffer was loaded on 12.5% SDS-PAGE, and electrophoresis was performed with a current of 30 mA. After electrophoresis, the PVDF membrane was transferred for 1 hour with a current of 160 mA. The PVDF membrane after transfer was blocked by reacting 5% skim milk at room temperature for 1 hour. Next, 2.5 ml of anti-MANSC1 polyclonal antibody (AVIVA systems, # ARP34359_P050) diluted 3000 times with 5% skim milk was reacted at room temperature for 1 hour. After the reaction, the PVDF membrane was washed 3 times with PBS containing 0.05% Tween 20 (hereinafter referred to as PBS-T), and 2.5 ml of anti-rabbit IgG-POD (MBL, # 458) diluted 5000 times with 5% skim milk was added. And allowed to react at room temperature for 1 hour. After the reaction, the PVDF membrane was washed 5 times with PBS-T, developed with a chromogenic substrate (Millipore, Immobilon Western # WBKLS0500), and exposed to the film for 15 seconds.

  In the culture supernatant of 293T introduced with MANSC1, MANSC1 signal was detected in the vicinity of 10 kDa when immunoprecipitated with the ACT35-51_1B4A7D antibody, but no signal was detected when immunoprecipitated with the isotype control (FIG. 4). ). In addition, in the culture supernatant of 293T into which only the vector was introduced, no signal was detected in both the isotype control and those immunoprecipitated with the ACT35-51_1B4A7D antibody (FIG. 4). These results also suggested that part of MANSC1 may be cleaved and secreted into the culture supernatant.

[Example 8] Effect of ACT35-51_1B4A7D monoclonal antibody on proliferation of cancer cells (MTT)
The effect of ACT35-51_1B4A7D antibody on cancer cell proliferation was analyzed using MTT assay. For various cancer cells, the prostate cancer cell line AsPC1 was inactivated with RPMI1640 medium (WAKO) containing 10% FBS (Equitech, the same applies hereinafter) and 1% P / S, and other cancer cells. DMEM medium (SIGMA) containing inactivated 10% FBS and 1% P / S solution was used. The medium for hybridoma producing the target antibody includes RPMI medium (Wako) containing the required amount of HT (Sigma-Aldrich, H0137, HT media supplement (50X) Hybri-Max), 15% FBS, 1% P / S solution. Was used.

  As controls, Mouse IgG1 (BECKMAN COULTER, 731581), IgG2a (MBL, M076-3), IgG2b (MBL, M077-3), IgG3 (MBL, M078-3) 1 μl each, 1 ml of hybridoma medium A mouse isotype control mixture dissolved in was used.

Each experiment was performed in 3 wells per antibody culture supernatant test. Various cancer cells were seeded in a 96-well plate (IWAKI) at 2 × 10 ³ cells in 100 μl medium per well and incubated at 37 ° C. for 24 hours under 5% CO ₂ condition.

  The culture supernatant containing ACT35-51_1B4A7D antibody or mouse isotype control was added at 100 μl per well to the 96-well plate after incubation, and incubated for 72 hours. To each cell obtained by removing the culture supernatant by centrifugation, add 100 μl of 5% WST-1 solution (vol./vol., Roche, 11 644 807 001) prepared by dissolving in fresh medium, After incubating for 1 to 4 hours, the color development of WST-1 was measured with a microplate reader (BIO-RAD) every 1 hour.

  The graphed measurement results are shown in FIG. This data showed the color development of WST-1 after 3 hours as O.D. value (O.D450nm-O.D.650nm). In the MTT assay, the ACT35-51_1B4A7D antibody suppressed GCIY cell proliferation to approximately 25% compared to the isotype control (FIG. 5B). That is, there was a growth suppression effect of about 75%. Furthermore, the cell growth inhibitory effect of the ACT35-51_1B4A7D antibody was remarkably observed in glioma-derived T98G cells, and its growth was suppressed to about 7% compared to the isotype control (FIG. 5C). That is, an about 93% growth inhibitory effect was observed.

[Example 9] Effect of antibody by tail vein administration on mouse tumor bearing model
In vivo antitumor effect of ACT35-51_1B4A7D antibody was examined using a mouse tumor bearing model.

A cell suspension is prepared so that GCIY cells become 5 × 10 ⁶ cells / 0.2 ml saline / mouse individual subcutaneously on the back of the neck of 6-week-old male SCID mice (purchased from CLEA Japan at 5 weeks of age). Transplanted. Measure the size of the tumor that engrafted 3 weeks after transplantation, and control the tumor-bearing mice, control group, positive control group, ACT35-51_1B4A7D antibody group so that the average tumor volume of each group is about 55 ± 5 mm ³ The three groups were divided into 4 groups.

Sample administration to each group was performed on the tail vein of cancer-bearing mice from 3 weeks after cell transplantation. Saline (Otsuka raw diet) for the control group, Taxotere (600 μg per mouse, sanofi-aventis) for the positive control group, and ACT35-51_1B4A7D antibody (10 mg / kg) for the antibody administration group once a week It was administered via the tail vein (3 times in total for 3 consecutive weeks). In addition, body weight measurement and tumor measurement were performed immediately before each administration. An animal balance was used for body weight measurement, and for tumor measurement, the major axis and minor axis were measured with a digimatic caliper, and the tumor volume was determined by the formula "tumor volume (mm ³ ) = 0.5 x major axis x minor axis x minor axis". .

  The tumor volume data was compared with the control group to examine the antitumor effect over time. As a result, in the control group, the tumor increased with time, and at the end of the experiment 3 weeks after the start of administration, the tumor volume was about 25 times that at the start. In contrast, in the group administered Taxotere, tumor growth suppression was observed from the second week after the start of administration, and in the third week after the end of the experiment, it remained at about 11 times the start of the experiment, about 59 times that of the control group. % Tumor volume suppression was observed. In addition, in the group administered ACT35-51_1B4A7D antibody, a tendency to suppress tumor growth was observed with respect to the control group from the first week after the start of administration, and at the end of the experiment, the tumor volume remained about 16 times that at the start of administration. The tumor volume of the control group was suppressed by about 44% (FIG. 6).

  Necropsy was performed 3 weeks after the start of administration of each sample. The tumor-bearing mice were subjected to ether anesthesia lethality, and the tumors were removed subcutaneously from the back of the neck and their weights were measured. The tumor weights of each group were compared. The excised tumor weights were 1.33 g for the control group, 0.87 g for the positive control group, and 0.83 g for the ACT35-51_1B4A7D antibody group, respectively. In the group to which the ACT35-51_1B4A7D antibody was administered, the excised tumor weight was reduced by about 38% compared to the control group, and the same tumor growth inhibitory effect as that of the positive control group was observed (FIG. 7).

  Regarding the body weight transition, a significant decrease in body weight was observed in the positive control group 2 weeks after the start of administration, but no decrease in body weight was observed in the ACT35-51_1B4A7D antibody group. Also in the calculated body weight obtained by subtracting the tumor weight from the body weight at the time of necropsy, a decrease in the body weight of the positive control group was obvious, but an increase in the body weight was observed in the ACT35-51_1B4A7D antibody group (FIG. 8).

  Statistical tests were performed by one-way analysis of variance (ANOVA), and when there was a difference at p <0.05, a significant difference test was performed by Tukey's multiple comparison method. When the risk rate was p <0.05, the control group was evaluated as having a significant difference. (*: p <0.05, **: p <0.01)

[Example 10] Antibody variable region determination method
To clarify the gene sequence of the variable region of ACT35-51_1A4B7D antibody, ACT35-51_1B4A7D antibody-producing cell hybridoma cell 2 × 10 ⁶ was suspended in 1 ml of Trizol (invitrogen, # 15596-026) and left for 5 minutes, and chloroform was 200 μl. The suspension was added and suspended for 15 seconds, followed by centrifugation at 12,000 × g for 15 minutes to obtain a supernatant. This supernatant was mixed with 500 μl of isopropanol, and then centrifuged at 12,000 × g for 10 minutes. The obtained pellet was washed with 80% ethanol to obtain 40 μg of total RNA. The whole amount was dissolved in 20 μl of water. Among them, using a solution corresponding to 5 μg of total RNA, double-stranded cDNA was prepared from total RNA using SuperScript ^™ Choice System (invitorgen, # 18090-019). The obtained double-stranded cDNA was precipitated with ethanol, and the 5 ′ end and 3 ′ end of the double-stranded cDNA were bound using LigationHigh (TOYOBO # LGK-201), and PCR was performed using 1 μl of this as a template. Primers designed for the heavy and light chain constant regions were used. Primer sequences are as follows.

Heavy chain 5 ′ side gtccacgaggtgctgcacaat (SEQ ID NO: 18)
Heavy chain 3 ′ side gtcactggctcagggaaataacc (SEQ ID NO: 19)
Light chain 5 ′ side aagatggatacagttggtgc (SEQ ID NO: 20)
Light chain 3 ′ side tgtcaagagcttcaacagga (SEQ ID NO: 21)

The PCR product was electrophoresed on a 1.5% gel, and then excised and purified. Sequencing was performed using the purified DNA. For the light chain, the purified DNA was cloned and then sequenced. The determined light chain variable region base sequence is SEQ ID NO: 9, the amino acid sequence is SEQ ID NO: 10, the heavy chain variable region base sequence is SEQ ID NO: 11, and the amino acid sequence is SEQ ID NO: 12. Show.

  In addition, the amino acid sequences of these variable regions are numbered using sequence analysis (http://www.bioinf.org.uk/abysis/tools/analyze.cgi) on UCL's “Andrew CR Martin's Bioinformatics Group” site. The CDR regions were identified according to the criteria described in “Table of CDR Definitions” (http://www.bioinf.org.uk/abs/#kabatnum). The results of CDR prediction and the light chain and heavy chain signal sequences are shown in FIG. The light chain CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NOs: 3-5, and the heavy chain CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NOs: 6-8.

[Example 11] Epitope analysis of ACT35-51_1B4A7D antibody
In order to specify the epitope of the ACT35-51_1B4A7D antibody, Ba / F3 cells expressing several types of MANSC1 peptides with different chain lengths were prepared, and the reactivity with the antibody was evaluated.

MANSC1, extramembranous region 60aa (from N-terminal, the same applies hereinafter), 153aa, 186aa, 219aa, 250aa, 285aa, 385aa (extracellular full length) were used as peptides to be analyzed. Using the cDNA library subjected to the GCIY signal sequencing strap shown in Example 1 as a template, using the DNA of the following sequence as a primer, PrimeSTAR MAX DNA polymerase (TaKaRa, # R045A) as a polymerase, seven kinds of genes Was isolated.
Forward primer (common to two genes): (SEQ ID NO: 16)
Reverse primer (The number added to R means the chain length of the peptide encoded by the amplification product)

R60: (SEQ ID NO: 22) ttttccttttgcggccgcttgagttgaagtatatacgg
R153: (SEQ ID NO: 23) ttttccttttgcggccgctaggggagtgactgcttgtg
R186: (SEQ ID NO: 24) ttttccttttgcggccgcaaatagtttctccaagtgat
R219: (SEQ ID NO: 25) ttttccttttgcggccgccagcagatgcgctatttctt
R250: (SEQ ID NO: 26) ttttccttttgcggccgcggtgggtagaagggtggcgg
R285: (SEQ ID NO: 27) ttttccttttgcggccgctgtagaaatgagggtcgtgg
R385: (SEQ ID NO: 28) ttttccttttgcggccgcaagccatttttcaaatggaa

Each PCR product obtained was electrophoresed on a 1% agarose gel, cut out and purified, and then treated with restriction enzymes with EcoRI and NotI. pMX-SST was also digested with EcoRI and NotI and purified. Further, each was treated with LigationHigh (TOYOBO, # LGK-201), and then the same treatment as in Example 2 (after transformation to E. coli) was performed, and the transformed E. coli was applied to a 50 μg ampicillin-containing LB agarose plate. Was plated. PCR was performed from colonies obtained by overnight culture at 37 ° C. so as to contain the insert portion, and it was confirmed by sequencing whether it was a pMX-SST vector containing the desired sequence. The following oligonucleotides were used as PCR primers for sequencing.

SST3 'side 5'-ggcgcgcagctgtaaacggtag-3' (SEQ ID NO: 29)
SST5 'side 5'-cgggggtggaccatcctcta-3' (SEQ ID NO: 30)

Thereafter, Ba / F3 cells containing MANSC1 gene sequences of various chain lengths were prepared in the same manner as in Example 1 (4) (after virus packaging). Furthermore, the reactivity between Ba / F3 cells expressing MANSC1 molecules of various chain lengths and the ACT35-51_1B4A7D antibody was analyzed with a flow cytometer in the same manner as in Example 4 (FIG. 10). As a result, the ACT35-51_1B4A7D antibody does not react with clones expressing MANSC1 molecules up to 1-60, but reacts with clones expressing longer than 1 to 153 region. From these results, it has been clarified that an antibody epitope is contained between positions 61 to 153 from the N-terminus of the MANSC1 molecule.

  Since the monoclonal antibody of the present invention has excellent anticancer activity, it can be used for the treatment or prevention of cancer. In particular, it has a strong cell growth inhibitory effect on gastric cancer and glioma. The monoclonal antibody of the present invention is extremely useful for medical treatment because it is considered to have an excellent effect on Skills gastric cancer, which is very malignant and has been difficult to treat. In addition, the monoclonal antibody of the present invention can be applied to cancer diagnosis and cancer cell detection / selection.

Claims (17)

  Antibody that binds to human-derived MANSC1 protein and has anticancer activity   The antibody according to claim 1, which binds to an extracellular region of a human-derived MANSC1 protein.   The antibody according to claim 1, wherein the cancer is gastric cancer or glioma.   The antibody according to claim 1, which retains a light chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 3 to 5 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 6 to 8.   In the antibody according to claim 4, at least one of the amino acid sequences of SEQ ID NOs: 3 to 8, one or more amino acids are substituted, deleted, added and / or inserted, and claim 4 An antibody having the same activity as the antibody described in 1.   The antibody according to claim 1, which retains a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12.   In the antibody of claim 6, at least one of the amino acid sequences of SEQ ID NOs: 10 and 12, wherein one or more amino acids are substituted, deleted, added and / or inserted, and An antibody having the same activity as the antibody described in 1.   An antibody having a signal sequence removed from at least one of the amino acid sequences of SEQ ID NOs: 10 and 12 in the antibody of claim 6, and having an activity equivalent to that of the antibody of claim 6.   An antibody that binds to an epitope of the antibody according to claim 6 and has anticancer activity in a human-derived MANSC1 protein.   A peptide comprising the light chain of an antibody according to claim 1 or a variable region thereof, comprising the amino acid sequence of SEQ ID NOs: 3 to 5.   The peptide according to claim 10, comprising the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence.   A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the amino acid sequence set forth in SEQ ID NOs: 6 to 8.   The peptide according to claim 12, comprising the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence.   DNA encoding the antibody according to any one of claims 1 to 9 or the peptide according to any one of claims 10 to 13.   A hybridoma that produces the antibody according to any one of claims 1 to 9, or comprises the DNA according to claim 14.   The anticancer agent which uses the antibody in any one of Claim 1 to 9 as an active ingredient.   The anticancer agent according to claim 16, wherein the cancer is gastric cancer or glioma.