HK1148008A

HK1148008A - Anti-cldn6 antibody

Info

Publication number: HK1148008A
Application number: HK11102167.0A
Authority: HK
Inventors: 油谷浩幸; 堤修一; 西村邦裕; 作本裕史; 川合重人
Original assignee: 国立大学法人东京大学; 株式会社未来创药研究所
Priority date: 2008-01-11
Filing date: 2009-01-09
Publication date: 2011-08-26

Abstract

Disclosed is an antibody capable of binding to Claudin-6 (CLDN6) which is expressed on the surface of a cell membrane. The antibody can recognize human CLDN6 occurring on the surface of a cell membrane in its native form, and has a cytotoxic activity (e.g., an ADCC activity and a CDC activity) against a cancer cell line which expresses human CLDN6 at a high level. A conjugate of the antibody and a toxin has an inhibitory activity on the cell proliferation of a cancer cell line which expresses human CLDN6 at a high level. The expression of human CLDN6 is not observed in a normal tissue, but is increased in a tumor tissue (e.g., pulmonary adenocarcinoma, gastric cancer, ovarian cancer). The anti-CLDN6 antibody is expected to be highly accumulated in a tumor in which human CLDN6 has been expressed at a high level, and therefore can be used as an extremely effective anti-tumor agent.

Description

anti-CLDN 6 antibodies

Technical Field

The present invention relates to a general antibody drug. More particularly, the present invention relates to an anti-CLDN 6 antibody, and a cell growth inhibitor and an anticancer agent comprising the same.

Background

The Claudin family is a family of 4 transmembrane Cell membrane proteins constituting a tightly linked molecular weight of about 23kD, and includes 24 members in human and mouse, and each Claudin is known to exhibit a very unique expression pattern in each epithelial Cell (non-patent document 1 (Furute and Tsukuta, TRENDS in Cell Biology 2006, 16: 181); non-patent document 2(Wilcox, et al., Cell 2001, 104: 165); non-patent document 3(Rahner, et al., StroeNTEROGY 2001, 120: 411); non-patent document 4(Morita, et al., Proc.Natl.Acad.Sci.USA 1999, 96: 511)). It is known that in the epithelial cell layer, a mechanism for preventing the leakage (diffusion) of substances in the intercellular spaces plays a role, and that in the mechanism for preventing the leakage, a cell adhesion device called tight junction plays an important role as a "barrier".

High expression of human CLDN6 transcript in Cancer has been disclosed in non-patent document 5(Hewitt, et al, BMC Cancer 2006, 6: 186) or patent document 1(WO 2003/088808) and the like. In addition, expression of human and mouse CLDN6 at the protein level in Cancer is described in non-patent document 6(Osanai, et al, Cancer Sci.2007, 98: 1557) and non-patent document 7(Azadeh Arabzadeh, et al, BMC Cancer 2007, 7: 196). Non-patent document 6 discloses western blot analysis using MCF7, which is a breast cancer cell line. The document claims: as its title said, epigenetic silencing of human CLDN6 in breast cancer cell lines promotes anchorage-independent growth of cancer cells. Non-patent document 6 discloses that human CLDN6, which is an oncogene, in the MCF7 cell line is reduced in expression due to partial methylation of the promoter region, and as a result, apoptosis sensitivity is reduced, colony forming ability is reduced, infiltration ability of cancer cells is improved, metalloprotease activity is increased, Migration (Migration) ability of cancer cells is enhanced, and it contributes to malignancy of cancer.

However, western blotting of human CLDN6 in MCF7 cells performed in non-patent document 6 was used to confirm whether the knock-out system of human CLDN6 by siRNA was effective, and there was no description about materials, antibodies or methods used, and there was no experiment on how much the expression level of human CLDN6 protein in breast cancer cell line MCF7 was changed compared to normal tissues. The authors of non-patent document 6 pointed out that non-patent document 8(Quan and Lu, cartinogenesis 2003, 24: 1593), which is an earlier existing document than this document, was cited in this document, and further studies were conducted based on the description of non-patent document 8. The above non-patent document 8 has examined that human CLDN6 is a cancer suppressor gene in breast cancer because human CLDN6 has reduced mRNA expression in breast cancer cell lines BT-474 and MCF7 as compared with normal breast epithelial cells. That is, in non-patent document 6, the expression of human CLDN6 protein was reduced in breast cancer cell line MCF7 as compared with normal breast, and the following findings were made based on the above-mentioned viewpoint: epigenetic silencing of human CLDN6 in breast cancer cell lines promotes anchorage-independent growth of cancer cells.

Further, non-patent document 7 relates only to the following: changes in the expression profile of several mouse Claudin proteins, including the mouse CLDN6 protein, in mouse chemooncogenic tumors given DMBA/TPA were examined by immunohistological staining and indicated that mouse CLDN6 was expressed in the "upper basal compartment" in normal mice.

It should be noted that, as for the anti-CLDN 6 antibody, a monoclonal antibody capable of recognizing human CLDN6 on the surface of a cell membrane by flow cytometry, that is, capable of recognizing human CLDN6 present on the surface of a cell membrane in a natural form has not been reported.

[ patent document 1] WO2003/088808

[ non-patent document 1] Mikio Furuse and Shoichiro Tsukita: claudins in encapsulating joints of human and flies. trends in Cell Biology 2006, 16: 181

[ non-patent document 2] Edward R.Wilcox, Quianna L.Burton, Sadaf Naz, Saima Riazuddin, Tenesha N.Smith, Barbara Pplis, Inna Belylarseva, Tamar Ben-Yose, Nikkia.Liburd, Robert J.Morell, Bechara Kachar, Doris K.Wu, Andrew J.Griffith, Sheikh Riazuddin, and Thomas B.Friedman: details in the Gene Encoding light connecting Claudin-14 Cause automatic recovery DFNB29.cell 2001, 104: 165

[ non-patent document 3] Christoph Rahner, Laura L.Mitic, and James M.Anderson: heterology in expression and subunit Localization of Claudin 2, 3, 4, and 5 in the Rat Liver, Pancreas, and Gut. GASTROENTEROLOGY2001, 120: 411

[ non-patent document 4] Kazumasa Morita, Mikio Furuse, Kazushi Fujimoto, and Shoichiro Tsukita: claudin multigene family encoding four-transmission domain protein components of light junction strings, Proc. Natl. Acad. Sci. USA 1999, 96: 511

[ non-patent document 5] Kyle J Hewitt, Rachana Agarwal and Patrice J Morin: the claudin gene family: expression of in normal and neoplastic tissues s. bmccancer 2006, 6: 186

[ non-patent document 6] Makoto Osanai, Masaki Murata, Hideki Chiba, Takashi Kojima and Norimasa Sawada: cancer Sci 2007, 98: 1557

[ non-patent document 7] Azadeh Arabzadeh, Tammy-Claire Troy and Kursad Turksen: BMC Cancer 2007, 7: 196

[ non-patent document 8] Chengshi Quan and Shi-Jiang Lu: identification of genes expressed in a geographic intrinsic cells of a genetic ratio utilization and microorganisations. cartigenesis 2003, 24: 1593

[ non-patent document 9] Kohls MD, Lappi DA: Mab-ZAP: a tool for evaluating antibody efficacy for use in an immunological approach, BioTechniques 2000, 28 (1): 162

[ non-patent document 10] Nimmerjahn F, Ravetch JV.: dictionary immunoglobulin G subunit activity through selective Fc receptor binding. science.2005, 310: 1510

[ non-patent document 11] Nimmerjahn F, Ravetch JV.: fc γ Receptors: old friends and new family members, immunity, 2006, 24: 19

Disclosure of Invention

The present inventors have found that human CLDN6 mRNA is not expressed in all normal tissues of an adult but is expressed more strongly in tumor tissues (lung adenocarcinoma, gastric cancer, and ovarian cancer).

The present inventors also found that human CLDN6 protein is highly expressed in many cancer cell lines, and that the protein expression thereof matches the mRNA expression analysis result.

Furthermore, the present inventors have succeeded in producing the following monoclonal antibodies: a monoclonal antibody recognizing human CLDN6 present in a native form on the surface of a cell membrane; a monoclonal antibody that exhibits cytotoxicity by ADCC and/or CDC activity against a cancer cell line that highly expresses human CLDN 6; a monoclonal antibody having a cytostatic effect on a cancer cell line highly expressing human CLDN6 by binding to a toxin.

In addition, no expression of human CLDN6 was found in normal tissues, indicating that the tumor specificity of human CLDN6 was very high. Therefore, it is presumed that the anti-CLDN 6 antibody is highly aggregated in tumors highly expressed by human CLDN6 and found to be a very effective anti-tumor agent.

Namely, the present invention provides an antibody that binds to Claudin6(CLDN6) expressed on a cell membrane. The invention also provides an anti-CLDN 6 antibody that is cytotoxic. Preferably, the anti-CLDN 6 antibody of the present invention has ADCC activity and/or CDC activity. In addition, in a preferred embodiment, the anti-CLDN 6 antibody of the invention binds to a cytotoxic agent.

In other aspects, the present invention provides an antibody according to any one of the following (a) to (j):

(a) an antibody (AB3-1 heavy chain) comprising a heavy chain variable region having a CDR1 comprising the amino acid sequence represented by sequence No. 24, a CDR2 comprising the amino acid sequence represented by sequence No. 25, a CDR3 comprising the amino acid sequence represented by sequence No. 26;

(b) an antibody (AB3-1 light chain) comprising a light chain variable region having a CDR1 comprising the amino acid sequence represented by sequence No. 27, a CDR2 comprising the amino acid sequence represented by sequence No. 28, a CDR3 comprising the amino acid sequence represented by sequence No. 29;

(c) an antibody (AB3-1) comprising the heavy chain variable region of (a) and the light chain variable region of (b);

(d) an antibody (AE1-16, AE49-11 heavy chain) comprising a heavy chain variable region having CDR1 comprising the amino acid sequence represented by sequence No. 30, CDR2 comprising the amino acid sequence represented by sequence No. 31, CDR3 comprising the amino acid sequence represented by sequence No. 32;

(e) an antibody (AE1-16, AE49-11 light chain) comprising a light chain variable region having CDR1 comprising the amino acid sequence represented by sequence No. 33, CDR2 comprising the amino acid sequence represented by sequence No. 34, CDR3 comprising the amino acid sequence represented by sequence No. 35;

(f) an antibody (AE1-16, AE49-11) comprising the heavy chain variable region of (d) and the light chain variable region of (e);

(g) an antibody (AE3-20 heavy chain) comprising a heavy chain variable region having a CDR1 comprising the amino acid sequence represented by sequence number 40, a CDR2 comprising the amino acid sequence represented by sequence number 41, a CDR3 comprising the amino acid sequence represented by sequence number 42;

(h) an antibody (AE3-20 light chain) comprising a light chain variable region having a CDR1 comprising the amino acid sequence represented by sequence No. 43, a CDR2 comprising the amino acid sequence represented by sequence No. 44, a CDR3 comprising the amino acid sequence represented by sequence No. 45;

(i) an antibody (AE3-20) comprising the heavy chain variable region of (g) and the light chain variable region of (h);

(j) an antibody that recognizes the same epitope as that recognized by the antibody according to any one of (a) to (i).

In addition, in other aspects, the present invention provides a pharmaceutical composition comprising an anti-CLDN 6 antibody. Preferably, the pharmaceutical composition of the present invention is a cytostatic agent. It is also preferred that the pharmaceutical composition of the present invention is an anticancer agent. It is also preferable that the pharmaceutical composition of the present invention contains the above-described antibody of the present invention.

In still another aspect, the present invention provides a method for diagnosing cancer, the method comprising the steps of:

(a) a step of providing a sample taken from a subject;

(b) detecting the CLDN6 protein contained in the sample of (a).

CLDN6 protein is preferably detected using an anti-CLDN 6 antibody.

Drawings

Figure 1 shows the expression profile of human CLDN6 in normal tissue.

Fig. 2 shows the expression profile of human CLDN6 in lung cancer.

FIG. 3 shows the expression profile of human CLDN6 in gastric cancer.

Fig. 4 shows the expression profile of human CLDN6 in ovarian cancer.

FIG. 5 shows a Western blot using a goat polyclonal antibody against CLDN6 (Santa Cruz, C-20, code. sc-17669).

FIG. 6 shows the measurement of the binding of an anti-human CLDN6 antibody to human CLDN 6-forced expressing cells and parent strains (flow cytometry analysis).

FIG. 7 shows the measurement of the binding properties of an anti-human CLDN6 antibody to a lung adenocarcinoma cell line ABC-1 and a gastric cancer cell line AGS (flow cytometry analysis).

FIG. 8 is a graph showing ADCC activity of an anti-human CLDN6 antibody on lung adenocarcinoma cell line ABC-1.

FIG. 9 shows ADCC activity of an anti-human CLDN6 antibody on gastric cancer cell line AGS.

FIG. 10 is a graph showing CDC activity of an anti-human CLDN6 antibody against lung adenocarcinoma cell line ABC-1.

FIG. 11 is a graph showing the antitumor effect of an anti-human CLDN6 monoclonal antibody against a lung adenocarcinoma cell line ABC-1 using Mab-ZAP.

FIG. 12 is a graph showing the antitumor effect of an anti-human CLDN6 monoclonal antibody against a gastric cancer cell line AGS using Mab-ZAP.

FIG. 13 shows the results of immunostaining by a goat anti-CLDN 6 polyclonal antibody (Santa Cruz, sc-17669) (A: lung adenocarcinoma tumor tissue, B: lung adenocarcinoma non-tumor tissue).

FIG. 14 is a graph showing the results of evaluation of the antitumor activity of AE49-11 antibody in a PA-1 subcutaneous graft model.

FIG. 15 shows the results of evaluation of the antitumor activity of AE49-11 antibody in the NUGC-3 subcutaneous transplantation model (thin line: vehicle, iv, thick line: low fucose AE49-11(50mg/kg, iv)).

This specification includes the contents described in the specification of japanese patent application No. 2008-004423, which is the basis of priority of the present application.

Detailed Description

CLDN6

The amino acid sequence of Claudin6(CLDN6) and the gene sequence encoding the same are disclosed in GenBank accession Nos. NP-067018.1 and NM-021195.3 (SEQ ID NO: 22 and SEQ ID NO: 23), or GenBank accession Nos. NP-067018.2 and NM-021195.4 (SEQ ID NO: 46 and SEQ ID NO: 47).

In the present invention, the CLDN6 protein includes both full-length proteins and fragments thereof. Fragments are polypeptides comprising any region of the CLDN6 protein, and may not have the function of the native CLDN6 protein. Examples of fragments include fragments comprising the extracellular region of CLDN6 protein.

anti-CLDN 6 antibodies

The anti-CLDN 6 antibody of the present invention may be any antibody as long as it binds to CLDN6, regardless of its origin (mouse, rat, human, etc.), type (monoclonal, polyclonal antibody), shape (altered antibody, low molecular weight antibody, modified antibody, etc.), and the like.

Preferably, the anti-CLDN 6 antibody used in the present invention specifically binds to CLDN 6. In addition, preferably, the anti-CLDN 6 antibody used in the present invention is a monoclonal antibody.

Preferred anti-CLDN 6 antibodies of the present invention include antibodies capable of binding to CLDN6 expressed on cell membranes. CLDN6 expressed on the cell membrane is not particularly limited, and examples thereof include CLDN6 expressed on the cell membrane of cells forcibly expressing CLDN6 (for example, Ba/F3 cells and the like) or cancer cells expressing CLDN6 (for example, lung adenocarcinoma cell line ABC-1, gastric cancer cell line AGS and the like).

Whether an anti-CLDN 6 antibody binds to CLDN6 expressed on the cell membrane can be confirmed by flow cytometry or other methods well known to those skilled in the art.

Another preferred embodiment of the anti-CLDN 6 antibody of the present invention is an antibody having cytotoxicity. The antibody having cytotoxicity is not particularly limited, and examples thereof include an antibody having antibody-dependent cell-mediated cytotoxicity (ADCC), an antibody having complement-dependent cytotoxicity (CDC), and an antibody binding to a cytotoxic substance.

In the present invention, CDC activity refers to cytotoxicity caused by the complement system. On the other hand, ADCC activity refers to an activity in which, when a specific antibody is attached to a cell surface antigen of a target cell, a cell (e.g., an immune cell) having an Fc γ receptor binds to the Fc portion thereof via the Fc γ receptor, thereby destroying the target cell.

In the present invention, whether an antibody has ADCC activity or CDC activity can be determined by a known method (for example, Current protocols in Immunology, Chapter 7.Immunologic studios in humans, Editor, John E, Coligan et al, John Wiley & Sons, Inc. (1993)).

Specifically, first, effector cells, a complement solution, and target cells are prepared.

(1) Preparation of Effector cells

Spleens were excised from CBA/N mice and the like, and spleen cells were isolated in RPMI1640 medium (Invitrogen). After washing with the same medium containing 10% fetal bovine serum (FBS, Hyclone), the cell concentration was adjusted to 5X 10⁶(ii) ml, from which effector cells can be formulated.

(2) Preparation of complement solution

The Complement solution can be prepared by diluting Baby Rabbit supplement (CEDARLANE) 10-fold in a medium containing 10% FBS (Invitrogen).

(3) Preparation of target cells

Cells expressing CLDN6 protein were cultured in DMEM medium containing 10% FBS for 1 hour at 37 ℃ together with 0.2mCi of 51 Cr-sodium chromate (GE Healthcare Biosciences), whereby the target cells were radiolabeled. As cells expressing CLDN6 protein, cells transformed with a gene encoding CLDN6 protein, cancer cells (lung adenocarcinoma cells, gastric cancer cells, etc.), and the like can be used. After radiolabeling, cells were washed 3 times with RPMI1640 medium containing 10% FBS, and cell concentration was adjusted to 2X 10⁵And/ml, from which the target cells can be formulated.

The ADCC activity or CDC activity can be measured by the following method. For the measurement of ADCC activity, 50. mu.l each of target cells and anti-CLDN 6 antibody was added to a 96-well U-bottom plate (Becton Dickinson), and the mixture was reacted for 15 minutes on ice. Then, 100. mu.l of effector cells were added and cultured in a carbon dioxide incubator for 4 hours. The final concentration of antibody was made to be 0 or 10. mu.g/ml. After the culture, 100. mu.l of the supernatant was collected, and the radioactivity was measured by a GAMMA counter (COBRAII AUTO-GAMMA, MODEL D5005, manufactured by Packard Instrument Company). The cytotoxicity (%) was calculated according to the calculation formula (A-C)/(B-C). times.100 using the obtained values. A represents the radioactivity (cpm) of each sample, B represents the radioactivity (cpm) of a sample to which 1% NP-40 (manufactured by nacalai tesque) was added, and C represents the radioactivity (cpm) of a sample containing only target cells.

On the other hand, when CDC activity was measured, 50 μ l each of target cells and anti-CLDN 6 antibody was added to a 96-well flat-bottom plate (manufactured by Becton Dickinson), and the mixture was reacted for 15 minutes on ice. Then, 100. mu.l of complement solution was added thereto, and the mixture was incubated in a carbon dioxide incubator for 4 hours. The final concentration of antibody was 0 or 3. mu.g/ml. After the incubation, 100. mu.l of the supernatant was recovered and the radioactivity was measured by a gamma counter. Cytotoxicity can be calculated in the same manner as in ADCC activity assay.

When the anti-CLDN 6 antibody bound to a cytotoxic substance is incorporated into a cell, apoptosis of the cell into which the antibody is incorporated can be induced by the cytotoxic substance. Therefore, the cytotoxic agent-bound antibody more preferably has an internalization activity. In the present invention, the term "antibody having an internalization activity" refers to an antibody that can be transported into a cell (into cytoplasm, into a vesicle, or into another organ) when bound to CLDN6 on the cell surface.

Whether an antibody has an internalizing activity can be confirmed by a method known to those skilled in the art, for example, by contacting an anti-CLDN 6 antibody bound with a marker with a cell expressing CLDN6 to confirm whether the marker is incorporated into the cell; a method of contacting a cell expressing CLDN6 with an anti-CLDN 6 antibody to which a cytotoxic agent is bound, and confirming whether or not apoptosis of the cell expressing CLDN6 is induced, and the like. More specifically, whether or not the antibody has an internalizing activity can be confirmed according to the method described in the examples below and the like.

The cytotoxic substance used in the present invention may be any substance as long as it can induce apoptosis, and examples thereof include toxins, radioactive substances, and chemotherapeutic agents. The above-mentioned cytotoxic substance in the present invention includes a prodrug which is converted into an active cytotoxic substance in vivo. The prodrug may be activated by enzymatic conversion or non-enzymatic conversion.

In the present invention, the toxin refers to various proteins or polypeptides having cytotoxicity derived from microorganisms, animals or plants. Examples of the toxin used in the present invention include the following toxins: diphtheria toxin a Chain (dipheria toxin a Chain) (Langone j.j., et al., Methods in Enzymology, 93, 307-308, 1983); pseudomonas Exotoxin (Pseudomonas Exotoxin) (Nature Medicine, 2, 350-; ricin A Chain (Ricin A Chain) (Fulton R.J., et al., J.biol.Chem., 261, 5314. C.5319, 1986; Sivam G.et al., Cancer Res., 47, 3169. C.3173, 1987; Cumber A.J.et al., J.Immunol.methods, 135, 15-24, 1990; WawrynczakE.J., Cancer Res., 50, 7519. C.7562, 1990; Gheieite V.et al., J.Immunol.methods, 142, 223. C.230, 1991); deglycated Ricin a Chain (Thorpe p. e., et al., Cancer res., 47, 5924-; abrin a Chain (Abrin a Chain) (wawrynczak e.j., et al., br.j. Cancer, 66, 361-; gelonin (Gelonin) (Sivam G., et al., Cancer Res., 47, 3169-3173, 1987; Cumber A. J. et al., J. Immunol. methods, 135, 15-24, 1990; Wawrzynczake. J., et al., Cancer Res., 50, 7519-7562, 1990; Borognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); pokeweed anti-viral proteins (PAP-s; Pokeweed anti-viral protein from seeds) (Bolognesi A., et al., Clin. exp. Immunol., 89, 341-346, 1992); briodin (Bolognesi a., et al., clin. exp. immunol., 89, 341-346, 1992); saporin (Saporin) (blognesi a., et al., clin. exp. immunol., 89, 341- "346, 1992); momordica Charantia toxin protein (Momordin) (Cumber A.J., et al., J.Immunol.methods, 135, 15-24, 1990; Wawrynczak E.J., et al., Cancer Res., 50, 7519-; woodlousin (momocohin) (bologna a., et al., clin. exp. immunol., 89, 341-346, 1992); dianthin 32(Dianthin 32) (Bolognesi a., et al., clin. exp. immunol., 89, 341- & 346, 1992); dianthin 30(Dianthin 30) (stir f., Barbieri l., FEBS letter195, 1-8, 1986); syphilin ii (modeccin) (sirerpe f., Barbieri l., FEBS letter195, 1-8, 1986); mistletoe poison protein (Viscumin) (Stirpe F., Barbieri L., FEBS letter195, 1-8, 1986); syphilin i (volksein) (stir f., Barbieri l., FEBS letter195, 1-8, 1986); pokeweed poison protein (Dodecandrin) (Stirpe F., Barbieri L., FEBSletter 195, 1-8, 1986); wheat toxic protein (Tritin) (stir f., Barbieri l., FEBSletter 195, 1-8, 1986); cucurbitin (Luffin) (stir f., Barbieri l., FEBSletter 195, 1-8, 1986); trichokirin (Trichokirin) (Casellas P., et al, Eur. J. biochem.176, 581-588, 1988; Borognesi A., et al, Clin. exp. Immunol., 89, 341-346, 1992).

In the present invention, the radioactive substance refers to a substance containing a radioactive isotope. The radioisotope is not particularly limited, and any radioisotope can be used, for example, radioisotope can be used³²P、¹⁴C、¹²⁵I、³H、¹³¹I、¹⁸⁶Re、¹⁸⁸Re, and the like.

The chemotherapeutic agent in the present invention means a substance having cytotoxicity other than the above toxin and radioactive substance, and includes cytokines, antitumor agents, enzymes, and the like. The chemotherapeutic agent used in the present invention is not particularly limited, but is preferably a low molecular weight chemotherapeutic agent. It is considered that when the molecular weight is small, the possibility of interfering with the function of an antibody is low even after the antibody is bound thereto. In the present invention, the low molecular weight chemotherapeutic agent usually has a molecular weight of 100 to 2000, preferably 200 to 1000. The present invention is not particularly limited, and the following chemotherapeutic agents can be used, for example: melphalan (Melphalan) (Rowland g.f., et al, Nature 255, 487-488, 1975); cis-platinum (Cis-platinum) (Hurwitz E.and Haimovich J., Method In Enzymology 178, 369. 375, 1986; Schechter B.et al., int. Int.J. Cancer 48, 167. Qu. 172, 1991; Carboplatin (Carboplatin) (Ota, Y.et al., Asia-Oceania J.Obstet. Gynaec. 19, 449. Qu. 457, 1993); mitomycin C (mitomycin C) (Noguchi, A.et al., Bioconjugate Chem.3, 132. Bu 137, 1992); Adriamycin (Adriamycin) (Dorubicin) (Shih, L.B. Imal., Cancer Res. 4192-4198, 1991; Zwilwiz. J. 1995, Z. J. 3, Z. Zhen. J. 072, 267, 40. Zhen. J. 072, 267, 40, Z. Zhen. J. Cancer immunol. Immunother 40, 257. su 267, 1995); daunorubicin (Dalunoubicin) (Dillman, R.O., et al., Cancer Res.48, 6097-; bleomycin (Bleomycin) (Manabe, y., et al., biochem. biophysis. res. commun.115, 1009-; novel oncostatin (Neocarzinostatin) (Kitamura K., et al., Cancer Immunol. Immunother 36, 177-184, 1993; Yamaguchi T., et al., Jpn. J. Cancer Res.85, 167-171, 1994); methotrexate (Methotrexate) (Kralovic, J., et al., Cancer Immunol. Immunother29, 293. sub.302, 1989; Kulkarni, P.N., et al., Cancer Res.41, 2700. sub.2706, 1981; Shin, L.B., et al., int.J.cancer 41, 293. sub.839, 1988; Gamett M.C., et al., int.J.cancer 31, 670, 1983); 5-Fluorouridine (5-Fluorouridine) (Shin, l.b., int.j. cancer 46, 1101-; 5-Fluoro-2 '-deoxyuridine (5-Fluoro-2' -deoxyuridine) (Goerlach A., et al, Bioconjugate chem.2, 96-101, 1991); cytarabine (cytarabine arabine) (Hurwitz e., et al, j.med.chem.28, 137-; aminopterin (Aminopterin) (Kanellos j., et al., immunol. cell. biol.65, 483-493, 1987); vincristine (Vincristine) (Johnson j.r., et al, br.j.cancer 42, 17, 1980); desacetylvinblastine (Vindesine) (Johnson j.r., et al, br.j. cancer44, 472-; interleukin-2 (Interleukin-2); tumor necrosis factor (TNF α); interferon (INF); carboxypeptidase (Carboxypeptidase); alkaline Phosphatase (alkali Phosphatase); beta-lactamase (beta-lactamase); cytidine deaminase (cytidin deaminase).

The cytotoxic substance used in the present invention may be one kind, or two or more kinds of cytotoxic substances may be used in combination.

The binding of the anti-CLDN 6 antibody to the above cytotoxic agent may be performed by a covalent bond or a non-covalent bond. Methods for producing antibodies to which the above cytotoxic substances are bound are known.

The anti-CLDN 6 antibody may be directly bound to the cytotoxic substance via a linker or the like included in the antibody itself, or may be indirectly bound to the cytotoxic substance via a linker or an intermediate support or the like. Examples of the linking group for directly binding an anti-CLDN 6 antibody to a cytotoxic substance include disulfide bonds using SH groups. Specifically, the intramolecular disulfide bond in the Fc region of the antibody is reduced with a reducing agent such as dithiothreitol, and the disulfide bond in the cytotoxic substance is similarly reduced, and the two are bound by a disulfide bond. Before binding, either the antibody or the cytotoxic substance may be activated using an activation promoter, such as Ellman's reagent, to promote the formation of disulfide bonds between the two. Other methods for directly binding the anti-CLDN 6 antibody to a cytotoxic substance include, for example, a method using Schiff base, a carbodiimide method, an active ester method (N-hydroxysuccinimide method), a method using mixed anhydride (mixed anhydride), and a method using a diazo reaction.

The binding of an anti-CLDN 6 antibody to a cytotoxic agent may also be indirectly bound by other agents. The other substance for indirect binding is not particularly limited, and examples thereof include compounds having 2 or more groups selected from amino groups, carboxyl groups, mercapto groups, and the like, in any 1 kind or combination of 2 or more kinds; a peptide linker; a compound having a binding ability to an anti-CLDN 6 antibody, and the like. Examples of the compound having 2 or more groups selected from amino groups, carboxyl groups, mercapto groups and the like in any 1 or a combination of 2 or more include N-Succinimidyl3- (2-pyridyldithio) propionate (SPDP: N-Succinimidyl3- (2-pyridyldithio) propinate) (Wawrzynczak E.J., et al, Cancer Res., 50, 7519-one 7562, 1990; Thorpe P.E., et al, Cancer Res., 47, 5924-one 5931, 1987); succinimidyl 6-3- [ 2-pyridyldithio ] propionamide) hexanoate (LC-SPDP: succinimidyl 6-3- [ 2-pyridldithio ] propinamide) hexanate) (Hermanson G.T., BIOCONJUGATE Techniques, 230-232, 1996); thiosuccinimidyl 6-3- [ 2-pyridyldithio ] propionamide) hexanoate (Sulfo-LC-SPDP: sulfosuccinimidyl 6-3- [ 2-pyridldithio ] propinamide) hexanoate) (Hermanson G.T., BIOCONJUGATE technologies, 230- "232, 1996); N-Succinimidyl3- (2-pyridyldithio) butanoate (SPDB: N-Succinimidyl3- (2-pyridyldithio) butanoate) (Wawrzynczak E.J., et al, Br.J. cancer, 66, 361-366, 1992); succinimidyloxycarbonyl- α - (2-pyridyldithio) toluene (SMPT: succinimidyoxycarbonyi- α - (2-pyridyldithio) toruene) (Thorpe P.E., et al, Cancer Res., 47, 5924-5931, 1987); succinimidyl 6- (. alpha. -methyl- [ 2-pyridyldithio ] toluamide) hexanoate (LC-SMPT: Succinimidyl 6- (. alpha. -methyl- [ 2-pyridyldithio ] amine) hexanoate) (Hermanson G.T., BIOCONJUGATE Techniques, 232-; sulfosuccinimidyl 6- (. alpha. -methyl- [ 2-pyridyldithio ] toluamide) hexanoate (Sulfo-LC-SMPT: Sulfo succinimidyl 6- (. alpha. -methyl- [ 2-pyridyldithio ] toruamide) hexanoate) (Hermanson G.T., BIOCONJUGATETechniques, 232-ion 235, 1996); succinimidyl-4- (p-maleimidophenyl) butyrate (SMPB: Succinimidyl-4- (p-maleimidophenyl) butyrate) (Hermanson G.T., BIOCONJUGATE technologies, 242-243, 1996); thiosuccinimidyl-4- (p-maleimidophenyl) butyrate (Sulfo-SMPB: Sulfo-succinimide-4- (p-maleimidophenyl) butyrate) (Hermanson G.T., BIOCONJUGATE Techniques, 242-243, 1996); M-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS: m-Maleimidobenzoyl-N-hydroxysuccinimide ester) (Hermanson G.T., BIOCONJUGATETechniques, 237-; M-Maleimidobenzoyl-N-hydroxythiosuccinimide ester (Sulfo-MBS: m-Maleimidobenzoyl-N-hydroxysuccinimide ester) (Hermanson G.T., BIOCONJUGATETEChniques, 237-238, 1996); s-acetylmercaptosuccinic anhydride (SAMSA: S-Acetyl mercaptosuccinic anhydride) (Casellas P., et al., Eur.J. biochem, 176, 581-588, 1988); dimethyl 3, 3 '-dithiodipropionamide ester (DTBP: Dimethyl 3, 3' -dithiobispromimide) (Casellas P., et al., Eur.J. biochem, 176, 581 588, 1988); 2-iminothiolane (2-iminothiolane) (Thorpe p.e., et al, Cancer res., 47, 5924-.

Examples of other substances used for binding of the anti-CLDN 6 antibody to a cytotoxic substance include peptides, antibodies, poly-L-glutamic acid (PGA), carboxymethyl dextran, aminodextran, avidin-biotin, aconitic acid, glutamic dianhydride, Human Serum Albumin (HSA), and the like.

Further, a proteinaceous cytotoxic substance may also be bound to an antibody by genetic engineering methods. Specifically, for example, a recombinant vector incorporated into an expression vector can be constructed by in-frame fusion of a DNA encoding the above-mentioned cytotoxic substance peptide and a DNA encoding an anti-CLDN 6 antibody. The anti-CLDN 6 antibody to which the toxic peptide is bound is obtained as a fusion protein by introducing the vector into an appropriate host cell to obtain a transformed cell, and culturing the transformed cell to express the incorporated DNA. In obtaining a fusion protein with an antibody, a proteinaceous drug or toxin is usually disposed on the C-terminal side of the antibody. The antibody may also be linked to the proteinaceous drug or toxin by a peptide linker.

One of preferred embodiments of the anti-CLDN 6 antibody of the present invention is an antibody having CLDN6 bound thereto and having substantially no CLDN9 bound thereto. CLDN9 is generally considered to have high homology to CLDN6 and is the closest molecule to CLDN 6. Therefore, an antibody having CLDN6 bound thereto and CLDN9 not substantially bound thereto is considered to have very high specificity for CLDN6 and to be useful as a drug. The amino acid sequence of CLDN9 is well known, for example, the amino acid sequence of human CLDN9 is described in GenBank accession No. NP-066192.1 (SEQ ID NO: 48).

In the present invention, an antibody having CLDN6 bound thereto and having substantially no CLDN9 bound thereto means an antibody having CLDN9 binding activity of usually 50% or less, preferably 30% or less, and more preferably 10% or less, compared to CLDN6 binding activity.

In addition, one of preferred embodiments of the anti-CLDN 6 antibody of the present invention is an antibody having a binding to CLDN6 and substantially no binding to CLDN 3. The amino acid sequence of CLDN3 is well known, for example, the amino acid sequence of human CLDN3 is described in GenBank accession No. NP-001297.1 (SEQ ID NO: 49). In the present invention, an antibody having CLDN6 bound thereto and having substantially no CLDN3 bound thereto means an antibody having CLDN3 binding activity of usually 50% or less, preferably 30% or less, and more preferably 10% or less, compared to CLDN6 binding activity.

In addition, one of preferred embodiments of the anti-CLDN 6 antibody of the present invention is an antibody having a binding to CLDN6 and substantially no binding to CLDN 4. The amino acid sequence of CLDN4 is well known, for example, the amino acid sequence of human CLDN4 is described in GenBank accession No. NP-001296.1 (SEQ ID NO: 50). In the present invention, an antibody having CLDN6 bound thereto and having substantially no CLDN4 bound thereto means an antibody having CLDN4 binding activity of usually 50% or less, preferably 30% or less, and more preferably 10% or less, compared to CLDN6 binding activity.

In addition, one of preferred embodiments of the anti-CLDN 6 antibody of the present invention is an antibody having a binding to CLDN6 and substantially no binding to CLDN 1. The amino acid sequence of CLDN1 is well known, for example, the amino acid sequence of human CLDN1 is described in GenBank accession No. NP-066924.1 (SEQ ID NO: 51). In the present invention, an antibody having CLDN6 bound thereto and having substantially no CLDN1 bound thereto means an antibody having CLDN1 binding activity of usually 50% or less, preferably 30% or less, and more preferably 10% or less, compared to CLDN6 binding activity.

In the present invention, preferred examples of anti-CLDN 6 antibodies include antibodies that bind to human CLDN6 and do not substantially bind to human CLDN1 and human CLDN 3; an antibody that binds to human CLDN6 and does not substantially bind to human CLDN1, human CLDN3, and human CLDN 4; an antibody that binds to human CLDN6 and does not substantially bind to human CLDN1, human CLDN3, human CLDN4, and human CLDN 9.

Preferred examples of the anti-CLDN 6 antibody of the present invention include antibodies described in any one of the following (a) to (j):

The test antibody recognizes the same epitope as that recognized by a certain antibody, i.e., a common epitope, and can be confirmed by competition between the two epitopes. Competition between antibodies is detected by cross-blocking assay (cross-blocking assay) or the like. For example, a competitive ELISA assay is a preferred cross-blocking assay. Specifically, in the cross-blocking assay, the anti-CLDN 6 antibody of the present invention is added after pre-culturing CLDN6 protein coated on wells of a microtiter plate in the presence or absence of a candidate competitor antibody. The amount of anti-CLDN 6 antibody of the invention that binds to CLDN6 protein in a well is indirectly related to the binding capacity of a candidate competing antibody (test antibody) that competes for binding to the same epitope. That is, the greater the affinity of the test antibody for the same epitope, the lower the amount of binding of the anti-CLDN 6 antibody of the present invention to the hole coated with CLDN6 protein, and the higher the amount of binding of the test antibody to the hole coated with CLDN6 protein.

The amount of antibody bound to the wells can be readily determined by pre-labeling the antibody. For example, biotin-labeled antibodies can be assayed by using an avidin peroxidase conjugate and an appropriate matrix. Cross-blocking assays using enzymatic labels such as peroxidase are particularly referred to as competitive ELISA assays. The antibody may be labeled with other labels that can be detected or measured. Specifically, a radioactive label, a fluorescent label, or the like is known.

Furthermore, when the test antibody has a constant region from a species different from that of the anti-CLDN 6 antibody of the present invention, the amount of antibody bound to the well can also be measured by a labeled antibody recognizing the constant region of the antibody. Alternatively, if the antibodies are from the same species but the types are different, the amount of antibody bound to the well can be measured by recognizing the antibodies of the respective types.

A candidate competing antibody is an antibody that binds to substantially the same epitope as an anti-CLDN 6 antibody of the invention or an antibody that competes for binding to the same epitope if the candidate antibody is capable of blocking the binding of anti-CLDN 6 antibody by at least 20%, preferably at least 30%, more preferably at least 50%, compared to the binding activity obtained in a control assay performed in the absence of the candidate competing antibody.

Furthermore, in the present invention, 1 or more amino acids may be substituted, deleted, added, and/or inserted in the CDR sequences as long as they are functionally equivalent to the antibodies of (a) to (j) above. In the present invention, "functional equivalence" means equivalence of binding activity or cytotoxicity to CLDN 6. In the present invention, "equivalent" means that the antibody has an activity of at least 50%, preferably 70%, more preferably 90% or more, as compared with the above antibody. The upper limit of the activity is not particularly limited, and may have an activity higher than that of the above antibody. The binding activity and cytotoxicity can be measured by methods known to those skilled in the art, and for example, the methods described in examples can be used.

As Methods known to those skilled in the art for substitution, deletion, addition and/or insertion of amino Acids, for example, site-directed mutagenesis (Hashimoto-Gotoh, T.et al (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods enzymol.100, 468-500, Kramer, W.et al (1984) Nucleic Acids Res.12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods. enzymol.154, 350-367, Kunkel, TA (1985) Proc Natl AcSci USA.82, 488-492, Kunkel (1988) Methods enzymol.85, 2763-2766) and the like can be cited. In addition, amino acid variations can also occur in nature. As described above, the antibody of the present invention has an amino acid sequence in which 1 or more amino acids are varied in the amino acid sequence, and an antibody having an activity equivalent to that of the antibody is also included in the antibody of the present invention. It is considered that the number of variant amino acids in the above-mentioned variant is usually within 5 amino acids, preferably within 4 amino acids, and more preferably within 3 amino acids (for example, 1 or 2 amino acids) per one CDR.

The amino acid residue to be mutated is not particularly limited, but is preferably mutated to another amino acid residue having the property of an amino acid side chain retained. For example, based on the nature of the amino acid side chains, the following classifications are established:

a hydrophobic amino acid (A, I, L, M, F, P, W, Y, V);

a hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T);

an amino acid having an aliphatic side chain (G, A, V, L, I, P);

an amino acid having a hydroxyl-containing side chain (S, T, Y);

an amino acid having a sulfur atom-containing side chain (C, M);

having an amino acid (D, N, E, Q) comprising a carboxylic acid and an amide;

an amino acid having a base-containing side chain (R, K, H);

an amino acid having an aromatic-containing side chain (H, F, Y, W);

(the amino acids are indicated in parentheses by the single letter symbols).

It is known that a polypeptide having an amino acid sequence modified as described above maintains its biological activity by substitution, deletion, addition and/or substitution of 1 or more amino acid residues in the amino acid sequence (Mark, D.F.et al, Proc.Natl.Acad.Sci.USA (1984)81, 5662-. That is, in general, when amino acids classified into various groups are substituted for each other in an amino acid sequence constituting a certain polypeptide, there is a high possibility that the activity of the polypeptide is maintained.

Method for producing antibody

The anti-CLDN 6 antibody of the present invention can be obtained by a known method. As the anti-CLDN 6 antibody of the present invention, a monoclonal antibody derived from a mammal is particularly preferred. Monoclonal antibodies derived from mammals include monoclonal antibodies produced by hybridomas, monoclonal antibodies produced by hosts transformed with expression vectors containing antibody genes by genetic engineering methods, and the like.

Hybridomas that produce monoclonal antibodies can be produced by known techniques, for example, as described below. First, CLDN6 protein, cells expressing CLDN6, or a gene encoding CLDN6 was used as a sensitizing antigen, which was immunized according to a general immunization method. An immune cell obtained from an immunized animal is fused with a known parent cell by a conventional cell fusion method to obtain a hybridoma. Furthermore, a hybridoma producing an anti-CLDN 6 antibody can be selected by screening cells producing the target antibody from the hybridoma by a general screening method.

Specifically, the monoclonal antibody can be produced, for example, as follows. First, by expressing CLDN6 gene, CLDN6 protein can be obtained, which is used as a sensitizing antigen for obtaining antibodies. The base sequence of the human CLDN6 gene may use a sequence disclosed in GenBank accession No. NM _021195.3 (seq id No. 23) or GenBank accession No. NM _021195.4 (seq id No. 47), and the like. That is, after a gene sequence encoding CLDN6 is inserted into a known expression vector and an appropriate host cell is transformed, a target human CLDN6 protein can be purified from the host cell or culture supernatant by a known method. In addition, purified native CLDN6 protein may be used in the same way. The formation can be carried out by using a combination of 1 or more times of a plurality of types of chromatography such as ordinary ion chromatography and affinity chromatography. In addition, a fusion protein obtained by fusing a desired partial polypeptide of CLDN6 protein with a different polypeptide may also be used as an immunogen. For the preparation of a fusion protein as an immunogen, for example, an Fc fragment of an antibody, a peptide tag, or the like can be used. The vector for expressing the fusion protein can be prepared by in-frame fusion of genes encoding two or more desired polypeptide fragments and insertion of the fused genes into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning 2nd ed (Sambrook, J.et. al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab.Press, 1989).

The CLDN6 protein purified as shown above can be used as a sensitizing antigen for use in immunizing a mammal. In addition, partial peptides of CLDN6 may also be used as sensitizing antigens.

The mammal immunized with the sensitizing antigen is not particularly limited. In order to obtain a monoclonal antibody by the cell fusion method, it is preferable to select an immunized animal in view of compatibility with the parent cell used in cell fusion. In general, rodents are preferred as the immunized animals. Specifically, a mouse, rat, hamster, or rabbit may be used as the immunized animal. Furthermore, monkeys and the like may be used as the immunized animals.

The animal can be immunized by sensitizing antigen according to a known method. For example, as a general approach, a mammal may be immunized by intraperitoneal or subcutaneous injection of a sensitizing antigen. Specifically, the sensitizing antigen is administered to the mammal a plurality of times every 4 to 21 days. The sensitizing antigen can be diluted with PBS (Phosphate-Buffered Saline) or physiological Saline at an appropriate dilution ratio for immunization. Also, the sensitizing antigen may be administered with an adjuvant. For example, the antigen can be mixed with Freund's complete adjuvant (Freund's complete adjuvant) and emulsified to form a sensitized antigen. In addition, a suitable carrier may be used for immunization with the sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is preferable to immunize the antigen by binding the sensitizing antigen peptide to a carrier protein such as albumin or keyhole limpet hemocyanin (keyhole limpet hemocyanin).

After the mammal is immunized as described above and the increase in the amount of the desired antibody in the serum is confirmed, immune cells are collected from the mammal and cell fusion is performed. As the immune cell, spleen cells are particularly preferably used.

As the cells to be fused with the above immune cells, mammalian myeloma cells are used. Myeloma cells preferably have an appropriate selection marker for selection. The selection marker is a characteristic that it can survive (or cannot survive) under a specific culture condition. Known selectable markers include hypoxanthine guanine phosphoribosyl transferase kinase deletion (hereinafter abbreviated as HGPRT deletion) and thymidine kinase deletion (hereinafter abbreviated as TK deletion). HGPRT or TK-deleted cells have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter referred to as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium and die, but when fused with normal cells, can continue DNA synthesis using the salvage pathway (solution pathway) of normal cells, and therefore can proliferate in HAT selection medium.

HGPRT-deleted cells can be selected using a medium containing 6-mercaptoguanine, 8-azaguanine (hereinafter referred to as 8AG), while TK-deleted cells can be selected using a medium containing 5' -bromodeoxyuridine. Normal cells die by incorporating these pyrimidine analogs into DNA, but cells lacking these enzymes can survive in selective media because they do not incorporate the pyrimidine analogs. Another selection marker, called G418 resistance, confers resistance to 2-deoxystreptomycin (2-deoxystreptamine) antibiotics (gentamicin analogs) due to the presence of a neomycin resistance gene. A variety of myeloma cells suitable for cell fusion are known. For example, cells such as P3 (P3X 63Ag8.653) (J.Immunol. (1979)123, 1548-1550), P3X63 Ag8U.1(Current toppcs in Microbiology and Immunology (1978)81, 1-7), NS-1(Kohler, G.and Milstein, C.Eur.J.Immunol. (1976)6, 511-519), MPC-11(Margulies, D.H.et., Cell (1976)8, 405-415), SP2/0(Shulman, M.et., Nature (1978)276, 269-270), F0 (St.Groth, S.F.Immunol. (1980)35, 1-21), Trdowns (Tr.194, 277-269, J.Immunol.35, 1-21), myeloma cells (11, 11-11, D.H.et., myeloma cells, 11, 9, 11-11, 11-11, and so on.

The above-mentioned cell fusion of the immunocyte and the myeloma cell can be carried out by a known method, for example, a method of Kohler and Milstein et al (Kohler.G.and Milstein, C., MethodsEnzymol (1981)73, 3-46).

More specifically, for example, the cell fusion can be carried out in a normal nutrient medium in the presence of a cell fusion promoter. Examples of fusion promoters that can be used include polyethylene glycol (PEG) and sendai virus (HVJ). If desired, an auxiliary agent such as dimethyl sulfoxide may be added to improve the fusion efficiency.

The ratio of the immune cells to the myeloma cells to be used can be arbitrarily set. For example, it is preferable to make the immune cells 1 to 10 times as large as the myeloma cells. The culture medium used for the cell fusion may be, for example, an RPMI1640 culture medium or an MEM culture medium suitable for the proliferation of the myeloma cell line, or a common culture medium used for such cell culture. Furthermore, serum replacement (serum replacement) such as Fetal Calf Serum (FCS) may be added to the culture medium.

In the cell fusion, predetermined amounts of the above immune cells and myeloma cells are mixed well in the above culture solution, and a PEG solution heated to about 37 ℃ in advance is mixed to form target fused cells (hybridomas). In the cell fusion method, for example, PEG having an average molecular weight of about 1000 to 6000 may be added at a concentration of usually 30 to 60% (w/v). Then, the appropriate culture medium as exemplified above is added, centrifuged, and the supernatant is removed, and the above operation is repeated to remove the cell fusion agent and the like which are not preferable in the culture of hybridomas.

The hybridoma obtained as described above can be selected using a selection medium suitable for a selection marker possessed by myeloma cells used for cell fusion. For example, HGPRT or TK-deleted cells can be selected by culturing in HAT medium (medium containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully undergone cell fusion with normal cells can be selectively proliferated in HAT culture medium. The culture using the HAT medium is continued for a time sufficient to cause the death of cells other than the target hybridoma (non-fused cells). Specifically, in general, a target hybridoma can be selected by culturing for several days to several weeks. Then, by performing a commonly used limiting dilution method, it is possible to screen and monoclonal hybridomas producing the target antibody. Alternatively, an antibody recognizing CLDN6 can be produced according to the method described in International publication WO 03/104453.

The screening and the monoclonal screening of the target antibody are preferably carried out by a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to a carrier such as a bead made of polystyrene or a commercially available 96-well microtiter plate, and reacted with a culture supernatant of hybridoma. Subsequently, the carrier is washed, and then an enzyme-labeled secondary antibody or the like is reacted. If the culture supernatant contains the target antibody that reacts with the sensitizing antigen, the secondary antibody is bound to the carrier by this antibody. Finally, by detecting the secondary antibody bound to the carrier, it is possible to determine whether the target antibody is present in the culture supernatant. Hybridomas that produce a desired antibody having antigen-binding ability can be cloned by a limiting dilution method or the like. In this case, a protein for immunization and substantially the same CLDN6 protein can be preferably used as the antigen. For example, an oligopeptide including the extracellular domain of CLDN6, or a part of the amino acid sequence constituting the region may be used as an antigen.

In addition to the method of obtaining the above-mentioned hybridoma by immunizing an animal other than a human with an antigen, a target antibody can be obtained by sensitizing human lymphocytes with an antigen. Specifically, human lymphocytes were first sensitized in vitro with CLDN6 protein. The immunosensitized lymphocytes are then fused with an appropriate fusion partner (fusion partner). As the fusion partner, for example, myeloma cells derived from human and having the ability to divide permanently can be used (see Japanese patent publication No. Hei 1-59878). The anti-CLDN 6 antibody obtained by this method is a human antibody having binding activity to CLDN6 protein.

Furthermore, an anti-CLDN 6 human antibody can also be obtained by administering CLDN6 protein as an antigen to a transgenic animal having the entire gene profile of human antibody genes. Antibody-producing cells of an immunized animal can be immortalized by fusion with an appropriate fusion partner cell or treatment with Epstein-Barr virus (EB virus) infection or the like. Human antibodies to CLDN6 protein can be isolated from immortalized cells obtained as described above (see International publications WO94/25585, WO93/12227, WO92/03918, WO 94/02602). Furthermore, by cloning immortalized cells, it is also possible to clone cells that produce antibodies specific to the desired reaction. When the transgenic animal is used as an immunized animal, the immune system of the animal recognizes human CLDN6 as a foreign body. Therefore, human antibodies against human CLDN6 can be easily obtained. The monoclonal antibody-producing hybridoma prepared as described above can be subcultured in a conventional culture medium. In addition, the hybridoma can be stored in liquid nitrogen for a long period of time.

In addition, a technique of obtaining a human antibody by panning using a human antibody library is also known. For example, a human antibody can be expressed on the surface of a phage by phage display method using the V region of a human antibody as a single-chain antibody (scFv), and a phage that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After the DNA sequence of the scFv that binds to the antigen is determined, the V region sequence is fused in frame with the sequence of the desired human antibody C region, and then an appropriate expression vector is inserted, whereby an expression vector can be prepared. The human antibody can be obtained by introducing the expression vector into the above-mentioned preferred expression cells and expressing a gene encoding the human antibody. The above-mentioned methods are known (International publications WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO 95/15388).

The hybridoma is cultured by a usual method, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma may be administered to a mammal compatible with the hybridoma to proliferate the hybridoma, and the monoclonal antibody may be obtained from ascites of the mammal. The former method is suitable for obtaining an antibody with high purity.

Recombinant antibodies

The antibody of the present invention may be a recombinant antibody that can be produced using an antibody gene cloned from an antibody-producing cell. The antibody gene thus cloned can be introduced into a suitable vector and introduced into a host to express the antibody. Methods for the isolation of antibody genes, introduction into vectors, and transformation of host cells have been established (see, e.g., Vandamme, a.m.et al, eur.j.biochem. (1990)192, 767-775).

For example, cDNA encoding the variable region (V region) of an anti-CLDN 6 antibody can be obtained from hybridoma cells producing an anti-CLDN 6 antibody. For this purpose, usually total RNA is first extracted from the hybridomas. As a method for extracting mRNA from cells, for example, guanidine ultracentrifugation (Chirgwin, J.M.et. al., Biochemistry (1979)18, 5294-.

The extracted mRNA can be purified using an mRNA Purification Kit (manufactured by GEHealthcare Biosciences) or the like. Alternatively, a Kit for directly extracting total mRNA from cells, such as QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Biosciences), is also commercially available. Total mRNA can also be obtained from hybridomas using the kit described above. A cDNA encoding the V region of the antibody can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using, for example, the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Biochemical industries, Ltd.). In addition, for the synthesis and amplification of cDNA, 5 '-Ampli FINDER RACE Kit (manufactured by Clontech) and 5' -RACE method using PCR (Frohman, M.A.et. al., Proc.Natl.Acad.Sci.USA (1988)85, 8998-. Furthermore, in the above-mentioned cDNA synthesis process, the following appropriate restriction enzyme sites may be introduced at both ends of the cDNA.

The target cDNA fragment was purified from the resulting PCR product, and then ligated with a vector DNA. After the recombinant vector is prepared as described above and introduced into Escherichia coli or the like to select a colony, a desired recombinant vector can be prepared from the Escherichia coli forming the colony. Next, it can be confirmed by a known method, for example, the dideoxyribonucleic acid chain termination method, whether or not the recombinant vector has the nucleotide sequence of the cDNA of interest.

In order to obtain a gene encoding a variable region, a PCR method using a primer for variable region gene amplification may be used. First, cDNA is synthesized using the extracted mRNA as a template to obtain a cDNA library. For convenience, commercially available kits were used for the synthesis of cDNA libraries. In fact, since only a very small amount of mRNA is obtained from a small number of cells, the yield is low when the mRNA is directly purified. Therefore, usually, vector RNA which is clearly known to contain no antibody gene is added and then purified. Alternatively, when a certain amount of RNA can be extracted, even only RNA from antibody-producing cells can be efficiently extracted. For example, when RNA is extracted from 10 or more, or 30 or more, preferably 50 or more antibody-producing cells, it may not be necessary to add carrier RNA.

The antibody gene was amplified by PCR using the obtained cDNA library as a template. Primers for amplifying antibody genes by the PCR method are well known. For example, primers for amplifying human antibody genes can be designed according to the disclosure of the article (J.mol.biol. (1991)222, 581-597) and the like. The primer has a different base sequence depending on the subclass of immunoglobulin. Therefore, when a cDNA library of unknown subclass is used as a template, it is necessary to consider all possibilities for performing the PCR method.

Specifically, for example, in order to obtain a gene encoding human IgG, primers capable of amplifying genes encoding γ 1 to γ 5 as a heavy chain, a κ chain and a λ chain as a light chain may be used. For amplifying the variable region gene of IgG, a primer annealing to a portion corresponding to the hinge region is generally used as the 3' -side primer. On the other hand, primers suitable for each subclass can be used as primers on the 5' side.

PCR products generated from the primers for gene amplification of each subclass of heavy and light chains form independent gene pools, respectively. Using the synthetic gene library described above, immunoglobulins comprising a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin to CLDN6 was used as an index, and a target antibody could be screened.

As a method for screening an antibody using a binding activity as an index, a panning method using a phage vector can be used. When the antibody gene is obtained as a gene library of the subclasses of heavy and light chains as described above, a screening method using a phage vector is advantageous. The genes encoding the variable regions of the heavy and light chains may be joined by appropriate linker sequences to form a single chain fv (scFv). If a gene encoding scFv is inserted into a phage vector, a phage expressing scFv on the surface can be obtained. When this phage is brought into contact with a target antigen and the phage bound to the antigen is recovered, DNA encoding scFv having a target binding activity can be recovered. By repeating this operation as necessary, the scFv having the target binding activity can be concentrated.

After obtaining a cDNA encoding the V region of the target anti-CLDN 6 antibody, the cDNA was digested with restriction enzymes recognizing restriction enzyme sites inserted into both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that are less likely to occur in the nucleotide sequences constituting the antibody genes. Furthermore, in order to insert 1 copy of the digested fragment into the vector in the correct orientation, it is preferable to provide a restriction enzyme for the sticky ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-CLDN 6 antibody digested as described above into an appropriate expression vector. In this case, a gene encoding the constant region (C region) of the antibody and a gene encoding the V region are fused in frame to obtain a full-length antibody.

To produce the anti-CLDN 6 antibody of the present invention, the antibody gene may be incorporated into an expression vector to be expressed under the control of an expression control region. The expression control region for expressing the antibody includes, for example, an enhancer and a promoter. Then, by transforming an appropriate host cell with this expression vector, a recombinant cell expressing a DNA encoding an anti-CLDN 6 antibody can be obtained.

When the antibody gene is expressed, DNAs encoding the heavy chain (H chain) and the light chain (L chain) of the antibody may be incorporated into different expression vectors. Antibody molecules having both H chains and L chains can be expressed by simultaneous transformation (co-transfection) of vectors incorporating both H chains and L chains in the same host cell. Alternatively, the DNA encoding the H chain and the L chain may be incorporated into a single expression vector to transform the host cell (see International publication WO 94/11523).

Various combinations of hosts and expression vectors are known for producing antibodies by temporarily isolating antibody genes and introducing them into appropriate hosts. The above expression systems can be used in the present invention. When eukaryotic cells are used as the host, animal cells, plant cells, or fungal cells can be used. Specifically, examples of the animal cell that can be used in the present invention include a mammalian cell (CHO, COS, myeloma, bhk (baby hamster kidney), Hela, Vero, etc.), an amphibian cell (Xenopus oocytes, etc.), an insect cell (sf9, sf21, Tn5, etc.), and the like.

Alternatively, an expression system of an antibody gene produced by a cell of the genus Nicotiana such as Nicotiana tabacum is known as a plant cell. Transformation of plant cells cultured from callus may be used.

Further, as the fungal cell, yeast (genus Saccharomyces such as Saccharomyces cerevisiae, genus Pichia such as Pichia pastoris), filamentous fungi (genus Aspergillus such as Aspergillus niger) and the like (genus Aspergillus) can be used.

Alternatively, expression systems using antibody genes from prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli (E.coli) and Bacillus subtilis can be used in the present invention.

When mammalian cells are used, an expression vector can be constructed which functionally binds to a commonly used effective promoter, an expressed antibody gene, and a downstream polyA signal on the 3' side thereof. Examples of the promoter/enhancer include human cytomegalovirus immediate early promoter/enhancer (human cytomegalovirus immediate early promoter/enhancer).

In addition, as a promoter/enhancer that can be used for expression of the antibody of the present invention, a viral promoter/enhancer, a promoter/enhancer derived from mammalian cells such as human elongation factor 1 α (HEF1 α), or the like can be mentioned. Examples of viruses that can utilize promoters and enhancers include retroviruses, polyomaviruses, adenoviruses, and monkey kidney virus 40(SV 40).

When the SV40 promoter/enhancer is used, the method of Mullgan et al (Nature (1979)277, 108) can be used. In addition, the HEF1 α promoter/enhancer can be readily used for target gene expression according to the method of Mizushima et al (Nucleic Acids Res. (1990)18, 5322).

In the case of E.coli, the gene can be expressed by functionally combining a commonly used effective promoter, a signal sequence for antibody secretion, and an expressed antibody gene. Examples of the promoter include lacZ promoter and araB promoter. When the lacZ promoter is used, the method of Ward et al (Nature (1989)341, 544-242546; FASEBJ. (1992)6, 2422-2427) can be employed. Alternatively, the araB promoter can be used for expression of a target gene by the method of Better et al (Science (1988)240, 1041-1043).

As a signal sequence for antibody secretion, pelB signal sequence can be used when produced in the periplasm of escherichia coli (Lei, s.p.et al, j.bacteriol. (1987)169, 4379). Then, after the antibody produced in the periplasm is separated, a protein denaturing agent such as guanidine hydrochloride of urea is used, thereby reorganizing (refolding) the structure of the antibody to have a desired binding activity.

As the origin of replication of the inserted expression vector, those derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), etc. can be used. Furthermore, in order to amplify the gene copy number in the host cell system, a selection marker may be inserted into the expression vector. Specifically, selection markers such as aminoglycoside transferase (APH) gene, Thymidine Kinase (TK) gene, E.coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase (dhfr) gene can be used.

The above expression vector is introduced into a host cell, and the transformed host cell is cultured in vitro or in vivo to produce the target antibody. The host cell can be cultured according to a known method. For example, DMEM, MEM, RPMI1640, IMDM may be used as the culture medium, and a serum supplement such as Fetal Calf Serum (FCS) may be used in combination.

The antibody expressed and produced as described above can be purified by a known method used for the purification of a general protein, alone or in a suitable combination. For example, Antibodies can be isolated and purified by appropriately selecting and combining an affinity column such as a protein A column, a chromatography column, filtration, ultrafiltration, salting out, dialysis, and the like (Antibodies A Laboratory Manual. Ed Harbor, David Lane, Cold Spring Harbor Laboratory, 1988).

In addition, in the production of recombinant antibodies, transgenic animals may be used in addition to the above host cells. That is, the antibody can be obtained from an animal into which a gene encoding the target antibody has been introduced. For example, an antibody gene is inserted in-frame into a gene encoding a protein inherently produced in milk to construct a fusion gene. As the protein secreted in milk, for example, goat β casein or the like can be used. The DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into an embryo of a goat, and the injected embryo is introduced into a female goat. A transgenic goat (or its offspring) is produced from the goat which received the embryo, and the desired antibody can be obtained from the milk produced from the transgenic goat as a fusion protein with a milk protein. In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be appropriately administered to the transgenic goat (Ebert, K.M.et. al., Bio/Technology (1994)12, 699-702).

Sugar chain modificationDecorative antibody

The anti-CLDN 6 antibody of the present invention may also be an antibody modified with a sugar chain. It is known that the cytotoxicity of an antibody can be enhanced by modifying the sugar chain of the antibody.

Examples of the antibody modified with a sugar chain include an antibody modified by glycosylation (e.g., WO 99/54342), an antibody lacking fucose attached to a sugar chain (e.g., WO00/61739, WO 02/31140, WO2006/067847, WO2006/067913), and an antibody having a sugar chain containing a truncated GlcNAc (e.g., WO 02/79255).

Preferred examples of the sugar chain-modified antibody of the present invention include fucose-deficient antibodies. The sugar chain bound to the antibody includes an N-glycosyl-binding sugar chain bound to the N atom of the side chain of asparagine of the antibody molecule and an O-glycosyl-binding sugar chain bound to the hydroxyl group of the side chain of serine or threonine of the antibody molecule, and in the present invention, the presence or absence of fucose is related to the N-glycosyl-binding sugar chain.

In the present invention, the term "fucose-deficient antibody" means that 20% or more, preferably 50% or more, more preferably 70% or more, and still more preferably 90% or more of the N-glycosyl-linked sugar chains of the antibody in the composition are fucose-deficient.

Fucose-deficient antibodies can be produced by methods known to those skilled in the art, and can be produced, for example, by expressing antibodies in host cells that do not have or have a low ability to attach alpha-1, 6core fucose (alpha-1, 6 corefusose). Host cells having no or low fucose attachment ability are not particularly limited, and examples thereof include rat myeloma YB2/3 HL.P2.G11.1698g.20 cells (YB 2/0 cells for short) (deposited as ATCC CRL 1662), FTVIII knockout CHO cells (WO 02/31140), Lec13 cells (WO 03/035835), and fucose transporter deficient cells (WO2006/067847, WO 2006/067913).

The sugar chain can be analyzed by a method known to those skilled in the art. For example, N-glycosidase F (Roche) or the like is allowed to act on an antibody to separate a sugar chain from the antibody. Then, after desalting by solid phase extraction using cellulose column (Shimizu Y.et al, Carbohydrate Research 332(2001), 381-388), concentration to dry, fluorescence labeling with 2-aminopyridine was performed (Kondo A.et al, Agricultural and Biological Chemistry 54: 8(1990), 2169-2170). The resulting PA-modified sugar chain was subjected to reagent removal by solid phase extraction using a cellulose column, and then concentrated by centrifugation to form a purified PA-modified sugar chain. Then, reversed-phase HPLC analysis using ODS column was performed, whereby the measurement was possible. After the PA-formed sugar chains are prepared, the two-dimensional mapping may be performed by combining the reversed-phase HPLC analysis using an ODS column and the normal-phase HPLC analysis using an amine column.

Chimeric and humanized antibodies

As another preferred embodiment of the antibody of the present invention, a chimeric antibody or a humanized antibody can be mentioned. A chimeric antibody is an antibody in which regions derived from different sources are linked to each other. Generally, a chimeric antibody is composed of a V region of an antibody derived from an animal other than a human and a C region derived from a human antibody. For example, an antibody comprising the variable regions of the heavy chain and the light chain of a mouse antibody and the constant regions of the heavy chain and the light chain of a human antibody is a mouse-human xenochimeric antibody.

In contrast, a humanized antibody is composed of Complementarity Determining Regions (CDRs) of an antibody derived from an animal other than a human, Framework Regions (FRs) derived from a human antibody, and C regions derived from a human antibody. Since the humanized antibody has low antigenicity in humans, it is useful as an active ingredient of the therapeutic agent of the present invention. Humanized antibodies are also known as reshaped (reshaped) human antibodies. Specifically, humanized antibodies and the like in which CDRs of an animal other than a human, for example, a mouse antibody are grafted onto a human antibody are known. General genetic recombination methods for obtaining humanized antibodies are also known.

Specifically, as a method for grafting CDRs of a mouse antibody into human FRs, for example, overlap Extension pcr (overlap Extension pcr) is known. In overlap extension PCR, a base sequence encoding a CDR of a mouse antibody to be grafted is attached to a primer for synthesizing a FR of a human antibody. Primers were prepared for each of the 4 FRs. In general, in grafting mouse CDRs into human FRs, it is advantageous to select human FRs having high homology with mouse FRs for maintaining the CDR functions. That is, it is generally preferable to use a human FR comprising an amino acid sequence having high homology with the amino acid sequence of the FR adjacent to the mouse CDR to be grafted.

The joined nucleotide sequences are designed to be connected to each other in frame. Human FRs were synthesized separately using each primer. As a result, DNA encoding mouse CDRs was attached to each FR. The base sequences of each product encoding the mouse CDR were designed to overlap each other. Next, a product is synthesized using a human antibody gene as a template, and overlapping CDR portions of the product are annealed to each other, thereby carrying out a complementary strand synthesis reaction. Using this reaction, human FRs are linked by the sequence of mouse CDRs.

Finally, the 3 CDR and 4 FR linked V region genes were amplified over their full length by primers that anneal at the 5 'and 3' ends and attach appropriate restriction enzyme recognition sequences. The DNA obtained as described above and a DNA encoding a human antibody C region are inserted into an expression vector and fused in frame, whereby a human antibody expression vector can be prepared. After the recombinant vector is introduced into a host and recombinant cells are established, the recombinant cells are cultured, and the humanized antibody is produced in the culture of the cultured cells by expressing a DNA encoding the humanized antibody (see European patent publication EP239400, International publication WO 96/02576).

By qualitatively or quantitatively measuring and evaluating the binding activity of the humanized antibody produced as described above to the antigen, it is possible to appropriately select the human antibody FR in which the CDR forms a good antigen-binding site when the antibody is bound by the CDR. If necessary, the amino acid residues of the FR may be substituted so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site. For example, a mutation of an amino acid sequence can be introduced into an FR by applying a PCR method used for grafting a mouse CDR into a human FR. Specifically, a mutation of a partial base sequence can be introduced into a primer annealing FR. Mutations of the nucleotide sequence were introduced into the FRs synthesized using the primers. The binding activity of a mutant antibody having substituted amino acids to an antigen is measured and evaluated by the above-described method, whereby a mutant FR sequence having desired properties can be selected (Sato, K.et al., Cancer Res, 1993, 53, 851-856).

As the C region of the humanized antibody, a human antibody C region can be used, for example, in the H chain, C γ 1, C γ 2, C γ 3, C γ 4, C μ, C δ, C α 1, C α 2, C ε and the like can be used, and in the L chain, C κ, C λ and the like can be used. In addition, the C region of a human antibody may be modified in order to improve the stability of the antibody or its production. The human antibody used for humanization may be any hetero-human antibody such as IgG, IgM, IgA, IgE, IgD, etc., but IgG is preferably used in the present invention. As IgG, IgG1, IgG2, IgG3, IgG4, and the like can be used.

After the humanized antibody is prepared, amino acids in the variable region (for example, CDR and FR) or the constant region may be substituted with other amino acids, deleted, added and/or inserted, and the humanized antibody of the present invention may include a humanized antibody obtained by substituting the above amino acids.

Bivalent antibody, low-molecular-weight antibody, and modified antibody

The anti-CLDN 6 antibody of the present invention may bind to CLDN6 protein, and includes not only bivalent antibodies represented by IgG but also monovalent antibodies or multivalent antibodies represented by IgM. The multivalent antibody of the present invention includes multivalent antibodies having all the same antigen binding sites, or multivalent antibodies having partially or all different antigen binding sites.

The antibody of the present invention is not limited to a full-length antibody, and may be a low-molecular antibody or a modified product thereof as long as it binds to CLDN6 protein.

The low molecular weight antibody includes an antibody fragment in which a part of a full-length antibody (e.g., a whole IgG or the like) is deleted. So long as it has the ability to bind to CLDN6 antigen, partial deletion of the antibody molecule is allowed. The antibody fragment of the present invention preferably contains either or both of a heavy chain variable region (VH) and a light chain variable region (VL). The amino acid sequence of VH or VL may include substitutions, deletions, additions and/or insertions. Further, any one or both of VH and VL may be partially deleted as long as it has a binding ability to CLDN6 antigen. In addition, the variable region may be chimeric or humanized. Specific examples of the antibody fragment include Fab, Fab ', F (ab') 2, Fv, and the like. Specific examples of the antibody having a reduced molecular weight include Fab, Fab ', F (ab') 2, Fv, scFv (single chain Fv), Diabody (Diabody), sc (Fv)2 (single chain (Fv)2), and the like. Multimers (e.g., dimers, trimers, tetramers, polymers) of the above-described antibodies are also included in the low molecular weight antibodies of the present invention.

The fragment of the antibody can be obtained by treating the antibody with an enzyme to produce an antibody fragment. As an enzyme for producing an antibody fragment, for example, papain, pepsin, plasmin, or the like is known. Alternatively, genes encoding the above antibody fragments can be constructed and, upon introduction into an expression vector, expressed in an appropriate host cell (see, e.g., Co, M.S. et al, J.Immunol, (1994)152, 2968-2976, Better, M. & Horwitz, A.H.methods in Enzymology (1989)178, 476-496, Plueckthun, A. & Skerra, A.methods in Enzymology (1989)178, 476-496, Lamori, E., Methods in Enzymology (1989)121, 652-663, Rousseaux, J.et al, Methods in Enzymology (1989)121, 663-669, 137-rd, R.E.et al, TISSeaux, ECH (1991) 132).

A diabody is a bivalent (bivalent) antibody fragment constructed by gene fusion (Holliger P et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993), EP404,097, WO93/11161, etc.). Diabodies are dimers consisting of 2 polypeptide chains. Usually, the polypeptide chains constituting the dimer are each bound via a linker to VL and VH in the same chain. Generally, the linker in a diabody is short in the region where VL and VH cannot bind to each other. Specifically, the linker is composed of about 5 amino acid residues, for example. Thus, VL and VH, encoded on the same polypeptide chain, cannot form single chain variable fragments, and form dimers with other single chain variable fragments. As a result, the diabody has 2 antigen-binding sites.

scFv is obtained by linking the H chain V region and the L chain V region of an antibody. In scFv, the H chain V region and the L chain V region are linked by a linker, preferably a peptide linker (Huston, J.S.et al, Proc.Natl.Acad.Sci.U.S.A, 1988, 85, 5879-. The H chain V region and the L chain V region in the scFv are described as antibodies in the present specification, but may be derived from any antibodies. The peptide linker connecting the V regions is not particularly limited. For example, any single-chain peptide containing about 3 to 25 residues can be used as a linker.

sc (fv)2 is a low molecular weight antibody in which 2 VH and 2 VL are bound to form a single chain by a linker or the like (Hudson et al, J immunol. methods 1999; 231: 177-189). sc (fv)2 can be produced by, for example, linking an scFv to a linker.

The antibody of the present invention may be used in the form of an antibody-modified product that is bound to various molecules such as polyethylene glycol (PEG). The modified antibody can be obtained by chemically modifying the antibody of the present invention. Methods for modifying antibodies are well established in the art.

Furthermore, the antibody of the present invention may be a bispecific antibody (bispecific antibody). A bispecific antibody is an antibody having variable regions that recognize different epitopes in the same antibody molecule, and the epitopes may be present in different molecules or may be present in the same molecule. That is, in the present invention, the bispecific antibody may have antigen binding sites that recognize different epitopes on CLDN6 protein. In the bispecific antibody, 2 molecules of the antibody can be bound to 1 molecule of CLDN 6. As a result, stronger cytotoxicity can be expected. The "antibody" in the present invention also includes the above-mentioned antibodies.

In the present invention, bispecific antibodies recognizing antigens other than CLDN6 may be combined. For example, bispecific antibodies recognizing an antigen different from CLDN6, which is the same antigen specifically expressed on the cell surface of target cancer cells as CLDN6, may be combined.

Methods for making bispecific antibodies are well known. For example, bispecific antibodies can be prepared by binding 2 antibodies recognizing different antigens. The antibody to be bound may be 1/2 molecules having an H chain and an L chain, or 1/4 molecules having only an H chain. Alternatively, a bispecific antibody-producing fused cell can be prepared by fusing hybridomas producing different monoclonal antibodies. Further, bispecific antibodies can be produced by genetic engineering methods.

Pharmaceutical composition

The present invention provides a pharmaceutical composition comprising the above-mentioned anti-CLDN 6 antibody as an active ingredient. The present invention also relates to an anticancer agent containing the anti-CLDN 6 antibody as an active ingredient. The anticancer agent of the present invention is preferably administered to a subject suffering from cancer or a subject who may have relapses.

In addition, the present invention provides a method for preventing or treating cancer, comprising the step of administering an anti-CLDN 6 antibody to a subject to be treated, in addition to an anti-cancer agent containing an anti-CLDN 6 antibody as an active ingredient, or the use of an anti-CLDN 6 antibody in the manufacture of an anti-cancer agent.

The type of cancer to be treated with the anticancer agent of the present invention is not particularly limited, and cancer in which CLDN6 protein is expressed is generally preferably lung adenocarcinoma, stomach cancer or ovarian cancer. In addition, the cancer to be treated by the anticancer agent of the present invention is not particularly limited, but is preferably a cancer highly expressing CLDN6 protein.

In the present invention, the phrase "comprising an anti-CLDN 6 antibody as an active ingredient" means that the antibody having the anti-CLDN 6 as a main active ingredient is contained, and the content of the monoclonal antibody is not limited.

In addition, the pharmaceutical composition, cytostatic agent or anticancer agent of the present invention may contain a plurality of antibodies as necessary. For example, by making a mixture of multiple anti-CLDN 6 antibodies, it may be possible to enhance cytotoxicity to cells expressing CLDN 6. Alternatively, in addition to the anti-CLDN 6 antibody, the therapeutic effect can be improved by combining with an antibody recognizing another tumor-associated antigen.

The pharmaceutical composition, cytostatic agent or anticancer agent of the present invention may be administered to a patient by any of oral administration and non-oral administration. Non-oral administration is preferred. Specific examples of the administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration. As an example of administration by injection, the pharmaceutical composition of the present invention can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. In addition, an appropriate administration method can be selected according to the age and symptoms of the patient. As the dose, for example, the dose is selected in the range of 0.0001mg to 1000mg per 1kg body weight per one administration. Alternatively, for example, the amount to be administered is selected in the range of 0.001 to 100000mg/body per patient. However, the pharmaceutical composition of the present invention is not limited to the above dosage.

The Pharmaceutical composition of the present invention can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, last edition, Mark Publishing Company, Easton, U.S. A), and can also contain a carrier or an additive acceptable for the drug. Examples of the surfactant include a surfactant, an excipient, a colorant, a perfume, a preservative, a stabilizer, a buffer, a suspending agent, an isotonic agent, a binder, a disintegrating agent, a lubricant, a fluidity enhancer, a flavoring agent, and the like. Further, the present invention is not limited to this, and other commonly used carriers may be used as appropriate. Specific examples of the carrier include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate (polyvinyl acetate), polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethylcellulose, corn starch, inorganic salts, and the like.

In addition, the present invention provides a method of disrupting a CLDN 6-expressing cell or inhibiting cell proliferation by contacting a CLDN 6-expressing cell with an anti-CLDN 6 antibody. anti-CLDN 6 antibodies were as described above. The cell to which the anti-CLDN 6 antibody binds is not particularly limited as long as it is a cell expressing CLDN 6. Preferred CLDN 6-expressing cells of the invention are cancer cells. Preferred cancer cells include lung adenocarcinoma cells, stomach cancer cells, ovarian cancer cells, and the like.

In the present invention, "contacting" may be performed in vitro or in vivo. For example, the contacting is performed by adding an antibody to a culture solution of CLDN 6-expressing cells cultured in a test tube. In this case, the shape of the antibody to be added may be appropriately a solution or a solid obtained by freeze drying or the like. When added as an aqueous solution, the solution may be an aqueous solution containing only the antibody, or may be a solution containing, for example, the above-mentioned surfactant, excipient, colorant, perfume, preservative, stabilizer, buffer, suspending agent, isotonic agent, binder, disintegrant, lubricant, fluidity promoter, flavoring agent, and the like. The concentration to be added is not particularly limited, but is preferably in the range of 1pg/ml to 1g/ml, more preferably 1ng/ml to 1mg/ml, and still more preferably 1. mu.g/ml to 1mg/ml, as the final concentration in the culture solution.

In addition, in the present invention, "contacting" may also be performed in other embodiments by administering to a non-human animal in which cells expressing CLDN6 are transplanted in vivo, or an animal inherently having cancer cells expressing CLDN 6. The administration method may be carried out by any of oral administration and non-oral administration. Particularly preferred is a method of administration by parenteral administration, and specific examples of the method of administration include injection administration, nasal administration, transpulmonary administration, and transdermal administration. Examples of the administration by injection include systemic or local administration of the pharmaceutical composition of the present invention, such as intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, as well as cell growth inhibitor and anticancer agent. In addition, an appropriate administration method can be selected according to the age and symptoms of the animal to be tested. When the antibody is administered as an aqueous solution, the antibody may be simply contained in the aqueous solution, or may be contained in a solution containing, for example, the above-mentioned surfactant, excipient, colorant, perfume, preservative, stabilizer, buffer, suspending agent, isotonic agent, binder, disintegrating agent, lubricant, fluidity promoter, taste corrigent, and the like. As the dose, for example, the dose can be selected in the range of 0.0001mg to 1000mg per 1kg body weight per administration. Alternatively, for example, the amount to be administered is selected in the range of 0.001 to 100000mg/body per patient. However, the amount of the antibody of the present invention to be administered is not limited to the above amount.

Diagnostic method

Further, the present invention provides a method for diagnosing cancer using the anti-CLDN 6 antibody. The cancer diagnosed by the method of the present invention is not particularly limited as long as CLDN6 is expressed, but is preferably lung adenocarcinoma, gastric cancer, or ovarian cancer.

The diagnostic method of the present invention may be carried out in vitro or in vivo, but is preferably carried out in vitro.

The method for diagnosing cancer using the anti-CLDN 6 antibody of the present invention is, for example, a method comprising the following steps.

(a) A step of providing a sample taken from a subject;

(b) detecting the CLDN6 protein contained in the sample of (a).

In the present invention, the term "detection" includes quantitative or qualitative detection. Examples of the qualitative detection include a measurement of the presence or absence of CLDN6 protein, a measurement of the presence or absence of a certain amount or more of CLDN6 protein, and a measurement of comparing the amount of CLDN6 protein with other samples (e.g., control samples). The quantitative detection includes, for example, measurement of the concentration of CLDN6 protein, measurement of the amount of CLDN6 protein, and the like.

The test sample of the present invention is not particularly limited as long as it is a sample that may contain CLDN6 protein. Specifically, a sample collected from the body of a living body such as a mammal is preferable. More preferably, the sample is a sample taken from a human body. Specific examples of the test sample include blood, interstitial fluid (interstitial fluid), plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph fluid, saliva, urine, and tissue. The sample is preferably a sample obtained from a test sample such as a tissue or cell-immobilized specimen or a cell culture solution collected from a living body.

Detection of CLDN6 protein may be performed by methods known to those skilled in the art, and may be performed, for example, by Radioimmunoassay (RIA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), Luminescence Immunoassay (LIA), Immunoprecipitation (IP), immunoturbidimetry (TIA), Western Blotting (WB), Immunohistochemistry (IHC), immunodiffusion (SRID), and the like.

In the present invention, when CLDN6 protein is detected (for example, when CLDN6 protein is contained in a test sample more than a control sample, when CLDN6 protein is contained in a test sample more than a certain amount, or the like), it can be determined as cancer or the possibility of cancer is high.

Examples

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[ example 1]Expression analysis of human CLDN6 mRNA Using human Exon 1.0ST Array

In order to elucidate the expression distribution of human CLDN6 mRNA in clinical cancers, cancer cell lines, and various normal organs, expression analysis was performed using human Exon 1.0ST Array (Affymetrix) originally developed for splice variant analysis. The advantage of performing expression analysis using the human Exon 1.0ST array is that, compared with the conventional Affymetrix expression array having only one probe set on the 3' side of each gene, at least one probe set is set for each Exon of the gene in the human Exon 1.0ST array, and therefore, when expression analysis of each gene is performed using this array, expression data of a plurality of probe sets can be obtained for each gene, and the reliability of expression data of each gene is improved.

In this expression analysis, total RNA was used from 22 tumor sites of enucleated tissues of lung adenocarcinoma, 2 normal sites of enucleated tissues of lung adenocarcinoma, 13 tumor sites of enucleated tissues of stomach cancer, 20 tumor sites of enucleated tissues of ovarian cancer, 19 lung adenocarcinoma Cell lines, 4 small Cell lung cancer Cell lines, 10 stomach cancer Cell lines, 20 ovarian cancer Cell lines, and 65 normal tissues (purchased from Clontech Laboratories, Ambion, STRATAGENE, Cell APPLICATION, Panomics, CHEMICON, and Bio chain Institute).

Total RNA was extracted from tumor sites and normal sites of all clinical cancer-excised tissues (informed consent) and cancer cell lines (purchased from ATCC, JCRB, Limo BIOSOURCE CENTER CELL BANK) by Trizol (Invitrogen) according to the method attached to the product. Gene expression analysis experiments were carried out using 1. mu.g of the above total RNA according to GeneChip White Transcript (WT) sensor Target laboratory Manual (Affymetrix), and the digitization of human Exon 1.0ST Array Date was carried out using ExACT (Exon Array Computational tool) software supplied by Affymetrix.

There were 3 core probe testers for human Exon 1.0ST Array of human CLDN6 with core probe tester IDs 3677351, 3677352, 3677353. The expression data of the 3 probe tester IDs in normal tissues are shown in fig. 1, the expression data in tumor sites of lung adenocarcinoma cell lines, small cell lung cancer cell lines, and lung adenocarcinoma extirpated tissues are shown in fig. 2, the expression data in tumor sites of gastric cancer cell lines and gastric cancer extirpated tissues are shown in fig. 3, and the expression data in tumor sites of ovarian cancer cell lines and ovarian cancer extirpated tissues are shown in fig. 4.

As is clear from fig. 1 to 4, human CLDN6 transcript was not expressed in normal tissues except in fetal lung (compared with tumor tissues, it was not expressed in adult normal tissues examined this time, but it was negligible), but it was highly expressed in lung cancer, gastric cancer and ovarian cancer, although the frequency was low. This result indicates that an antitumor agent targeting human CLDN6 is not concerned at all about side effects in normal tissues, and that the pharmacological effect of the agent can be expected to be kept away from side effects.

[ example 2]Analysis of expression of human CLDN6 protein in cancer cell lines

Expression analysis of human CLDN6 protein in cancer cell lines was performed using western blotting of cell line lysates.

Human CLDN6 mRNA expression analysis was performed by human Exon 1.0ST Array and human Genome U133 Set Array, and based on the results, 2 strains, i.e., lung adenocarcinoma cell line ABC-1 and gastric cancer cell line AGS, were used in the experiment as cell lines highly expressing human CLDN6 mRNA; as cell lines not expressing the expression of human CLDN6 mRNA, 4 lines, namely, lung adenocarcinoma cell line NCI-H2347, lung small cell lung cancer cell line NCI-H209, lung small cell lung cancer cell line NCI-H1672 and lung small cell lung cancer cell line NCI-H1184 were used. ABC-1 was purchased from JCRB Cell Bank, AGS, NCI-H2347, NCI-H209, NCI-H1672, and NCI-H1184 were purchased from ATCC and used.

The cells were detached from the vessel with 1mM EDTA/PBS (-) to 1X 10⁶To each cell, 50uL NP40 lysine Buffer [ 0.5% Nonidet P40(v/v), 50mM Tris-HCl (pH 7.5), 150mM NaCl, 5mM EDTA, 1 tablet/10mL Complete mini EDTA free (Roche 04693159001), 100ug/mL P-APMSF (P-amidinophenyl) -methanesulfonyl fluoride hydrochloride (Wako Pure Chemical Industries, 014 10391)]After the cells were lysed by pipette, they were allowed to stand on iceAfter 30 minutes, the cell line lysate was centrifuged at 15000rpm at 4 ℃ for 30 minutes, and the resulting supernatant was used.

The lysate prepared as described above was prepared at 1: 1 using 2 × sample buffer (SIGMA 3401-IVL), incubated at room temperature for 15 minutes, and 10uL (1 × 10)⁵Aliquots of cell lysate) were used for western blotting. In western blotting, 15-25% polyacrylamide is used; anti-claudin-6 goat polyclonal antibody (C-20) (Santa Cruz code.sc-17669 lot.ht. h2605), which is a polyclonal antibody to the C-terminal peptide of human CLDN6, was used 1 time at 1/200 dilution; the secondary antibody was a porcine anti-goat Ig' srp conjugate (BIOSOURCE code. aci3404 lot.4101) used at 1/20000 dilution. The developed film was exposed to Hyperfilm ECL (GEHealthcare Corp. code.28-9068-36) using an ECL Plus Western Blotting Detection System (GEHealthcare code. RPN2132).

As shown in FIG. 5, the protein expression results were in good correlation with the analysis results of the transcript profile (transcriptome) shown in [ example 1 ]. From this result, it can be concluded that human CLDN6 protein expression is very consistent with human CLDN6 mRNA expression. The analysis result of the transcript profile obtained by using the Exon Array of [ example 1] was substantially identical to the analysis result of the protein expression. According to the above conclusions, it was first found that human CLDN6 protein is not substantially expressed in normal tissues of adults and is expressed in tumors in elevated levels.

[ example 3]Production of antibody recognizing human CLDN6 on surface of cancer cell membrane and use of the antibody Determination of antitumor Activity

As shown in examples 1 and 2, the expression of human CLDN6 protein has a good correlation with the expression of mRNA, and in addition, it was shown that the expression of human CLDN6 mRNA in adult normal tissues was very little or essentially absent compared to tumor tissues. Therefore, it is presumed that human CLDN6 protein is not substantially expressed also in adult normal tissues compared to tumor tissues. This means that an antibody recognizing the human CLDN6 protein expressed on the surface of cancer cells is an antibody with extremely high tumor specificity. When the above antibody is used as an antitumor agent, the drug effect and side effects are expected to be greatly separated, indicating that human CLDN6 has a very high potential as a target of an antitumor agent.

Therefore, an antibody recognizing human CLDN6 on the surface of a cancer cell membrane was actually prepared, and the anti-tumor effect of the above antibody was evaluated.

3-1 cloning of human CLDN6 cDNA

When an antibody against human CLDN6 was prepared, a sequence comprising an open reading frame of human CLDN6(refseqaccess No. nm — 021195.3) cDNA was cloned. The cDNA of human CLDN6 was cloned using primers represented by SEQ ID Nos. 1 and 2, using Marathon-Ready cDNA Fetal Lung (Clontech code.639333) as a template. Specifically, a solution containing 5. mu.L of 10 XKOD Buffer, 5. mu.L of 2mM dNTPs, and 3. mu.L of 25mM MgSO 2 was prepared using KOD plus DNA polymerase (TOYOBO Co.)₄1.5uL of 10. mu.M primer of SEQ ID NO. 1, 1.5uL of 10. mu.M primer of SEQ ID NO. 2, 2uLTemplate total Long cDNA, 1uL of KOD plus DNA polymerase, and 31uL of nuclease-free water were subjected to PCR amplification in a pattern of 94 ℃ for 2min, {94 ℃, 15 seconds, 58 ℃, 30 seconds, 68 ℃, 1 minute }. times.30 cycles. Next, amplification was performed again using the amplification product as a template, using primers represented by SEQ ID Nos. 3 and 4, or using the same enzyme, in the same manner and with the same composition, in a pattern of 94 ℃ for 2min, {94 ℃ for 15 seconds, 58 ℃ for 30 seconds, 68 ℃ for 1 minute }20 cycles. The amplified fragment was digested with Hind III and Nhe I and cloned into the Hind III and Nhe I sites of the pMCN-flag vector.

3-2 preparation of human CLDN 6-expressing CHO (DG44) and human CLDN 6-expressing Ba/F3 cells Making

pCOS2 vector was used as an expression vector in mammals for making human CLDN 6-expressing CHO cells (DG44, purchased from Invitrogen corporation) and human CLDN 6-expressing Ba/F3 cells. The pCOS2 vector has EF1 alpha promoter-enhancer sequence as promoter for inducing the expression of target gene, and target gene cDNA sequence inserted under the action of the promoter-enhancer, so that the expression of target gene may be induced when the vector is introduced into cell. In addition, since a neomycin resistance gene is incorporated into this vector, the vector can be selected for introduction into cells using a neomycin selection vector.

PCR was performed using a plasmid obtained by cloning the human CLDN6 cDNA described in [ example 3-1] as a template, using a primer represented by sequence No.5 (EcoRI recognition sequence-Kozak sequence-5 'end sequence of the open reading frame of human CLDN6(Refseq Access No. NM-021195.3)) and a primer represented by sequence No. 6 (Not I recognition sequence-3' end sequence of the open reading frame of human CLDN 6). The PCR amplification product was cloned into pCR 2.1-TOPO vector using TOPO TA Cloning (Invitrogen). This vector was digested with EcoRI and NotI, and the obtained fragment of human CLDN6 was incorporated into EcoRI and NotI sites of pCOS2 vector, thereby constructing an expression vector of human CLDN6/pCOS 2.

Human CLDN6/pCOS2 was digested with PvuI, and the resulting digests were electroporated (using the BIO-RAD GenePulser II) into CHO (DG44) cells and Ba/F3 cells. Human CLDN 6-constantly expressing CHO (DG44) cells and human CLDN 6-constantly expressing Ba/F3 cells were constructed using 500ug/mL Geneticin-induced cell lines.

In addition, this human CLDN6/pCOS2 vector can also be used for DNA immunization as described below.

3-3 preparation of anti-CLDN 6 antibody

To produce an anti-human CLDN6 antibody, mice were immunized by combining DNA immunization using Helios Gene Gun (BIO-RAD corporation) with cellular immunization of Ba/F3 cells forcibly expressed by human CLDN6, and monoclonal antibodies were screened by flow cytometry using human CLDN6 expressing CHO (DG44) cells.

Mice used for immunization were obtained from Charles River, Japan, under the name of BALB/cAnNCrlCrlj and MRL/MpJ-Tnfrff 6< lpr >/Crlj Genotype: ipr/lpr mice were immunized with DNA using Gene Gun by coating gold particles with plasmid DNA using the human CLDN6/pCOS2 vector described in example 3-2, according to BIO-RAD "HELIOS GENEGUN simple instructions ver.2.1" of BIO-RAD. The process of DNA immunization was performed as follows: each mouse is treated 2 times, 1 to 3 times in 1 week, and 8 to 17 times in total. Antibody titers in mouse serum were determined periodically by flow cytometry using cell lines that forcibly express human CLDN 6. After confirming that the antibody titer was increased by DNA immunization, cellular immunization with human CLDN 6-forced expression of Ba/F3 cell line was performed through the tail vein, and 2 to 3 days after the final cellular immunization, spleen cells were extracted and antibody-producing immortalized hybridomas (antibodies) were prepared by cell fusion with mouse cell line myeloma P3x63ag8u.1 (purchased from P3U1, ATCC). When spleen cells of a mouse were fused with mouse myeloma cell line p3x63ag8u.1 cells, the two cells were mixed at a ratio of 1 p3x63ag8u.1 cell to 2 to 4 spleen cells, PEG1500(roche diagnostics) was carefully and slowly added to the mixed cells, and then PEG1500 was diluted in RPMI1640 medium and centrifuged to remove PEG 1500. Then, the cells were suspended in HAT medium (RPMI 1640 medium containing 10% fetal bovine serum (Roche Diagnostics), 1 XPicillin-streptomycin (Invitrogen), 1 XPHAT media Supplement (Sigma), and 0.5 XPBM-conditioner H1hybrid Cloning Supplement (Roche Diagnostics)), and the fused cells were seeded on 10 to 30 96-well plates. After culturing at 37 ℃ in a carbon dioxide incubator for 7 to 10 days, the hybridoma culture supernatant was used for screening. The screening was performed by measuring the binding activity of the antibody to human CLDN6 forced expression CHO cells using a flow cytometer (Becton Dickinson). Since a plurality of hybridomas may be present in the positive well, the limited dilution method is used to perform monoclonal analysis of the hybridomas. After monoclonality, hybridoma clones producing antibodies having strong binding activity to human CLDN 6-forced expression CHO cells and human CLDN 6-forced expression Ba/F3 cells were selected, thereby constructing antibody-producing hybridomas recognizing human CLDN6 on the surface of cell membranes.

Among them, 18 kinds of hybridomas were selected in particular, and the antibodies produced by the 18 kinds of hybridomas were strong in binding activity to a human CLDN 6-forced expression cell line in flow cytometry and were classified into IgG type. Hybridomas were cultured using HAT medium containing Ultra Low IgG FBS (Invitrogen) instead of FBS, and antibodies were purified from the culture supernatants using HiTrap protein prep 1mL column (GE Healthcare). The degree of purification of the antibody was confirmed to be a sufficient level by SDS-PAGE and CBB staining. Note that isotripp (Roche) was used for typing of antibodies. The concentration of the purified antibody was measured using Dc Protein Assay Kit I (BIO-RAD) using bovine gamma globulin attached thereto as a control. The antibody concentration was expressed by conversion to bovine gamma globulin concentration. The antibody purification, typing, and protein quantification were performed according to the attached product guidelines.

3-4. anti-human CLDN6 monoclonal antibody forcibly expresses Ba/F3 on human CLDN6 cell surface Determination of binding Activity of human CLDN6

The binding of 18 purified anti-human CLDN6 monoclonal antibodies described in [ examples 3-3] to hCLDN 6-forced-expression Ba/F3 cells and the parental strain, Ba/F3, was evaluated by flow cytometry with the adjustment of antibody concentration.

The suspension was suspended in FACS buffer (0.5% BSA, 1PBS (-), 0.1% NaN₃)1 × 10 in (1)⁵Each cell was injected into a 96-well U-plate (FALCON 353910), and each antibody was added to a final concentration of 10, 2, 0.4, 0.08, 0. mu.g/mL, mixed and cultured at 4 ℃ for 1 hour. After centrifugation, removal of the reaction solution by aspiration, and washing of the cells by addition of 200 uL/well of FACSBuffer, FITC-labeled Goat F (ab')₂Fragmentanti-mouse IgG (Fc gamma) (BECKMAN COULTER) was added to the cells diluted 100-fold with FACS buffer. After incubation at 4 ℃ for 30 minutes, it was suspended in FACS buffer100uL containing propidium iodide (SIGMA) at a concentration of 10. mu.g/mL for flow cytometry.

In flow cytometry, X-axis: forward scattered light (forwad scanner), Y-axis: scattergram of side scatter light (side scatter), and X-axis: forward scattered light (forwad scanner), Y-axis: in a scattergram of fluorescence of propidium iodide (FL-3), a population of living cells was gated out.

As shown in fig. 6, the antibody of the present invention did not bind to Ba/F3 cells as a parent strain, strongly bound to human CLDN 6-forced-expression Ba/F3 cells, and was a human CLDN 6-specific antibody.

3-5 measurement of binding Activity of anti-human CLDN6 antibody on human CLDN6 on cancer cell Membrane surface Stator

Polyclonal antibodies recognizing the C-terminal intracellular peptide sequence of human CLDN6 are known, but no antibodies recognizing the extracellular portion of human CLDN6 occurring in a natural form on the surface of cancer cell membranes exist. Therefore, using the anti-human CLDN6 monoclonal antibody of the present invention prepared in [ example 3-3], it was evaluated by flow cytometry whether the above antibody recognizes not only a cell lysate of a human CLDN6 forced expression cell strain, but actually also human CLDN6 on the surface of a cancer cell membrane.

Based on the results of gene and protein expression analyses of [ example 1] and [ example 2], a lung adenocarcinoma cell line ABC-1 and a gastric cancer cell line AGS were used as a human CLDN 6-positive cancer cell line.

The suspension was suspended in FACS buffer (0.5% BSA, 1 XPBS (-), 0.1% NaN₃)1 × 10 in (1)⁵Each cell was injected into a 96-well U-plate (FALCON 353910), and each antibody was added to a final concentration of 10, 1 or 0. mu.g/mL, mixed, and cultured at 4 ℃ for 1 hour. After centrifugation, the reaction solution was aspirated off, and the cells were washed by adding 200 uL/well of FACS buffer, FITC-labeled Goat F (ab')₂Fragment Anti-mouseigG (Fc γ) (BECKMAN COULTER Co.) was added to the cells diluted 100-fold with FACS buffer. After incubation at 4 ℃ for 1 hour, washed with the same FACS buffer as above, suspended in 120uL FACS buffer and used for flow cytometry.

In flow cytometry, on the X-axis: forward scattered light (forwad scanner), Y-axis: live cell populations were gated out in a scatter plot of side scatter (side scatter).

As shown in FIG. 7, the antibodies produced in [ examples 3-3] were slightly different depending on the antibody, but all of the 18 antibodies were concentration-dependently bound to ABC-1, which is a human CLDN 6-expressing cancer cell line, and AGS cells.

3-6 determination of Antibody Dependent Cellular Cytotoxicity (ADCC) against human CLDN6 antibody

The ADCC activity of the anti-human CLDN6 monoclonal antibody on lung adenocarcinoma cell strains ABC-1 and gastric cancer cell strains AGS is researched by a chromium release method. ABC-1 or AGS was inoculated into a 96-well plate and allowed to adhere to the plate, and then chromium 51 was added thereto and the culture was continued for several hours. The culture medium was removed, and after washing the cells with the culture medium, a new culture medium was added. Then, an antibody was added to each well, effector cells (recombinant cells (japanese patent application 2007-20155)) about 5 times as many as the target cells were added to the wells, so that NK-92(ATCC, CRL-2407) was allowed to express a chimeric protein comprising an extracellular region of mouse Fc γ receptor 3(NM — 010188) and a transmembrane region and an intracellular region of human γ chain (NM — 004106) under control, and the plate was left to stand at 37 ℃ for 4 hours in a 5% carbon dioxide incubator. After standing, the plates were centrifuged, a certain amount of supernatant was recovered from each well, radioactivity was measured by a gamma counter Wallac 1480, and specific chromium release (%) was determined using the following formula.

Specific chromium release rate (%) - (A-C). times.100/(B-C)

Here, A represents the radioactivity in each well, B represents the average value of radioactivity released into the medium by cell lysis with a final concentration of 1% Nonidet P-40, and C represents the average value of radioactivity in the case where only the medium was added.

As a result, as shown in FIGS. 8 and 9, among the anti-human CLDN6 monoclonal antibodies of the present invention used in the test, in particular, AB3-1, AE1-16, AE49-11, AE3-20, and AC2-40 induced extremely strong ADCC activity against ABC-1 and AGS. This result indicates that it is very useful for antibody treatment of tumors targeting human CLDN 6.

3-7 determination of Complement Dependent Cytotoxicity (CDC) of anti-human CLDN6 antibody

CDC activity of the anti-human CLDN6 monoclonal antibody on lung adenocarcinoma cell strain ABC-1 was studied by using a chromium release method. ABC-1 cells were seeded on a 96-well plate and allowed to attach, and then chromium-51 was added thereto and the culture was continued for several hours. The culture medium was removed, and after washing the cells with the culture medium, a new culture medium was added. Then, the anti-human CLDN6 monoclonal antibodies of the invention (AB3-1, AC2-40, AD12-47, AE1-16, AE2-4, AE3-20, AE49-11) and the control mouse IgG1 antibody (Cat. No.553453, BD Biosciences Pharmingen) were added thereto to a final concentration of 10. mu.g/mL. Young rabbit complement (cat. No. cl3441, Cedarlane) was then added to a final concentration of 25%, 5% or 1%. The plate was allowed to stand in a 5% carbon dioxide incubator at 37 ℃ for 1.5 hours. After standing, the plate was centrifuged, a predetermined amount of supernatant was collected from each well, and the radioactivity was measured by a gamma counter Wallac 1480 to determine the specific chromium release rate (%) in the same manner as in 3-6.

As a result, as shown in FIG. 10, among the anti-human CLDN6 monoclonal antibodies of the present invention used in the test, AE1-16, AE3-20 and AE49-11 in particular induced strong CDC activity. On the other hand, the mouse IgG1 antibody used as a control did not exhibit CDC activity.

3-8 evaluation of anti-tumor Effect of anti-human CLDN6 antibody Using Mab-ZAP

Mab-ZAP (Advanced Targeting Systems) was used to evaluate whether immunotoxins Targeting human CLDN6 could exhibit anti-tumor activity. Mab-ZAP is a labeled antibody to the saporin (saporin) of goat anti-mouse IgG. Saporin is a proteinaceous toxin in ribosomes that acts as a mechanism for inhibiting protein synthesis. Not all antibodies are suitable for the preparation of immunotoxins, and it is known that immunotoxins including antibodies having a strong potency also include antibodies having a weak potency (non-patent document 9; Kohls and Lappi, BioTechniques 2000, 28 (1): 162). Thus, the potential of the 18 anti-human CLDN6 antibodies obtained this time as immunotoxins was evaluated using Mab-ZAP.

Lung adenocarcinoma cell line ABC-1 and gastric cancer cell line AGS were used as target cancer cell lines. For ABC-1, on day 0, at 5X 10 on 96-well plates³Cells were seeded at 100. mu.L/well, and each anti-human CLDN6 monoclonal antibody was added on day 1 to a final concentration of 100 ng/200. mu.L medium/well or 0 ng/200. mu.L medium/well, followed by Mab-ZAP to a final concentration of 100 ng/200. mu.L medium/well, and the cells were cultured in a carbon dioxide incubator at 37 ℃. On day 9, 20. mu.L/well of a reagent for measuring viable cells SF (Nacalai Tesque) was added thereto, and after incubation at 37 ℃ for 30 minutes in a carbon dioxide incubator, absorbance at 450 to 650nm was measured. For AGS, 1X 10 on 96-well plates on day 0³Cells were seeded at 100. mu.L/well and each anti-human CLDN6 monoclonal antibody was added on day 1 to a final concentration of 100 ng/200. mu.L medium/well or 0 ng/200. mu.L medium/well. Then, Mab-ZAP was added to a final concentration of 100 ng/200. mu.L medium/well, and the culture was carried out in a carbon dioxide incubator at 37 ℃. On day 7, 20. mu.L/well of living cell assay reagent SF (Nacalai Tesque Co.) was added, and after incubation at 37 ℃ for 30 minutes in a carbon dioxide incubator, absorbance at 450 to 650nm was measured.

The results for ABC-1 and AGS are shown in FIG. 11 and FIG. 12, respectively. While no antitumor effect was observed with either Mab-ZAP alone or the antibody alone, a very strong antitumor effect was observed with ABC-1 and AGS in the presence of the AE1-16 antibody or AE49-11 antibody in combination with Mab-ZAP.

The above results indicate that immunotoxins targeting human CLDN6 are very useful as anti-tumor agents.

[ example 4]]Determination of Gene sequences of variable regions of anti-human CLDN6 antibody

Based on the above results, 3 kinds of anti-human CLDN6 antibodies were selected from among the antibodies obtained this timeADCC, CDC, and antibodies (AB3-1, AE1-16, AE49-11, AE3-20) having strong antitumor activity as immunotoxins in the presence of Mab-ZAP were identified. Hybridomas producing various antibodies were cultured and cultured from 1X 10 using RNeasy (QIAGEN Co.)⁶Total RNA was purified from individual cells. PCR Amplification was performed using 1. mu.g of the purified total RNA, using SMART RACE cDNA Amplification Kit (Clontech), and synthetic oligonucleotide MHC-IgG1 (SEQ ID NO: 7) complementary to the constant region sequence of mouse IgG1, synthetic oligonucleotide MHC-IgG2b (SEQ ID NO: 8) complementary to the constant region sequence of mouse IgG2b, or synthetic oligonucleotide mKappaR (SEQ ID NO: 9) complementary to the base sequence of the constant region of mouse kappa chain, from the positions corresponding to the above-mentioned oligonucleotide sequences to the 5' -cDNA ends of the constant regions of 3 kinds of antibody H chain and L chain cDNAs. The amplified fragment was cloned into pTA2 vector (TOYOBO) and the cDNA sequence was determined. The H chain variable region of AB3-1 has the base sequence shown in SEQ ID NO. 10 and the amino acid sequence shown in SEQ ID NO. 11; the L chain variable region has the base sequence shown in SEQ ID NO. 12 and the amino acid sequence shown in SEQ ID NO. 13. The base sequence of the H chain variable region of AE1-16 is represented by SEQ ID NO. 14, and the amino acid sequence is represented by SEQ ID NO. 15; the L chain variable region has the nucleotide sequence shown in SEQ ID NO. 16 and the amino acid sequence shown in SEQ ID NO. 17. The base sequence of the H chain variable region of AE49-11 is represented by SEQ ID NO. 18, and the amino acid sequence is represented by SEQ ID NO. 19; the L chain variable region has the base sequence shown in SEQ ID NO. 20 and the amino acid sequence shown in SEQ ID NO. 21. The base sequence of the H chain variable region of AE3-20 is represented by SEQ ID NO. 36, and the amino acid sequence is represented by SEQ ID NO. 37; the L chain variable region has the base sequence shown in SEQ ID NO. 38 and the amino acid sequence shown in SEQ ID NO. 39.

In addition, the amino acid sequences of the CDRs of the above variable regions are shown in the following table.

[ Table 1]

[ example 5]Anti-human CLDN6 monoclonal antibodies to human CLDN1, CLDN3, CLDN4,

Evaluation of binding Activity of CLDN9 molecule

The binding activity of 4 anti-human CLDN6 monoclonal antibodies (AB3-1, AE1-16, AE49-11, AE3-20) having variable region amino acid sequences determined in [ example 4] to human CLDN1, CLDN3, CLDN4, and CLDN9 molecules was evaluated by preparing a Ba/F3 cell line forcibly expressing each molecule, adjusting the antibody concentration, and flow cytometry.

The suspension was suspended in FACS buffer (0.5% BSA, 1 XPBS (-), 0.1% NaN₃)1 × 10 in (1)⁵Each cell was injected into a 96-well U-plate (FALCON 353910), and each antibody was added to a final concentration of 10, 2, 0.4, 0.08, 0. mu.g/mL, mixed and cultured at 4 ℃ for 1 hour. After the reaction solution was removed by aspiration and FACSBuffer was added at 200 uL/well to wash the cells by centrifugation, FITC-labeled Goat F (ab')₂Fragmentanti-mouse IgG (Fc gamma) (BECKMAN COULTER) was added to the cells diluted 100-fold with FACS buffer. After incubation at 4 ℃ for 30 minutes, the cells were washed with the same FACSBuffer as described above, suspended in 100uL FACSBuffer containing propidium iodide (SIGMA) at a concentration of 10. mu.g/mL, and used for flow cytometry.

In flow cytometry, on the X-axis: forward scattered light (forwad scanner), Y-axis: scattergram of side scatter light (side scatter), and X-axis: forward scattered light (forwad scanner), Y-axis: in a scattergram of fluorescence of propidium iodide (FL-3), a population of living cells was gated out.

As shown in table 2, AE3-20, which is an antibody of the present invention, was an antibody that substantially specifically binds to human CLDN6, AE1-16 and AE49-11 were antibodies that cross-reacted moderately with human CLDN9, antibodies that cross-reacted weakly with human CLDN4, and AB3-1 was an antibody that cross-reacted with human CLDN 9.

[ Table 2]

	hCLDN6	hCLDN9	hCLDN4	hCLDN3	hCLDN1
						AE3-20	+++	+-	-	-	-
AE1-16	+++	++	+	-	-
						AE49-11	+++	++	+	-	-
AB3-1	++	++	-	-	-

[ example 6]Detection of CLDN6 in lung adenocarcinoma tissue by immunohistological staining

The expression of CLDN6 protein in lung adenocarcinoma tissue and the local presence on the cancer cell membrane were confirmed by immunohistological staining. In immunohistological staining, CLDN6 transcript was first quantified according to real-time PCR using total RNA extracted from lung adenocarcinoma clinical tissue, using cases with high expression of CLDN6 transcript. Frozen sections were fixed with 4% PFA and immunohistological staining was performed using a Ventana HX Discovery System (Ventana Medical Systems, Inc.) according to the conventional LSAB method. Goat anti-CLDN 6 polyclonal antibody (Code No. sc-17669 Lot. H2605, Santa Cruz Co.) was used at 12.5. mu.g/mL for 1 antibody in immunohistological staining. As a result, positive reactions were observed in the cell membrane and cytoplasm of lung adenocarcinoma tissue. On the other hand, in the non-tumor tissue, positive reactions were observed in macrophages, type II alveolar epithelium and bronchiole epithelium, but the staining intensity was slight, and no positive reaction was observed in the tumor tissue with respect to the cell membrane. The staining intensity of cell membranes in lung tumor tissue is higher than that of normal lung tissue. The invention discloses the expression detection of protein level in cell membrane of human tumor tissue for the first time

EXAMPLE 7 evaluation of anti-tumor Activity of anti-CLDN 6 antibody

Evaluation of antitumor Activity of AE49-11 antibody

The subclass of AE49-11 antibody is IgG2b, and it has been reported in the current research that IgG2a has a strong ADCC activity (non-patent documents [10] and [11]), and therefore, in order to enhance the drug efficacy, a vector expressing AE49-11 antibody (referred to as "AE 49-11/mIgG2 a"), H chain amino acid sequence (SEQ ID NO: 52 ") and L chain amino acid sequence (SEQ ID NO: 53") was constructed, expressed and purified using CHO-DG44 cells, and the AE49-11 antibody converted the Fc region of the antibody into IgG2 a. The binding activity of this AE49-11/mIgG2a antibody was confirmed to be substantially equivalent to that of the original IgG2b antibody by flow cytometry, and an in vivo antitumor experiment was conducted using this antibody as follows.

(1) PA-1 subcutaneous transplantation model

PA-1 cells were conditioned to 5X 10 cells using Hanks' Balanced Salt Solution (HBSS)⁷Cells/ml 200. mu.l of the PA-1 cells were transplanted under the abdominal skin of SCID mice (female, 9-week-old, Charles River, Japan) to which 100. mu.l of an anti-asialo GM1 antibody (Wako pure chemical industries, Ltd., 1 vial was dissolved in 1ml of distilled water for injection, and 4ml of physiological saline was added) was administered intraperitoneally the previous day. Antibodies to AE49-11/mIgG2a were administered 1 time per week via the tail vein from day 23 post-transplantation for 4 weeks. The antibody was adjusted to 5mg/ml with physiological saline and administered at 50 mg/kg. As a negative control, physiological saline (vehicle) was given in the same manner. Only experiments were performed in 1 group 5. Tumor volume was used to evaluate antitumor activity. Tumor volume (mm)³) The amount of change in tumor volume and the tumor growth inhibitory effect (%) were calculated as follows.

Tumor volume (mm)³) Long tumor diameter × short tumor diameter × 1/2

Tumor volume change (mm)³) Tumor volume at time of measurement-tumor volume at the beginning of administration

Tumor growth inhibition rate (%) {1- (mean value of change in tumor volume in drug-administered group/mean value of change in tumor volume in vehicle-administered group) } × 100

As a result of the experiment, AE49-11/mIgG2a antibody showed a tendency to inhibit tumor proliferation in the 50mg/kg administration group relative to the vehicle administration group. The tumor growth inhibition rates 1, 2, 3, and 4 weeks after administration of the drug were 49.5%, 31.1%, 29.9%, and 17.9%, and the tumor growth inhibition effect tends to be strong at the early stage of administration.

(2) NUGC-3 subcutaneous transplantation model

Next, the efficacy of the NUGC-3 subcutaneous transplantation model was studied. In the case of the efficacy test in this model, the efficacy test was carried out using an antibody (low fucose type AE49-11/mIgG2a antibody) obtained by expressing and purifying the AE49-11/mIgG2a antibody in a fucose transporter-knocked-out CHO-DXB11S cell.

NUGC-3 cells were adjusted to 5X 10 cells using Hanks' Balanced Salt Solution (HBSS)⁷Cells/ml, 200. mu.l of the NUGC-3 cells were transplanted subcutaneously into the abdomen of SCID mice (female, 12-week-old, Charles River, Japan). The mice were divided into 2 groups by tumor volume and body weight on day 11 after transplantation, and low fucose AE49-11/mIgG2a antibody and vehicle were administered to each group via tail vein on day 11, day 17 and day 24 after transplantation. The antibody was adjusted to 5mg/ml with a vehicle and administered at 50 mg/kg. As the carrier, a solution obtained by subjecting a mixed solution of 100mM glycine (pH 2.7) and 1/10 amount of 1M Tris-HCl (pH 9.0) to buffer substitution using an elusion buffer as D-PBS (-), using a PD-10 column, and subjecting the resulting solution to 0.22 μ M filter sterilization was used.

The test was performed in 1 group of 8. The life-prolonging effect was used to evaluate antitumor activity.

The test results showed that the low fucose type AE49-11/mIgG2a antibody had a life prolonging effect relative to the group to which the vector was administered.

The above results suggest that anti-CLDN 6 antibodies may show anti-tumor activity in human clinic.

Availability on production

The anti-CLDN 6 antibody of the present invention is useful as an antibody drug, particularly as a cell growth inhibitor and an anticancer agent.

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if fully set forth.

Sequence listing

<110> future institute of drug creation/national university of college court Tokyo university, Kabushiki Kaisha

<120> anti-CLDN 6 antibody

<130>PCG-9024WO

<150>JP2008-004423

<151>2008-01-11

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<211>38

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>5

ccggaattcc caccatggcc tctgccggaa tgcagatc 38

<210>6

<211>35

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>6

gcggccgctc agacgtaatt cttggtaggg tactc 35

<210>7

<211>21

<212>DNA

<213> mice

<400>7

gggccagtgg atagacagat g 21

<210>8

<211>24

<212>DNA

<213> mice

<400>8

caggggccag tggatagact gatg 24

<210>9

<211>27

<212>DNA

<213> mice

<400>9

ggcacctcca gatgttaact gctcact 27

<210>10

<211>354

<212>DNA

<213> mice

<400>10

gaggtccagc tgcaacagtc tggacctgag ttggtgaagc ctggaggttc aatgaagata 60

tcctgcaagg cttctggcta ctcattcact ggctacacca tgaactgggt gaagcagagc 120

catggaaaga accttgagtg gattggactt attaatcctt acaatggcgg tattagttac 180

aaccagaaat tcaaagacaa ggccacacta actatggaca agtcatccag cacagcctac 240

atggagctcc tcagtctgac atctgaggac tctgcaatct atttctgtgc aagagactat 300

aggtacgagg gctttgatta ctggggccaa gggactctgg tcactgtctc tgca 354

<210>11

<211>118

<212>PRT

<213> mice

<400>11

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Met Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Phe Cys

85 90 95

Ala Arg Asp Tyr Arg Tyr Glu Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210>12

<211>324

<212>DNA

<213> mice

<400>12

caaattgttc tcacccagtc tccagcaatc atgtctgcat ctctagggga acgggtcacc 60

atgacctgta ctgccagttc agttgtaatt tccacttact tgcactggta ccagcagaag 120

ccaggatcct cccccaaact ctggatttat agcacatcca acctggcttc tggagtccca 180

gttcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 240

gctgaagatg ctgccactta ttactgccac cagtatcatc gttccccgtg gacgttcggt 300

ggaggcacca agctggaaat caaa 324

<210>13

<211>108

<212>PRT

<213> mice

<400>13

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Val Val Ile Ser Thr

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro

85 90 95

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>14

<211>369

<212>DNA

<213> mice

<400>14

gaggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60

tcctgcaagg cttctggtta ctcatttact ggctacttta tgaactgggt gaaacagagc 120

catggaaaga gccttgagtg gattggacgt attaatcctt acaatggtga tactttctac 180

aaccagaagt tcaagggcaa ggccacatta actgtagaca aatcctctaa tacagcccac 240

atggagctcc ggagcctgac atctgaggac tctgcagtct attattgtgc aagagtcctc 300

ttccttgatt tcgacgaccc ctatcttatg gactattggg gtcaaggaac ctcagtcacc 360

gtctcctca 369

<210>15

<211>123

<212>PRT

<213> mice

<400>15

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Phe Leu Asp Phe Asp Asp Pro Tyr Leu Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>16

<211>318

<212>DNA

<213> mice

<400>16

gaaaatgtgc tcacccagtc tccagcaatc atgtctgcaa ctcttgggga gaaggtcacc 60

atgagctgca gggccacctc aaatgtaaag tacatgtact ggtaccagca gaagtcaggt 120

gcctccccca aactatggat ttattacaca tccaacctgg cttctggagt cccagctcgc 180

ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagcgt ggaggctgca 240

gatgctgcca cttattactg ccagcagttt actagttccc catccacgtt cggtgctggg 300

accaagctgg agctgaaa 318

<210>17

<211>106

<212>PRT

<213> mice

<400>17

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Thr Leu Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ala Thr Ser Asn Val Lys Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Tyr Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Ala

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Ser Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210>18

<211>369

<212>DNA

<213> mice

<400>18

gaggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60

tcctgcaagg cttctggtta ctcatttact ggctacttta tgaactgggt gaagcagagc 120

catggaaaga gccttgagtg gattggacgt attaatcctt acaatggtga tactttctac 180

aaccagaagt tcaagggcaa ggccacatta actgtagaca aatcctctag cacagcccac 240

atggagctcc ggagcctgac atctgaggac tctgcagtct attattgtgc aagagtcctc 300

ttccttgatt tcgacgaccc ctatcttatg gactattggg gtcaaggaac ctcagtcacc 360

gtctcctca 369

<210>19

<211>123

<212>PRT

<213> mice

<400>19

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala His

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Phe Leu Asp Phe Asp Asp Pro Tyr Leu Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>20

<211>318

<212>DNA

<213> mice

<400>20

gaaaatgtgc tcacccagtc tccagcaatc atgtctgcaa ctcttgggga gaaggtcacc 60

atgagctgca gggccacctc aaatgtaaag tacatgtact ggtaccagca gaagtcaggt 120

gcctccccca aactatggat ttattacaca tccaacctgg cttctggagt cccagctcgc 180

ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagcgt ggaggctgca 240

gatgctgcca cttattactg ccagcagttt actagttccc catccacgtt cggtgctggg 300

accaagctgg agctgaaa 318

<210>21

<211>106

<212>PRT

<213> mice

<400>21

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Thr Leu Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ala Thr Ser Asn Val Lys Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Tyr Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Ala

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Ser Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210>22

<211>220

<212>PRT

<213> Intelligent (homo sapiens)

<400>22

Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu

1 5 10 15

Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val

20 25 30

Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu

35 40 45

Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

50 55 60

Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

65 70 75 80

Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu

85 90 95

Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp

100 105 110

Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser

115 120 125

Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Val Ile

130 135 140

Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu

145 150 155 160

Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly

180 185 190

Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser

195 200 205

Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val

210 215 220

<210>23

<211>1363

<212>DNA

<213> Intelligent people

<400>23

atctccttcg cagtgcagct ccttcaacct cgccatggcc tctgccggaa tgcagatcct 60

gggagtcgtc ctgacactgc tgggctgggt gaatggcctg gtctcctgtg ccctgcccat 120

gtggaaggtg accgctttca tcggcaacag catcgtggtg gcccaggtgg tgtgggaggg 180

cctgtggatg tcctgcgtgg tgcagagcac cggccagatg cagtgcaagg tgtacgactc 240

actgctggcg ctgccacagg acctgcaggc tgcacgtgcc ctctgtgtca tcgccctcct 300

tgtggccctg ttcggcttgc tggtctacct tgctggggcc aagtgtacca cctgtgtgga 360

ggagaaggat tccaaggccc gcctggtgct cacctctggg attgtctttg tcatctcagg 420

ggtcctgacg ctaatccccg tgtgctggac ggcgcatgcc gtcatccggg acttctataa 480

ccccctggtg gctgaggccc aaaagcggga gctgggggcc tccctctact tgggctgggc 540

ggcctcaggc cttttgttgc tgggtggggg gttgctgtgc tgcacttgcc cctcgggggg 600

gtcccagggc cccagccatt acatggcccg ctactcaaca tctgcccctg ccatctctcg 660

ggggccctct gagtacccta ccaagaatta cgtctgacgt ggaggggaat gggggctccg 720

ctggcgctag agccatccag aagtggcagt gcccaacagc tttgggatgg gttcgtacct 780

tttgtttctg cctcctgcta tttttctttt gactgaggat atttaaaatt catttgaaaa 840

ctgagccaag gtgttgactc agactctcac ttaggctctg ctgtttctca cccttggatg 900

atggagccaa agaggggatg ctttgagatt ctggatcttg acatgcccat cttagaagcc 960

agtcaagcta tggaactaat gcggaggctg cttgctgtgc tggctttgca acaagacaga 1020

ctgtccccaa gagttcctgc tgctgctggg ggctgggctt ccctagatgt cactggacag 1080

ctgcccccca tcctactcag gtctctggag ctcctctctt cacccctgga aaaacaaatg 1140

atctgttaac aaaggactgc ccacctccgg aacttctgac ctctgtttcc tccgtcctga 1200

taagacgtcc accccccagg gccaggtccc agctatgtag acccccgccc ccacctccaa 1260

cactgcaccc ttctgccctg cccccctcgt ctcaccccct ttacactcac atttttatca 1320

aataaagcat gttttgttag tgcaaaaaaa aaaaaaaaaa aaa 1363

<210>24

<211>5

<212>PRT

<213> mice

<400>24

Gly Tyr Thr Met Asn

1 5

<210>25

<211>17

<212>PRT

<213> mice

<400>25

Leu Ile Asn Pro Tyr Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210>26

<211>9

<212>PRT

<213> mice

<400>26

Asp Tyr Arg Tyr Glu Gly Phe Asp Tyr

1 5

<210>27

<211>12

<212>PRT

<213> mice

<400>27

Thr Ala Ser Ser Val Val Ile Ser Thr Tyr Leu His

1 5 10

<210>28

<211>7

<212>PRT

<213> mice

<400>28

Ser Thr Ser Asn Leu Ala Ser

1 5

<210>29

<211>9

<212>PRT

<213> mice

<400>29

His Gln Tyr His Arg Ser Pro Trp Thr

1 5

<210>30

<211>5

<212>PRT

<213> mice

<400>30

Gly Tyr Phe Met Asn

1 5

<210>31

<211>17

<212>PRT

<213> mice

<400>31

Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210>32

<211>14

<212>PRT

<213> mice

<400>32

Val Leu Phe Leu Asp Phe Asp Asp Pro Tyr Leu Met Asp Tyr

1 5 10

<210>33

<211>10

<212>PRT

<213> mice

<400>33

Arg Ala Thr Ser Asn Val Lys Tyr Met Tyr

1 5 10

<210>34

<211>7

<212>PRT

<213> mice

<400>34

Tyr Thr Ser Asn Leu Ala Ser

1 5

<210>35

<211>9

<212>PRT

<213> mice

<400>35

Gln Gln Phe Thr Ser Ser Pro Ser Thr

1 5

<210>36

<211>357

<212>DNA

<213> mice

<400>36

gaagtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcaat agctatacca tgtcttgggt tcgccagact 120

ccggcgaaga ggctggagtg ggtcgtaacc attagtagtg gtggaggtcg cacctactat 180

ccagacagtg tgaagggccg attcaccatc tccagagaca atgccaggaa caccctgtac 240

ctacaaatga gcagtctgag gtctgaagac acggccatgt attactgtat aaggggggac 300

tataggtacg acgggtttgc ttactggggc caggggactc tggtcactgt ctctaca 357

<210>37

<211>119

<212>PRT

<213> mice

<400>37

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Thr Pro Ala Lys Arg Leu Glu Trp Val

35 40 45

Val Thr Ile Ser Ser Gly Gly Gly Arg Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ile Arg Gly Asp Tyr Arg Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Thr

115

<210>38

<211>321

<212>DNA

<213> mice

<400>38

gacatccaga tgactcagtc tccagcttcc ctgtctgcat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtga gaatattgac agttatttag catggtatca gcagaaacag 120

ggaaaatctc ctcaactcct ggtctatgct tcaacactct tagtagatgg tgtgccatca 180

aggttcagtg gcagtagatc aggcacacag ttttctctca aaatcaacag cctgcagtct 240

gaagatgttg cgagatatta ctgtcaacat tattatagta ttccgtatac gttcggatcg 300

gggaccaagc tggaaataaa a 321

<210>39

<211>107

<212>PRT

<213> mice

<400>39

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Ala Ser Thr Leu Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Ile Pro Tyr

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210>40

<211>5

<212>PRT

<213> mice

<400>40

Ser Tyr Thr Met Ser

1 5

<210>41

<211>17

<212>PRT

<213> mice

<400>41

Thr Ile Ser Ser Gly Gly Gly Arg Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210>42

<211>10

<212>PRT

<213> mice

<400>42

Gly Asp Tyr Arg Tyr Asp Gly Phe Ala Tyr

1 5 10

<210>43

<211>11

<212>PRT

<213> mice

<400>43

Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala

1 5 10

<210>44

<211>7

<212>PRT

<213> mice

<400>44

Ala Ser Thr Leu Leu Val Asp

1 5

<210>45

<211>9

<212>PRT

<213> mice

<400>45

Gln His Tyr Tyr Ser Ile Pro Tyr Thr

1 5

<210>46

<211>220

<212>PRT

<213> Intelligent people

<400>46

Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu

1 5 10 15

Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val

20 25 30

Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu

35 40 45

Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

50 55 60

Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

65 70 75 80

Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu

85 90 95

Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp

100 105 110

Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser

115 120 125

Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile

130 135 140

Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu

145 150 155 160

Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly

180 185 190

Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser

195 200 205

Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val

210 215 220

<210>47

<211>1389

<212>DNA

<213> Intelligent people

<400>47

cgacactcgg cctaggaatt tcccttatct ccttcgcagt gcagctcctt caacctcgcc 60

atggcctctg ccggaatgca gatcctggga gtcgtcctga cactgctggg ctgggtgaat 120

ggcctggtct cctgtgccct gcccatgtgg aaggtgaccg ctttcatcgg caacagcatc 180

gtggtggccc aggtggtgtg ggagggcctg tggatgtcct gcgtggtgca gagcaccggc 240

cagatgcagt gcaaggtgta cgactcactg ctggcgctgc cacaggacct gcaggctgca 300

cgtgccctct gtgtcatcgc cctccttgtg gccctgttcg gcttgctggt ctaccttgct 360

ggggccaagt gtaccacctg tgtggaggag aaggattcca aggcccgcct ggtgctcacc 420

tctgggattg tctttgtcat ctcaggggtc ctgacgctaa tccccgtgtg ctggacggcg 480

catgccatca tccgggactt ctataacccc ctggtggctg aggcccaaaa gcgggagctg 540

ggggcctccc tctacttggg ctgggcggcc tcaggccttt tgttgctggg tggggggttg 600

ctgtgctgca cttgcccctc gggggggtcc cagggcccca gccattacat ggcccgctac 660

tcaacatctg cccctgccat ctctcggggg ccctctgagt accctaccaa gaattacgtc 720

tgacgtggag gggaatgggg gctccgctgg cgctagagcc atccagaagt ggcagtgccc 780

aacagctttg ggatgggttc gtaccttttg tttctgcctc ctgctatttt tcttttgact 840

gaggatattt aaaattcatt tgaaaactga gccaaggtgt tgactcagac tctcacttag 900

gctctgctgt ttctcaccct tggatgatgg agccaaagag gggatgcttt gagattctgg 960

atcttgacat gcccatctta gaagccagtc aagctatgga actaatgcgg aggctgcttg 1020

ctgtgctggc tttgcaacaa gacagactgt ccccaagagt tcctgctgct gctgggggct 1080

gggcttccct agatgtcact ggacagctgc cccccatcct actcaggtct ctggagctcc 1140

tctcttcacc cctggaaaaa caaatgatct gttaacaaag gactgcccac ctccggaact 1200

tctgacctct gtttcctccg tcctgataag acgtccaccc cccagggcca ggtcccagct 1260

atgtagaccc ccgcccccac ctccaacact gcacccttct gccctgcccc cctcgtctca 1320

ccccctttac actcacattt ttatcaaata aagcatgttt tgttagtgca aaaaaaaaaa 1380

aaaaaaaaa 1389

<210>48

<211>217

<212>PRT

<213> Intelligent people

<400>48

Met Ala Ser Thr Gly Leu Glu Leu Leu Gly Met Thr Leu Ala Val Leu

1 5 10 15

Gly Trp Leu Gly Thr Leu Val Ser Cys Ala Leu Pro Leu Trp Lys Val

20 25 30

Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu

35 40 45

Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

50 55 60

Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

65 70 75 80

Arg Ala Leu Cys Val Ile Ala Leu Leu Leu Ala Leu Leu Gly Leu Leu

85 90 95

Val Ala Ile Thr Gly Ala Gln Cys Thr Thr Cys Val Glu Asp Glu Gly

100 105 110

Ala Lys Ala Arg Ile Val Leu Thr Ala Gly Val Ile Leu Leu Leu Ala

115 120 125

Gly Ile Leu Val Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile

130 135 140

Gln Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Leu Lys Arg Glu Leu

145 150 155 160

Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ala Ala Leu Leu Met Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Pro Pro Gln Val Glu Arg

180 185 190

Pro Arg Gly Pro Arg Leu Gly Tyr Ser Ile Pro Ser Arg Ser Gly Ala

195 200 205

Ser Gly Leu Asp Lys Arg Asp Tyr Val

210 215

<210>49

<211>220

<212>PRT

<213> Intelligent people

<400>49

Met Ser Met Gly Leu Glu Ile Thr Gly Thr Ala Leu Ala Val Leu Gly

1 5 10 15

Trp Leu Gly Thr Ile Val Cys Cys Ala Leu Pro Met Trp Arg Val Ser

20 25 30

Ala Phe Ile Gly Ser Asn Ile Ile Thr Ser Gln Asn Ile Trp Glu Gly

35 40 45

Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys Lys

50 55 60

Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Arg

65 70 75 80

Ala Leu Ile Val Val Ala Ile Leu Leu Ala Ala Phe Gly Leu Leu Val

85 90 95

Ala Leu Val Gly Ala Gln Cys Thr Asn Cys Val Gln Asp Asp Thr Ala

100 105 110

Lys Ala Lys Ile Thr Ile Val Ala Gly Val Leu Phe Leu Leu Ala Ala

115 120 125

Leu Leu Thr Leu Val Pro Val Ser Trp Ser Ala Asn Thr Ile Ile Arg

130 135 140

Asp Phe Tyr Asn Pro Val Val Pro Glu Ala Gln Lys Arg Glu Met Gly

145 150 155 160

Ala Gly Leu Tyr Val Gly Trp Ala Ala Ala Ala Leu Gln Leu Leu Gly

165 170 175

Gly Ala Leu Leu Cys Cys Ser Cys Pro Pro Arg Glu Lys Lys Tyr Thr

180 185 190

Ala Thr Lys Val Val Tyr Ser Ala Pro Arg Ser Thr Gly Pro Gly Ala

195 200 205

Ser Leu Gly Thr Gly Tyr Asp Arg Lys Asp Tyr Val

210 215 220

<210>50

<211>209

<212>PRT

<213> Intelligent people

<400>50

Met Ala Ser Met Gly Leu Gln Val Met Gly Ile Ala Leu Ala Val Leu

1 5 10 15

Gly Trp Leu Ala Val Met Leu Cys Cys Ala Leu Pro Met Trp Arg Val

20 25 30

Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ser Gln Thr Ile Trp Glu

35 40 45

Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

50 55 60

Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

65 70 75 80

Arg Ala Leu Val Ile Ile Ser Ile Ile Val Ala Ala Leu Gly Val Leu

85 90 95

Leu Ser Val Val Gly Gly Lys Cys Thr Asn Cys Leu Glu Asp Glu Ser

100 105 110

Ala Lys Ala Lys Thr Met Ile Val Ala Gly Val Val Phe Leu Leu Ala

115 120 125

Gly Leu Met Val Ile Val Pro Val Ser Trp Thr Ala His Asn Ile Ile

130 135 140

Gln Asp Phe Tyr Asn Pro Leu Val Ala Ser Gly Gln Lys Arg Glu Met

145 150 155 160

Gly Ala Ser Leu Tyr Val Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Asn Cys Pro Pro Arg Thr Asp Lys Pro

180 185 190

Tyr Ser Ala Lys Tyr Ser Ala Ala Arg Ser Ala Ala Ala Ser Asn Tyr

195 200 205

Val

<210>51

<211>211

<212>PRT

<213> Intelligent people

<400>51

Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu

1 5 10 15

Gly Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile

20 25 30

Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala Gln Ala Met Tyr Glu

35 40 45

Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr Gly Gln Ile Gln Cys

50 55 60

Lys Val Phe Asp Ser Leu Leu Asn Leu Ser Ser Thr Leu Gln Ala Thr

65 70 75 80

Arg Ala Leu Met Val Val Gly Ile Leu Leu Gly Val Ile Ala Ile Phe

85 90 95

Val Ala Thr Val Gly Met Lys Cys Met Lys Cys Leu Glu Asp Asp Glu

100 105 110

Val Gln Lys Met Arg Met Ala Val Ile Gly Gly Ala Ile Phe Leu Leu

115 120 125

Ala Gly Leu Ala Ile Leu Val Ala Thr Ala Trp Tyr Gly Asn Arg Ile

130 135 140

Val Gln Glu Phe Tyr Asp Pro Met Thr Pro Val Asn Ala Arg Tyr Glu

145 150 155 160

Phe Gly Gln Ala Leu Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu

165 170 175

Leu Gly Gly Ala Leu Leu Cys Cys Ser Cys Pro Arg Lys Thr Thr Ser

180 185 190

Tyr Pro Thr Pro Arg Pro Tyr Pro Lys Pro Ala Pro Ser Ser Gly Lys

195 200 205

Asp Tyr Val

210

<210>52

<211>453

<212>PRT

<213> Artificial

<400>52

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala His

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Leu Phe Leu Asp Phe Asp Asp Pro Tyr Leu Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Thr Ala

115 120 125

Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser

130 135 140

Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr

180 185 190

Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala

195 200 205

His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly

210 215 220

Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val

245 250 255

Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val

260 265 270

Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val

275 280 285

Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser

290 295 300

Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met

305 310 315 320

Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala

325 330 335

Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro

340 345 350

Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln

355 360 365

Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr

370 375 380

Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr

385 390 395 400

Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu

405 410 415

Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser

420 425 430

Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser

435 440 445

Arg Thr Pro Gly Lys

450

<210>53

<211>213

<212>PRT

<213> Artificial

<400>53

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Thr Leu Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ala Thr Ser Asn Val Lys Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Tyr Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Ala

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Ser Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

115 120 125

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

130 135 140

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

145 150 155 160

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

180 185 190

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

195 200 205

Asn Arg Asn Glu Cys

210

Claims

1. An antibody that binds to Claudin6(CLDN6) expressed on a cell membrane.

2. An anti-CLDN 6 antibody that is cytotoxic.

3. The anti-CLDN 6 antibody of claim 1 or 2 having ADCC activity.

4. The anti-CLDN 6 antibody of claim 1 or 2, having CDC activity.

5. The anti-CLDN 6 antibody of any one of claims 1-4 wherein a cytotoxic agent is bound.

6. The antibody according to any one of the following (a) to (j):

(a) an antibody comprising a heavy chain variable region having CDR1 comprising the amino acid sequence represented by sequence No. 24, CDR2 comprising the amino acid sequence represented by sequence No. 25, CDR3 comprising the amino acid sequence represented by sequence No. 26;

(b) an antibody comprising a light chain variable region having CDR1 comprising the amino acid sequence represented by sequence No. 27, CDR2 comprising the amino acid sequence represented by sequence No. 28, CDR3 comprising the amino acid sequence represented by sequence No. 29;

(c) an antibody comprising the heavy chain variable region of (a) and the light chain variable region of (b);

(d) an antibody comprising a heavy chain variable region having CDR1 comprising the amino acid sequence represented by sequence No. 30, CDR2 comprising the amino acid sequence represented by sequence No. 31, CDR3 comprising the amino acid sequence represented by sequence No. 32;

(e) an antibody comprising a light chain variable region having CDR1 comprising the amino acid sequence represented by sequence No. 33, CDR2 comprising the amino acid sequence represented by sequence No. 34, CDR3 comprising the amino acid sequence represented by sequence No. 35;

(f) an antibody comprising the heavy chain variable region of (d) and the light chain variable region of (e);

(g) an antibody comprising a heavy chain variable region having CDR1 comprising the amino acid sequence represented by sequence No. 40, CDR2 comprising the amino acid sequence represented by sequence No. 41, CDR3 comprising the amino acid sequence represented by sequence No. 42;

(h) an antibody comprising a light chain variable region having CDR1 comprising the amino acid sequence represented by sequence No. 43, CDR2 comprising the amino acid sequence represented by sequence No. 44, CDR3 comprising the amino acid sequence represented by sequence No. 45;

(i) an antibody comprising the heavy chain variable region of (g) and the light chain variable region of (h);

7. A pharmaceutical composition comprising an anti-CLDN 6 antibody.

8. The pharmaceutical composition of claim 7, which is a cytostatic agent.

9. The pharmaceutical composition of claim 8, which is an anticancer agent.

10. A pharmaceutical composition according to any one of claims 7 to 9, comprising an antibody according to any one of claims 1 to 6.

11. A method, which is a method of diagnosing cancer, comprising the steps of:

(a) a step of providing a sample taken from a subject;

(b) detecting the CLDN6 protein contained in the sample of (a).

12. The method of claim 11 wherein a CLDN6 protein is detected with an anti-CLDN 6 antibody.