HK1133661A - Pharmaceutical composition comprising anti-hb-egf antibody as active ingredient - Google Patents

Abstract

Disclosed is an anti-HB-EGF antibody having an internalizing activity. Preferably, the anti-HB-EGF antibody has a cytotoxic substance attached thereto. Also disclosed is a cancer therapeutic agent or a cell proliferation inhibitor comprising the antibody as an active ingredient. Further disclosed is a cancer treatment method or a cancer diagnosis method characterized by administering the antibody. The cancer to be treated by the cancer therapeutic agent includes pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, uterine cervical cancer, breast cancer, bladder cancer, brain tumor and hematological cancer.

Description

Pharmaceutical composition comprising anti-HB-EGF antibody as active ingredient

Technical Field

The present invention relates to methods of treating cancer and anticancer agents.

Background

An epidermal growth factor-like growth factor that binds heparin, or HB-EGF, is one of the growth factors belonging to the EGF ligand family. HB-EGF gene knockout mice show very deleterious phenotypes such as heart failure with cardiac hypertrophy and rapid death after birth (non-patent document 1). This suggests that HB-EGF makes a great contribution to the formation of the heart during pregnancy. On the other hand, in adults, the expression thereof is distributed in a wide range of tissues such as lung, heart, brain and skeletal muscle (non-patent document 2), and HB-EGF plays a very important role not only during pregnancy but also in maintaining the biological function of adults (non-patent document 3).

HB-EGF exists in vivo in two different structures: the membrane expressed on the cell surface of the HB-EGF-expressing cell binds to HB-EGF (hereinafter referred to as proHB-EGF) and a cell-free secretory type (hereinafter referred to as sHB-EGF or active HB-EGF). FIG. 1 schematically shows the structures of proHB-EGF and sHB-EGF. The proHB-EGF precursor protein consists of 208 amino acids, starting from the N-terminus, consisting of a signal peptide, a propeptide, a heparin-binding domain, an EGF-like domain, a juxtamembrane domain, a transmembrane domain, and a cytoplasmic domain. Cleavage of the signal peptide from the proHB-EGF precursor protein results in expression of proHB-EGF as a type 1 transmembrane protein. Subsequently, proHB-EGF is subjected to digestion by a protease, which is called ectodomain shedding, and sHB-EGF consisting of 73-87 amino acid residues is released into the extracellular environment. This sHB-EGF consists of only two domains, the heparin-binding domain and the EGF-like domain, and binds as active ligands to EGF receptor (Her1) and EGF receptor 4(Her 4). This leads to the induction of proliferation in various cells such as NIH3T3 cells, smooth muscle cells, epithelial cells, keratinocytes, renal tubular cells, and the like, through the downstream ERK/MAPK signaling pathway (non-patent document 4). Since mutations are introduced into the region involved in the shedding of the extracellular domain, cells expressing only proHB-EGF experience a substantial reduction in proliferative capacity. Furthermore, a transgenic mouse expressing only proHB-EGF has the same phenotype as an HB-EGF knockout mouse. Based on these findings, the function of HB-EGF as a growth factor is considered to be mainly possessed by the secretory HB-EGF (non-patent documents 5 and 6).

On the other hand, proHB-EGF is also known to have a unique function in vivo other than that of sHB-EGF. That is, proHB-EGF has been known to function as a receptor for Diphtheria Toxin (DT) in the beginning (non-patent documents 7 and 8). However, subsequent studies demonstrated that proHB-EGF binds to molecules such as DRAP27/CD9 and integrin alpha on the cell surface₃β₁And heparin sulfate form complexes and are involved in cell adhesion and migration. It has also been shown that proHB-EGF inhibits the growth of neighboring cells through the action of EGF receptor (hereinafter referred to as EGFR) via a mechanism of near secretionAnd induce death of neighboring cells. Therefore, with respect to the effect of HB-EGF as an EGFR ligand, it is known that membrane-bound proHB-EGF and secretory sHB-EGF transmit completely opposite signals (non-patent documents 5 and 8).

HB-EGF has a strong promoting activity on cell proliferation, cell movement and infiltration of various cell lines such as cancer cells. In addition, it has been reported that a wide range of cancer types (for example, pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, bladder cancer and brain tumor) are increased in expression of HB-EGF in normal tissues, suggesting that HB-EGF is deeply involved in cancer proliferation or malignant transformation (non-patent documents 4 and 10).

Therefore, based on these findings, studies for inhibiting the growth of cancer cells by inhibiting the HB-EGF activity were continued. For the efforts to inhibit the effect of HB-EGF using an anti-HB-EGF neutralizing antibody, the following effects were particularly reported: inhibition of DNA synthesis in 3T3 cells (non-patent document 11), inhibition of keratinocyte growth (non-patent document 12), inhibition of glioma cell growth (non-patent document 13), and inhibition of DNA synthesis in myeloma cells (non-patent document 14).

Meanwhile, the use of attenuated diphtheria toxin (CRM197) specifically binding to HB-EGF as an HB-EGF inhibitor has also been continuously studied. In fact, the group receiving CRM197 exhibited a better tumor shrinkage effect in a validity test on a mouse xenograft model (transplanted with an ovarian cancer cell line) (non-patent document 15). In addition, clinical trials have also been conducted on cancer patients using CRM197 (non-patent document 16).

The references cited in this specification are listed below. The contents of these documents are incorporated herein by reference in their entirety. These documents are not admitted to be prior art to the present invention:

non-patent document 1: iwamoto R, Yamazaki S, Asakura M et al, Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function, proc.natl.acad.sci.usa, 2003; 100: 3221-6.

Non-patent document 2: abraham JA, Damm D, Bajardi A, Miller J, Klagsbrunm, Ezekowitz RA, heparin-binding EGF-like growth factor: characterization of rat and mouse cDNA clones, protein domain servation across sites, and transcript expression in tissues. biochem Biophys Res Commun, 1993; 190: 125-33.

Non-patent document 3: karen M., Frontiers in Bioscience, 3, 288-.

Non-patent document 4: raab G, Klagsbrun M.heparin-binding EGF-likegrowth factor.Biochim Biophys Acta, 1997; 1333: f179-99.

Non-patent document 5: yamazaki S, Iwamoto R, Saeki K et al, Mice with features in HB-EGF ectomin rendering show segment depth evaluation and analysis methods. J Cell Biol, 2003; 163: 469-75.

Non-patent document 6: ongusaha P., Cancer Res, (2004)64, 5283-.

Non-patent document 7: iwamoto R., Higashiyama S., EMBO J.13, 2322-2330(1994).

Non-patent document 8: naglich JG., Metherall JE., Cell, 69, 1051-.

Non-patent document 9: iwamoto R, hand K, Mekada e.contact-dependent growth inhibition and apoptosis of Epidermal Growth Factor (EGF) receptor-expressing cells by the membrane-expressed for of heparin-binding EGF-like growth factor.j.biol.chem.1999; 274: 25906-12.

Non-patent document 10: miyamoto S, Cancer Sci.97, 341-347(2006).

Non-patent document 11: blotnick S., Proc.Natl.Acad.Sci.USA, (1994)91, 2890-.

Non-patent document 12: hashimoto K., J.biol.chem. (1994)269, 20060-.

Non-patent document 13: mishima K., Act neuropathohol (1998)96, 322-328.

Non-patent document 14: wang yd. oncogene, (2002)21, 2584-.

Non-patent document 15: miyamoto S., Cancer Res. (2004)64, 5720-

Non-patent document 16: buzzi S., Cancer Immunol Immunother, (2004)53, 1041-.

Disclosure of Invention

An object of the present invention is to provide a novel pharmaceutical composition comprising an anti-HB-EGF antibody. More specific object is to provide a novel method for treating cancer using an anti-HB-EGF antibody, a novel cell proliferation inhibitor comprising the anti-HB-EGF antibody, a novel anti-cancer agent comprising the anti-HB-EGF antibody, and a novel anti-HB-EGF antibody.

The present inventors have found that an antibody against HB-EGF, which is a protein highly expressed in cancer cells, shows an internalizing activity. The present inventors have also found that the anti-HB-EGF antibody exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cell-mediated cytotoxicity (CDC). Based on these findings, the present inventors also found that an anti-HB-EGF antibody is effective for treating a cancer having an up-regulated expression of HB-EGF (most notably ovarian cancer), thereby completing the present invention.

When the present inventors produced a monoclonal antibody by immunizing a mouse with the HB-EGF protein, they found that the obtained antibody had an internalizing activity. In addition, when a cytotoxic substance was bound to the obtained internalizing anti-HB-EGF antibody and cell death-inducing activity was measured, significant cell death-inducing activity was noted. Further, when the ADCC activity and CDC activity of the obtained anti-HB-EGF antibody are measured, it is found that the anti-HB-EGF antibody exhibits ADCC activity and/or CDC activity.

Accordingly, the present application provides monoclonal antibodies and low molecular weight antibody derivatives selected from the group consisting of (1) to (24) below:

(1) an anti-HB-EGF antibody having internalizing activity;

(2) an anti-HB-EGF antibody conjugated with a cytotoxic substance;

(3) the antibody of (2) having an internalization activity;

(4) an anti-HB-EGF antibody having ADCC activity or CDC activity;

(5) the antibody of any one of (1) to (4) further having internalization activity;

(6) an antibody selected from the group consisting of [1] to [13] below:

[1] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 14, SEQ ID NO as CDR 2: 16 and the amino acid sequence of SEQ ID NO: 18, or a heavy chain variable region of the amino acid sequence of seq id no;

[2] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 20, SEQ ID NO as CDR 2: 22 and the amino acid sequence of SEQ ID NO: 24, or a light chain variable region of the amino acid sequence of seq id no;

[3] an antibody comprising the heavy chain of [1] and the light chain of [2 ];

[4] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 26, the amino acid sequence of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 30, or a heavy chain variable region of the amino acid sequence of seq id no;

[5] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 32, the amino acid sequence of SEQ ID NO: 34 and the amino acid sequence of SEQ ID NO: 36, or a light chain variable region of the amino acid sequence of seq id no;

[6] an antibody comprising the heavy chain of [4] and the light chain of [5 ];

[7] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 76, SEQ ID NO as CDR 2: 77 and the amino acid sequence of SEQ ID NO: 78 (HE-39H chain);

[8] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 79, as CDR2, SEQ ID NO: 80 and the amino acid sequence of SEQ ID NO: 81 (HE-39L chain-1);

[9] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 82, SEQ ID NO as CDR 2: 83 and SEQ ID NO: 84 (HE-39L chain-2);

[10] an antibody comprising the heavy chain of [7] and the light chain of [8 ];

[11] an antibody comprising the heavy chain of [7] and the light chain of [9 ];

[12] an antibody having an activity equivalent to that of the antibody according to any one of [1] to [11 ]; and

[13] an antibody that binds to the same epitope as the antibody described in any one of [1] to [12 ].

(7) A pharmaceutical composition comprising the antibody of any one of (1) to (6);

(8) a pharmaceutical composition comprising a cytotoxic agent bound to an antibody of any one of (1) to (6);

(9) the pharmaceutical composition according to (7) or (8), which is an inhibitor of cell proliferation;

(10) the pharmaceutical composition according to (9), which is an anticancer agent;

(11) the pharmaceutical composition according to (10), wherein the cancer is pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, bladder cancer, brain tumor or hematological cancer;

(12) a method for delivering a cytotoxic substance into a cell using an anti-HB-EGF antibody;

(13) a method for inhibiting cell proliferation using a cytotoxic substance bound to an anti-HB-EGF antibody;

(14) the method of (13), wherein the cell is a cancer cell;

(15) the method of any one of (12) - (14), wherein the cytotoxic substance is a chemotherapeutic agent, a radioactive substance, or a toxic peptide;

(16) use of an anti-HB-EGF antibody for transporting a cytotoxic substance into a cell;

(17) use of an anti-HB-EGF antibody having internalization activity for inhibiting cell proliferation;

(18) the use of (17), wherein the anti-HB-EGF antibody further comprises neutralizing activity;

(19) the use according to (18), wherein the anti-HB-EGF antibody further comprises ADCC activity or CDC activity;

(20) the use of any one of (16) - (19), wherein the cell is a cancer cell;

(21) the use according to any one of (16) to (19), wherein the cytotoxic substance is bound to an anti-HB-EGF antibody;

(22) a method of preparing a pharmaceutical composition comprising the steps of:

(a) providing an anti-HB-EGF antibody;

(b) determining whether the antibody of (a) has internalizing activity;

(c) selecting an antibody having internalization activity; and

(d) binding a cytotoxic agent to the antibody selected in (c);

(23) the production method according to (22), wherein the pharmaceutical composition is an anticancer agent;

(24) a method for diagnosing cancer using an anti-HB-EGF antibody;

(25) the diagnostic method as described in (24), comprising using an anti-HB-EGF antibody bound with a labeling substance;

(26) the diagnostic method according to (24) or (25), wherein the anti-HB-EGF antibody incorporated into the cell is detected;

(27) an anti-HB-EGF antibody bound with a labeling substance;

(28) the antibody of (27), which has an internalizing activity.

Brief Description of Drawings

FIG. 1 is a diagram schematically describing the structures of proHB-EGF, sHB-EGF and HB-EGF Fc used as an immunogen;

FIG. 2a is a graph schematically depicting the effect of HB-EGF binding to EGFR Ba/F3 cells;

FIG. 2b is a graph showing the dependence of EGFR Ba/F3 cell proliferation on HB-EGF concentration;

FIG. 3a is a graph showing the neutralization activity of HB-EGF antibodies (HA-1, HA-3, HA-9, HA-10 and HA-20) on HB-EGF-dependent growth of EGFR _ Ba/F3 cells;

FIG. 3b is a graph showing the neutralization activity of HB-EGF antibodies (HB-10, HB-13, HB-20, HB-22 and HC-74) against HB-EGF-dependent growth of EGFR Ba/F3 cells;

FIG. 3c is a graph showing the neutralization activity of HB-EGF antibodies (HC-15, HC-19, HC-26 and HC-42) against HB-EGF-dependent growth of EGFR _ Ba/F3 cells;

FIG. 4 is a comparison of the sequences of the variable regions of the HB-EGF neutralizing antibodies;

FIG. 5 is a graph showing the binding activity of antibodies HA-20, HB-20 and HC-15 to the active type HB-EGF;

FIG. 6 is a graph showing the binding activity of antibodies HA-20, HB-20 and HC-15 to proHB-EGF;

FIG. 7 is a schematic diagram showing that an HB-EGF antibody inhibits binding between HB-EGF and EGFR on a solid phase;

FIG. 8 is a schematic diagram showing an ELISA-based analytical model for EGFR/HB-EGF binding patterns;

FIG. 9 is a graph showing an HB-EGF concentration curve detected in an ELISA-based analytical model for EGFR/HB-EGF binding patterns;

FIG. 10 is a graph showing that the antibodies HA-20, HB-20 and HC-15 inhibit the binding of HB-EGF to EGFR;

FIG. 11 is a graph comparing the inhibition of the growth of EGFR _ Ba/F3 cells by antibodies HA-20, HB-20, and HC-15;

FIG. 12a is a graph showing that antibodies HA-20, HB-20 and HC-15 inhibit the growth of ovarian cancer cell line RMG-1 in medium containing 8% FCS;

FIG. 12b is a graph showing that antibodies HA-20, HB-20 and HC-15 inhibit the growth of ovarian cancer cell line RMG-1 in medium containing 2% FCS;

FIG. 13 is a schematic diagram of a process of inducing cell death by internalizing an antigen-binding antibody (complex of HB-EGF targeting antibody and saporin-labeled antibody) into a cell;

FIG. 14 is a graph showing internalization-mediated activity of HA-20, HB-20, and HC-15 antibodies to induce cell death in SKOV-3 cells (naive cell line) and HB-EGF _ SKOV3 cells (HB-EGF high expressing SKOV-3 cells);

FIG. 15 is a graph showing the binding activity of HA-20, HB-20 and HC-15 antibodies to HB-EGF on ES-2 cells;

FIG. 16 is a graph showing internalization-mediated cell death-inducing activity of HA-20, HB-20, and HC-15 on ES-2 ovarian cancer cells;

FIG. 17 is a view showing FACS analysis of expression of HB-EGF in ovarian cancer cell lines (RMG-1, MCAS);

FIG. 18 is a graph analyzing the neutralizing activity of antibodies HA-20 and HC-15 against RMG-1 cell proliferation in a soft agar colony formation assay;

FIG. 19 is a graph that analyzes the internalization-mediated proliferation-inhibiting activity of antibodies HA-20 and HC-15 on RMG-1 ovarian cancer cells in a soft agar colony formation assay;

FIG. 20 is a graph that analyzes the internalization-mediated proliferation-inhibiting activity of antibodies HA-20 and HC-15 on MCAS ovarian cancer cells in a soft agar colony formation assay;

FIG. 21 is a graph showing FACS analysis of several blood cancer cell lines expressing HB-EGF;

FIG. 22 shows internalization-mediated inhibition of proliferation of several blood cancer cell lines by HA-20 and HC-15 antibodies;

FIG. 23a is a graph analyzing the proliferation inhibitory activity of saporin-tagged HA-20 antibody (HA-SAP), saporin-tagged HC-15 antibody (HC-SAP) and saporin-tagged control antibody (IgG-SAP) against various solid cancer cell lines and normal human endothelial cells;

FIG. 23b is a graph showing the analysis of proliferation inhibitory activity of saporin-labeled HA-20 antibody (HA-SAP) and saporin-labeled HC-15 antibody (HC-SAP) against various blood cancer cell lines;

FIG. 24 is a graph comparing the binding activity of the antibody HE-39 to the active type HB-EGF with the binding activity of the antibodies HA-20, HB-20 and HC-15 to the active type HB-EGF;

FIG. 25 is a graph showing the binding activity of antibody HE-39 and antibodies HA-20, HB-20 and HC-15 to proHB-EGF overexpressed in DG44 cells;

FIG. 26 is a graph showing the ability of HA-20, HB-20, HC-15 and HE-39 antibodies to inhibit binding of HB-EGF to EGFR;

FIG. 27 shows a graph comparing the growth inhibitory activity shown by HA-20, HB-20, HC-15 and HE-39 antibodies against EGFR _ Ba/F3 cells;

FIG. 28 is a comparison of the variable region sequences of HE-39 antibodies;

FIG. 29a shows the binding activity of monoclonal antibodies (HE39-1, HE39-5, HE39-14) obtained by additional limiting dilution of the HE-39 antibody on proHB-EGF overexpressed in DG44 cells;

FIG. 29b is a photograph in which the expression of each variable region (VH, VL-1, VL-2) was confirmed by RT-PCR for monoclonal antibodies (HE39-1, HE39-5, HE39-14) obtained by additional limiting dilution of the HE-39 antibody;

FIG. 30 is a graph showing the internalization-mediated cell death-inducing activity of the antibodies HE-39, HA-20 and HC-15 against HB-EGF _ DG44 cells;

FIG. 31a is a diagram schematically showing the structure of HB-EGF (upper panel), the structure of a fusion protein between GST protein and mature HB-EGF or each domain (heparin-binding domain, EGF-like domain) (lower panel);

FIG. 31b shows the results of SDS-PAGE and CBB staining (left) of GST fusion proteins expressed in E.coli (E.coli) and Western blot analysis with HE-39 antibody (right);

FIG. 32a shows the amino acid sequence of EGF-like domain (EGFD) of HB-EGF and the amino acid sequence of EGF-like domain divided into 3 fragments (EGFD5, EGFD6, EGFD 7); these sequences were fused to the C-terminus of the GST protein for epitope mapping;

FIG. 32b shows the results of SDS-PAGE and CBB staining (left panel) of GST fusion proteins (GST-EGFD, GST-EGFD5, GST-EGFD6, GST-EGFD7) expressed in E.coli and Western blot analysis (right panel) with HE-39 antibody;

FIG. 33a is a FACS analysis graph showing the binding activity of various anti-HB-EGF antibodies to HB-EGF overexpressed in Ba/F3 cells; fluorescence intensity as a G-average value is shown on the vertical axis; and

FIG. 33b shows ADCC activity against HB-EGF _ Ba/F3 cells (upper panel) exhibited by various anti-HB-EGF antibodies and CDC activity against HB-EGF _ Ba/F3 cells (lower panel) exhibited by various anti-HB-EGF antibodies; the vertical axis shows the amount of chromium released from the cells due to ADCC-mediated or CDC-mediated cytotoxicity.

Preferred embodiments of the invention

Molecular forms of HB-EGF

HB-EGF is a growth factor belonging to the EGF ligand family; the sequence of the gene encoding human HB-EGF is disclosed in GenBank accession No. NM-001945 (SEQ ID NO: 49), and the amino acid sequence of HB-EGF is disclosed in GenBank accession No. NP-001936 (SEQ ID NO: 50). In the scope of the present invention, the "HB-EGF protein" is a term including the full-length protein and fragments thereof. In the scope of the present invention, "fragment" means a polypeptide comprising any region of the HB-EGF protein, wherein the fragment may not exhibit the function of the naturally occurring HB-EGF protein.

The sHB-EGF used herein as a specific embodiment of the fragment is a molecule consisting of 73 to 87 amino acid residues and is produced in vivo when proHB-EGF expressed on the cell surface of an HB-EGF-expressing cell is cleaved by a protease in a process called ectodomain shedding. Various sHB-EGF molecules are known; these sHB-EGF molecules have the following structures: wherein the carboxy terminus is a proline residue at position 149 in a proHB-EGF molecule consisting of the amino acid sequence of SEQ ID NO: 50 and the amino terminus is an asparagine residue at position 63 in the proHB-EGF molecule, an arginine residue at position 73 in the proHB-EGF molecule, a valine residue at position 74 in the proHB-EGF molecule, or a serine residue at position 77 in the proHB-EGF molecule.

anti-HB-EGF antibody

The anti-HB-EGF antibody of the present invention is an antibody that can bind to the HB-EGF protein, but there is no limitation on the origin (mouse, rat, human, etc.), type (monoclonal antibody, polyclonal antibody) and configuration (engineered antibody, low molecular weight antibody, modified antibody, etc.) thereof.

The anti-HB-EGF antibody used in the present invention preferably specifically binds to HB-EGF. The anti-HB-EGF antibody used in the present invention is also preferably a monoclonal antibody.

Antibodies with internalizing activity are a preferred embodiment of the antibodies for use in the present invention. "antibody having an internalizing activity" refers to an antibody that is transported into a cell (cytoplasm, vesicle, other organelles, etc.) upon binding to HB-EGF on the cell surface.

The presence/absence of internalization activity of an antibody can be determined using methods known to those skilled in the art. For example, internalization activity can be determined by: contacting an anti-HB-EGF antibody binding to the marker with a cell expressing HB-EGF, and checking whether the marker is incorporated into the cell; or contacting an anti-HB-EGF antibody binding to the cytotoxin with an HB-EGF-expressing cell, and examining whether cell death is induced in the HB-EGF-expressing cell. More specifically, for example, the presence/absence of internalization activity of an antibody can be determined using the methods described in the examples provided below.

In the case where the anti-HB-EGF antibody has an internalizing activity, the anti-HB-EGF antibody is preferably an antibody capable of binding to proHB-EGF, more preferably an antibody that binds proHB-EGF more strongly than to sHB-EGF.

Cytotoxic substance

Another preferred embodiment of the antibody for use in the present invention is an antibody conjugated with a cytotoxic substance. Such antibodies with bound cytotoxic agents can be incorporated into cells, resulting in the induction of cytotoxic agent-mediated cell death of the incorporated antibodies. Thus, an antibody that is conjugated to a cytotoxic agent preferably also has internalization activity.

The cytotoxic substance used in the present invention may be any substance capable of inducing cell death of cells, and may include toxins, radioactive substances, chemotherapeutic agents, and the like. The cytotoxic agents useful in the present invention include prodrugs, which undergo conversion to the active cytotoxic agent in vivo. Prodrug activation may be performed by enzymatic or non-enzymatic conversion.

In the context of the present invention, toxin refers to various cytotoxic protein polypeptides and the like of microbial, animal or plant origin. Toxins for use in the present invention may include the following: diphtheria toxin A chain (Langon J. et al, Methods in Enzymology, 93, 307-308, 1983), Pseudomonas exotoxin (Nature Medicine, 2, 350-353, 1996), ricin A chain (Fulton R. J. et al, J. biol. chem., 261, 5314-5319, 1986; Sivam G. et al, Cancer Res. 47, 3169-3173, 1987; Cumber A. J. et al, J. Immunol. Methods, 135, 15-24, 1990; Wawrynczak E. J. et al, Cancer Res. 50, 7519-7562, 1990; Gheeite V. et al, J. Immunol. Methoid, 142, 223-230, 1991), deglycosylation of ricin A. E. et al, ricin A. 19819, 19819-7519, 1987; Warne. 19-31, 19819-31, 1987; Warne. 19-31, 1987; Warne. K. E. K. et al, 1987; Warne. K. et al, 1983, 1987, 1983; Warne. K. Cancer Res., 47, 5924-; cumber a.j. et al, j.immunol.methods, 135, 15-24, 1990; wawrzynczak E.J., et al, Cancer Res., 50, 7519-7562, 1990; blognesi a. et al, clin. exp. immunol., 89, 341- & 346, 1992), PAP-s (pokeweed antiviral protein from seeds; blognesi a. et al, clin. exp. immunol., 89, 341-plus 346, 1992), bronodin (blognesi a. et al, clin. exp. immunol., 89, 341-plus 346, 1992), saporin (blognesi a. et al, clin. exp. immunol., 89, 341-plus 346, 1992), momordin (momordin) (Cumber a. j. et al, j. immunol. methods, 135, 15-24, 1990; wawrzynczak e.j, et al, cancer res, 50, 7519-; borognesi A. et al, protein Exp. Immunol., 89, 341-346, 1992), momordica cochinchinensis (molochin A. et al, protein Exp. Immunol., 89, 341-346, 1992), carnation protein (dianthin)32(Bolognesi A. et al, protein Exp. Immunol., 89, 341-346, 1992), carnation protein 30(Stirpe F., Barbieri L. FEBS Letter, 195, 1-8, 1986), syphilin II (Stigmodulcin F., Barbieri L., 195, 1-8, 1986), parasitic toxin protein (viscumulin) (Stirpe F., Stigmipe F., 195, 1-8, FE, 1986), Lebidir protein (Leriber F., Leriber L., Leriber L., FEI-195, Leriber L. FE, Leriber L., FEI, Leriber L. E, Leriber L. 1-8, Leriber L. FEI, Leriber L., FEI, Leriber L. E, Leriber, P. 1-6, Leriber, Le, 195, 1-8, 1986), cucurbitin (luffin) (stirpef, Barbieri l, FEBS Letter, 195, 1-8, 1986) and trichosanthes seed poison (trichokirin) (Casellas p., et al, eur.j. biochem., 176, 581-; blognesi a. et al, clin. exp. immunol., 89, 341-.

The radioactive substance in the present invention refers to a substance containing a radioactive isotope. The radioisotope is not particularly limited, and any radioisotope can be used. Examples of useful radioisotopes are³²P、¹⁴C、¹²⁵I、³H、¹³¹I、¹⁸⁶Re、¹⁸⁸Re, and the like.

Chemotherapeutic agents in the present invention refer to cytotoxic substances other than the toxins and radioactive substances listed above, including: such as cytokines, antineoplastic agents, enzymes, and the like. The chemotherapeutic agent used in the present invention is not particularly limited, but a low molecular weight chemotherapeutic agent is preferable. It is believed that low molecular weight chemotherapeutic agents have a lower likelihood of interfering with antibody function, even after the chemotherapeutic agent has bound the antibody. In the context of the present invention, a low molecular weight chemotherapeutic agent generally refers to a molecular weight of 100-2000, preferably 200-1000. The chemotherapeutic agent in the present invention is not particularly limited, and examples of useful chemotherapeutic agents are as follows: melphalan (melphalan) (Rowland G.F. et al, Nature, 255, 487-488, 1975), cisplatin (cis-platinum) (Hurwitz E. and Haimovich J., Method in Enzymology, 178, 369-375, 1986; Schechter B. et al, int.J.cancer, 48, 167-172, 1991), carboplatin (beta Y. et al, Asia-Oceania J.Obstet. Gynaecol., 19, 449-457, 1993), mitomycin C (mitomycin C) (noguchi A. et al, Bioconjugate chem., 3, 132-137, 1992), doxorubicin (doxorubicin) (Shih L.B. et al, Cancer Res 261, 92, 4192, 4178, 1995, Zuch J. et al, 1995, J. Scech., 78, 1995, J. et al, J. Scech. J. et al, J. 40, 257, 267, 1995; zhu Z et al, Cancer Immunol. Immunother., 40, 257-267, 1995), daunorubicin (dallumubicin) (Dillman R.O. et al, Cancer Res., 48, 6097-6102, 1988; hudecz F. et al, Bioconjugate chem., 1, 197-204, 1990; tukada Y. et al, J.Natl.cancer Inst., 75, 721, 729, 1984), bleomycin (bleomycin) (Manabe Y. et al, biochem.Biophys.Res.Commun., 115, 1009, 1014, 1983), neocarzinostatin (neocarzinostatin) (Kitamura K. et al, Cancer Immunol.Immunother., 36, 177, 184, 1993; yamaguchi t. et al, jpn.j. Cancer res., 85, 167-; kulkarni p.n. et al, Cancer res, 41, 2700-; shin L.B. et al, int.J. cancer, 41, 832-charge 839, 1988; gamett m.c. et al, int.j.cancer, 31, 661-.

The present invention may use one cytotoxic agent or a combination of two or more cytotoxic agents.

The above cytotoxic substance may be bound or coupled to the anti-HB-EGF antibody by a covalent bond or a non-covalent bond. Methods for producing antibodies that bind to these cytotoxic substances are well known.

The cytotoxic substance may directly bind to the anti-HB-EGF antibody through, for example, a linking group present in these substances themselves, or may indirectly bind to the anti-HB-EGF antibody through another substance such as a linker or an intermediate support (intermediate support). In the case where the anti-HB-EGF antibody is directly bound to a cytotoxic substance, the linking group includes a disulfide bond based on the utilization of an SH group. In particular, intramolecular disulfide bonds within the Fc region of an antibody can be reduced with a reducing agent such as dithiothreitol; disulfide bonds in cytotoxic substances can be similarly reduced; and the two substances may be linked to each other by a disulfide bond. The formation of disulfide bonds between two substances can be promoted by primary activation of the antibody or cytotoxic substance with an activation promoter such as Ellman's reagent. Examples of other methods for achieving direct binding between the anti-HB-EGF antibody and the cytotoxic substance are as follows: a method using Schiff base (Schiff base), a carbodiimide method, an active ester method (N-hydroxysuccinimide method), a method using mixed anhydrides, and a method using a diazo reaction.

The binding between the anti-HB-EGF antibody and the cytotoxic substance can also be achieved by indirect binding via another substance. There is no particular limitation on the other substance for indirect binding, and the other substance may include a compound having at least two groups (including a single type or a combination of two or more types) selected from amino groups, carboxyl groups, mercapto groups, and the like, and may also include a peptide linker and a compound having the ability to bind to an anti-HB-EGF antibody. The following are examples of compounds having at least two groups (including a single type or a combination of two or more types) selected from amino, carboxyl, mercapto, and the like: n-succinimidyl 3- (2-pyridyldithio) propionate (SPDP; Wawrzynczak E.J., et al, Cancer Res., 50, 7519-K7562, 1990; Thorpe P.E. et al, Cancer Res., 47, 5924-K5931, 1987), succinimidyl 6- (3- [ 2-pyridyldithio ] propionamido) hexanoate (LC-SPDP; Hermanson G.T., BIOCONJUGTechniques, 230-K232, 1996), sulfosuccinimidyl 6- (3- [ 2-pyridyldithio ] propionamido) hexanoate (sulfo-LC-SPDP; Hermanson G.T., BIOCONJUGUGH, 230-K232, 1996), N-succinimidyl 3- (2-pyridyldithio) butyrate (DB; Wawrzak E.J., Wawraze. J., 366, J. Br, 66-K, N-succinimidyl 3- (2-pyridyldithio) butyrate (SMyncBr; S. Br, S. J. E. 366, S. D. E. 366, S.,366, T.,366, N-Succinimidyl 6- (2-pyridyl) propionate, cancer res, 47, 5924-5931, 1987), succinimidyl 6- (α -methyl- [ 2-pyridinedithio ] toluamide) hexanoate (LC-SMPT; hermanson G.T., BIOCONJUGATE technologies, 232- & 235, 1996), sulfosuccinimidyl 6- (α -methyl- [ 2-pyridyldithio ] toluamide) hexanoate (sulfo-LC-SMPT; hermanson G.T., BIOCONJUGATE technologies, 232- & 235, 1996), succinimidyl-4- (p-maleimidophenyl) butyrate (SMPB; hermanson G.T., BIOCONJUGA TE Techniques, 242-243, 1996), sulfo-succinimidyl-4- (p-maleimidophenyl) butyrate (sulfo-SMPB; hermanson g.t., bioconjuugate technologies, 242-243, 1996), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; hermanson g.t., bioconjuugate technologies, 237-; hermanson g.t., bioconjuugate technologies, 237-; caselas p. et al, eur.j. biochem., 176, 581-588, 1988), dimethyl 3, 3' -dithiodipropionimidate (DTBP; casellas P. et al, Eur.J. biochem., 176, 581-.

Examples of other substances that can be used to bind the HB-EGF antibody to a cytotoxic substance are peptides, antibodies, poly-L-glutamic acid (PGA), carboxymethyl dextran, aminodextran, avidin-biotin, aconitic acid, glutamic acid dihydrazide, Human Serum Albumin (HSA), and the like.

Alternatively, cytotoxic proteins may be bound to antibodies by genetic engineering techniques. As a specific example, the above-mentioned DNA encoding a cytotoxic peptide may be fused in frame with a DNA encoding an anti-HB-EGF antibody, and a recombinant vector may be constructed by incorporating into an expression vector. The vector is transfected into a suitable host cell, and the resulting transformed cell is cultured to express the incorporated DNA and obtain a fusion protein comprising the anti-HB-EGF antibody bound to the toxic peptide. In preparing fusion proteins with antibodies, the drug protein or protein toxin is typically located on the C-terminal side of the antibody. In addition, a peptide linker may be interposed between the antibody and the drug protein or protein toxin.

Neutralizing Activity

The anti-HB-EGF antibody used in the present invention may have a neutralizing activity.

Neutralizing activity generally refers to the ability to inhibit the biological activity of a ligand that exhibits biological activity on a cell, an agonist being one example of such a ligand. Therefore, a substance having a neutralizing activity refers to a substance that binds to such a ligand or to a receptor that binds to the ligand, thereby inhibiting the binding of the ligand or the receptor. Receptors that are prevented from binding to the ligand due to neutralizing activity are then unable to exhibit biological activity through the receptor. Antibodies exhibiting such neutralizing activity are generally referred to as neutralizing antibodies. The neutralizing activity of a particular test substance can be determined by comparing the biological activity in the presence of the ligand and the test substance to the biological activity in the presence of the ligand and in the absence of the test substance.

EGF receptor is considered to be the main receptor for HB-EGF as described herein. In this case, tyrosine kinase (which is a self-domain in the cell) is activated due to binding of the ligand to form a dimer. Activated tyrosine kinases lead to the formation of phosphorylated tyrosine-containing peptides by autophosphorylation, to which various signal transduction helper molecules are bound. Mainly, PLC γ (phospholipase C γ), Shc, Grb2, and the like are included. Of these accessory molecules, the first two are additionally phosphorylated by tyrosine kinases of the EGF receptor. The major pathway of signal transduction from the EGF receptor is phosphorylation of the pathway of sequential transduction along Shc, Grb2, Sos, Ras, Raf/MAPK kinase/MAP kinase. It is also believed that there is a path from PLC γ to PKC, which is the second path. This intracellular signaling cascade is different in each cell type and thus the appropriate target molecule can be determined for each desired target cell. There is no limitation to the above factors. Neutralization activity can be assessed by measuring in vivo signal activation. Commercially available kits for measuring in vivo signal activation (e.g., protein kinase C activation assay system from ge healthcare Biosciences) can be suitably used.

In vivo signal activation can also be detected by induction of transcription of target genes that are concentrated downstream of the in vivo signaling cascade. The reporter assay concept is used to detect changes in the transcriptional activity of a target gene. Specifically, a reporter gene (e.g., Green Fluorescent Protein (GFP) or luciferase) may be located downstream of a transcription factor or promoter region of a target gene, and a change in transcription activity may be determined from the reporter gene activity by measuring the reporter gene activity.

In addition, since signal transduction through the EGF receptor generally acts in a direction promoting cell growth, the neutralizing activity can be evaluated by measuring the growth activity of the target cell.

The antibody having neutralizing activity and internalizing activity can be a very effective anticancer agent against cancers highly expressing HB-EGF.

ADCC activity and/or CDC activity

The anti-HB-EGF antibody used in the present invention may have antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).

In the present invention, CDC activity refers to a cell destruction activity caused by a complement system. On the other hand, ADCC activity refers to an activity as described below: in which a specific antibody is attached to a cell surface antigen on a target cell, an Fc γ receptor-presenting cell (immune cell or the like) binds to an Fc region of the antigen-binding antibody via its Fc γ receptor, and then attacks the target cell.

Known methods can be used in the present invention to determine whether an antibody exhibits ADCC activity and whether an antibody exhibits CDC activity (e.g., Current Protocols in immunology. Chapter 7: Immunologic students in humans. editor: John E.Coligan et al, John Wiley & Sons, Inc. (1993), etc.).

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

(1) Preparation of Effector cells

For example, spleens were removed from CBA/N mice and splenocytes isolated in RPMI1640 medium (Invitrogen Corporation). Effector cells were then prepared by: washing with the same medium containing 10% fetal bovine serum (FBS, HyClone), followed by adjustment of the cell concentration to 5X 10⁶Per milliliter.

(2) Preparation of complement solution

The complement solution can be prepared by 10-fold dilution of young rabbit complement (Cedarlane Laboratories Ltd.) with a medium containing 10% FBS (Invitrogen corporation).

(3) Preparation of target cells

For radiolabeling target cells, 0.2mCi was used in DMEM medium containing 10% FBS at 37 deg.C⁵¹Cr^-Cells expressing the HB-EGF protein were cultured for 1 hour with sodium chromate (GE Healthcare Biosciences). For example, a cancer cell (e.g., ovarian cancer cell) or a cell transformed with a gene encoding an HB-EGF protein can be used as the cell expressing the HB-EGF protein. After radiolabelling, cells were washed 3 times with RPMI1640 medium containing 10% FBS and adjusted to a cell concentration of 2X 10⁵The target cells were prepared per ml.

ADCC activity and CDC activity can be measured by the following methods. To determine ADCC activity, 50. mu.L of target cells and 50. mu.L of anti-HB-EGF antibody were added to a 96-well U-shaped plate (Becton, Dickinson and Company) and reacted on ice for 15 minutes. Then 100. mu.L of effector cells were added and incubated in CO₂Incubate for 4 hours. Final antibody concentrations of 0 or 10 μ g/mL were used. After the culture, 100. mu.L of the supernatant was collected and the radioactivity was measured by a GAMMA counter (COBRA II AUTO-GAMMA, MODEL D5005, packard Instrument Company). Using the values obtained, cytotoxicity (%) was calculated from the formula (A-C)/(B-C) x100, where A is the radioactivity (cpm) of the specific sample, B is the radioactivity (cpm) of the sample to which 1% NP-40(Nacalai Tesque, Inc.) was added, and C is the radioactivity (cpm) of the sample containing only the target cells.

On the other hand, when the CDC activity is to be measured, 50. mu.L of the target cells and 50. mu.L of the anti-HB-EGF antibody are added to a 96-well flat-bottom plate (Becton, Dickinson and Company), and reacted on ice for 15 minutes. Then 100. mu.L of complement solution was added and the mixture was incubated in CO₂Incubate for 4 hours. Final antibody concentrations of 0 or 3 μ g/mL were used. After incubation, 100. mu.L of the supernatant was recovered and radioactivity was measured by a gamma counter. Cytotoxicity can be calculated in the same manner as ADCC activity is measured.

The antibody having internalization activity and ADCC activity and/or CDC activity can be a very effective anticancer agent against HB-EGF-highly expressing cancers. In addition, an antibody having neutralizing activity and ADCC activity and/or CDC activity can be a very effective anticancer agent against cancers highly expressing HB-EGF. Furthermore, an antibody having an internalization activity plus a neutralization activity plus an ADCC activity and/or a CDC activity can be a very effective anticancer agent against cancers that highly express HB-EGF.

Production of antibodies

The monoclonal anti-HB-EGF antibody of the present invention can be obtained using a known method. The anti-HB-EGF antibody of the present invention is particularly preferably a monoclonal antibody of mammalian origin. The monoclonal antibodies of mammalian origin include, in particular, monoclonal antibodies produced by hybridomas and monoclonal antibodies produced by hosts that have been transformed with expression vectors containing antibody genes by genetic engineering techniques.

Hybridomas producing monoclonal antibodies can be prepared by using known techniques, for example, the techniques described below. First, an animal is immunized with the HB-EGF protein as a sensitizing antigen according to a usual immunization method. The immune cells obtained from the immunized animal are fused with known companion cells by a usual cell fusion technique to obtain hybridomas. Among these hybridomas, a hybridoma producing an anti-HB-EGF antibody is selected by screening cells producing a desired antibody using a usual screening technique.

Specifically, the production of monoclonal antibodies can be performed, for example, as follows. First, an HB-EGF protein serving as a sensitizing antigen for obtaining an antibody can be obtained by expression of the HB-EGF gene. For example, the base sequence of the human HB-EGF gene as GenBank accession No. NM-001945 (SEQ ID NO: 49) is disclosed. Thus, the gene sequence encoding HB-EGF is inserted into a known expression vector, and then a suitable host cell is transformed with the expression vector; the desired human HB-EGF protein is then purified from within the host cell or from the culture supernatant. The purified natural HB-EGF protein can also be used in the same manner. The protein may be purified using a common chromatographic technique or combination thereof, e.g., ion chromatography, affinity chromatography, etc., using one run or multiple runs. The immunogen used in the present invention may also be a fusion protein obtained by fusing a desired partial polypeptide derived from the HB-EGF protein with a different polypeptide. For example, a peptide tag or Fc fragment from an antibody can be used to generate a fusion protein to be used as an immunogen. Vectors for expressing the fusion protein can be prepared by fusing the genes encoding the desired two or more polypeptide fragments in frame and inserting the fusion gene into the above-described expression vector. Methods for producing fusion proteins are described in Molecular Cloning, 2 nd edition (Sambrook, J. et al, Molecular Cloning, 2 nd edition, 9.47-9.58, Cold Spring harbor laboratory Press, 1989).

The HB-EGF protein purified in the manner can be used as a sensitizing antigen for immunizing a mammal. A partial peptide derived from HB-EGF can also be used as an sensitizing antigen. For example, the following peptides can be used as sensitizing antigens:

a peptide obtained from the amino acid sequence of human HB-EGF by chemical synthesis;

a peptide obtained by incorporating a part of the human HB-EGF gene into an expression vector and expressing the part; and

a peptide obtained by degrading a human HB-EGF protein with a protein-degrading enzyme.

There is no limitation on the size of the HB-EGF region or partial peptide used as the partial peptide. A preferred region may be selected from the amino acid sequences constituting the extracellular domain of HB-EGF (positions 22-149 in the amino acid sequence of SEQ ID NO: 50). The number of amino acids constituting the peptide to be used as a sensitizing antigen is preferably at least 3, for example at least 5 or at least 6. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

There is no particular limitation on the mammal that can be immunized with the sensitizing antigen described above. In order to obtain a monoclonal antibody by the cell fusion technique, an immunized animal is preferably selected in consideration of compatibility with a partner cell to be used in cell fusion. Rodents are generally preferred as immunized animals. In particular, a mouse, rat, hamster, or rabbit may be used as the immunizing animal. Monkeys can also be used as immunized animals.

The animals can be immunized with the sensitizing antigen according to known methods. For example, as a general method, a mammal may be immunized by subcutaneous or intraperitoneal injection of a sensitizing antigen. Specifically, the sensitizing antigen can be administered to the mammal multiple times on a 4-21 day schedule. For example, the sensitizing antigen is diluted to an appropriate dilution factor with Phosphate Buffered Saline (PBS) or physiological saline. The sensitizing antigen can also be administered in combination with an adjuvant. For example, the sensitizing antigen can be prepared by mixing with Freund's complete adjuvant and emulsifying. Suitable carriers may also be used for sensitizing antigen immunization. Particularly in the case of using a partial peptide of low molecular weight as an sensitizing antigen, it is desirable to use a peptide such as albumin, keyhole limpetThe sensitizing peptide antigen combined with protein carriers such as hemocyanin realizes immunity.

After immunization of the animals in the manner described and observation of the expected increase in serum antibody titer, immune cells were collected from the mammals and subjected to cell fusion. Splenocytes are particularly preferred immune cells.

Mammalian myeloma cells are used as the cells to be fused with the above immune cells. Myeloma cells preferably have appropriate selectable markers to support screening. A selectable marker refers to a trait that may (or may not) occur under specific culture conditions. Known selectable markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as KT deficiency). HGPRT-deficient or TK-deficient cells show hypoxanthine-aminopurine-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT sensitive cells are unable to synthesize DNA on HAT selection medium and die; however, when fused to normal cells, DNA synthesis can continue using salvage pathways of normal cells and growth can also occur on HAT selection media.

HGPRT-deficient cells can be selected on media containing 6-thioguanine or 8-azaguanine (8AG), while TK-deficient cells can be selected on media containing 5' -bromodeoxyuridine. Normal cells incorporate these pyrimidine analogs into their DNA and die, while cells deficient in these enzymes do not incorporate these pyrimidine analogs and are able to survive on selective media. Another selectable marker, known as G418 resistance, provides resistance to 2-deoxystreptamine-type antibiotics (gentamicin analogs) based on the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known. For example, the following myeloma cells may be used: p3(P3x63Ag8.653) (J.Immunol. (1979)123, 1548-1550), P3x63Ag8U.1(Current Topics 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 al, 1976 (1976)8, 405-Cell 415), SP2/0(Shulman, M. et al, Nature (1978)276, 122-270), FO (St.Groth, S.F. et al, J.Immunol.methods (35, 1-21), S194 (Trobrge, I.313. S.313. 277) 35, 1-21), S.11 (Trowr.11, III.J.S.H. 269, 23, 9-Med.23, 9, Med.H. 67, et al.

Cell fusion between the above immune cells and myeloma cells can be carried out according to known Methods, for example, according to the Methods of Kohler and Milstein (Kohler, G., and Milstein, C., Methods Enzymol (1981)73, 3-46).

More specifically, for example, cell fusion can be carried out in a normal nutrient culture liquid in the presence of a cell fusion promoter. For example, polyethylene glycol (PEG) or Sendai virus (HVJ) can be used as the fusion promoter. If necessary, an adjuvant such as dimethyl sulfoxide may be added in order to improve the fusion efficiency.

The ratio of immune cells to myeloma cells can be freely chosen. For example, it is preferred to use immune cells at 1X-10X relative to myeloma cells. For example, the culture medium for cell fusion may be RPMI1640 medium or MEM medium, which is well suited for the growth of the above-mentioned myeloma cell lines, or a commonly used medium for this type of cell culture. Serum supplements such as Fetal Calf Serum (FCS) may also be added to the medium.

Desired fused cells (hybridomas) are formed by cell fusion by thoroughly mixing predetermined amounts of immune cells and myeloma cells in the above culture solution and mixing a PEG solution that has been preheated to about 37 ℃. For example, PEG having an average molecular weight of 1000-6000 may be added to the cell fusion process at a concentration of typically 30-60% (w/v). Then, the cell fusion agent and the like unnecessary for the growth of the hybridoma are removed by repeating the process of adding the appropriate culture solution, centrifugation and removal of the supernatant as described above.

Hybridomas obtained in this manner can be selected by using a selective medium suitable for the selection marker exhibited by myeloma for cell fusion. For example, HGPRT-deficient or TK-deficient cells can be selected by culturing on HAT medium (hypoxanthine, aminopurine and thymidine containing medium). Thus, when HAT sensitive myeloma cells are used for cell fusion, cells resulting from cell fusion with normal cells can be selectively grown on HAT medium. The culture on HAT medium is continued for a period of time sufficient to allow the cells other than the desired hybridoma (unfused cells) to die. Specifically, the desired hybridoma can be selected generally by culturing for several days to several weeks. A typical limiting dilution method can be used to screen for and monoclonal produce hybridomas with the desired antibodies. Alternatively, an antibody recognizing HB-EGF can also be produced by the method described in WO 03/104453.

Screening and monoclonality of the desired antibody can be appropriately performed by a screening procedure based on a known antigen-antibody reaction. For example, the antigen can be bound to a carrier (e.g., beads such as polystyrene or commercially available 96-well microtiter plates) and then reacted with hybridoma culture supernatants. Subsequently, after washing the carrier, the cells are reacted with, for example, an enzyme-labeled secondary antibody. If the desired sensitizing antigen-reactive antibody is present in the culture supernatant, the second antibody will be bound to the carrier by the antibody. The presence/absence of the desired antibody in the culture supernatant can be finally determined by detecting the secondary antibody bound to the carrier. For example, hybridomas producing desired antigen-binding antibodies can be cloned by limiting dilution. Herein, substantially the same HB-EGF proteins are suitably used as an antigen, including those used for immunization. For example, an oligopeptide comprising the extracellular domain of HB-EGF or comprising a partial amino acid sequence from this region can be used as an antigen.

In addition to the above-described method for producing hybridomas by immunizing non-human animals with antigens, desired antibodies can also be obtained by antigen sensitization of human lymphocytes. Specifically, human lymphocytes were first sensitized with HB-EGF protein in vivo. The immunosensitized lymphocytes are then fused with a suitable fusion partner. For example, human-derived myeloma cells having the ability to permanently divide cells can be used as fusion partners (refer to Japanese patent laid-open No. H1-59878). The anti-HB-EGF antibody obtained by this method is a human antibody having an activity of binding to the HB-EGF protein.

A human anti-HB-EGF antibody can also be obtained by administering an HB-EGF protein as an antigen to a transgenic animal having the complete human antibody gene repertoire (superhaire). Antibody-producing cells from immunized animals can be immortalized by cell fusion with a suitable fusion partner or by treatment such as infection with epstein barr virus. A human antibody against HB-EGF protein can be isolated from the immortalized cells produced (see International publications WO 94/25585, WO93/12227, WO 92/03918 and WO 94/02602). Furthermore, cells producing antibodies with the desired reaction specificity can also be cloned by cloning immortalized cells. When a transgenic animal is used as the immunized animal, the animal's immune system recognizes human HB-EGF as exogenous. This enables a human antibody against human HB-EGF to be easily obtained. The monoclonal antibody-producing hybridoma constructed in such a manner can be subcultured in a usual medium. Hybridomas can also be stored for long periods in liquid nitrogen.

The above-mentioned hybridomas can be cultured according to a usual method, and desired monoclonal antibodies can be obtained from the resulting culture supernatant. Alternatively, the hybridoma can be injected into a mammal compatible with the cell, and the monoclonal antibody can be obtained from ascites of the mammal. The foregoing methods are well suited for producing antibodies of high purity.

The present invention may also use an antibody encoded by an antibody gene cloned from an antibody-producing cell. Expression of the antibody can be achieved by incorporating the cloned antibody gene into a suitable vector followed by transfection of the host. Methods for isolating and inserting antibody genes into vectors and transforming host cells have been established (see, e.g., Vandamm, A.M. et al, Eur.J.biochem. (1990)192, 767-775).

For example, cDNA encoding the variable region (V region) of the anti-HB-EGF antibody can be obtained from a hybridoma cell producing the anti-HB-EGF antibody. Total RNA is generally first extracted from the hybridoma. Methods which can be used for the extraction of mRNA from cells are, for example, the guanidine ultracentrifugation (Chirgwin, J.M. et al, Biochemistry (1979)18, 5294-.

The extracted mRNA can be purified using, for example, an mRNA purification kit (GE Healthcare Biosciences). Alternatively, kits for extracting mRNA directly from cells are also commercially available, such as the QuickPrep mRNA purification kit (GE healthcare biosciences). Kits such as these can also be used to obtain total mRNA from hybridomas. A cDNA encoding the V region of the antibody can be synthesized from the obtained mRNA using reverse transcriptase. For example, cDNA can be synthesized using AMV reverse transcriptase first strand cDNA Synthesis kit (Seikagaku corporation). In addition, the 5 '-Ampli FINDER RACE kit (Clontech) and the PCR-based 5' -RACE method (Frohman, M.A. et al, Proc. Natl. Acad. Sci. USA (1988)85, 8998-9002; Belyavsky, A., et al, Nucleic Acids Res. (1989)17, 2919-2932) can also be used to synthesize and amplify cDNA. In addition, the following appropriate restriction sites may be introduced into both ends of the cDNA in such a cDNA synthesis method.

Purifying a target cDNA fragment from the obtained PCR product, and then connecting with a vector DNA; the recombinant vector prepared in this manner is transfected into, for example, Escherichia coli, and colonies are selected; the desired recombinant vector can be prepared from E.coli which exhibits colony formation. In addition, a known method such as the dideoxynucleotide chain termination method can be used to confirm whether or not the recombinant vector has the base sequence of the target cDNA.

In order to obtain a gene encoding a variable region, PCR using a variable region gene amplification primer may also be used. First, in order to obtain a cDNA library, cDNA is synthesized using the extracted mRNA as a template. Commercially available kits can be conveniently used to synthesize the cDNA library. In fact, the amount of mRNA obtained from only a small number of cells is very small, and thus direct purification has a low yield. Thus, purification is generally performed after adding vector RNA that apparently does not contain an antibody gene. Alternatively, in those cases where a certain amount of RNA can be extracted, efficient extraction can be achieved even with only RNA from the antibody-producing cells. For example, in some cases it is not necessary to add carrier RNA to the RNA extraction from at least 10 or at least 30, preferably at least 50, antibody-producing cells.

Using the obtained cDNA library as a template, antibody genes can be amplified by PCR. Primers for PCR-based amplification of antibody genes are known. For example, primers for amplifying human antibody genes can be designed based on information in the literature (e.g., J.mol.biol. (1991)222, 581-597). These primers have base sequences varying with the subclass of immunoglobulin. Therefore, when a cDNA library of an unknown subclass is used as a template, PCR is performed in consideration of all possibilities.

Specifically, for example, when the objective is to obtain a gene encoding human IgG, primers having the ability to amplify genes encoding the κ chain and the λ chain of γ 1 to γ 5 of the heavy chain and the light chain may be used. For amplifying the IgG variable region gene, a primer that anneals to a region corresponding to the hinge region is generally used for the 3' -side primer. On the other hand, for the 5' -side primer, a primer suitable for each subclass can be used.

PCR products were prepared as independent libraries based on gene amplification primers for each heavy and light chain subclass. Using the library so synthesized, immunoglobulins comprising a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin to HB-EGF can be used as an index to screen a desired antibody.

The binding of the antibody of the present invention to HB-EGF is more preferably specific binding. For example, screening for an antibody that binds to HB-EGF can be performed by:

(1) contacting the HB-EGF with an antibody comprising a V region encoded by a cDNA obtained from the hybridoma;

(2) detecting binding of HB-EGF to the antibody; and

(3) selecting an antibody that binds to HB-EGF.

Methods for detecting binding of an antibody to HB-EGF are known. Specifically, the test antibody can be reacted with HB-EGF immobilized on a carrier, and then reacted with a labeled antibody recognizing the antibody. After washing, the labeled antibody on the carrier can be detected as an indication that the test antibody binds to HB-EGF. A fluorescent substance such as FITC or an enzyme protein such as peroxidase or β -galactoside may be used as the label. The HB-EGF-expressing cells in immobilized form can also be used to evaluate the binding activity of the antibody.

Panning (Panning) using phage vectors can also be employed as an antibody screening method using binding activity as an indicator. When antibody genes are obtained as a library of heavy chain subclasses and light chain subclasses as described above, screening using phage vectors is advantageous. The genes encoding the variable regions of the heavy and light chains can be made into single chain fv (scFv) by ligation with appropriate linker sequences. Genes encoding the scFv can be inserted into a phage vector to obtain a phage that expresses the scFv on its surface. Contacting the phage with a target antigen and recovering the antigen-bound phage enables recovery of DNA encoding the scFv having the desired binding activity. If desired, the scFv having the desired binding activity can be enriched by repeating this process.

In the present invention, the polynucleotide encoding the antibody may encode the full length of the antibody or may encode a portion of the antibody. The portion of the antibody may be any portion of an antibody molecule. Antibody fragments are the following terms used in some instances to refer to a portion of an antibody. Preferred antibody fragments of the invention comprise Complementarity Determining Regions (CDRs). More preferred antibody fragments of the invention comprise all 3 CDRs which make up the variable region.

Once a cDNA encoding the V region of the anti-HB-EGF antibody of interest is obtained, the cDNA is digested with a restriction enzyme recognizing a restriction enzyme site that has been inserted into both ends of the cDNA. Preferred restriction enzymes recognize and digest base sequences that are less likely to occur among the base sequences constituting the antibody gene. For inserting 1 copy of the digested fragment in the correct orientation in the vector, a restriction enzyme that produces a sticky end is preferred. The antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-HB-EGF antibody digested as described above into an appropriate expression vector. In this regard, chimeric antibodies can be obtained by fusing in-frame the gene encoding the constant region (C region) of an antibody with the aforementioned gene encoding the V region. Herein, a chimeric antibody refers to a product in which the constant region and the variable region have different origins. Thus, in the context of the present invention, "chimeric antibodies" also include human-human allogeneic chimeric antibodies (allochimeric antibodies) in addition to, for example, mouse-human xenochimeric antibodies (heterochimeric antibodies). A chimeric antibody expression vector can also be constructed by inserting the aforementioned V region gene into an expression vector already carrying a constant region.

Specifically, for example, a restriction enzyme recognition sequence of a restriction enzyme for digesting the aforementioned V region gene may be provided in advance on the 5' side of an expression vector carrying a DNA encoding a desired antibody constant region (C region). The two were digested with the same restriction enzyme combination and fused in-frame, resulting in the construction of a chimeric antibody expression vector.

To produce the anti-HB-EGF antibody of the present invention, the antibody gene may be incorporated into an expression vector in such a manner that expression takes place under the control of the expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. A recombinant cell expressing a DNA encoding the anti-HB-EGF antibody can then be obtained by transforming a suitable host cell with the expression vector.

For expression of the antibody gene, a DNA encoding the heavy chain (H chain) of the antibody and a DNA encoding the light chain (L chain) of the antibody may be incorporated into separate expression vectors. Antibody molecules having both H chains and L chains can be expressed by simultaneously transforming (co-transfecting) the same host cell with a vector incorporating H chains and a vector incorporating L chains. Alternatively, the DNA encoding the H chain and the L chain may be incorporated into one expression vector, and the host cell may be transformed (International publication WO 94/11523).

For the production of antibodies by isolating antibody genes and transfecting appropriate hosts, a number of host/expression vector combinations are known. Any of these expression systems may be applied to the present invention. When eukaryotic cells are used as hosts, animal cells, plant cells or fungal cells may be used. Specific examples of the animal cell that can be used in the present invention are mammalian cells (e.g., CHO, COS, myeloma, Baby Hamster Kidney (BHK), Hela, Vero, etc.), amphibian cells (e.g., Xenopus laevis (Xenopus laevis) oocyte, etc.), and insect cells (e.g., sf9, sf21, Tn5, etc.).

For plant cells, an antibody gene expression system based on cells from the genus Nicotiana such as tobacco (Nicotiana tabacum) or the like is known. Cells cultured from callus may be used for plant cell transformation.

The following may be used, for example, as fungal cells: yeasts (e.g., Saccharomyces such as Saccharomyces cerevisiae, Pichia such as Pichia pastoris, etc.) and filamentous fungi (e.g., Aspergillus such as Aspergillus niger).

Antibody gene expression systems using prokaryotic cells are also known. As the bacterium, for example, Escherichia coli, Bacillus subtilis and the like can be used in the present invention.

When mammalian cells are used, an expression vector can be constructed by functionally linking an effective, commonly used promoter, an antibody gene to be expressed, and a polyA signal located downstream of the 3' -end of the antibody gene. An example of a promoter/enhancer is the human cytomegalovirus immediate early promoter/enhancer.

Other promoters/enhancers that may be used to express the antibodies of the invention are, for example, viral promoters/enhancers and promoters/enhancers derived from mammalian cells, such as human elongation factor 1 alpha (HEF1 alpha). Specific examples of promoters/enhancers that can be provided are retroviruses, polyomaviruses, adenoviruses and simian virus 40(SV 40).

The SV40 promoter/enhancer can be used according to the method of Mullingan et al (Nature (1979)277, 108). Furthermore, the HEF1 alpha promoter/enhancer can be easily used for desired gene expression according to the method of Mizushima et al (Nucleic acids SRes. (1990)18, 5322).

For E.coli, expression of the gene in question can be achieved by functionally linking an efficient, commonly used promoter, a signal sequence for antibody secretion and the antibody gene to be expressed. For example, the promoter may be lacZ promoter or araB promoter. The lacZ promoter can be used according to the method of Ward et al (Nature (1989)341, 544-546; FASEBJ. (1992)6, 2422-2427). Alternatively, the araB promoter can be used for the desired gene expression according to the method of Better et al (Science (1988)240, 1041-1043).

As for the signal sequence for antibody secretion, the pelB signal sequence (Lei, s.p. et al, j.bacteriol. (1987)169, 4379) can be used in the case of production in the periplasm of e. After the antibodies produced in the periplasm have been isolated, the antibody structure can be engineered (refolded) to exhibit the desired binding activity by using protein denaturants such as guanidine hydrochloride and urea.

For example, the origin of replication inserted into the expression vector may be derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), or the like. In addition, a selectable marker may be inserted into the expression vector in order to amplify the copy number of the gene in the host cell system. Specifically, useful selectable markers are an aminoglycoside transferase (APH) gene, a Thymidine Kinase (TK) gene, an E.coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, and the like.

The target antibody can be produced by transfecting the expression vector of interest into a host cell and culturing the transformed host cell in vitro or in vivo. Host cell culture can be performed according to known methods. For example, DMEM, MEM, RPMI1640, or IMDM can be used as the medium; serum supplements such as Fetal Calf Serum (FCS) may also be added.

The antibody expressed and produced as described above can be purified by a commonly used method known for protein purification; one such method or a suitable combination of these methods may be used. Suitable selections and combinations of the following methods can be used to isolate and purify the antibody: for example, affinity columns (e.g., protein A columns), column chromatography, filtration, ultrafiltration, salting out, dialysis, and the like (Antibodies: A Laboratory Manual. Ed Harlow, David Lane, Cold spring Harbor Laboratory, 1988).

In addition to the aforementioned host cells, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody under investigation can be obtained from an animal into which a gene encoding the target antibody has been introduced. For example, a fused gene can be prepared by inserting an antibody gene in-frame into a gene encoding a protein naturally produced in milk. For example, goat beta-casein can be used as a protein secreted into milk. A DNA fragment containing a fusion gene incorporating an antibody gene may be injected into a goat embryo, and the injected embryo may be introduced into a female goat. The desired antibody can be obtained as a fusion protein with a milk protein from the milk produced by a transgenic goat born by the goat implanted with the embryo (or its offspring). In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be suitably used for the transgenic goat (Ebert, K.M. et al, Bio/Technology (1994)12, 699-702).

The C region derived from an animal antibody can be used as the C region of the recombinant antibody of the present invention. Mouse antibody H chain C regions designated C γ 1, C γ 2a, C γ 2b, C γ 3, C μ, C δ, C α 1, C α 2, and C ε may be used, and L chain C regions designated C κ and C λ may also be used. In addition to mouse antibodies, animal antibodies derived from, for example, rat, rabbit, goat, sheep, camel, monkey, and the like may also be used as the animal antibodies. These sequences are known. The C region may be modified in order to improve the stability of the antibody or its production. When the antibody is to be administered to a human, an artificially engineered recombinant antibody can be prepared in the present invention, for example, in order to reduce foreign antigenicity in a human body. Such recombinant antibodies include, for example, chimeric antibodies and humanized antibodies.

These engineered antibodies can be produced using known methods. A chimeric antibody refers to an antibody in which a variable region is linked to a constant region having a different origin from the variable region. For example, an antibody having a heavy chain variable region and a light chain variable region from a mouse antibody and a heavy chain constant region and a light chain constant region from a human antibody is a mouse-human-xenochimeric antibody. A recombinant vector for expressing a chimeric antibody can be constructed by ligating a DNA encoding a mouse antibody variable region with a DNA encoding a human antibody constant region and incorporating it into an expression vector. The recombinant cells transformed with the vector are then cultured to allow expression of the incorporated DNA, and the produced chimeric antibody in the culture medium can then be recovered. The C region of a human antibody is used for the C region of a chimeric antibody and a humanized antibody. For the H chain, for example, C γ 1, C γ 2, C γ 3, C γ 4, C μ, C δ, C α 1, C α 2 and C ε may be used for the C region. For the L chain, C.kappa.and C.lambda.can be used for the C region. The amino acid sequences of these C regions are known, and the base sequences encoding these amino acid sequences are also known. In addition, the C region of a human antibody may be modified in order to improve the antibody itself or to increase the stability of antibody production.

Chimeric antibodies are generally constructed from the V region of an antibody of non-human animal origin and the C region of an antibody of human origin. In contrast, a humanized antibody is composed of the Complementarity Determining Regions (CDRs) of an antibody of non-human animal origin, the Framework Regions (FRs) of an antibody of animal origin, and the C regions of an antibody of human origin. In order to reduce antigenicity in humans, humanized antibodies can be used as an active ingredient in the therapeutic agent of the present invention.

The variable region of an antibody typically consists of 3 CDRs sandwiched between 4 FRs. CDRs are regions that substantially determine the binding specificity of an antibody. The amino acid sequences of the CDRs are rich in diversity. On the other hand, the amino acid sequences constituting the FRs often show high homology even between antibodies having different binding specificities. For this reason, the binding specificity of one antibody can be transferred to another antibody, typically by CDR grafting.

Humanized antibodies are also known as reshaped human antibodies. Specifically, for example, humanized antibodies in which CDRs from a non-human animal antibody such as a mouse antibody have been grafted into a human antibody are known. General genetic recombination techniques for obtaining humanized antibodies are also known.

Specifically, for example, overlap extension PCR is known as a method for grafting a mouse antibody CDR into a human FR. In overlap extension PCR, a base sequence encoding a CDR of a mouse antibody to be grafted is added to a primer for synthesizing a FR of a human antibody. Primers were prepared for each of the 4 FRs. Selection of human FRs that show high homology to mouse FRs generally facilitates preservation of CDR function in grafting mouse CDRs onto human FRs. Therefore, it is generally preferred to use human FRs whose amino acid sequences show high homology with the amino acid sequences of FRs adjacent to the mouse CDR to be grafted.

In addition, the base sequences to be ligated are designed so as to be capable of being ligated in frame with each other. Human FRs were synthesized using the respective primers. In this way, a product in which DNA encoding mouse CDRs was attached to each FR was obtained. The base sequences encoding the mouse CDRs in each product were designed so as to overlap each other. Then, overlapping CDR regions of the product synthesized using the human antibody gene as a template anneal to each other, and a complementary strand synthesis reaction is performed. This reaction results in the ligation of human FRs via mouse CDR sequences.

Finally, the variable region gene comprising 4 FRs linked to 3 CDRs is subjected to full-length amplification by annealing primers to the 5 'and 3' ends of which an appropriate restriction enzyme recognition sequence has been added. An expression vector for a human antibody is constructed by inserting the DNA obtained as described above and a DNA encoding a human antibody C region into an expression vector and fusing them in frame. Inserting the thus-prepared vector into a host and establishing a recombinant cell; culturing the recombinant cell to express DNA encoding the humanized antibody; thereby producing a humanized antibody in the medium in which the cells are cultured (refer to European patent publication EP 239,400 and International publication WO 96/02576).

The human antibody FRs capable of allowing the CDRs to form a high-quality antigen-binding site when connected across the CDRs can be appropriately selected by qualitatively and quantitatively measuring and evaluating the binding activity of the humanized antibody constructed as described above to an antigen. Amino acid substitutions may also be made on the FRs, if necessary, to allow the CDRs of the reshaped human antibody to form a well adapted antigen binding site. For example, mutations in the amino acid sequence can be introduced into the FR using PCR methods for grafting mouse CDRs onto human FRs. Specifically, a partial base sequence mutation may be introduced into a primer to be annealed to an FR. Then, base sequence mutations were introduced into FRs synthesized using these primers. By assaying and evaluating the antigen binding activity of the mutated, amino acid-substituted antibody using the methods described above, one can select a mutated FR sequence having the desired properties (Sato, K. et al, Cancer Res., 1993, 53, 851-856).

Methods for obtaining human antibodies are also known. For example, human lymphocytes can be sensitized in vitro with a desired antigen or cells expressing a desired antigen. Then, a desired human antibody capable of binding to an antigen can be obtained by fusing the sensitized lymphocytes with human myeloma cells (refer to Japanese patent laid-open No. H1-59878). For example, U266 can be used for human myeloma cells that are used as fusion partners.

The desired human antibody can also be obtained by immunizing a transgenic animal having the complete human antibody gene repertoire (supertoxin) with the desired antigen (see international publications WO93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735). Techniques for obtaining human antibodies by panning using human antibody libraries are also known. For example, a human antibody V region as a single chain antibody (scFv) can be expressed on the surface of a phage by a phage display method, and a phage that binds to an antigen can be selected. The DNA sequence encoding the V region of the human antibody that binds the antigen can then be determined by analyzing the genes of the selected phage. Once the DNA sequence of the scFv that binds the antigen has been determined, the V region sequence can be fused in frame with the sequence of the desired human antibody C region, after which an expression vector can be constructed by inserting it into a suitable expression vector. The expression vector can be transfected into an appropriate expression cell as described above, and a human antibody can be obtained by expression of a gene encoding the human antibody. Such processes are known (International publications 92/01047, WO92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388).

To the extent that binding to the HB-EGF protein occurs, the antibody of the present invention includes not only a bivalent antibody represented by IgG but also a multivalent antibody and a monovalent antibody represented by IgM. Multivalent antibodies of the invention include multivalent antibodies in which all antigen binding sites are the same, and multivalent antibodies in which some or all antigen binding sites are different. The antibody of the present invention is not limited to the full-length antibody molecule, but also includes low molecular weight antibodies and modifications thereof as long as they are capable of binding to HB-EGF protein.

Low molecular weight antibodies include antibody fragments produced by deleting a portion of an intact antibody (e.g., an intact IgG). Partial deletion of the antibody molecule is permissible as long as there is an ability to bind to the HB-EGF antigen. The antibody fragment for use in the present invention preferably comprises a heavy chain variable region (VH) or a light chain variable region (VL) or both. The amino acid sequence of VH or VL may include substitutions, additions and/or insertions. Further, a part of VH or VL or both may be deleted as long as the ability to bind to HB-EGF antigen still remains. The variable regions may also be chimeric or humanized. Specific examples of antibody fragments are Fab, Fab ', F (ab') 2 and Fv. Specific examples of low molecular weight antibodies are Fab, Fab ', F (ab') 2, FV, scFv (single chain FV), diabody, and sc (FV)2 (single chain (FV) 2). The low molecular weight antibodies of the invention also include multimers (e.g., dimers, trimers, tetramers, multimers) of these antibodies.

Antibody fragments can also be obtained by producing antibody fragments by treating the antibodies with enzymes. For example, papain, pepsin, plasmin, and the like are known as enzymes for producing antibody fragments. Alternatively, genes encoding such antibody fragments can be constructed and inserted into expression vectors and then expressed by suitable host cells (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; Lamoyi, E.methods in Enzymology (1989)121, 652-663; Rousseaux, J. et al, Methods in Enzymology (1989)121, 663-669; and rd, R.E. et al, TIE. E. et al, TIH (1991)9, ECH 132).

The digestive enzymes cleave specific antibody fragment sites to produce antibody fragments having specific structures as described below. When genetic engineering techniques are applied to these enzymatically produced antibody fragments, any portion of the antibody may be deleted.

And (3) papain digestion: f (ab)2 or Fab

And (3) pepsin digestion: f (ab ') 2 or Fab'

Fibrinolytic enzyme digestion: facb

Diabodies represent bivalent antibody fragments constructed by gene fusion (Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90, 6444-6448(1993), EP 404,097, WO93/11161 et al). Diabodies are dimers consisting of two polypeptide chains. Generally, each polypeptide chain constituting a diabody is VL and VH connected as one identical chain by a linker. Linkers for diabodies are generally short enough that VL and VH cannot bind to each other. Specifically, for example, about 5 amino acid residues constitute a linker. For this reason, VL and VH encoded on the same polypeptide chain cannot form single chain variable fragments, and form dimers with single chain variable fragments alone. Thus, diabodies have two antigen binding sites.

scFv was obtained by linking the H chain V region to the L chain V region of an antibody. The H chain V region and the L chain V region in the scFv are linked to each other by a linker, and preferably by a peptide linker (Huston, J.S. et al, Proc. Natl.Acad.Sci.USA 85, 5879-. The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies described herein. There is no particular limitation on the peptide linker connecting the V regions. For example, any single peptide chain having about 3-25 residues can be used as a linker.

For example, the V regions can be ligated using the PCR techniques described previously. In order to link the V regions by PCR, first, a DNA encoding all or a desired part of the amino acid sequence derived from the DNA sequence encoding the H chain or H chain V region of the antibody and a DNA encoding all or a desired part of the amino acid sequence derived from the DNA sequence encoding the L chain or L chain V region of the antibody are used as templates.

The DNA encoding the H chain V region and the DNA encoding the L chain V region are amplified by PCR using a primer pair having sequences corresponding to both ends of the DNA to be amplified, respectively. DNA encoding the peptide linker region is then prepared. DNA encoding the peptide linker may also be synthesized using PCR. Before the 5' side of the primers used, a base sequence capable of being ligated to each of the V region amplification products synthesized separately was added. Then, PCR reaction was carried out using the assembly PCR primers and each of the DNAs for [ H chain V region DNA ] - [ peptide linker DNA ] - [ L chain V region DNA ]. The assembly PCR primer is a combination of a primer that anneals to the 5 '-side of [ H chain V region DNA ] and a primer that anneals to the 3' -side of [ L chain V region DNA ]. That is, the PCR primers are assembled to form a primer set capable of amplifying DNA encoding the full-length sequence of the scFv to be synthesized. On the other hand, a base sequence that can be ligated to each V region DNA is added to the [ peptide linker DNA ]. As a result, these DNAs are ligated together, and finally, full-length scFv is produced as an amplification product by assembling PCR primers. Once DNA encoding scFv has been produced, an expression vector containing the DNA and a recombinant cell transformed with the expression vector can be obtained by a usual method. In addition, the recombinant cells thus obtained may be cultured, and scFv may be obtained by expression of DNA encoding scFv.

sc (fv)2 is a low molecular weight antibody in which two VH and two VL are connected to a single chain, for example, by a linker (Hudson et al, J.Immunol. methods, 231, 177-189 (1999)). For example, sc (fv)2 can be prepared by linking an scFv with a linker.

Preferably, the antibody characterized by having two VH and two VL arranged in a sequence is VH, VL, VH, VL ([ VH ] linker- [ VL ] linker- [ VH ] linker- [ VL ]) from the N-terminal side of the single-chain polypeptide.

The sequences of two VH and two VL are not particularly limited to the above arrangement, and they may be arranged into any sequence. The following sequence may be provided as an example.

[ VL ] linker- [ VH ] linker- [ VL ]

[ VH ] linker- [ VL ] linker- [ VH ]

[ VH ] linker- [ VL ]

[ VL ] linker- [ VH ]

[ VL ] linker- [ VH ] linker- [ VL ] linker- [ VH ]

The linker to which the variable region of the antibody is attached may be, for example, a peptide linker which can be inserted by genetic Engineering or a synthetic compound linker, for example, as disclosed in Protein Engineering, 9(3), 299-305 (1996). In the present invention, a peptide linker is preferred. The length of the peptide linker is not particularly limited and may be appropriately selected by those skilled in the art in consideration of the intended application. Generally, there are 1 to 100 amino acid residues, preferably 3 to 50 amino acid residues, more preferably 5 to 30 amino acid residues, and particularly preferably 12 to 18 amino acid residues (e.g., 15 amino acid residues) in the peptide linker.

The amino acid sequence of the peptide linker can be any sequence that does not interfere with the binding action of the scFv.

Alternatively, synthetic chemical linkers (chemical crosslinkers) can be used to link the V regions. Crosslinking agents typically used to crosslink such peptide compounds may be used in the present invention. For example, the following compounds may be used: n-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), disuccinimidyl suberate (BS3), dithiobissuccinimide propionate (DSP), dithiobissulfosuccinimidyl propionate (DTSSP), ethylene glycol bissuccinimide succinate (EGS), ethylene glycol bissulfosuccinimidyl succinate (sulfo-EGS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfo-DST), bis [2- (succinimidyoxycarbonyloxy) ethyl ] sulfone (BSOCOES), bis [2- (sulfosuccinimidyoxycarbonyloxy) ethyl ] (sulfo-BSOCOCOES), and the like.

When linking 4 antibody variable regions, usually 3 linkers are required. The plurality of linkers may be the same as each other, or different linkers may be used. Diabodies and sc (FV)2 are preferred low molecular weight antibodies of the invention. To obtain such low molecular weight antibodies, the antibodies may be treated with enzymes (e.g., papain, pepsin, etc.) to produce antibody fragments, or DNA encoding these antibody fragments may be constructed and inserted into expression vectors, followed by expression in suitable host cells (see, e.g., Co, M.S. et al, J.Immunol. (1994)152, 2968-2976; Better, M. and Horwitz, A.H.methods Enzymol. (1989)178, 476-496; Plueckthun, A. and Skerra, A.methods Enzymol. (1989)178, 497 515; Lamoyi, E.methods Enzymol. (1986) 663; Rousseaux, J.et al, Methods Enzymol. (1986)121, Methods; 198121; Trench, 121, and Walsh @ 132, Biotech. (1991) 121).

In addition, the antibody of the present invention may be used in the form of a modified antibody to which various molecules such as polyethylene glycol (PEG) are bonded. These modified antibodies can be obtained by chemical modification of the antibodies of the invention. Antibody modification methods have been established in the art.

The antibodies of the invention may also be bispecific antibodies. Bispecific antibodies are antibodies that have variable regions within the same antibody molecule that recognize different epitopes, where the epitopes may be present in different molecules or may be present in one molecule. Thus, in the scope of the present invention, the bispecific antibody may have an antigen binding site recognizing different epitopes on the HB-EGF molecule. Using such a bispecific antibody, two antibody molecules can bind to one HB-EGF molecule. Therefore, stronger cytotoxicity can be expected. The "antibodies" of the present invention also include these antibodies.

The present invention also includes bispecific antibodies recognizing antigens other than HB-EGF. For example, the present invention includes a bispecific antibody recognizing an antigen different from HB-EGF, wherein the antigen is specifically expressed on the cell surface of a cancer cell as the same target as HB-EGF.

Methods of producing bispecific antibodies are known. For example, bispecific antibodies can be produced by linking two antibodies that recognize different antigens. Each antibody linked may be a half molecule with H and L chains or may be a quarter molecule with only H chains. Alternatively, a fused cell producing a bispecific antibody can also be produced by fusing hybridomas producing different monoclonal antibodies. Alternatively, bispecific antibodies can be produced by genetic engineering techniques.

The antibody of the present invention may be an antibody having a modified sugar chain. It is known that the cytotoxicity of an antibody can be improved by engineering the sugar chain on the antibody.

The following are examples of antibodies with engineered sugar chains: glycosylation-engineered antibodies (e.g., WO 99/54342), antibodies in which fucose present in sugar chains has been deleted (e.g., WO 00/61739, WO 02/3140, WO 2006/067847, WO2006/067913), and antibodies carrying sugar chains having bisecting GlcNAc (e.g., WO 02/79255).

A fucose-negative antibody is an example of a preferred sugar chain-modified antibody of the present invention. The sugar chain attached to the antibody may be an N-glycoside-linked sugar chain attached to the side chain N atom of asparagine in the antibody molecule, or may be an O-glycoside-linked sugar chain attached to the side chain hydroxyl group of serine or threonine in the antibody molecule; however, in the present invention, the presence/absence of fucose is a problem associated with an N-glycoside-linked sugar chain.

In the context of the present invention, a fucose-negative antibody means that fucose has been deleted in at least 20%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the N-glycoside-linked sugar chains based on the N-glycoside-linked sugar chains on the antibody in the particular composition.

Fucose-negative antibodies can be prepared by methods well known to those skilled in the art; for example, can be produced by expressing the antibody protein in a host cell that has little or no ability to add α -1, 6 core fucose. Host cells with little or no ability to add fucose include, but are not limited to: YB2/3hl. p2.g 11.16ag.20 rat myeloma cells (abbreviated as YB2/0 cells, deposited as ATCC CRL 1662), FTVIII knockout CHO cells (WO 02/31140), Lec13 cells (WO 03/035835), and fucose transporter negative cells (e.g., WO 2006/067847, WO 2006/067913).

Sugar chains can be analyzed by methods known to those skilled in the art. For example, sugar chains can be released from the antibody by, for example, the action of N-glycosidase F (Roche) on the antibody. The samples were then desalted by solid phase extraction using cellulose columns (Shimizu Y. et al, Carbohydrate Research 332(2001), 381-. After removing the reagent from the PA-labeled sugar chains obtained by solid phase extraction using a cellulose column, purified PA-labeled sugar chains were obtained by concentration using a centrifuge, and determined by reverse phase HPLC analysis using an ODS column. In addition, after the preparation of the PA-labeled sugar chains, two-dimensional mapping (in which reverse phase HPLC analysis using an ODS column is combined with normal phase HPLC analysis using an amine column) may also be performed.

The antibodies described in the following [1] to [13] are examples of the antibody which can be used in the present invention.

[1] Comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 14, SEQ ID NO as CDR 2: 16 and the amino acid sequence of SEQ ID NO: 18, or a heavy chain variable region of the amino acid sequence of seq id no;

[2] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 20, SEQ ID NO as CDR 2: 22 and the amino acid sequence of SEQ ID NO: 24, or a light chain variable region of the amino acid sequence of seq id no;

[3] an antibody comprising the heavy chain of [1] and the light chain of [2 ];

[4] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 26, the amino acid sequence of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 30, or a heavy chain variable region of the amino acid sequence of seq id no;

[5] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 32, the amino acid sequence of SEQ ID NO: 34 and the amino acid sequence of SEQ ID NO: 36, or a light chain variable region of the amino acid sequence of seq id no;

[6] an antibody comprising the heavy chain of [4] and the light chain of [5 ];

[7] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 76, SEQ ID NO as CDR 2: 77 and the amino acid sequence of SEQ ID NO: 78, or a heavy chain variable region of the amino acid sequence of seq id no; (HE-39 heavy chain)

[8] Comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 79, as CDR2, SEQ ID NO: 80 and the amino acid sequence of SEQ ID NO: 81, or a light chain variable region of the amino acid sequence of seq id no; (HE-39L chain-1)

[9] Comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 82, SEQ ID NO as CDR 2: 83 and SEQ ID NO: 84, an antibody to the light chain variable region of the amino acid sequence of seq id no; (HE-39L chain-2)

[10] An antibody comprising the heavy chain of [7] and the light chain of [8 ];

[11] an antibody comprising the heavy chain of [7] and the light chain of [9 ];

[12] an antibody having an activity equivalent to that of the antibody according to any one of [1] to [11 ]; and

[13] an antibody that binds to the same epitope as the antibody described in any one of [1] to [12 ].

With respect to the antibody of the above [12], the "comparable activity" means that the binding activity to HB-EGF is at least 70%, preferably at least 80%, more preferably at least 90% of the binding activity of the antibody of any one of [1] to [11], or, in the case of binding to a cytotoxic substance, the anti-tumor activity is at least 70%, preferably at least 80%, more preferably at least 90% of the anti-tumor activity of the antibody of any one of [1] to [11 ].

Introduction of mutations into polypeptides is a method well known to those skilled in the art for producing polypeptides functionally equivalent to a particular polypeptide. For example, it is well known to those skilled in the art that antibodies exhibiting activity comparable to that of the present invention can be produced by introducing appropriate mutations into the antibodies of the invention using site-specific mutagenesis (Hashimoto-Gotoh, T. et al (1995) Gene 152, 271-275; Zoller, M.J. and Smith, M. (1983) Methods enzymol.100, 468-500; Kramer, W. et al (1984) Nucleic Acids Res.12, 9441-9456; Kramer, W. and Fritz, H.J. (1987) Methods enzymol.154, 350-367; Kunkel, T.A. (1985) Proc.Natl.Acad.Sci.USA 82, USA 492; and Kunkel (1988) Methods 2785, 2763). Amino acid mutations may also be generated by natural mutations. The antibodies of the invention also include antibodies that: which has an amino acid sequence generated by mutation of one or more amino acids in the amino acid sequence of the antibody of the present invention and shows an activity equivalent to that of the antibody of the present invention. Regarding the number of amino acids mutated in such mutants, it is conceivable that generally not more than 50 amino acids, preferably not more than 30 amino acids, more preferably not more than 10 amino acids (for example, not more than 5 amino acids) are included.

Preferably, the amino acid residue is mutated to another amino acid residue that retains the characteristics of the amino acid side chain. For example, the following classifications have been established based on the characteristics of amino acid side chains.

Hydrophobic amino acids (A, I, L, M, F, P, W, Y, V)

Hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T)

Amino acids having aliphatic side chains (G, A, V, L, I, P)

Amino acids having hydroxyl-containing side chains (S, T, Y)

Amino acids having sulfur-containing side chains (C, M)

Having carboxyl-or amide-containing side chains (D, N, E, Q)

Amino acids having a base-containing side chain (R, K, H)

Amino acids having aryl-containing side chains (H, F, Y, W)

(the amino acid is given in parentheses in the single letter symbols)

For a polypeptide having a modified amino acid sequence produced by deletion from and/or addition of one or more amino acid residues to a specific amino acid sequence and/or by replacement of one or more amino acid residues in the specific amino acid sequence with another amino acid, such a polypeptide is known to retain its biological activity (Mark, D.F. et al, Proc. Natl.Acad.Sci.USA (1984)81, 5662-. That is, in general, when an amino acid in a specific class is replaced with another amino acid in the class in the amino acid sequence of a specific polypeptide, the possibility of maintaining the activity of the specific polypeptide is high. Among the above-provided classifications of amino acids, substitutions between amino acids in the same classification are referred to herein as conservative substitutions.

In the above [13], the present invention also provides an antibody that binds to the same epitope as the anti-HB-EGF antibody disclosed in the present invention. Such an antibody can be obtained, for example, by the following method.

Whether a test antibody and a particular antibody have the same epitope can be determined based on their competition for the same epitope. For example, competition between antibodies can be detected by a mutual blocking assay (recipcalbocking assay). For example, competitive ELISA assays are one preferred mutual blocking assay. Specifically, in the mutual blocking assay, an HB-EGF protein was coated on a well of a microtiter plate; pre-incubation in the presence or absence of a candidate competitive antibody; then, the anti-HB-EGF antibody of the present invention is added. The amount of the anti-HB-EGF antibody of the invention that has bound to the HB-EGF protein in the well is indirectly related to the binding activity of the candidate competitive antibody (test antibody) that competes for binding to the same epitope. That is, the higher the affinity of the test antibody for the same epitope, the less the anti-HB-EGF antibody of the present invention binds to the HB-EGF protein-coated well, and the greater the amount of the test antibody bound to the HB-EGF protein-coated well.

The amount of antibody bound to the wells can be conveniently determined by labeling the antibody in advance. For example, a biotin-labeled antibody can be assayed using an avidin-peroxidase conjugate and a suitable substrate. Mutual blocking assays based on enzyme labels such as peroxidase are especially known as competitive ELISA assays. The antibody may be labeled with some other detectable or detectable label. In particular, radioactive labels and fluorescent labels are also known.

In addition, when the test antibody has a constant region derived from a species different from that of the anti-HB-EGF antibody of the present invention, the amount of the antibody bound to the well can also be determined using a labeled second antibody recognizing the constant region of the antibody. Alternatively, even when the antibodies are derived from the same species but different species, the amount of antibody bound to the wells can be determined using a second antibody that can distinguish between the individual species.

Such candidate competitive antibody is an antibody that binds to substantially the same epitope or an antibody that competes for binding to the same epitope as the anti-HB-EGF antibody of the present invention, when the candidate antibody can block binding of at least 20%, preferably at least 20% -50%, even more preferably at least 50% of the anti-HB-EGF antibody, in contrast to the binding activity obtained in a control test conducted in the absence of the candidate competitive antibody.

For example, an antibody recognizing a region having the sequence APSCICHPGYHGERCHGLSL in the HB-EGF protein is a preferable example of an antibody that binds to the same epitope as the antibody in [10] or [11 ].

Binding Activity of antibodies

The antigen binding activity of Antibodies can be determined using known methods (Antibodies: antibody Manual. Ed Harlow, David Lane, Cold Spring harbor laboratory, 1988). For example, enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Radioimmunoassay (RIA) or immunofluorescence may be used. Antibodies: the method described in A Laboratory Manual, p 359-420, is an example of a method for determining the binding activity of an antibody to an antigen expressed in a cell.

In addition, methods, in particular those using flow cytometry, can suitably be used to determine suspension in, for example, a bufferAnd an antibody against the antigen expressed on the cell surface of (a). Examples of flow cytometers that can be used are as follows: FACSCANTO^TM II、FACSAria^TM、FACSArray^TM、FACSVantage^TMSE and FACSCalibur^TM(the above devices are from BD Biosciences) and EPICS ALTRA HyPerSort, Cytomics FC 500, EPICS XL-MCL ADC EPICS XL ADC and Cell LabQuanta/Cell Lab Quanta SC (the above devices are from Beckman Coulter).

In an example of a convenient method for measuring the binding activity of the test HB-EGF antibody to an antigen, the test antibody is reacted with cells expressing HB-EGF and stained with a FITC-labeled secondary antibody recognizing the test antibody. Fluorescence intensity was measured using a FACSCalibur (Becton, Dickinson and Company) and analyzed using CELL QUEST software (Becton, Dickinson and Company).

Proliferation inhibitory Activity

The following method can be conveniently used to evaluate or measure the cell proliferation inhibitory effect caused by the anti-HB-EGF antibody. In a method which can be used for evaluating or measuring the effect of inhibition of cell proliferation in vitro, the pair of living cells added to a medium is measured³H]Uptake of labeled thymidine is an indicator of DNA replication ability. More convenient methods include the MTT method and the dye exclusion method, in which the ability of cells to exclude dyes (e.g., trypan blue) is examined using a microscope. The MTT method makes use of the fact that: the living cells have the function of converting the tetrazolium salt MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) into blue formazansThe capacity of the product. More specifically, a ligand and a test antibody are added to a culture solution of a test cell, and after a certain period of time has elapsed, an MTT solution is added to the culture solution, and MTT is incorporated into the cell by standing for a certain period of time. As a result, the yellow compound MTT is converted into a blue compound by succinate dehydrogenase in the mitochondria of the cell. Dissolving the blue product to provide a coloration, absorbing itThe measurement of the degree provides an indication of the viable cell count. In addition to MTT, reagents such as MTS, XTT, WST-1, WST-8, and the like are also commercially available (Nacalai Tesque, Inc.) and can be suitably used. In the activity assay, a control antibody was used in the same manner as the anti-HB-EGF antibody; the control antibody is a binding antibody having the same isotype as the anti-HB-EGF antibody without the above cell proliferation inhibitory activity. When the anti-HB-EGF antibody shows a stronger cell proliferation inhibitory activity than the control antibody, the antibody has a cell proliferation inhibitory activity.

Mouse models carrying tumors can also be used as a means of evaluating or measuring cell proliferation inhibitory activity in vivo. For example, cancer cells whose growth is promoted by HB-EGF can be transplanted into a non-human test animal subcutaneously or intradermally, after which the test antibody can be administered intravenously or intraperitoneally, starting on the day of transplantation or the next day, or at intervals of multiple days. Cell proliferation inhibitory activity can be assessed by measuring tumor size over time. As with the in vitro evaluation, a control antibody having the same isotype was administered, and the antibody had cytostatic activity when the tumor size of the group receiving the anti-HB-EGF antibody was significantly smaller than that of the group receiving the control antibody. When a mouse is used as a non-human test animal, a nude (nu/nu) mouse is suitably employed; nude (nu/nu) mice lack T-lymphocyte function due to genetic deletion of the thymus. The use of this type of mouse makes it possible to exclude the contribution of T-lymphocytes in test animals when evaluating or measuring the cell proliferation inhibitory activity due to the administered antibody.

Method for inhibiting cell proliferation

The present invention provides a method for inhibiting the proliferation of a cell expressing HB-EGF by contacting the cell with the antibody of the present invention. The antibody of the present invention present in the cell proliferation inhibitor of the present invention is the above-mentioned antibody that binds to HB-EGF protein. The cells that can be contacted with the anti-HB-EGF antibody are not particularly limited except that these cells are required to express HB-EGF, but are preferably disease-related cells. Cancer cells are a preferred example of disease-related cells. The cancer is preferably pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, bladder cancer, brain tumor or hematological cancer. Hematological cancers include, for example, myeloma, lymphoma, and leukemia.

Delivery methods using anti-HB-EGF antibodies

The present invention relates to a method for delivering a cytotoxic substance into a cell using an anti-HB-EGF antibody. The antibody used in this method is the above-mentioned cytotoxic-bound anti-HB-EGF antibody. Delivery of the cytotoxic substance can be achieved by contacting a cell expressing HB-EGF with an anti-HB-EGF antibody bound with the cytotoxic substance. In the present invention, there is no particular limitation on cells to which toxic substances are delivered, but disease-related cells are preferred. Cancer cells are an example of disease-related cells. The cancer is preferably pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, bladder cancer, brain tumor or hematological cancer. Hematological cancers include, for example, myeloma, lymphoma, and leukemia.

The contacting of the invention may be performed in vitro or in vivo. For the state in which the antibody is added, for example, a solid or a solution obtained by freeze-drying may be suitably used. In those cases where the antibody is added in the form of an aqueous solution, it may be an aqueous solution containing only pure antibody, or may be a solution containing, for example, a surfactant, excipient, colorant, perfume, preservative, stabilizer, buffer, suspending agent, tonicity agent, binder, disintegrant, lubricant, fluidity improver, taste-masking agent, or the like. There is no particular limitation on the concentration to be added, and a suitable final concentration in the culture solution is preferably 1pg/mL-1g/mL, more preferably 1ng/mL-1mg/mL, even more preferably 1. mu.g/mL-1 mg/mL.

In the present invention, the "contact" in vivo can also be performed by administering to a non-human animal into which a cell expressing HB-EGF has been implanted or transplanted, or by administering to an animal carrying a cancer cell expressing HB-EGF. The mode of administration may be oral or parenteral. Parenteral administration is particularly preferred, and the corresponding routes of administration may include injection, nasal administration, pulmonary administration, transdermal administration, and the like. As for the example of injection administration, the pharmaceutical composition of the present invention as a cell proliferation inhibitor or an anticancer agent may be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection. The appropriate administration mode can be selected according to the age and symptoms of the subject animal. In the case of administering an aqueous solution, the solution may be an aqueous solution containing only a pure antibody, or may be a solution containing, for example, a surfactant, an excipient, a colorant, a perfume, a preservative, a stabilizer, a buffer, a suspending agent, a tonicity agent, a binder, a disintegrant, a lubricant, a fluidity improver, a taste-masking agent, or the like. The dosage may be selected, for example, from 0.0001mg to 1000mg per kg body weight per administration. Alternatively, the dosage may be selected from 0.001-100000mg per patient. However, the dose of the antibody of the present invention is not limited to the above dose.

Pharmaceutical composition

In another aspect, the invention features a pharmaceutical composition that includes an antibody that binds to an HB-EGF protein. Another feature of the present invention is a cell proliferation inhibitor, particularly an anticancer agent, which comprises an antibody that can bind to HB-EGF protein. The cell proliferation inhibitor of the present invention and the anticancer agent of the present invention are preferably administered to a subject suffering from or at risk of cancer.

In the present invention, a cell proliferation inhibitor comprising an antibody that can bind to an HB-EGF protein also relates to a method for inhibiting cell proliferation comprising the step of administering to a subject an antibody that can bind to an HB-EGF protein, and the use of an antibody that can bind to an HB-EGF protein for producing a cell proliferation inhibitor.

In addition, in the present invention, an anticancer agent comprising an antibody that can bind to HB-EGF protein relates to a method for preventing or treating cancer comprising the step of administering to a subject an antibody that can bind to HB-EGF protein, and the use of an antibody that can bind to HB-EGF protein in producing an anticancer agent.

The antibody present in the pharmaceutical composition of the present invention (e.g., cell proliferation inhibitor or anticancer agent; the same applies hereinafter) is not particularly limited except that the antibody is required to have the ability to bind to the HB-EGF protein, and any antibody provided herein as an example may also be used.

The mode of administration of the pharmaceutical composition of the present invention may be oral administration or parenteral administration. Parenteral administration is particularly preferred, and the corresponding routes of administration may include injection, nasal administration, pulmonary administration, transdermal administration, and the like. As for the injection administration, the pharmaceutical composition of the present invention can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection. The appropriate administration mode can be selected according to the age and symptoms of the patient. The dosage may be selected, for example, from 0.0001mg to 1000mg per kg body weight per administration. Alternatively, the dosage may be selected from 0.001-100000mg per patient. However, the pharmaceutical composition of the present invention is not limited to the above-mentioned dosage.

The Pharmaceutical composition of the present invention can be prepared according to a conventional method (e.g., Remington's Pharmaceutical Science, latest edition, Mack publishing company, Easton, USA), and can comprise a pharmaceutically acceptable carrier and a pharmaceutically acceptable additive. Examples are surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity enhancers, taste masking agents, and the like, but there is no limitation to the foregoing, and other commonly used carriers may be suitably used. Specific examples thereof include light silicic anhydride (light silicic anhydride), lactic acid, microcrystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hardened castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like.

Production ofMethod for producing a pharmaceutical product

The present invention further provides a method of producing a pharmaceutical composition, in particular an anti-cancer agent, comprising the steps of:

(a) providing an anti-HB-EGF antibody;

(b) determining whether the antibody of (a) has internalizing activity;

(c) selecting an antibody having internalization activity; and

(d) binding a cytotoxic agent to the antibody selected in (c).

The presence/absence of internalization activity can be determined by the methods described above. In addition, the anti-HB-EGF antibody and the cytotoxic substance may be the above-mentioned anti-HB-EGF antibody and the cytotoxic substance.

Cancer diagnosis

In view of the fact that HB-EGF expression is increased in a wide range of cancers such as pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, bladder cancer, brain tumor and hematologic cancer, the present invention provides in another aspect a method for diagnosing a disease using the anti-HB-EGF antibody, particularly a method for diagnosing cancer using the anti-HB-EGF antibody.

The diagnostic method of the present invention can be carried out by detecting an anti-HB-EGF antibody which has been incorporated into cells. The anti-HB-EGF antibody used in the present invention preferably has an internalizing activity, and is preferably labeled with a labeling substance.

Therefore, a preferred embodiment of the diagnostic method of the present invention is a diagnostic method using an anti-HB-EGF antibody which has been labeled with a labeling substance and has an internalizing activity. The above-mentioned anti-HB-EGF antibody can be used for an anti-HB-EGF antibody to be bound to a labeling substance.

The labeling substance bound to the anti-HB-EGF antibody is not particularly limited, and those known to those skilled in the art, for example, fluorescent dyes, enzymesCoenzymes, chemiluminescent substances, radioactive substances, and the like. Specific examples are radioisotopes (e.g.,³²p、¹⁴C、¹²⁵I、³H、¹³¹i, etc.), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, beta-galactosidase, beta-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, carbohydrate oxidase, microperoxidase, biotin, etc. When biotin is used as the labeling substance, it is preferable to add a biotin-labeled antibody, followed by addition of biotin attached to an enzyme such as alkaline phosphatase. The labeling substance may be attached to the anti-HB-EGF antibody using a known method, for example, glutaraldehyde method, maleimide method, pyridine disulfide method, periodic acid method, etc. The labeling substance can be attached to the antibody by procedures known to those skilled in the art.

When the cancer is a disease diagnosed by the method of the present invention, there is no particular limitation on the type of cancer, but the cancer is preferably pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, bladder cancer, brain tumor, or hematological cancer. Hematological cancers include, for example, myeloma, lymphoma, and leukemia.

The diagnosis of the present invention may be performed in vivo or in vitro.

For example, in vitro diagnosis can be performed according to a method comprising the steps of:

(a) providing a sample collected from a subject;

(b) contacting the sample from (a) with an anti-HB-EGF antibody bound with a labeling substance; and

(c) detecting the antibody that has been incorporated into the cells.

The collected sample is not particularly limited and may include cells collected from a subject and tissues collected from a subject. The sample used in the present invention also includes a secondary sample obtained from a test sample, for example, a cell culture solution or a specimen prepared by fixing tissue or cells collected from a living organism.

For example, in vivo diagnosis may be performed according to a method comprising the steps of:

(a) administering a labeled anti-HB-EGF antibody to the subject; and

(b) detecting the antibody that has been incorporated into the cancer cell.

The dose of the anti-HB-EGF antibody can be appropriately set by those skilled in the art depending on factors such as the type of the labeling substance, the disease to be diagnosed, and the like. The labeled anti-HB-EGF antibody can be prepared using the above-described method.

The present invention further provides a method for producing a diagnostic agent, particularly a cancer diagnostic agent, comprising the steps of:

(a) providing an anti-HB-EGF antibody;

(b) determining whether the antibody of (a) has internalizing activity;

(c) selecting an antibody having internalization activity; and

(d) binding a labeling substance to the antibody selected in (c).

The presence/absence of internalization activity can be determined by the methods described above. In addition, the anti-HB-EGF antibody and the labeling substance may be the above-mentioned anti-HB-EGF antibody and the labeling substance.

The contents of all patents and references explicitly cited in the specification are hereby incorporated by reference in their entirety. The contents of the description and drawings of Japanese patent applications 2006-286824 and 2007-107207, which form the basis of the priority cited in this application, are also incorporated herein by reference in their entirety.

Examples

The present invention is described in more detail by the examples provided below, but the present invention is not limited to these examples.

Immunization

1-1 Generation of immunogens

Construction of HB-EGF expression vector

To construct an HB-EGF expression vector, first, the HB-EGF gene was cloned as follows. RT-PCR was performed using human cardiac cDNA (human Marathon Ready cDNA, Clontech laboratories, Inc.) as a template and Pyrobest Taq polymerase (Takara BioInc.), and the full-length HB-EGF gene was cloned.

EGF-1：ATGAAGCTGCTGCCGTCGGTG(SEQ ID NO：51)

EGF-2：TCAGTGGGAATTAGTCATGCCC(SEQ ID NO：52)

(94 ℃/30 seconds, 65 ℃/30 seconds, 72 ℃/60 seconds: 35 cycles)

Using the obtained PCR product as a template, a second PCR was performed under the conditions given below to obtain a full-length HB-EGF cDNA fragment to which SalI and NotI restriction enzyme sequences were added at the 5 'and 3' ends, respectively.

EGF-3：TAAGTCGACCACCATGAAGCTGCTGCCGTCGGTG(SEQ ID NO：53)

EGF-4：

TTTGCGGCCGCTCACTTGTCATCGTCGTCCTTGTAGTCGTGGGAATTAGTCATGCCCAAC(SEQ ID NO：54)

(94 ℃/30 seconds, 65 ℃/30 seconds, 72 ℃/60 seconds: 25 cycles)

This fragment was digested with SalI and NotI, and inserted into an expression vector for animal cells (pMCN) which had also been digested with SalI and NotI, thereby constructing an HB-EGF expression vector (pMCN _ HB-EGF).

Construction of HB-EGF _ Fc fusion protein expression vector

A fusion protein of the extracellular domain of HB-EGF and the Fc region of mouse IgG2a (HB-EGF _ Fc) was used as an immunogen for obtaining an HB-EGF neutralizing antibody. The structure of the fusion protein used for immunization is shown in FIG. 1.

An expression vector for the mouse Fc region/HB-EGF fusion protein was constructed as follows. First, PCR was performed using Pyrobest Taq polymerase (Takara Bio Inc.) under the following conditions using an HB-EGF expression vector (pMCN _ HB-EGF) as a template.

EGF-5：AAAGAATTCCACCATGAAGCTGCTGCCGTC(SEQ IDNO：55)

EGF-6：

TATCGGTCCGCGAGGTTCGAGGCTCAGCCCATGACACCTC(SEQID NO：56)

(94 ℃/30 sec, 68 ℃/30 sec, 72 ℃/30 sec: 25 cycles)

The obtained PCR product was then digested with EcoRI and CpoI. The resulting DNA fragment was inserted between EcoRI and CpoI of an animal cell expression vector containing mouse IgG2a _ Fc (pMCDN _ mIgG2a _ Fc) to construct an HB-EGF-Fc expression vector (pMCDN _ HB-EGF-Fc).

1-1-3. production of HB-EGF _ Fc-producing Strain

15 μ gHB-EGF-Fc expression vector pMCDN _ HB-EGF-Fc, which had been linearized by digestion with pvuI, was transfected into DG44 cells (1X 10 of 1. times.10) suspended in PBS (-) by electroporation at 1.5kV, 25 μ F (gene pulser from Bio-Rad Laboratories, Inc.)⁷cells/mL, 800 μ L). After dilution to appropriate cell counts with growth medium containing penicillin/streptomycin (PS) (CHO-S-SFM II, Invitrogen Corporation), cells were seeded onto 96-well plates and 500. mu.g/mL G418 (Geneticin, Invitrogen Corporation) was added the next day. After about 2 weeks, wells with single clones were selected under a microscope and subjected to SDS-PAGE using 10. mu.L of culture supernatant per well. Using PVDF membrane and goat anti-HB-EGF antibody (AF-259-NA, R)&DSystems, Inc.) and HRP-anti-goat antibody (ACI3404, BioSource), HB-EGF-Fc producing cell lines were screened by Western blotting. The highest producing cell line was selected and expanded.

Purification of HB-EGF _ Fc protein

The HB-EGF _ Fc Protein was purified from the culture supernatant of the obtained HB-EGF _ Fc-producing strain using a Hi Trap Protein G HP 1mL column (Amersham Biosciences # 17-0404-01). The culture supernatant was adsorbed at a flow rate of 1mL/min, followed by washing with 20mL of 20mM phosphate buffer (pH7.0), and then eluting with 3.5mL of 0.1M glycine-HCl (pH 2.7). The eluate was recovered in 0.5mL fractions in Eppendorf tubes, each of which contained 50. mu.L of 1M Tris-HCl (pH 9.0). Determination of OD_280nm. Fractions containing the target protein were pooled, PBS (-) was added to reach a total of 2.5mL, and then the buffer was replaced with PBS (-) using a PD-10 column (Amersham Biosciences # 17-0851-01). The purified protein was passed through a 0.22 μm filter (Millipore # SLGV033RS) and stored at 4 ℃.

1-2. immunization

An emulsion of the HB-EGF _ Fc protein was prepared with complete adjuvant (DIFCO DF263810) for the first immunization and with incomplete adjuvant (DIFCO DF263910) for the second and subsequent immunization. Three animals [ (MRL/lpr, male, mouse-aged: 4 weeks) (balb/c, female, mouse-aged: 6 weeks), all purchased from Charles River Japan ] were immunized by subcutaneous injection (1mL Thermo syringe, 26 gauge needle) at a dose of 50. mu.g/mouse. Two weeks after the first immunization, a second immunization was given, and a total of 4-5 immunizations were given at one week intervals. For the last immunization, HB-EGF _ Fc (50 μ g) was suspended in 100 μ L PBS and injected into the tail vein; cell fusion was performed 3 days later.

1-3 Generation of hybridomas

Cell fusion was performed as follows. Spleens were removed from mice under sterile conditions and single cell suspensions were prepared by trituration in culture medium 1(RPMI1640+ PS). The suspension was passed through a 70 μm nylon sieve (Falcon) to remove adipose tissues and the like, and the cells were counted. Mixing the obtained B cells with mouse myeloma cells (P3U1 cells) at a cell count ratio of about 2: 1; 1mL of 50% PEG (Roche, cat # 783641) was added; and cell fusion was performed. The fused cells were suspended in medium 2(RPMI1640+ PS, 10% FCS, HAT (Sigma, H0262), 5% BM conditioned H1(Roche #1088947)), dispensed in an appropriate number of 96-well plates (10 plates) at 200. mu.L/well, and cultured at 37 ℃. One week later, the culture supernatant was used to screen hybridomas, and the hybridomas were analyzed. Hybridomas derived from two Balb/c mice were designated as HA series and HB series, respectively, and hybridomas derived from one Mrl/lpr mouse were designated as HC series.

Screening of anti-HB-EGF neutralizing antibody

2-1 production of human cell line expressing HB-EGF

Production of HB-EGF _ DG44 Strain

A DG44 cell line expressing HB-EGF was established as follows. First, 15. mu.g of the HB-EGF expression vector pMCN _ HB-EGF constructed as described in 1-1-1 was digested with pvuI and transfected into DG44 cells by electroporation using the same procedure as described in 1-1-3. Then, G418-resistant strains were selected, and the cells were stained with goat anti-HB-EGF antibody (R & DSsystems, Inc.) and FITC-labeled anti-goat IgG antibody. HB-EGF expressed on the cell surface was analyzed with FACSCalibur (Becton, Dickinson and Company), and highly expressed clones were selected.

Production of HB-EGF _ Ba/F3 Strain

A Ba/F3 cell line expressing HB-EGF on the cell membrane was established as follows. The HB-EGF expressed on the cell membrane was treated with protease and excised into the medium. Thus, an expression vector for proHB-EGF mutated at the protease cleavage site was first constructed.

Using pMCN-HB-EGF as a template, separate PCRs were performed using the following two sets of conditions and PyrobestTaq polymerase (Takara Bio Inc.).

PCR reaction 1

EGF-3：TAAGTCGACCACCATGAAGCTGCTGCCGTCGGTG(SEQ ID NO：53)

EGF-7：

CGATTTTCCACTGTGCTGCTCAGCCCATGACACCTCTC(SEQ IDNO：57)

(94 ℃/30 sec, 68 ℃/30 sec, 72 ℃/30 sec: 20 cycles)

PCR reaction 2

EGF-8：

TGGGCTGAGCAGCACAGTGGAAAATCGCTTATATACCTA(SEQID NO：58)

EGF-4：

TTTGCGGCCGCTCACTTGTCATCGTCGTCCTTGTAGTCGTGGGAATTAGTCATGCCCAAC(SEQ ID NO：54)

(94 ℃/30 sec, 68 ℃/30 sec, 72 ℃/30 sec: 20 cycles)

Then mixing the two DNA fragments obtained by PCR reactions 1 and 2; the recombination reaction was performed using Pyrobest Taq polymerase (Takara Bio Inc.) (94 ℃/30 seconds, 72 ℃/60 seconds: 5 cycles); then, PCR was performed under the following conditions using 1. mu.L of the aforementioned reaction solution as a template.

EGF-3：TAAGTCGACCACCATGAAGCTGCTGCCGTCGGTG(SEQ ID NO：53)

EGF-4：

TTTGCGGCCGCTCACTTGTCATCGTCGTCCTTGTAGTCGTGGGAATTAGTCATGCCCAAC(SEQ ID NO：54)

(94 ℃/30 sec, 68 ℃/30 sec, 72 ℃/60 sec: 22 cycles)

The obtained PCR product was digested with SalI and NotI, followed by inserting the fragment into an expression vector for animal cells (pMCN) which had also been digested with SalI and NotI, to construct a proHB-EGF expression vector (pMCN-MHB-EGF).

Then, a Ba/F3 cell line expressing proHB-EGF was produced as follows. Mu.g of the proHB-EGF expression vector pMCN-MHB-EGF constructed above was digested with pvuI, and then transfected into Ba/F3 cells (1X 10) suspended in PBS (-) by electroporation at 0.33kV, 950. mu.F (gene pulser from Bio-Rad Laboratories, Inc.)⁷cells/mL, 800 μ L). Then in 96-well plates containing 1ng/mThese cells were cultured in a medium (RPMI140, 10% FCS, PS) containing IL-3 and 500. mu.g/mLG 418, and after two weeks, G418-resistant strains were selected. With goat anti-HB-EGF antibody (R)&D Systems, Inc.) and FITC-labeled anti-mouse IgG antibody (Beckman Coulter, PN IM0819) stained the cells and clones exhibiting high levels of expression of cell surface HB-EGF were selected according to FACS (Becton, Dickinson and Company).

2-2 Generation of SKOV-3 cells expressing HB-EGF

A SKOV-3 cell line expressing HB-EGF was established as follows. An ovarian cancer cell line SKOV-3 (purchased from ATTC) was cultured in growth medium (McCoy' S5A medium, Invitrogen corporation) containing 10% FCS and penicillin/streptomycin (P/S).

Mu.g of the HB-EGF expression vector pMCN _ HB-EGF constructed in 1-1-1 was digested with pvuI. It was then transfected into SKOV-3 cells (1X 10) suspended in PBS (-) by electroporation at 1.5kV at 25. mu.F (Gene pulser from Bio-Rad laboratories, Inc.)⁷cells/mL, 800 μ L). After dilution to the appropriate cell count using the growth medium described above, the cells were seeded into 96-well plates. The next day G418 (Geneticin, Invitrogen Corporation) was added in an amount of 500. mu.g/mL. After about 2 weeks, G418-resistant monoclonals were selected and HB-EGF-expressing cell lines were selected by Western blotting. The highest producing cell line was selected and used in subsequent experiments.

2-3 production of EGFR _ Ba/F3 cell line showing HB-EGF-dependent growth

Construction of pCV-hEGFR/G-CSFR

In order to evaluate the activity of the antibody of the present invention, a vector expressing a chimeric receptor (hEGFR/mG-CSFR) consisting of the extracellular region of human EGFR and the extracellular region of mouse G-CSFR was constructed. When HB-EGF is bound to a cell expressing the chimeric receptor, the effect on the cell is shown in FIG. 2 a.

To clone a gene encoding the extracellular region of human Epidermal Growth Factor Receptor (EGFR), PCR was performed using human liver cDNA (Marathon Ready cDNA, Clontech Laboratories, Inc.) as a template, using the primer set listed below. SEQ ID NO: 59 and SEQ ID NO: 60 shows the nucleotide sequence (MN _005228) and the amino acid sequence (NP _005219) of human EGFR, respectively.

EGFR-1：ATGCGACCCTCCGGGACGGC(SEQ ID NO：61)

EGFR-2：CAGTGGCGATGGACGGGATCT(SEQ ID NO：62)

(94 ℃/30 seconds, 65 ℃/30 seconds, 72 ℃/2 minutes: 35 cycles)

The amplified cDNA (about 2Kb) was excised from the agarose gel and inserted into pCR-TOPO vector (Invitrogen Corporation). The base sequence of the fragment inserted into the plasmid was analyzed, and it was confirmed that the obtained EGFR gene had the correct sequence. Then, PCR was performed using the plasmid obtained above as a template, using the following primer set.

EGFR-5：TTGCGGCCGCCACCATGCGACCCTCCGGGACGGC(SEQ ID NO：63)

EGFR-6：

ACCAGATCTCCAGGAAAATGTTTAAGTCAGATGGATCGGACGGGATCTTAGGCCCATTCGT(SEQ ID NO：64)

(94 ℃/30 sec, 68 ℃/30 sec, 72 ℃/2 min: 25 cycles)

A gene fragment encoding the extracellular region of EGFR and having a 5 'NotI site and a 3' BglII site was obtained. This fragment was digested with NotI-BglII and inserted between NotI-BamHI in pCV _ mG-CSFR.

The expression plasmid vector pCV was constructed by replacing the poly (A) addition signal of pCOS1 (International publication No. WO 98/13388) with the poly (A) addition signal from human G-CSF. pEF-BOS (Mizushima S. et al, Nuc. acids Res.18, 5322(1990)) was digested with EcoRI and XbaI to obtain poly (A) addition signal fragment derived from human G-CSF. This fragment was inserted into the EcoRI/XbaI site of pBacPAK8(Clontech Laboratories, Inc.). After digestion with EcoRI, blunt-ended treatment and digestion of both ends with BamHI, a fragment containing a poly (A) addition signal derived from human G-CSF having a BamHI site added to the 5 'end and a blunt-ended 3' end was generated. This fragment was signalized at the BamHI/EcoRV site with the poly (A) addition of pCOS1, resulting in an expression plasmid vector designated pCV.

pCV _ mG-CSFR contains the mouse G-CSF receptor from the asparagine residue at position 623 to the C-terminus in pCV, which is the intracellular domain. In SEQ ID NO: 65 (M58288), the base sequence of mouse G-CSF receptor, as shown in SEQ ID NO: the amino acid sequence of the mouse G-CSF receptor (AAA37673) is shown in 66. However, since a BamHI site (restriction enzyme site) was generated in the cDNA sequence encoding the N-terminal region of the insert sequence of pCV _ mG-CSFR, SEQ ID NO: the glycine residue at position 632 in position 66 is replaced with a glutamic acid residue.

The construction of vector pCV _ hEGFR/mG-CSFR expressing chimeric receptor hEGFR/mG-CSFR consisting of extracellular region of human EGFR and intracellular region of mouse G-CSFR was completed by confirming the base sequence of the gene fragment inserted into pCV _ mG-CSFR.

In SEQ ID NO: 67 and SEQ ID NO: 68, the base sequence and amino acid sequence of the protein expressed by the expression vector (i.e., human EGFR/mouse G-CSFR chimeric receptor), respectively.

Generation of HB-EGF dependent cell lines

15 μ g of hEGFR/mG-CSFR chimeric receptor expression vector pCV _ hEGFR/mG-CSFR linearized by digestion with pvuI was transfected into Ba/F3 cells by electroporation (Gene pulser, Bio-Rad laboratories, Inc.) at 0.33kV, 950 μ F. These cells were cultured for 2 weeks in a medium (RPMI1640, 10% FCS, PS) containing 10ng/mL HB-EGF and 500. mu.g/mL G418, and colonies which appeared were picked out.

It was then determined in the following experiment whether the obtained cell strain showed growth depending on the concentration of HB-EGF. At 0-100ng/mL HB-EGF (R)&D Systems, inc., 259-HE) at 1 × 10³Cell/well Density EGFR _ Ba/F3 cells were seeded onto 96-well plates and cultured for 3 days.Cell counts were then determined using WST-8 reagent (cell counting kit-8, Dojindo Laboratories) according to the instructions.

The results showed that the growth of the established EGFR _ Ba/F3 cell line was promoted in a manner dependent on the concentration of HB-EGF (FIG. 2 b).

2-4 hybridoma selection

2-4-1 screening for antibody binding to HB-EGF (Primary screening)

To obtain an anti-HB-EGF neutralizing antibody, an antibody that binds to HB-EGF is first screened. Binding antibodies were screened using ELISA and FACS.

2-4-1-1.ELISA

Hybridoma culture supernatants were purified by passage of the HB-EGF protein (R) at 1. mu.g/mL&D Systems, inc., 259-HE) coated ELISA plates (NUNC) were incubated for 1 hour for reaction. Followed by reaction with Alkaline Phosphatase (AP) -labeled anti-mouse IgG (Zymed Laboratories, Inc., #62-6622) for 1 hour, followed by color development by addition of 1mg/mL substrate (Sigma, S0942-50 TAB). OD was measured with a plate reader (Bio-Rad Laboratories, Inc.)₄₀₅And ELISA positive wells were selected.

2-4-1-2.FACS

To HB-EGF _ Ba/F3 cells (approximately 1X 10)⁵Cells) were added to hybridoma culture supernatant and incubated at 4 ℃ for 1 hour. FITC-labeled anti-mouse IgG antibody (Beckman Coulter, PN IM0819) was then added and incubated at 4 ℃ for 30 minutes. Each hybridoma culture supernatant was then analyzed for the binding activity to cell surface HB-EGF by FACS (Becton, Dickinson and Company).

2-4-1-3. limiting dilution

In order to classify clones showing HB-EGF binding activity according to ELISA or FACS analysis as single clones, Limiting Dilution (LD) was performed. Cell counts in positive wells were determined and seeded onto 96-well plates to provide 3 cells/well. After approximately 10 days of culture, binding activity was again analyzed by ELISA or FACS on the culture supernatants of wells in which colonies had appeared. Using this series of steps, 5 monoclonals showing HB-EGF binding activity were obtained in the HA series, 4 monoclonals showing HB-EGF binding activity were obtained in the HB series, and 5 monoclonals showing HB-EGF binding activity were obtained in the HC series.

2-4-1-4. subtype determination

Antibody subtypes were determined using isosttip (Roche #1,493,027). Hybridoma culture supernatants diluted 10-fold with PBS (-) were used for subtype determination.

TABLE 1 characterization of the isolated antibodies

2-4-2 antibody purification

The antibody was purified from 80mL of the culture supernatant of the obtained monoclonal hybridoma using a HiTrap Protein G HP 1mL column (Amersham Biosciences # 17-0404-01). The hybridoma supernatant was adsorbed at a flow rate of 1mL/min, followed by washing with 20mL of 20mM phosphate buffer (pH7.0), and then eluting with 3.5mL of 0.1M glycine-HCl (pH 2.7). The eluate was recovered in 0.5mL fractions in Eppendorf tubes, each of which contained 50. mu.L of 1M Tris-HCl (pH 9.0). Determination of OD_280nm. Antibody-containing fractions were pooled, PBS (-) was added to reach a total volume of 2.5mL, and then the buffer was replaced with PBS (-) using a PD-10 column (Amersham Biosciences # 17-0851-01). The purified antibodies were passed through a 0.22 μm filter (Millipore # SLGV033RS) and the properties of each purified antibody were investigated in detail as follows.

2-4-3 analysis of growth-neutralizing Activity in EGFR _ Ba/F3 cells (second Screen)

Each purified antibody was analyzed for HB-EGF-dependent growth neutralizing activity of EGFR _ Ba/F3. In the presence of HB-EGF (80ng/mL) at 2X 10⁴Cell/well Density EGFR _ Ba/F3 cells were seeded onto 96-well plates and added at 0-200ng/mLAnd (4) determining the purified antibody. After 3 days of culture, cell count was determined using WST-8 (cell count kit-8).

The results showed that HA-20 in the HA series, HB-20 in the HB series, and HC-20 in the HC series showed strong neutralizing activity (FIGS. 3a-3 c).

Analysis of Properties of HB-EGF neutralizing antibodies (HA-20, HB-20, HC-15)

Cloning and determination of amino acid sequence of variable regions of HA-20, HB-20 and HC-15

From about 5X 10 using Trizol (#15596-⁶Total RNA was purified from each hybridoma. Full-length cDNA synthesis was performed from 1. mu.g of the obtained total RNA using SMART RACE cDNA amplification kit (Clontech laboratories, Inc., # PT3269-1) according to the manual provided by the kit. For each antibody, genes encoding the heavy chain variable region (VH) and the light chain variable region (VL) were amplified using the obtained cDNA as a template and using an Advantage 2PCR Enzyme System (Clontech laboratories, Inc. # PT 3281-1).

Cloning primer for light chain variable region

UPM-k(VL-k)

UPM: kit provision

VL-k：GCT CAC TGG ATG GTG GGA AGA TG(SEQ ID NO：69)

Cloning primer for heavy chain variable region

HA-20：UPM-VH-G1

HB-20，HC-15：UPM-VH-2a

UPM: kit provision

VH-G1：GGG CCA GTG GAT AGA CAG ATG(SEQ ID NO：70)

VH-2a：CAG GGG CCA GTG GAT AGA CCG ATG(SEQ ID NO：71)

94 deg.C/5 sec, 72 deg.C/2 min, 5 cycles

94 deg.C/5 sec, 70 deg.C/10 sec, 72 deg.C/2 min for 5 cycles

94 ℃/5 sec, 68 ℃/10 sec, 72 ℃/2 min, 27 cycles

The gene fragment amplified in the foregoing step was TA-cloned into pCRII-TOPO (Invitrogen TOPO TA-cloning kit, #45-0640), and the base sequence of each inserted fragment was determined. The sequences of the variable regions determined are shown in FIG. 4.

3-2 analysis of binding Activity of active type HB-EGF

In order to compare the ability of the 3 antibodies (HA-20, HB-20, HC-15) thus obtained to bind to the active type HB-EGF protein, the following experiment was conducted. HA-20, HB-20 or HC-15 antibody at 1. mu.g/mL HB-EGF protein (R)&D Systems, inc., 259-HE) coated plates (NUNC) were reacted at various concentrations. Then reacted with Alkaline Phosphatase (AP) -labeled anti-mouse IgG (Zymed Laboratories, Inc., #62-6622) for 1 hour, and 1mg/mL of substrate (Sigma, S0942-50TAB) was added for color development. OD measurement with plate reader₄₀₅And the concentration of antibody to 50% binding (ED) was calculated from the binding curve obtained with the particular antibody₅₀). As for the binding activity to the active type HB-EGF, ED of 0.2 to 1.4nM is observed₅₀Values, and thus strong binding activity was found to be present in all cases (fig. 5).

TABLE 2 ED that antibodies HA-20, HB-20, and HC-15 bind to HB-EGF₅₀Value mAb HB-EGF binding (ED50, nmol/L)

3-3 analysis of binding Activity of proHB-EGF

The obtained 3 antibodies were then analyzed for binding activity to proHB-EGF. RMG1 cells (ovarian cancer cell line, purchased from Japan Health sciences Foundation) were cultured in growth medium (Ham's F12 medium, Invitrogen Corporation) containing 10% FCS, which is known to internally express HB-EGF. Each antibody (10. mu.g/mL) was reacted at 4 ℃ with RMG1 cells intrinsically expressing HB-EGF and Ba/F3 cells (HB-EGF _ Ba/F3), DG44 cells expressing HB-EGF (HB-EGF _ DG44) and SKOV-3 cells (HB-EGF _ SKOV-3) (the latter three being cells overexpressing HB-EGF) for 1 hour, followed by staining with FITC-labeled anti-mouse IgG antibody (BeckmanCoulter, PN IM 0819). The binding of each antibody to the cell surface HB-EGF was then analyzed by FACS (Becton, Dickinson and company).

FIG. 6 shows graphs comparing the binding activities of HA-20, HB-20 and HC-15 antibodies to proHB-EGF expressed internally in RMG1 cells and proHB-EGF overexpressed in Ba/F3, DG44 and SKOV-3 cells according to FACS analysis. The gray waveform shows the staining pattern in the absence of primary antibody (control), while the solid line shows the staining pattern in the presence of specific antibody. The horizontal axis represents staining intensity, and the vertical axis represents cell number. As shown in FIG. 6, HB-20 and HC-15 recognized HB-EGF overexpressed on the cell membrane and HB-EGF overexpressed in the cell membrane of ovarian cancer cell line, whereas HA-20 did not bind at all or only very weakly. These results indicate that HA-20 is an antibody that strongly binds to the active type HB-EGF without recognizing proHB-EGF.

3-4 analysis of neutralizing Activity

3-4-1 solid phase assay for the ability to inhibit EGFR/HB-EGF binding

Production of EGFR-Fc protein

In order to construct an ELISA system that can examine the binding between HB-EGF and its receptor (EGFR) under solid phase conditions, a fusion protein of the extracellular region of EGFR and the Fc region of human IgG1 (EGFR-Fc) was first prepared as a receptor protein. The pattern of inhibition of binding between HB-EGF and EGFR on a solid phase by the HB-EGF antibody is shown in FIG. 7.

First, an EGFR-Fc expression vector is constructed. PCR was performed using the following primers and using pCV _ hEGFR/mG-CSFR constructed in example 2-3-1 as a template.

EGFR-7：GTTAAGCTTCCACCATGCGACCCTCCGGGAC(SEQID NO：72)

EGFR-8：GTTGGTGACCGACGGGATCTTAGGCCCATTCGTTG(SEQ ID NO：73)

(94 ℃/30 seconds, 72 ℃/30 seconds: 25 cycles)

The amplified gene fragment encoding the extracellular region of EGFR was digested with BstEII and HindIII and inserted between BstEII-HindIII in pMCDN 2-Fc. The base sequence of the inserted gene fragment was confirmed to complete the construction of a vector (pMCDN2_ EGFR-Fc) expressing a fusion protein (EGFR-Fc) of the extracellular region of human EGFR with the Fc region of human IgG 1. The base sequence and amino acid sequence of the protein expressed by the expression vector (i.e., EGFR-Fc) are set forth in SEQ ID NO: 74 and SEQ ID NO: shown at 75.

Cell lines producing the EGFR-Fc protein were then established as follows. The 15 μ g EGFR-Fc expression vector pMCDN2_ EGFR-Fc was first digested with pvuI and then transfected into DG44 cells by electroporation. The EGFR-Fc protein produced in the culture supernatant of the G418-resistant strain was subsequently analyzed by Western blotting. Thus, 10 μ L of a particular culture supernatant was separated by SDS-PAGE; imprinting on a PVDF membrane; and the target protein was detected with an HRP-labeled anti-human IgG antibody (Amersham, NA 933V). The clone producing the highest level was selected, expanded, and the culture supernatant was recovered.

Purification of the EGFR-F protein was performed as follows. The culture supernatant of the obtained EGFR-Fc producing strain was adsorbed on a HiTrap Protein G HP 1mL column (Amersham Biosciences #17-0404-01) at a flow rate of 1 mL/min. After washing with 20mL of 20mM phosphate buffer (pH7.0), the protein was eluted with 3.5mL of 0.1M glycine-HCl (pH 2.7). To identify fractions containing the target protein, 10 μ L each of the recovered fractions was separated by SDS-PAGE, followed by Western blotting and Coomassie blue staining. The buffer was replaced with PBS (-) using a PD-10 column (Amersham Biosciences # 17-0851-01). The purified protein was passed through a 0.22 μm filter (Millipore # SLGV033RS) and stored at 4 ℃.

3-4-1-2 analysis of binding between HB-EGF and EGFR Using ELISA

The purified EGFR-Fc was reacted at a concentration of 0.5. mu.g/mL for 1 hour in an anti-human IgG antibody-coated ELISA plate. 0-250ng/mL of HB-EGF (R & D Systems, Inc., 259-HE) was reacted for 1 hour, followed by detection of HB-EGF protein binding to EGFR-Fc with biotin-labeled anti-HB-EGF antibody (R & DSystems, Inc., BAF259) and AP-labeled streptavidin (Zymed, # 43-8322). A model for analyzing the binding pattern of EGFR/HB-EGF using ELISA is shown in FIG. 8. The results showed that binding of HB-EGF to EGFR could be detected with the solid phase system from a concentration of about 4ng/mL (FIG. 9).

3-4-1-3 analysis of antibody-mediated inhibitory Activity of HB-EGF/EGFR binding

The inhibitory activity of the antibody obtained in 2-4-2 on HB-EGF/EGFR binding was analyzed using the aforementioned solid phase system. Each antibody and HB-EGF (50ng/mL) were added to an ELISA plate on which EGFR-Fc was immobilized, and reacted at room temperature for 1 hour. The plate was washed with TBS-T and EGFR-binding HB-EGF was detected by the previous procedure (FIG. 10).

Concentration-dependent binding inhibition activity was observed for all antibodies, confirming that HA-20, HB-20, and HC-15 had particularly strong binding inhibition.

3-4-2 growth inhibitory Activity on EGFR _ Ba/F3 cells

HA-20, HB-20 and HC-15 were compared for their HB-EGF-dependent growth of EGFR _ Ba/F3 cells. As described above, in the presence of HB-EGF (80ng/mL), at 2X 10⁴Cell/well Density EGFR _ Ba/F3 cells were seeded into 96-well plates and specific purified antibodies were added. After 3 days of culture, cell count was measured using WST-8 (cell count kit-8), and a growth curve was constructed. From the results obtained, the antibody concentration (EC) at 50% of the maximal inhibitory effect was calculated₅₀Value).

According to the results, HC-15 showed the strongest growth inhibitory Effect (EC) on EGFR _ Ba/F3 cells₅₀3.8nM), followed by HA-20 (EC)₅₀＝32.6nM) and HB-20 (EC)₅₀40.3nM) (fig. 11).

TABLE 3 ED growth inhibitory Effect of HA-20, HB-20, and HC-15 antibodies on EGFR _ Ba/F3 cells₅₀Value of

3-4-3 growth inhibitory Activity on RMG-1 cells

The neutralizing activity on RMG-1 cells was analyzed as follows. RMG-1 cells (6X 10)³Cells/well) were inoculated into Ham's F12 medium containing 8% or 2% FCS in 96-well plates, followed by addition of specific antibodies. After one week of culture, cell counts were determined using WST-8 reagent.

According to the results, HA-20 inhibited the growth of RMG-1 cells in an antibody concentration-dependent manner (FIG. 12). Growth inhibitory activity was particularly pronounced at 2% FCS concentration.

3-5 analysis of cytotoxicity mediated by the internalizing Activity of antibodies

3-5-1. evaluation System for cell death Induction mediated by internalization Activity

The activity of inducing cell death by antibody internalization was evaluated using saporin (toxin) -labeled anti-mouse IgG antibodies (Mab-ZAP, Advanced Targeting Systems). An indirect toxin-labeled antibody was first prepared by mixing the primary antibody and Mab-ZAP and reacting for 15 minutes at room temperature, and added to the target cells. When the added antibody is internalized into the cell, the Mab-ZAP is also incorporated into the cell along with the primary antibody, resulting in cellular death induced by saporin released inside the cell. This is shown schematically in fig. 13.

3-5-2 Induction of internalization-mediated cell death in cell lines highly expressing HB-EGF

Induction of cells by internalization Activity Using HC-15 detection antibodiesThe ability to die. At 2X 10³Cell/well Density SKOV-3 cells and HB-EGF _ SKOV3 cells (HB-EGF overexpressing SKOV-3 cells) were seeded into 96-well plates. After overnight culture, Mab-ZAP was reacted with the obtained anti-HB-EGF antibody (100 ng/well) in an amount of 100 ng/well, and added to the cells. After 4 days of antibody addition, viable cells were counted using WST-8. For the original SKOV-3 cells that only weakly express HB-EGF, no ability to induce cell death was observed for any of the antibodies; however, for SKOV-3 cells overexpressing HB-EGF, cell death-inducing activity was observed in the presence of Mab-ZAP for each antibody. In particular, strong cell death-inducing activity of HB-20 and HC-15 binding to proHB-EGF was observed (FIG. 14).

3-5-3. internalization-mediated cell death induction in ovarian cancer strains

3-5-3-1 analysis of cytotoxicity against ovarian cancer Strain (ES-2)

3-5-3-1-1 binding Activity of Each antibody on ES-2

Then, the ability to induce cell death in an ovarian cell line (ES-2) internally expressing HB-EGF was investigated. ES-2 cells (ovarian cancer cell line, purchased from ATTC) were cultured in growth medium (McCoy' S5A medium, Invitrogen Corporation) containing 10% FCS and penicillin/streptomycin (P/S).

The ability of each antibody to bind to the cell surface of ES-2 cells was first analyzed using FACS. Stripping the cells with 1mM EDTA; FACS buffer (containing 2% FCS and 0.05% NaN) at 4 ℃₃PBS), cells were reacted with a specific antibody (10. mu.g/mL) for 1 hour; and stained with FITC-labeled anti-mouse IgG antibody (Beckman Coulter, PNIM0819) at 4 ℃ for 30 minutes. FACS (Becton, Dickinson and company) was used to analyze the antibody binding to HB-EGF expressed on the cell surface.

FIG. 15 provides graphs comparing the binding activity of HA-20, HB-20, and HC-15 antibodies to ES-2 cells via FACS analysis. The gray waveform indicates the staining pattern in the absence of primary antibody (control), while the solid line indicates the staining pattern in the presence of specific antibody. The horizontal axis represents staining intensity, and the vertical axis represents cell number. As shown in FIG. 15, in particular for HC-15, binding to HB-EGF expressed on the cell membrane of ES-2 cells was detected.

3-5-3-1-2 ability to induce cell death in ES-2

Internalization-mediated cytotoxicity of ES-2 cells was studied for each antibody. At 2X 10³Cell/well Density ES-2 cells were seeded into 96-well plates. After overnight incubation, specific antibodies (100 ng/well) were reacted with Mab-ZAP (100 ng/well) and added to the cells. After 3 days, viable cells were counted using WST-8. According to the results shown in FIG. 16, for HC-15 showing the strongest HB-EGF binding activity, cell death-inducing activity was observed in the presence of Mab-ZAP.

3-5-3-2 analysis of the ability to inhibit the growth of ovarian cancer cell lines (RMG-1, MCAS)

3-5-3-2-1 binding Activity of HC-15 on MCAS and RMG-1

The ability of the antibodies to inhibit growth was then investigated using individual ovarian cancer cell lines (RMG-1, MCAS). MCAS cells (purchased from JCRB) were cultured in growth medium (Eagle essential medium, Invitrogen Corporation) containing 20% FCS.

To investigate whether and to what extent MCAS and RMG-1 express HB-EGF on the cell surface, FACS analysis was performed using HC-15 antibody. Stripping the cells with 1mM EDTA; FACS buffer (containing 2% FCS and 0.05% NaN) at 4 ℃₃PBS), cells were reacted with HC-15 antibody (10. mu.g/mL) for 1 hour; and stained with FITC-labeled anti-mouse IgG antibody (Beckman Coulter, PN IM0819) at 4 ℃ for 30 minutes. FACS (Becton, Dickinson and Company) was used to analyze the antibody binding to HB-EGF expressed on the cell surface.

FIG. 17 provides graphs comparing the binding activity of HC-15 antibody on RMG-1 and MCAS cells by FACS analysis. It was shown that HB-EGF was expressed on the cell surface of both RMG-1 cells and MCAS cells.

3-5-3-2-2 analysis of the ability of the antibodies to inhibit proliferation of RMG-1 and MCAS Using the Soft agar colony formation assay

The activity of the antibodies on anchorage-independent proliferation of RMG-1 and MCAS cells was then studied using a soft agar colony formation assay. The soft agar colony formation assay was performed as follows.

To prepare agar bottoms, MEM medium containing 0.6% agar (3: 1NuSieve, Cambrex) was added to each well of a 96-well plate at a concentration of 100. mu.L/well. Cells were then suspended in medium containing 0.3% agar at a density of 8000 cells/well. Mixing each test substance (antibody, Mab-ZAP) with cells into agar; the preparation was added dropwise to the agar plate at a concentration of 100. mu.L/well. After 3 weeks to 1 month of incubation at 37 ℃, the colonies that appeared were stained with 1% iodonium chloride nitrotetrazolium salt (Sigma, 18377) and examined under a microscope.

First, the effect of each antibody (HA-20, HC-15) on the colony formation of RMG-1 cells was analyzed. HA-20 or HC-15 antibody was mixed into RMG-1 cells to provide 50. mu.g/mL, and colonies formed in agar were observed after about 3 weeks. According to the results, colony formation of RMG-1 cells was inhibited in the HC-15 antibody-added group compared with the non-added group (FIG. 18). Thus, it was found that HC-15 antibody can inhibit anchorage-independent colony formation of RMG-1 cells only by its neutralizing activity alone.

The HA-20 and HC-15 antibodies were then assayed for toxin-mediated colony formation-inhibiting activity. Mab-ZAP (1. mu.g/mL) was mixed into agar along with RMG-1 cells, MCAS cells and HA-20 or HC-15 antibody (10. mu.g/mL) and cultured for about 3 weeks to 1 month. The colonies formed were stained and observed by microscopy.

According to the results, it was observed that inhibition of colony formation was caused by the simultaneous addition of HC-15 antibody and Mab-ZAP to RMG-1 cells (FIG. 19) and MCAS cells (FIG. 20). From the foregoing, it was demonstrated that HC-15 antibody can inhibit the ability of ovarian cancer cells to form colonies not only by its neutralizing activity but also by its internalizing activity.

3-5-4. Induction of internalization mediated cell death in blood cancer cell lines

3-5-4-1 analysis of HB-EG expression in blood cancer cell lines

It was then examined whether internalization-mediated antitumor effects of HC-15 could also be observed for hematological cancers. The following cells were cultured in RPMI1640(Invitrogen Corporation) containing 10% FCS: RPMI8226 (multiple myeloma, from ATCC), Jurkat (acute T cell leukemia, from ATCC), HL-60 (acute myelogenous leukemia, from JCRB), THP-1 (acute monocytic leukemia, from JCRB), and U937 (monocytic leukemia, from JCRB).

FACS analysis was performed to investigate HB-EGF expression of these cells. HC-15 (10. mu.g/mL) antibody and specific cell line (2X 10)⁵Cells) were reacted on ice for 60 minutes and stained with FITC-labeled anti-mouse IgG antibody (Beckman Coulter, PN IM 0819). The binding of the antibody to HB-EGF expressed on the cell surface was then analyzed by FACS (Becton, Dickinson and Company).

FIG. 21 shows a graph comparing the expression of HB-EGF of each blood cancer cell line based on FACS analysis. It was shown that THP-1 and U937 showed particularly strong HB-EGF expression. In contrast, Jurkat and RPMI8226 were barely expressed.

3-5-4-2 analysis of cytotoxicity of blood cancer cell lines

At 1-2X 10⁴Cell/well Density specific blood cancer cell lines were seeded into 96-well plates. Then Mab-ZAP was reacted with a specific anti-HB-EGF antibody (100 ng/well) in an amount of 100 ng/well, and added to the cells. After 5 days of antibody addition, viable cell counts were determined using WST-8. Inhibition of proliferation by the simultaneous addition of HC-15 antibody and Mab-ZAP to U937 cells and THP-1 cells was observed. From these results, it was revealed that the internalization activity of HC-15 antibody as an antitumor agent is effective for several hematological cancers.

Assay for saporin-labeled antibody-induced cell death

4-1. saporin labeling of antibodies

HA-20 antibody directly labeled with toxin (HA-SAP) and HC-15 antibody directly labeled with toxin (HC-SAP) were used to study cytotoxicity mediated by the internalizing activity of the antibodies.

Purified HA-20 antibody and purified HC-15 antibody labeled with saporin were coated on Advanced Targeting Systems. Thus, antibodies consisting of HA-20 labeled with an average of 3 saporin molecules and HC-15 labeled with an average of 2.4 saporin molecules (designated HA-SAP and HC-SAP, respectively) were obtained. These antibodies were used in the study of the ability to induce cancer cell death.

4-2. analysis of cytotoxicity of saporin-labeled antibodies

4-2-1. analysis of cytotoxicity of saporin-labeled antibody on solid cancer cell lines

The following cancer cells were used in the assay: ES-2, MCAS (ovarian cancer), Capan-2 (pancreatic cancer, purchased from Japan Health Sciences Foundation), BxPC-3, 22Rv1 (prostate cancer, purchased from ATCC), and HUVEC (human endothelial cells, purchased from Takara BioInc.). These cells were cultured in each case using the culture conditions indicated by the instructions provided by the supplier.

Cytotoxicity was analyzed as follows. At 1-5X 10³Cell/well Density Each cell line was seeded into 96-well plates and cultured overnight. The next day, HA-SAP, HC-SAP or control antibody (saporin-labeled mouse IgG (IgG-SAP), Advanced targeting systems) was added to provide approximately 100nM-1fM, and cultured for 3-5 days. Viable cell counts were determined using WST-8.

According to the results shown in FIG. 23a, HC-SAP strongly induced cell death in the ovarian cancer cell lines ES-2 and MCAS. The HC-SAP activity was as follows: EC for ES-2 cells₅₀EC at 0.09nM for MCAS cells₅₀0.86 nM. On the other hand, it showed no effect at all on HUVEC (normal human endothelial cells).

4-2-2 analysis of cytotoxicity of blood cancer cell lines by saporin-labeled antibody

The following cell lines were cultured in RPMI1640(Invitrogen Corporation) containing 10% FCS: RPMI8226 (multiple myeloma, from ATCC), HL-60 (acute myelogenous leukemia, from JCRB), SKM-1 and THP-1 (acute monocytic leukemia, from JCRB) and U937 (monocytic leukemia, from JCRB).

The cytotoxicity of HA-SAP and HC-SAP on these blood cancer cell lines was examined as follows. At 1-5X 10³Cell/well Density Each cell strain was seeded in a 96-well plate, followed by the addition of HA-SAP or HC-SAP at approximately 100nM-1fM, and cultured for 3-5 days. Viable cell counts were then determined using WST-8.

According to the results shown in FIG. 23b, HC-SAP induced cell death substantially in U937, SKM-1 and THP-1 cells. The HC-SAP activity was as follows: for U937 cell EC₅₀EC for SKM-1 cells at 0.33nM₅₀EC for THP-1 cells 0.02nM₅₀0.01 nM. These results indicate that an antibody against HB-EGF labeled with, for example, a toxin is also effective for hematological cancer.

DNA immunization

5-1 construction of expression vector for secretory HB-EGF

An expression vector for secretory HB-EGF was constructed as follows. To amplify a fragment encoding the extracellular region of HB-EGF (amino acids 1-148), PCR was first performed under the following conditions using Pyrobest Taq polymerase (Takara Bio Inc.) and using HB-EGF expression vector pMCN _ HB-EGF as a template.

EGF-9：TCC GAA TTC CAC CAT GAA GCT GCT GCC GTCGGT G(SEQ ID NO：91)

EGF-10：TTT GCG GCC GCT AGA GGC TCA GCC CAT GACACC T(SEQ ID NO：92)

(94 ℃/30 seconds, 65 ℃/30 seconds, 72 ℃/30 seconds: 25 cycles)

The resulting PCR product was digested with EcoRI and NotI. The resulting DNA fragment was inserted between EcoRI and NotI in a pMCDN2 expression vector for animal cells to construct a pMCDN _ sHB-EGF expression vector for secretory HB-EGF.

DNA immunization

50 μ g of the secretory HB-EGF expression vector pMCDN _ sHB-EGF was coated on gold particles as described in the instructions provided by Bio-Rad Laboratories, Inc (# 165-2431). The DNA-bound gold particles obtained in this manner were coated in a catheter using Tubig Prep Station (Bio-Rad Laboratories, Inc.), and the catheter was cut into an appropriate length with a cutter, and stored at 4 ℃ as DNA for immunization.

Then DNA immunization was performed. DNA was introduced into the abdomen of 3 animals [ (MRL/lpr, male, age: 4 weeks) (balb/c, female, age: 6 weeks) purchased from Charles River Japan ] by bombardment using a Helios Gene gun (Bio-Rad Laboratories, Inc.). A total of 11 immunizations were then given by the same procedure at 3-4 day intervals. For the last immunization, 50. mu.g of HB-EGF _ Fc was suspended in 100. mu.L of PBS and injected into the tail vein. Cell fusion was performed 3 days later. Hybridomas were prepared by the same procedure as in 1-3.

A method for screening a candidate antibody by comparing the binding activity of the cloned HB-EGF and the neutralizing activity was carried out using the same procedure as described in 2-4. Limiting dilution, subtype determination and antibody purification were then performed by the methods described in 2-4. An antibody HE-39 showing a strong HB-EGF binding activity and neutralizing activity was finally obtained by DNA immunization.

Analysis of Properties of novel HB-EGF neutralizing antibody (HE-39)

6-1 analysis of the ability of HE-39 to bind to active type HB-EGF

In order to compare the obtained HE-39 binding ability to the active type HB-EGF protein with that of 3 previously obtained antibodies (HA-20, HB-20, HC)-15) ability to bind to the active type HB-EGF protein was compared, and the following experiment was conducted. HA-20, HB-20, HC-15 or HE-39 antibodies at 1. mu.g/mL HB-EGF protein (R)&D Systems, Inc., 259-HE) coated ELISA plates (NUNC) were reacted at various concentrations and with Alkaline Phosphatase (AP) -labeled anti-mouse IgG (Zymed Laboratories, Inc., #62-6622) for 1 hour. Color was developed by addition of 1mg/mL substrate (Sigma, S0942-50 TAB). OD measurement with plate reader₄₀₅And the concentration of antibody to 50% binding (ED) was calculated from the binding curve obtained with the particular antibody₅₀). As a result, it was revealed that HE-39 has an ED of about 0.016nM for the binding activity of the active type HB-EGF₅₀Values, thus indicating that HE-39 has much stronger binding activity than the other 3 antibodies (fig. 24).

TABLE 4 binding to HB-EGF (ED)₅₀，nmol/L)

HA20	HB20	HC15	HE39
				3.01	5.49	0.65	0.016

Assay of binding Activity of HE-39 for proHB-EGF

In order to compare the binding activity of HE-39 to proHB-EGF with that of 3 previously obtained antibodies (HA-20, HB-20, HC-15), the following experiment was conducted.

A specific antibody (10. mu.g/mL) was reacted with DG44 cells expressing HB-EGF (HB-EGF _ DG44) at 4 ℃ for 1 hour, followed by staining with an FITC-labeled anti-mouse IgG antibody (Beckman Coulter, PN IM 0819). The binding of specific antibodies to cell surface HB-EGF was then analyzed by FACS (Becton, Dickinson and Company).

FIG. 25 shows comparison of the binding activity of the antibodies HA-20, HB-20, HC-15 and HE-39 to proHB-EGF overexpressed in DG44 cells according to FACS analysis. The gray waveform indicates the staining pattern in the absence of primary antibody (control), while the solid line indicates the staining pattern in the presence of specific antibody. The horizontal axis represents staining intensity, and the vertical axis represents cell number. As shown in FIG. 25, similarly to HB-20 and HC-15, the HE-39 antibody is an antibody recognizing HB-EGF on the cell membrane.

6-3 analysis of neutralizing Activity

6-3-1. ability to inhibit binding of HB-EGF to EGFR

Using the solid phase evaluation system described in 3-4-1, the ability of the HE-39 antibody to inhibit the binding of HB-EGF to EGFR was compared with the binding inhibition ability of 3 previously obtained antibodies (HA-20, HB-20, HC-15). HB-EGF (50ng/mL) and serially diluted antibodies were added to an ELISA plate on which EGFR-Fc was immobilized, and reacted at room temperature for 1 hour. The plate was washed with TBS-T, and HB-EGF binding to EGFR was detected by the procedure described in 3-4-1 (FIG. 26).

The results demonstrate that the binding of HB-EGF to the receptor is strongly inhibited by HC-15 and HE-39 antibodies, among others.

TABLE 5 inhibition of HB-EGF binding to EGFR (EC)₅₀，nmol/L)

HA20	HB20	HC15	HE39
				9.52	9.51	2.06	0.83

6-3-2. ability to inhibit growth of EGFR _ Ba/F3 cells

The HE-39 antibody was then analyzed for neutralization activity against HB-EGF-dependent growth of EGFR _ Ba/F3 cells, and compared with the neutralization activity of HA-20, HB-20, and HC-15. The method described in 3-4-2, in the presence of HB-EGF (80ng/mL) at 2X 10⁴Cell/well Density EGFR _ Ba/F3 cells were seeded into 96-well plates and specific purified antibodies were added. After 3 days of culture, cell count was measured using WST-8 (cell count kit-8), and a growth curve was constructed. From the results obtained, the antibody concentration (EC) at 50% of the maximal inhibitory effect was calculated₅₀Value).

According to the results, HE-39 showed substantially better than HC-15 (EC)₅₀2.06nM) growth inhibitory Activity (EC)₅₀0.83nM), additionally showed the strongest growth inhibitory effect on EGFR _ Ba/F3 cells (fig. 27).

TABLE 6 inhibition of HB-EGF dependent growth (EC)₅₀，nmol/L)

HA20	HB20	HC15	HE39
				9.52	9.51	2.06	0.83

Cloning of HE-39 antibody variable regions

7.1 cloning of the variable regions

Cloning and analysis of amino acid sequence of HE-39 antibody variable region were performed according to the method described in 3-1. Since HE-39 is IgG1, the light chain variable region was cloned using the VL-k primer (SEQ ID NO: 69) and the heavy chain variable region was cloned using the VH-G1 primer (SEQ ID NO: 70).

The gene fragment amplified in the foregoing step was TA-cloned into pCRII-TOPO (Invitrogen TOPO TA-cloning kit, #45-0640), after which the base sequence of each inserted fragment was confirmed. The sequence of the confirmed variable strand is shown in FIG. 28.

7-2 identification of light chain variable region

As a result of cloning the variable region, two different genes (VL-1, VL-2) were present in the light chain variable region derived from HE-39 hybridoma. This leads to the following assumptions: HE-39 hybridomas are not completely monoclonal. Thus, single cloning was again performed by limiting dilution. HE-39 was seeded into 96-well plates to reach 1 cell/well. After approximately 10 days of culture, the culture supernatants of the wells in which colonies appeared were analyzed by FACS using Ba/F3 cells expressing HB-EGF. As a result, as shown in FIG. 29a, 3 monoclonal antibodies (HE39-1, HE39-5, HE39-14) showing HB-EGF binding activity were obtained.

Then, in order to determine which light chain variable regions (VL-1, VL-2) were expressed in these monoclonal hybridomas, the following experiment was performed.

RNA was purified from each hybridoma (HE39, HE39-1, HE39-5, HE39-14) and cDNA was synthesized using SuperScript III First Strand System (Invitrogen Corporation). To examine which light chains were expressed in the respective hybridomas, RT-PCR was performed using synthetic cDNA derived from each hybridoma as a template under the following conditions using primers specific to the HE-39 heavy chain (HE39VH) and primers specific to both light chain types (VL-1, VL-2) (HE39VL1, HE39VL 2).

Primers specific for HE-39 heavy chain variable region

VH-G1：GGG CCA GTG GAT AGA CAG ATG(SEQ ID NO：70)

HE39VH：CTG GGT CTT TCT CTT CCT CCT GTC A(SEQ IDNO：93)

Primers specific for HE-39 light chain variable region

VL-1

VL-k：GCT CAC TGG ATG GTG GGA AGA TG(SEQ ID NO：69)

HE39VL1：TGA GAT TGT GAT GAC CCA GAC TCC A(SEQ IDNO：94)

VL-2

VL-k：GCT CAC TGG ATG GTG GGA AGA TG(SEQ ID NO：69)

HE39VL2：TTC TCA CCC AGT CTC CAG CAA TCA(SEQ IDNO：95)

94 ℃/5 sec, 72 ℃/2 min: 5 cycles

94 ℃/5 sec, 70 ℃/10 sec, 72 ℃/2 min: 5 cycles

94 ℃/5 sec, 68 ℃/10 sec, 72 ℃/2 min: 27 cycles

From the results, it was confirmed that, as shown in FIG. 29b, not only in HE39, but also in hybridomas (HE39-1, HE39-5, HE39-14) that had been monoclonal by additional limiting dilution, two types of light chains (VL-1, VL-2) were expressed. These results indicate that two types of VL-1, VL-2 are present in the light chain of HE-39.

Assay for internalization Activity of HE-39 antibodies

8-1 assay for internalization Activity-mediated cytotoxicity of HE-39 antibodies

The presence/absence of internalization activity-mediated cytotoxicity of HE-39 antibodies obtained by DNA immunization was also investigated.

At 2X 10³Cell/well Density HB-EGF _ DG44 (DG 44 cells overexpressing HB-EGF) was seeded into 96-well plates. These cells were reacted with HA-20, HC-15 or HE-39 antibodies (100 ng/well or 1000 ng/well) and Mab-ZAP (100 ng/well), cultured for 4 days, and viable cell counts were determined using WST-8.

For the group to which the anti-HB-EGF antibody and Mab-ZAP were added, the ability to induce cell death was observed. A particularly strong ability to induce cell death was observed for HC-15 and HE-39 (FIG. 30).

Epitope analysis of HE-39 antibodies

Analysis of binding Domain of HE-39 antibody

HB-EGF has the structure shown in FIG. 31 a. The mature HB-EGF consists of two distinct domains (i.e., heparin-binding domain and EGF-like domain). Coli expression vectors for expression of GST fusion proteins were first constructed as described below, with the goal of determining which of the two domains is the domain recognized by HE-39 antibody.

9-1-1. preparation of GST fusion protein expression vector for epitope mapping

9-1-1-1. construction of GST-HBEGF mature expression vector

An expression vector for the GST protein/mature HB-EGF fusion protein was prepared as follows. In order to amplify a fragment encoding mature HB-EGF, PCR was performed using Pyrobest Taq polymerase (Takara Bio Inc.) under the following conditions, using the HB-EGF expression vector pMCN _ HB-EGF as a template.

EGF 11：TTGGATCCGTCACTTTATCCTCCAAGCCACA(SEQID NO：96)

EGF 12：TTCTCGAGGAGGCTCAGCCCATGACACCT(SEQ IDNO：97)

(94 ℃/30 seconds, 65 ℃/30 seconds, 72 ℃/30 seconds: 30 cycles)

The obtained PCR product was then digested with BamHI and XhoI and inserted downstream of the GST-coding region of E.coli GST fusion expression vector (pGEX-6P-1) which was also digested with BamHI and XhoI to construct a mature HB-EGF/GST fusion protein expression vector (mature pGEX-HBEGF).

9-1-1-2. construction of GST-HBEGF HBD expression vector

A vector expressing GST protein/HBD (heparin-binding domain of HB-EGF) fusion protein was prepared as follows. To amplify the fragment encoding the heparin-binding domain, PCR was first performed using Pyrobest Taq polymerase (Takara Bio Inc.) under the following conditions using the HB-EGF expression vector pMCN _ HB-EGF as a template.

EGF 11：TTGGATCCGTCACTTTATCCTCCAAGCCACA(SEQID NO：96)

EGF 13：TTCTCGAGCCGAAGACATGGGTCCCTCTT(SEQ IDNO：98)

(94 ℃/30 seconds, 65 ℃/30 seconds, 72 ℃/30 seconds: 30 cycles)

The obtained PCR product was then digested with BamHI and XhoI and inserted downstream of the GST coding region of E.coli GST fusion expression vector (pGEX-6P-1) which was also digested with BamHI and XhoI to construct HB-EGF heparin-binding domain/GST fusion protein expression vector (pGEX-HBEGF _ HBD).

9-1-1-3. construction of GST-HBEGF EGFD expression vector

A vector expressing GST protein/EGFD (EGF-like domain of HB-EGF) fusion protein was prepared as follows. To amplify a fragment encoding an EGF-like domain, PCR was first performed using Pyrobest Taq polymerase (Takara Bio Inc.) using the HB-EGF expression vector pMCN _ HB-EGF as a template under the following conditions.

EGF 14：TAGGATCCAAGAGGGACCCATGTCTTCGG(SEQ IDNO：99)

EGF 12：TTCTCGAGGAGGCTCAGCCCATGACACCT(SEQ IDNO：97)

(94 ℃/30 seconds, 65 ℃/30 seconds, 72 ℃/30 seconds: 30 cycles)

The obtained PCR product was then digested with BamHI and XhoI and inserted downstream of the GST-coding region of E.coli GST fusion expression vector (pGEX-6P-1) which was also digested with BamHI and XhoI to construct HB-EGF EGF-like domain/GST fusion protein expression vector (pGEX-HBEGF _ EGFD).

9-1-2 Induction of expression of respective GST fusion proteins

Various E.coli expression vectors constructed as described above were transformed into E.coli BL 21. Coli transformants were cultured in LB medium (1mL each) and IPTG (final concentration of 1mM) was added in the logarithmic growth phase to induce protein expression. Recovering the escherichia coli after 4-5 hours; lysates were prepared by lysis in SDS sample buffer (0.5 mL); SDS-PAGE was performed by standard methods using 5. mu.L of lysate, followed by Western blot analysis on PVDF membrane.

9-1-3 analysis of HE-39 recognition Domain on mature HB-EGF protein

Using the GST fusion protein prepared as described above, in which the respective regions (heparin-binding domain, EGF-like domain) of the mature HB-EGF protein were fused, the region of the mature HB-EGF protein recognized by the HE-39 antibody was investigated by Western blotting. The results of Western blot analysis shown in FIG. 31b demonstrate that the HE-39 antibody recognizes the EGF-like domain of the mature HB-EGF protein.

Analysis of epitopes in EGF Domain

Since HE-39 recognizes the EGF-like domain of the mature HB-EGF protein, in order to determine the epitope sequence, the EGF-like domain was more finely divided as shown in FIG. 32 a.

9-2-1 construction of GST-EGFD5, GST-EGFD6 and GST-EGFD7 expression vectors

Coli expression vectors of GST fusion proteins with EGF domains divided into 3 fragments (EGFD5, EGFD6, EGFD7) were prepared as follows.

To construct DNA fragments encoding the respective regions (EGFD5, EGFD6, EGFD7), the following two oligomers were first designed for each region.

Oligomers for EGFD5 synthesis

HEP9：

GAT CCA AGA GGG ACC CAT GTC TTC GGA AAT ACA AGGACT TCT GCA TCC ATG GAG AAT GCA AAT ATC(SEQ ID NO：100)

HEP10：

TCG AGA TAT TTG CAT TCT CCA TGG ATG CAG AAG TCCTTG TAT TTC CGA AGA CAT GGG TCC CTC TTG(SEQ ID NO：101)

Oligomers for EGFD6 synthesis

HEP 11：

GAT CCT GCA TCC ATG GAG AAT GCA AAT ATG TGA AGGAGC TCC GGG CTC CCT CCT GCA TCT GCC ACC CGC(SEQ IDNO：102)

HEP 12：

TCG AGC GGG TGG CAG ATG CAG GAG GGA GCC CGGAGC TCC TTC ACA TAT TTG CAT TCT CCA TGG ATG CAG(SEQID NO：103)

Oligomers for EGFD7 synthesis

HEP 13：

GAT CCG CTC CCT CCT GCA TCT GCC ACC CGG GTT ACCATG GAG AGA GGT GTC ATG GGC TGA GCC TCC(SEQ ID NO：104)

HEP 14：

TCG AGG AGG CTC AGC CCA TGA CAC CTC TCT CCA TGGTAA CCC GGG TGG CAG ATG CAG GAG GGA GCG(SEQ IDNO：105)

In each case, two oligomers were combined and annealed by standard methods to prepare double-stranded DNA fragments, which were inserted downstream of the GST coding region of E.coli GST fusion expression vector (pGEX-6P-1) that had been digested with BamHI and XhoI to generate individual constructs (pGEX-HBEGF _ EGFD5, pGEX-HBEGF _ EGFD6, pGEX-HBEGF _ EGFD 7).

9-2-2 Induction of expression of respective GST fusion proteins

Various E.coli expression vectors constructed as described above were transformed into E.coli BL 21. Coli transformants were cultured in LB medium (1mL each) and IPTG (final concentration of 1mM) was added in the logarithmic growth phase to induce protein expression. Recovering the escherichia coli after 4-5 hours; lysates were prepared by lysis in SDS sample buffer (0.5 mL); SDS-PAGE was performed by standard methods using 5. mu.L of lysate, followed by Western blot analysis on PVDF membrane.

Epitope mapping of 9-2-3.HE-39

Using GST fusion proteins (GST-EGFD5, GST-EGFD6, GST-EGFD7) prepared as described above, the recognition sequence of the HE-39 antibody was investigated by Western blotting using one of 3 fragments of EGF-like domain. The results of the Western blot analysis shown in FIG. 32b demonstrate that the HE-39 antibody recognizes sequences in the EGF-like domain due to binding of the HE-39 antibody to GST-EGFD7 [ APSCICHPGYHGERCHGLSL ].

Analysis of ADCC Activity mediated by anti-HB-EGF antibody

10-1 analysis of binding Activity of Each antibody for Membrane-expressed HB-EGF

The binding activity of the antibody obtained so far to membrane-expressed HB-EGF was compared by FACS analysis. The specific antibody (10. mu.g/mL) was reacted with HB-EGF _ Ba/F3 cells (Ba/F3 cells overexpressing HB-EGF) at 4 ℃ for 1 hour, followed by staining with FITC-labeled anti-mouse IgG antibody (Beckman Coulter, PN IM 0819). The binding of this specific antibody to the cell surface HB-EGF was analyzed by FACS (Becton, Dickinson and Company).

FIG. 33a shows the binding activity of each antibody to HB-EGF _ Ba/F3 as determined by FACS analysis. The G-average (geometric mean) on the vertical axis is a value obtained by converting the antibody-induced fluorescence intensity of the cells into a numerical value. The analysis results showed that HC-15 is an antibody having the strongest binding activity to HB-EGF on the cell membrane, and HE-39, HE-48 and HE-58 also have strong binding activity.

10-2 analysis of Antibody Dependent Cellular Cytotoxicity (ADCC) of anti-HB-EGF antibody

ADCC activity against HB-EGF _ Ba/F3 cells was analyzed using a chromium release method and antibodies (HB-10, HB-20, HB-22, HC-15, HE-39, HE-48, HE-58) which have shown binding activity to HB-EGF _ Ba/F3 cells.

To HB-EGF _ Ba/F3 cells propagated in a 96-well plate, chromium-51 was added and the culture was continued for several hours. Removing the culture medium; washing the cells with the culture medium; fresh medium was then added. Adding antibody to achieve a final concentration of 10 μ g/mL; effector cells (recombinant cells obtained by inducing the forced expression of mouse Fc-gamma receptor (NM-010188) in NK-92(ATCC, CRL-2407)) were also added to each well at about 5X or 10X relative to the target cells; the culture plate is kept at 37 DEG C、5％CO₂The incubator is kept still for 4 hours. After resting, the plates were centrifuged and a constant amount of supernatant was recovered from each well; radioactivity was measured using a Wallac 1480 γ counter; and the specific chromium release (%) was determined. According to the results shown in the upper panel of FIG. 33b, among the anti-HB-EGF monoclonal antibodies used in the test, HB-22, HC-15, HE-39, HE-48 and HE-58 induced particularly very strong ADCC activity. These results indicate that anti-tumor antibody treatment against HB-EGF is very useful.

The specific chromium release rate was calculated using the following formula.

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

Wherein: a is the radioactivity in the specific well; b is the average value of the radioactivity released into the medium by cell lysis with 1% final concentration of NonidetP-40; c is the average value of radioactivity added to the medium alone.

10-3 determination of complement dependent cytotoxicity (ADCC) of anti-HB-EGF antibody

HB-EGF _ Ba/F3 cells were recovered by centrifugation (1000rpm, 5 minutes, 4 ℃); the cell pellet was suspended in approximately 200. mu.L of medium and 3.7MBq chromium-51 (accession number CJS4, Amersham Pharmacia Biotech); and 5% CO at 37 deg.C₂Incubate for 1 hour. Then washing the cells 3 times with the culture medium; subsequently adjusting the cell density to 1X 10 with culture medium⁴Per mL; and 100. mu.L of the solution was added to each well of a 96-well flat-bottom plate.

anti-HB-EGF monoclonal antibodies (HB-10, HB-20, HB-22, HC-15, HE-39, HE-48, HE-58) and control mouse IgG2a antibody (catalog No. 553453, BD Biosciences Pharmingen) were diluted with the medium and then added in an amount of 50. mu.L/well. The antibody was adjusted to a final concentration of 10. mu.g/mL. Then, young rabbit complement (catalog number CL3441, Cedarlane) was added to each plate well to reach a concentration of 3% or 10%, followed by 5% CO at 37 ℃₂The incubator is kept still for 1.5 hours. After standing, the plates were centrifuged (1000rpm, 5 min, 4 ℃); recovering 100 μ L of supernatant from each well; and useA gamma counter (1480WIZARD3 ", Wallac) measures the radioactivity in the recovered supernatant.

According to the results shown in the lower graph of FIG. 33b, among the anti-HB-EGF monoclonal antibodies used in this test, HB-20, HB-22, HC-15 and HE-48 showed CDC activity. On the other hand, the mouse IgG2a antibody used as a control did not show CDC activity at the same concentration.

Industrial applicability

The antibodies of the invention and pharmaceutical compositions comprising the antibodies of the invention are useful for the treatment and diagnosis of cancer.

Sequence listing

<110> future institute for drug discovery by Kabushiki Kaisha (Foreruner Pharma Research Co., Ltd.)

<120> anti-HB-EGF antibody

<130>PCG-9018WO

<150>JP 2006-286824

<151>200610-20

<150>JP 2007-107207

<151>2007-04-16

<160>105

<170>PatentIn version 3.1

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Glu Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Val Trp Ser Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr

115 120 125

Leu Thr Val Ser Ser

130

<210>39

<211>378

<212>DNA

<213> mice

<400>39

atgcagatta tcagcttgct gctaatcagt gtcacagtca tagtgtctaa tggagaaatt 60

gtgctcaccc agtctccaac caccatggct gcatctcccg gggagaagat cactatcacc 120

tgcagtgcca gctcaagtat aagttccaat tacttgcatt ggtatcagca gaagccagga 180

ttctccccta aactcttgat ttataggaca tccaatctgg cttctggagt cccagctcgc 240

ttcagtggca gtgggtctgg gacctcttac tctctcacaa ttggcaccat ggaggctgaa 300

gatgttgcca cttactactg ccagcagggt agtagtatac cattcacgtt cggctcgggg 360

acaaagttgg aaataaaa 378

<210>40

<211>126

<212>PRT

<213> mice

<400>40

Met Gln Ile Ile Ser Leu Leu Leu Ile Ser Val Thr Val Ile Val Ser

1 5 10 15

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

20 25 30

Pro Gly Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser

35 40 45

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

50 55 60

Leu Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val pro Ala Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr

85 90 95

Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser

100 105 110

Ile Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210>41

<211>411

<212>DNA

<213> mice

<400>41

atggctgtcc tggcattact cttctgcctg gtaacattcc caagctgtat cctttcccag 60

gtgcagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcaca 120

tgcaccgtct cagggttctc attaaccggc tatggtataa actgggttcg ccagcctcca 180

ggaaagggtc tggagtggct gggaatgatc tggggtgatg gaagcgcaga ctataattca 240

gctctcaaat ccagactgag catccgcaag gacaactcca agagccaagt tttcttagaa 300

atgaacagtc tgcaaactga tgacacagcc aggtactact gtgccagagg ggattactac 360

ggctacaggt tttcttactg gggccaaggg actctggtca ctgtctctgc a 411

<210>42

<211>137

<212>PRT

<213> mice

<400>42

Met Ala Val Leu Ala Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys

1 5 10 15

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

20 25 30

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

35 40 45

Thr Gly Tyr Gly Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Met Ile Trp Gly Asp Gly Ser Ala Asp Tyr Asn Ser

65 70 75 80

Ala Leu Lys Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln

85 90 95

Val Phe Leu Glu Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr

100 105 110

Tyr Cys Ala Arg Gly Asp Tyr Tyr Gly Tyr Arg Phe Ser Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ala

130 135

<210>43

<211>396

<212>DNA

<213> mice

<400>43

atggaatcac agactcaggt cttcctctcc ctgctgctct gggtatctgg tacctttggg 60

aacattatgc tgacacagtc gccatcatct ctggctgtgt ctgcaggaga aaaggtcact 120

atgagctgta agtccagtca aagtgtttta tacagttcaa atcagaagaa cttcttggcc 180

tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 240

gaatctggtg tccctgatcg cttcgcaggc agtggatctg ggacagattt tactcttacc 300

atcagcagtg tacaaactga agacctggca gtttattact gtcatcaata cctctcctcg 360

tatacgttcg gaggggggac caagctggaa ataaaa 396

<210>44

<211>132

<212>PRT

<213> mice

<400>44

Met Glu Ser Gln Thr Gln Val Phe Leu Ser Leu Leu Leu Trp Val Ser

1 5 10 15

Gly Thr Phe Gly Asn Ile Met Leu Thr Gln Ser Pro Ser Ser Leu Ala

20 25 30

Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Val Leu Tyr Ser Ser Asn Gln Lys Asn Phe Leu Ala Trp Tyr Gln Gln

50 55 60

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

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Ala Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Thr Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys His Gln Tyr Leu Ser Ser Tyr Thr Phe Gly Gly Gly Thr Lys

115 120 125

Leu Glu Ile Lys

130

<210>45

<211>411

<212>DNA

<213> mice

<400>45

atgggatgga actggatctt tattttaatc ctgtcagtaa ctacaggtgt ccactctgag 60

gtccagctgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaagatatcc 120

tgcaaggctt ctggttactc attcactggc tactacatgc actgggtgaa gcaaagtcct 180

gaaaagagac ttgagtggat tggagagatt aatcctagaa ctggtattac tacctacaac 240

cagaagttca aggccaaggc cacattgact gtagacaaat cctccagcac agcctacatg 300

cagctcaaga gcctgacatc tgaggactct gcagtctatt actgtgcaag agttggcagc 360

tcgggccctt ttacgtactg gggccaaggg actctggtca ctgtctctgc a 411

<210>46

<211>137

<212>PRT

<213> mice

<400>46

Met Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr Thr Gly

1 5 10 15

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

20 25 30

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

35 40 45

Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser Pro Glu Lys Arg Leu

50 55 60

Glu Trp Ile Gly Glu Ile Asn Pro Arg Thr Gly Ile Thr Thr Tyr Asn

65 70 75 80

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

85 90 95

Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Val Gly Ser Ser Gly Pro Phe Thr Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ala

130 135

<210>47

<211>384

<212>DNA

<213> mice

<400>47

atggacatga gggctcctgc tcaggttttt ggcttcttgt tgctctggtt tccaggtgcc 60

agatgtgaca tccagatgac ccagtctcca tcctccttat ctgcctctct gggagaaaga 120

gtcagtctca cttgccgggc aagtcaggac attcatggtt atttaaactt gtttcagcag 180

aaaccaggtg aaactattaa acacctgatc tatgaaacat ccaatttaga ttctggtgtc 240

ccgaaaaggt tcagtggcag taggtctggg tcagattatt ctctcattat cggcagcctt 300

gagtctgaag attttgcaga ctattactgt ctacaatatg ctagttcgct cacgttcggt 360

gctgggacca agctggagct gaaa 384

<210>48

<211>128

<212>PRT

<213> mice

<400>48

Met Asp Met Arg Ala Pro Ala Gln Val Phe Gly Phe Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser

35 40 45

Gln Asp Ile His Gly Tyr Leu Asn Leu Phe Gln Gln Lys Pro Gly Glu

50 55 60

Thr lle Lys His Leu Ile Tyr Glu Thr Ser Asn Leu Asp Ser Gly Val

65 70 75 80

Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Ile

85 90 95

Ile Gly Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Ser Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

<210>49

<211>627

<212>DNA

<213> Intelligent (homo sapiens)

<400>49

atgaagctgc tgccgtcggt ggtgctgaag ctctttctgg ctgcagttct ctcggcactg 60

gtgactggcg agagcctgga gcggcttcgg agagggctag ctgctggaac cagcaacccg 120

gaccctccca ctgtatccac ggaccagctg ctacccctag gaggcggccg ggaccggaaa 180

gtccgtgact tgcaagaggc agatctggac cttttgagag tcactttatc ctccaagcca 240

caagcactgg ccacaccaaa caaggaggag cacgggaaaa gaaagaagaa aggcaagggg 300

ctagggaaga agagggaccc atgtcttcgg aaatacaagg acttctgcat ccatggagaa 360

tgcaaatatg tgaaggagct ccgggctccc tcctgcatct gccacccggg ttaccatgga 420

gagaggtgtc atgggctgag cctcccagtg gaaaatcgct tatataccta tgaccacaca 480

accatcctgg ccgtggtggc tgtggtgctg tcatctgtct gtctgctggt catcgtgggg 540

cttctcatgt ttaggtacca taggagagga ggttatgatg tggaaaatga agagaaagtg 600

aagttgggca tgactaattc ccactga 627

<210>50

<211>208

<212>PRT

<213> Intelligent people

<400>50

Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Phe Leu Ala Ala Val

1 5 10 15

Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly

20 25 30

Leu Ala Ala Gly Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr Asp

35 40 45

Gln Leu Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu

50 55 60

Gln Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro

65 70 75 80

Gln Ala Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg Lys Lys

85 90 95

Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Tyr

100 105 110

Lys Asp Phe Cys Ile His Gly Glu Cys Lys Tyr Val Lys Glu Leu Arg

115 120 125

Ala Pro Ser Cys Ile Cys His Pro Gly Tyr His Gly Glu Arg Cys His

130 135 140

Gly Leu Ser Leu Pro Val Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr

145 150 155 160

Thr Ile Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu

165 170 175

Val Ile Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr

180 185 190

Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His

195 200 205

<210>51

<211>21

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>51

atgaagctgc tgccgtcggt g 21

<210>52

<211>22

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>52

tcagtgggaa ttagtcatgc cc 22

<210>53

<211>34

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>53

taagtcgacc accatgaagc tgctgccgtc ggtg 34

<210>54

<211>60

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>54

tttgcggccg ctcacttgtc atcgtcgtcc ttgtagtcgt gggaattagt catgcccaac 60

<210>55

<211>30

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>55

aaagaattcc accatgaagc tgctgccgtc 30

<210>56

<211>40

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>56

tatcggtccg cgaggttcga ggctcagccc atgacacctc 40

<210>57

<211>38

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>57

cgattttcca ctgtgctgct cagcccatga cacctctc 38

<210>58

<211>39

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>58

tgggctgagc agcacagtgg aaaatcgctt atataccta 39

<210>59

<211>3633

<212>DNA

<213> Intelligent people

<400>59

atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60

gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag 120

ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg 180

gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag 240

accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct 300

ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca 360

gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta 420

caggaaatcc tgcatggcgc cgtgcggttc agcaacaacc ctgccctgtg caacgtggag 480

agcatccagt ggcgggacat agtcagcagt gactttctca gcaacatgtc gatggacttc 540

cagaaccacc tgggcagctg ccaaaagtgt gatccaagct gtcccaatgg gagctgctgg 600

ggtgcaggag aggagaactg ccagaaactg accaaaatca tctgtgccca gcagtgctcc 660

gggcgctgcc gtggcaagtc ccccagtgac tgctgccaca accagtgtgc tgcaggctgc 720

acaggccccc gggagagcga ctgcctggtc tgccgcaaat tccgagacga agccacgtgc 780

aaggacacct gccccccact catgctctac aaccccacca cgtaccagat ggatgtgaac 840

cccgagggca aatacagctt tggtgccacc tgcgtgaaga agtgtccccg taattatgtg 900

gtgacagatc acggctcgtg cgtccgagcc tgtggggccg acagctatga gatggaggaa 960

gacggcgtcc gcaagtgtaa gaagtgcgaa gggccttgcc gcaaagtgtg taacggaata 1020

ggtattggtg aatttaaaga ctcactctcc ataaatgcta cgaatattaa acacttcaaa 1080

aactgcacct ccatcagtgg cgatctccac atcctgccgg tggcatttag gggtgactcc 1140

ttcacacata ctcctcctct ggatccacag gaactggata ttctgaaaac cgtaaaggaa 1200

atcacagggt ttttgctgat tcaggcttgg cctgaaaaca ggacggacct ccatgccttt 1260

gagaacctag aaatcatacg cggcaggacc aagcaacatg gtcagttttc tcttgcagtc 1320

gtcagcctga acataacatc cttgggatta cgctccctca aggagataag tgatggagat 1380

gtgataattt caggaaacaa aaatttgtgc tatgcaaata caataaactg gaaaaaactg 1440

tttgggacct ccggtcagaa aaccaaaatt ataagcaaca gaggtgaaaa cagctgcaag 1500

gccacaggcc aggtctgcca tgccttgtgc tcccccgagg gctgctgggg cccggagccc 1560

agggactgcg tctcttgccg gaatgtcagc cgaggcaggg aatgcgtgga caagtgcaac 1620

cttctggagg gtgagccaag ggagtttgtg gagaactctg agtgcataca gtgccaccca 1680

gagtgcctgc ctcaggccat gaacatcacc tgcacaggac ggggaccaga caactgtatc 1740

cagtgtgccc actacattga cggcccccac tgcgtcaaga cctgcccggc aggagtcatg 1800

ggagaaaaca acaccctggt ctggaagtac gcagacgccg gccatgtgtg ccacctgtgc 1860

catccaaact gcacctacgg atgcactggg ccaggtcttg aaggctgtcc aacgaatggg 1920

cctaagatcc cgtccatcgc cactgggatg gtgggggccc tcctcttgct gctggtggtg 1980

gccctgggga tcggcctctt catgcgaagg cgccacatcg ttcggaagcg cacgctgcgg 2040

aggctgctgc aggagaggga gcttgtggag cctcttacac ccagtggaga agctcccaac 2100

caagctctct tgaggatctt gaaggaaact gaattcaaaa agatcaaagt gctgggctcc 2160

ggtgcgttcg gcacggtgta taagggactc tggatcccag aaggtgagaa agttaaaatt 2220

cccgtcgcta tcaaggaatt aagagaagca acatctccga aagccaacaa ggaaatcctc 2280

gatgaagcct acgtgatggc cagcgtggac aacccccacg tgtgccgcct gctgggcatc 2340

tgcctcacct ccaccgtgca gctcatcacg cagctcatgc ccttcggctg cctcctggac 2400

tatgtccggg aacacaaaga caatattggc tcccagtacc tgctcaactg gtgtgtgcag 2460

atcgcaaagg gcatgaacta cttggaggac cgtcgcttgg tgcaccgcga cctggcagcc 2520

aggaacgtac tggtgaaaac accgcagcat gtcaagatca cagattttgg gctggccaaa 2580

ctgctgggtg cggaagagaa agaataccat gcagaaggag gcaaagtgcc tatcaagtgg 2640

atggcattgg aatcaatttt acacagaatc tatacccacc agagtgatgt ctggagctac 2700

ggggtgaccg tttgggagtt gatgaccttt ggatccaagc catatgacgg aatccctgcc 2760

agcgagatct cctccatcct ggagaaagga gaacgcctcc ctcagccacc catatgtacc 2820

atcgatgtct acatgatcat ggtcaagtgc tggatgatag acgcagatag tcgcccaaag 2880

ttccgtgagt tgatcatcga attctccaaa atggcccgag acccccagcg ctaccttgtc 2940

attcaggggg atgaaagaat gcatttgcca agtcctacag actccaactt ctaccgtgcc 3000

ctgatggatg aagaagacat ggacgacgtg gtggatgccg acgagtacct catcccacag 3060

cagggcttct tcagcagccc ctccacgtca cggactcccc tcctgagctc tctgagtgca 3120

accagcaaca attccaccgt ggcttgcatt gatagaaatg ggctgcaaag ctgtcccatc 3180

aaggaagaca gcttcttgca gcgatacagc tcagacccca caggcgcctt gactgaggac 3240

agcatagacg acaccttcct cccagtgcct gaatacataa accagtccgt tcccaaaagg 3300

cccgctggct ctgtgcagaa tcctgtctat cacaatcagc ctctgaaccc cgcgcccagc 3360

agagacccac actaccagga cccccacagc actgcagtgg gcaaccccga gtatctcaac 3420

actgtccagc ccacctgtgt caacagcaca ttcgacagcc ctgcccactg ggcccagaaa 3480

ggcagccacc aaattagcct ggacaaccct gactaccagc aggacttctt tcccaaggaa 3540

gccaagccaa atggcatctt taagggctcc acagctgaaa atgcagaata cctaagggtc 3600

gcgccacaaa gcagtgaatt tattggagca tga 3633

<210>60

<211>1210

<212>PRT

<213> Intelligent people

<400>60

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

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

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

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

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

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

645 650 655

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

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

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

690 695 700

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

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

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

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe

1010 1015 1020

Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu

1025 1030 1035

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

1040 1045 1050

Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg

1055 1060 1065

Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp

1070 1075 1080

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro

1085 1090 1095

Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln

1100 1105 1110

Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro

1115 1120 1125

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln

1130 1135 1140

Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala

1145 1150 1155

Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln

1160 1165 1170

Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys

1175 1180 1185

Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

1190 1195 1200

Ser Ser Glu Phe Ile Gly Ala

1205 1210

<210>61

<211>20

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>61

atgcgaccct ccgggacggc 20

<210>62

<211>21

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>62

cagtggcgat ggacgggatc t 21

<210>63

<211>34

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>63

ttgcggccgc caccatgcga ccctccggga cggc 34

<210>64

<211>61

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>64

accagatctc caggaaaatg tttaagtcag atggatcgga cgggatctta ggcccattcg 60

t 61

<210>65

<211>2514

<212>DNA

<213> mice

<400>65

atggtagggc tgggagcctg caccctgact ggagttaccc tgatcttctt gctactcccc 60

agaagtctgg agagctgtgg acacatcgag atttcacccc ctgttgtccg cct gggggac 120

cctgtcctgg cctcttgcac catcagccca aactgcagca aactggacca acaggcaaag 180

atcttatgga gactgcaaga tgagcccatc caacctgggg acagacagca tcatctgcct 240

gatgggaccc aagagtccct catcactctg cctcacttga actacaccca ggccttcctc 300

ttctgcttag tgccatggga agacagcgtc caactcctgg atcaagctga gcttcacgca 360

ggctatcccc ctgccagccc ctcaaaccta tcctgcctca tgcacctcac caccaacagc 420

ctggtctgcc agtgggagcc aggtcctgag acccacctgc ccaccagctt catcctaaag 480

agcttcagga gccgcgccga ctgtcagtac caaggggaca ccatcccgga ttgtgtggca 540

aagaagaggc agaacaactg ctccatcccc cgaaaaaact tgctcctgta ccagtatatg 600

gccatctggg tgcaagcaga gaatatgcta gggtccagcg agtccccaaa gctgtgcctc 660

gaccccatgg atgttgtgaa attggagcct cccatgctgc aggccctgga cattggccct 720

gatgtagtct ctcaccagcc tggctgcctg tggctgagct ggaagccatg gaagcccagt 780

gagtacatgg aacaggagtg tgaacttcgc taccagccac agctcaaagg agccaactgg 840

actctggtgt tccacctgcc ttccagcaag gaccagtttg agctctgcgg gctccatcag 900

gccccagtct acaccctaca gatgcgatgc attcgctcat ctctgcctgg attctggagc 960

ccctggagcc ccggcctgca gctgaggcct accatgaagg cccccaccat cagactggac 1020

acgtggtgtc agaagaagca actagatcca gggacagtga gtgtgcagct gttctggaag 1080

ccaacgcccc tgcaggaaga cagtggacag atccagggct acctgctgtc ctggaattcc 1140

ccagatcatc aagggcagga catacacctt tgcaacacca cgcagctcag ctgtatcttc 1200

ctcctgccct cagaggccca gaacgtgacc cttgtggcct acaacaaagc agggacctct 1260

tcacctacta cagtggtttt cctggagaac gaaggtccag ctgtgaccgg actccatgcc 1320

atggcccaag accttaacac catctgggta gactgggaag cccccagcct tctgcctcag 1380

ggctatctca ttgagtggga aatgagttct cccagctaca ataacagcta taagtcctgg 1440

atgatagaac ctaacgggaa catcactgga attctgttaa aggacaacat aaatcccttt 1500

cagctctaca gaattacagt ggctcccctg tacccaggca tcgtgggacc ccctgtaaat 1560

gtctacacct tcgctggaga gagagctcct cctcatgctc cagcgctgca tctaaagcat 1620

gttggcacaa cctgggcaca gctggagtgg gtacctgagg cccctaggct ggggatgata 1680

cccctcaccc actacaccat cttctgggcc gatgctgggg accactcctt ctccgtcacc 1740

ctaaacatct ccctccatga ctttgtcctg aagcacctgg agcccgccag tttgtatcat 1800

gtctacctca tggccaccag tcgagcaggg tccaccaata gtacaggcct taccctgagg 1860

accctagatc catctgactt aaacattttc ctgggcatac tttgcttagt actcttgtcc 1920

actacctgtg tagtgacctg gctctgctgc aaacgcagag gaaagacttc cttctggtca 1980

gatgtgccag acccagccca cagtagcctg agctcctggt tgcccaccat catgacagag 2040

gaaaccttcc agttacccag cttctgggac tccagcgtgc catcaatcac caagatcact 2100

gaactggagg aagacaagaa accgacccac tgggattccg aaagctctgg gaatggtagc 2160

cttccagccc tggttcaggc ctatgtgctc caaggagatc caagagaaat ttccaaccag 2220

tcccagcctc cctctcgcac tggtgaccag gtcctctatg gtcaggtgct tgagagcccc 2280

accagcccag gagtaatgca gtacattcgc tctgactcca ctcagcccct cttggggggc 2340

cccaccccta gccctaaatc ttatgaaaac atctggttcc attcaagacc ccaggagacc 2400

tttgtgcccc aacctccaaa ccaggaagat gactgtgtct ttgggcctcc atttgatttt 2460

cccctctttc aggggctcca ggtccatgga gttgaagaac aagggggttt ctag 2514

<210>66

<211>837

<212>PRT

<213> mice

<400>66

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

1 5 10 15

Leu Leu Leu Pro Arg Ser Leu Glu Ser Cys Gly His Ile Glu Ile Ser

20 25 30

Pro Pro Val Val Arg Leu Gly Asp Pro Val Leu Ala Ser Cys Thr Ile

35 40 45

Ser Pro Asn Cys Ser Lys Leu Asp Gln Gln Ala Lys Ile Leu Trp Arg

50 55 60

Leu Gln Asp Glu Pro Ile Gln Pro Gly Asp Arg Gln His His Leu Pro

65 70 75 80

Asp Gly Thr Gln Glu Ser LeuIle Thr Leu Pro Hi s Leu Asn Tyr Thr

85 90 95

Gln Ala Phe Leu Phe Cys Leu Val Pro Trp Glu Asp Ser Val Gln Leu

100 105 110

Leu Asp Gln Ala Glu Leu His Ala Gly Tyr Pro Pro Ala Ser Pro Ser

115 120 125

Asn Leu Ser Cys Leu Met His Leu Thr Thr Asn Ser Leu Val Cys Gln

130 135 140

Trp Glu Pro Gly Pro Glu Thr His Leu Pro Thr Ser Phe Ile Leu Lys

145 150 155 160

Ser Phe Arg Ser Arg Ala Asp Cys Gln Tyr Gln Gly Asp Thr Ile Pro

165 170 175

Asp Cys Val Ala Lys Lys Arg Gln Asn Asn Cys Ser Ile Pro Arg Lys

180 185 190

Asn Leu Leu Leu Tyr Gln Tyr Met Ala Ile Trp Val Gln Ala Glu Asn

195 200 205

Met Leu Gly Ser Ser Glu Ser Pro Lys Leu Cys Leu Asp Pro Met Asp

210 215 220

Val Val Lys Leu Glu Pro Pro Met Leu Gln Ala Leu Asp Ile Gly Pro

225 230 235 240

Asp Val Val Ser His Gln Pro Gly Cys Leu Trp Leu Ser Trp Lys Pro

245 250 255

Trp Lys Pro Ser Glu Tyr Met Glu Gln Glu Cys Glu Leu Arg Tyr Gln

260 265 270

Pro Gln Leu Lys Gly Ala Asn Trp Thr Leu Val Phe His Leu Pro Ser

275 280 285

Ser Lys Asp Gln Phe Glu Leu Cys Gly Leu His Gln Ala Pro Val Tyr

290 295 300

Thr Leu Gln Met Arg Cys Ile Arg Ser Ser Leu Pro Gly Phe Trp Ser

305 310 315 320

Pro Trp Ser Pro Gly Leu Gln Leu Arg Pro Thr Met Lys Ala Pro Thr

325 330 335

Ile Arg Leu Asp Thr Trp Cys Gln Lys Lys Gln Leu Asp Pro Gly Thr

340 345 350

Val Ser Val Gln Leu Phe Trp Lys Pro Thr Pro Leu Gln Glu Asp Ser

355 360 365

Gly Gln Ile Gln Gly Tyr Leu Leu Ser Trp Asn Ser Pro Asp His Gln

370 375 380

Gly Gln Asp Ile His Leu Cys Asn Thr Thr Gln Leu Ser Cys Ile Phe

385 390 395 400

Leu Leu Pro Ser Glu Ala Gln Asn Val Thr Leu Val Ala Tyr Asn Lys

405 410 415

Ala Gly Thr Ser Ser Pro Thr Thr Val Val Phe Leu Glu Asn Glu Gly

420 425 430

Pro Ala Val Thr Gly Leu His Ala Met Ala Gln Asp Leu Asn Thr Ile

435 440 445

Trp Val Asp Trp Glu Ala Pro Ser Leu Leu Pro Gln Gly Tyr Leu Ile

450 455 460

Glu Trp Glu Met Ser Ser Pro Ser Tyr Asn Asn Ser Tyr Lys Ser Trp

465 470 475 480

Met Ile Glu Pro Asn Gly Asn Ile Thr Gly Ile Leu Leu Lys Asp Asn

485 490 495

Ile Asn Pro Phe Gln Leu Tyr Arg Ile Thr Val Ala Pro Leu Tyr Pro

500 505 510

Gly Ile Val Gly Pro Pro Val Asn Val Tyr Thr Phe Ala Gly Glu Arg

515 520 525

Ala Pro Pro His Ala Pro Ala Leu His Leu Lys His Val Gly Thr Thr

530 535 540

Trp Ala Gln Leu Glu Trp Val Pro Glu Ala Pro Arg Leu Gly Met Ile

545 550 555 560

Pro Leu Thr His Tyr Thr Ile Phe Trp Ala Asp Ala Gly Asp His Ser

565 570 575

Phe Ser Val Thr Leu Asn Ile Ser Leu His Asp Phe Val Leu Lys His

580 585 590

Leu Glu Pro Ala Ser Leu Tyr His Val Tyr Leu Met Ala Thr Ser Arg

595 600 605

Ala Gly Ser Thr Asn Ser Thr Gly Leu Thr Leu Arg Thr Leu Asp Pro

610 615 620

Ser Asp Leu Asn Ile Phe Leu Gly Ile Leu Cys Leu Val Leu Leu Ser

625 630 635 640

Thr Thr Cys Val Val Thr Trp Leu Cys Cys Lys Arg Arg Gly Lys Thr

645 650 655

Ser Phe Trp Ser Asp Val Pro Asp Pro Ala His Ser Ser Leu Ser Ser

660 665 670

Trp Leu Pro Thr Ile Met Thr Glu Glu Thr Phe Gln Leu Pro Ser Phe

675 680 685

Trp Asp Ser Ser Val Pro Ser Ile Thr Lys Ile Thr Glu Leu Glu Glu

690 695 700

Asp Lys Lys Pro Thr His Trp Asp Ser Glu Ser Ser Gly Asn Gly Ser

705 710 715 720

Leu Pro Ala Leu Val Gln Ala Tyr Val Leu Gln Gly Asp Pro Arg Glu

725 730 735

Ile Ser Asn Gln Ser Gln Pro Pro Ser Arg Thr Gly Asp Gln Val Leu

740 745 750

Tyr Gly Gln Val Leu Glu Ser Pro Thr Ser Pro Gly Val Met Gln Tyr

755 760 765

Ile Arg Ser Asp Ser Thr Gln Pro Leu Leu Gly Gly Pro Thr Pro Ser

770 775 780

Pro Lys Ser Tyr Glu Asn Ile Trp Phe His Ser Arg Pro Gln Glu Thr

785 790 795 800

Phe Val Pro Gln Pro Pro Asn Gln Glu Asp Asp Cys Val Phe Gly Pro

805 810 815

Pro Phe Asp Phe Pro Leu Phe Gln Gly Leu Gln Val His Gly Val Glu

820 825 830

Glu Gln Gly Gly Phe

835

<210>67

<211>2583

<212>DNA

<213> Artificial

<220>

<223> chimeric protein

<400>67

atgcgacctt ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60

gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag 120

ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg 180

gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag 240

accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct 300

ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca 360

gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta 420

caggaaatcc tgcatggcgc cgtgcggttc agcaacaacc ctgccctgtg caatgtggag 480

agcatccagt ggcgggacat agtcagcagt gactttctca gcaacatgtc gatggacttc 540

cagaaccacc tgggcagctg ccaaaagtgt gatccaagct gtcccaatgg gagctgctgg 600

ggtgcaggag aggagaactg ccagaaactg accaaaatca tctgtgccca gcagtgctcc 660

gggcgctgcc gtggcaagtc ccccagtgac tgctgccaca accagtgtgc tgcaggctgc 720

acaggccccc gggagagcga ctgcctggtc tgccgcaaat tccgagacga agccacgtgc 780

aaggacacct gccccccact catgctctac aaccccacca cgtaccagat ggatgtgaac 840

cccgagggca aatacagctt tggtgccacc tgcgtgaaga agtgtccccg taattatgtg 900

gtgacagatc acggctcgtg cgtccgagcc tgtggggccg acagctatga gatggaggaa 960

gacggcgtcc gcaagtgtaa gaagtgcgaa gggccttgcc gcaaagtgtg taacggaata 1020

ggtattggtg aatttaaaga ctcactctcc ataaatgcta cgaatattaa acacttcaaa 1080

aactgcacct ccatcagtgg cgatctccac atcctgccgg tggcatttag gggtgactcc 1140

ttcacacata ctcctcctct ggatccacag gaactggata ttctgaaaac cgtaaaggaa 1200

atcacagggt ttttgctgat tcaggcttgg cctgaaaaca ggacggacct ccatgccttt 1260

gagaacctag aaatcatacg cggcaggacc aagcaacatg gtcagttttc tcttgcagtc 1320

gtcagcctga acataacatc cttgggatta cgctccctca aggagataag tgatggagat 1380

gtgataattt caggaaacaa aaatttgtgc tatgcaaata caataaactg gaaaaaactg 1440

tttgggacct ccggtcagaa aaccaaaatt ataagcaaca gaggtgaaaa cagctgcaag 1500

gccacaggcc aggtctgcca tgccttgtgc tcccccgagg gctgctgggg cccggagccc 1560

agggactgcg tctcttgccg gaatgtcagc cgaggcaggg aatgcgtgga caagtgcaac 1620

cttctggagg gtgagccaag ggagtttgtg gagaactctg agtgcataca gtgccaccca 1680

gagtgcctgc ctcaggccat gaacatcacc tgcacaggac ggggaccaga caactgtatc 1740

cagtgtgccc actacattga cggcccccac tgcgtcaaga cctgcccggc aggagtcatg 1800

ggagaaaaca acaccctggt ctggaagtac gcagacgccg gccatgtgtg ccacctgtgc 1860

catccaaact gcacctacgg atgcactggg ccaggtcttg aaggctgtcc aacgaatggg 1920

cctaagatcc cgtccgatcc atctgactta aacattttcc tggagatcct ttgcttagta 1980

ctcttgtcca ctacctgtgt agtgacctgg ctctgctgca aacgcagagg aaagacttcc 2040

ttctggtcag atgtgccaga cccagcccac agtagcctga gctcctggtt gcccaccatc 2100

atgacagagg aaaccttcca gttacccagc ttctgggact ccagcgtgcc atcaatcacc 2160

aagatcactg aactggagga agacaagaaa ccgacccact gggattccga aagctctggg 2220

aatggtagcc ttccagccct ggttcaggcc tatgtgctcc aaggagatcc aagagaaatt 2280

tccaaccagt cccagcctcc ctctcgcact ggtgaccagg tcctctatgg tcaggtgctt 2340

gagagcccca ccagcccagg agtaatgcag tacattcgct ctgactccac tcagcccctc 2400

ttggggggcc ccacccctag ccctaaatct tatgaaaaca tctggttcca ttcaagaccc 2460

caggagacct ttgtgcccca acctccaaac caggaagatg actgtgtctt tgggcctcca 2520

tttgattttc ccctctttca ggggctccag gtccatggag ttgaagaaca agggggtttc 2580

tag 2583

<210>68

<211>860

<212>PRT

<213> Artificial

<220>

<223> chimeric protein

<400>68

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

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

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

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

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys Ile Pro Ser Asp Pro Ser Asp Leu Asn Ile Phe Leu Glu Ile

645 650 655

Leu Cys Leu Val Leu Leu Ser Thr Thr Cys Val Val Thr Trp Leu Cys

660 665 670

Cys Lys Arg Arg Gly Lys Thr Ser Phe Trp Ser Asp Val Pro Asp Pro

675 680 685

Ala His Ser Ser Leu Ser Ser Trp Leu Pro Thr Ile Met Thr Glu Glu

690 695 700

Thr Phe Gln Leu Pro Ser Phe Trp Asp Ser Ser Val Pro Ser Ile Thr

705 710 715 720

Lys Ile Thr Glu Leu Glu Glu Asp Lys Lys Pro Thr His Trp Asp Ser

725 730 735

Glu Ser Ser Gly Asn Gly Ser Leu Pro Ala Leu Val Gln Ala Tyr Val

740 745 750

Leu Gln Gly Asp Pro Arg Glu Ile Ser Asn Gln Ser Gln Pro Pro Ser

755 760 765

Arg Thr Gly Asp Gln Val Leu Tyr Gly Gln Val Leu Glu Ser Pro Thr

770 775 780

Ser Pro Gly Val Met Gln Tyr Ile Arg Ser Asp Ser Thr Gln Pro Leu

785 790 795 800

Leu Gly Gly Pro Thr Pro Ser Pro Lys Ser Tyr Glu Asn Ile Trp Phe

805 810 815

His Ser Arg Pro Gln Glu Thr Phe Val Pro Gln Pro Pro Asn Gln Glu

820 825 830

Asp Asp Cys Val Phe Gly Pro Pro Phe Asp Phe Pro Leu Phe Gln Gly

835 840 845

Leu Gln Val His Gly Val Glu Glu Gln Gly Gly Phe

850 855 860

<210>69

<211>23

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>69

gctcactgga tggtgggaag atg 23

<210>70

<211>21

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>70

gggccagtgg atagacagat g 21

<210>71

<211>24

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>71

caggggccag tggatagacc gatg 24

<210>72

<211>31

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>72

gttaagcttc caccatgcga ccctccggga c 31

<210>73

<211>35

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>73

gttggtgacc gacgggatct taggcccatt cgttg 35

<210>74

<211>1146

<212>DNA

<213> Artificial

<220>

<223> chimeric protein

<400>74

atgaagctgc tgccgtcggt ggtgctgaag ctctttctgg ctgcagttct ctcggcactg 60

gtgactggcg agagcctgga gcggcttcgg agagggctag ctgctggaac cagcaacccg 120

gaccctccca ctgtatccac ggaccagctg ctacccctag gaggcggccg ggaccggaaa 180

gtccgtgact tgcaagaggc agatctggac cttttgagag tcactttatc ctccaagcca 240

caagcactgg ccacaccaaa caaggaggag cacgggaaaa gaaagaagaa aggcaagggg 300

ctagggaaga agagggaccc atgtcttcgg aaatacaagg acttctgcat ccatggagaa 360

tgcaaatatg tgaaggagct ccgggctccc tcctgcatct gccacccggg ttaccatgga 420

gagaggtgtc atgggctgag cctcgaacct cgcggaccga caatcaagcc ctgtcctcca 480

tgcaaatgcc cagcacctaa cctcttgggt ggaccatccg tcttcatctt ccctccaaag 540

atcaaggatg tactcatgat ctccctgagc cccatagtca catgtgtggt ggtggatgtg 600

agcgaggatg acccagatgt ccagatcagc tggtttgtga acaacgtgga agtacacaca 660

gctcagacac aaacccatag agaggattac aacagtactc tccgggtggt cagtgccctc 720

cccatccagc accaggactg gatgagtggc aaggagttca aatgcaaggt caacaacaaa 780

gacctgccag cgcccatcga gagaaccatc tcaaaaccca aagggtcagt aagagctcca 840

caggtatatg tcttgcctcc accagaagaa gagatgacta agaaacaggt cactctgacc 900

tgcatggtca cagacttcat gcctgaagac atttacgtgg agtggaccaa caacgggaaa 960

acagagctaa actacaagaa cactgaacca gtcctggact ctgatggttc ttacttcatg 1020

tacagcaagc tgagagtgga aaagaagaac tgggtggaaa gaaatagcta ctcctgttca 1080

gtggtccacg agggtctgca caatcaccac acgactaaga gcttctcccg gactccgggt 1140

aaatga 1146

<210>75

<211>381

<212>PRT

<213> Artificial

<220>

<223> chimeric protein

<400>75

Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Phe Leu Ala Ala Val

1 5 10 15

Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly

20 25 30

Leu Ala Ala Gly Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr Asp

35 40 45

Gln Leu Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu

50 55 60

Gln Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro

65 70 75 80

Gln Ala Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg Lys Lys

85 90 95

Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Tyr

100 105 110

Lys Asp Phe Cys Ile His Gly Glu Cys Lys Tyr Val Lys Glu Leu Arg

115 120 125

Ala Pro Ser Cys Ile Cys His Pro Gly Tyr His Gly Glu Arg Cys His

130 135 140

Gly Leu Ser Leu Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro

145 150 155 160

Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile

165 170 175

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

180 185 190

Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln

195 200 205

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

210 215 220

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

225 230 235 240

Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys

245 250 255

Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys

260 265 270

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

275 280 285

Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr

290 295 300

Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys

305 310 315 320

Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly

325 330 335

Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val

340 345 350

Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn

355 360 365

His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

370 375 380

<210>76

<211>5

<212>PRT

<213> mice

<400>76

Asp Tyr Tyr Met Asn

1 5

<210>77

<211>17

<212>PRT

<213> mice

<400>77

Arg Val Asn Pro Asn Asn Gly Gly Thr Ser Tyr Ser Gln Lys Phe Lys

1 5 10 15

Asp

<210>78

<211>7

<212>PRT

<213> mice

<400>78

Ile Tyr Tyr Gly Gly Ser Asp

1 5

<210>79

<211>16

<212>PRT

<213> mice

<400>79

Lys Ser Ser Gln Ser Leu Leu Tyr Thr Thr Gly Lys Thr Tyr Leu Asn

1 5 10 15

<210>80

<211>7

<212>PRT

<213> mice

<400>80

Gln Val Ser Lys Leu Val Pro

1 5

<210>81

<211>9

<212>PRT

<213> mice

<400>81

Leu Gln Gly Thr Tyr Tyr Pro His Thr

1 5

<210>82

<211>11

<212>PRT

<213> mice

<400>82

Ala Ser Ser Ser Val Ser Ser Met Tyr Leu His

1 5 10

<210>83

<211>7

<212>PRT

<213> mice

<400>83

Gly Thr Ser Asn Leu Ala Ser

1 5

<210>84

<211>9

<212>PRT

<213> mice

<400>84

Gln Gln Tyr His Ser Asp Pro Phe Thr

1 5

<210>85

<211>444

<212>DNA

<213> mice

<400>85

atgtcctctc cacagacact gaacacactg actctaaaca tgggatggag ctgggtcttt 60

ctcttcctcc tgtcaggaac tgcaggtgtc cactctgagg tccagctgca acagtctgga 120

cctgagctga tgaagcctgg ggcttcagtg aagatgtcct gtaaggcttc tggatacatt 180

ttcactgact attacatgaa ctgggtgaag cagagtcatg gaaagagcct tgaatggatt 240

ggacgtgtta atcctaacaa tggtggaact agctacagcc agaagttcaa ggacaaggcc 300

acattgacag tagacaaatc cctcaacaca gcctacatgc aggtcaacag cctgacatct 360

gaggactctg cggtctatta ctgtgcaaga atctactatg gtggttcgga ctggggccaa 420

ggcaccactc tcacagtctc ctca 444

<210>86

<211>148

<212>PRT

<213> mice

<400>86

Met Ser Ser Pro Gln Thr Leu Asn Thr Leu Thr Leu Asn Met Gly Trp

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Gly Arg Val Asn Pro Asn Asn Gly Gly Thr Ser Tyr Ser Gln Lys php

85 90 95

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Leu Asn Thr Ala Tyr

100 105 110

Met Gln Val Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

115 120 125

Ala Arg Ile Tyr Tyr Gly Gly Ser Asp Trp Gly Gln Gly Thr Thr Leu

130 135 140

Thr Val Ser Ser

145

<210>87

<211>396

<212>DNA

<213> mice

<400>87

atgatgagtc ctgtccagtt cctgtttctg ttaatgctct ggattcagga atccaacggt 60

gagattgtga tgacccagac tccactgtct ttgtcggtta ccattggaca accagcctct 120

atctcttgca agtcaagtca gagcctctta tatactactg gaaagacata tttgaattgg 180

ttacaacaga ggcctggcca ggctccaaaa cacctgatgt atcaggtgtc caaactggtc 240

cctggcatcc ctgacaggtt cagtggcagt ggatcagaaa cagattttac acttaaaatc 300

agcagagtgg aggctgaaga tttgggagtt tattactgct tgcaaggtac atattatcct 360

catacgttcg gatcggggac caagctggaa ataaaa 396

<210>88

<211>132

<212>PRT

<213> mice

<400>88

Met Met Ser Pro Val Gln Phe Leu Phe Leu Leu Met Leu Trp Ile Gln

1 5 10 15

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

20 25 30

Val Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Tyr Thr Thr Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg

50 55 60

Pro Gly Gln Ala Pro Lys His Leu Met Tyr Gln Val Ser Lys Leu Val

65 70 75 80

Pro Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe

85 90 95

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr

100 105 110

Cys Leu Gln Gly Thr Tyr Tyr Pro His Thr Phe Gly Ser Gly Thr Lys

115 120 125

Leu Glu Ile Lys

130

<210>89

<211>390

<212>DNA

<213> mice

<400>89

atggattttc aagtgcagat tttcagcttc ttgctgatca gtgcctcagt cataatgacc 60

agaggacaaa atgttctcac ccagtctcca gcaatcatgt ctgcctctcc aggggagaag 120

gtcaccatga cctgcagtgc cagctcaagt gtaagttcca tgtacttgca ctggtaccag 180

cagaagtcag gagcctcccc caaactctgg atttatggca catccaacct ggcttctgga 240

gtccctactc gcctcagtgg cagtgggtct gggacctctt actctctcac aatcagcagc 300

gtggaggctg aaaatgctgc cacttattac tgccagcagt atcatagtga cccattcacg 360

ttcggcacgg ggacaaaatt ggaaataaaa 390

<210>90

<211>130

<212>PRT

<213> mice

<400>90

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

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

20 25 30

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

35 40 45

Ser Ser Val Ser Ser Met Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly

50 55 60

Ala Ser Pro Lys Leu Trp Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly

65 70 75 80

Val Pro Thr Arg Leu Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

85 90 95

Thr Ile Ser Ser Val Glu Ala Glu Asn Ala Ala Thr Tyr Tyr Cys Gln

100 105 110

Gln Tyr His Ser Asp Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu

115 120 125

Ile Lys

130

<210>91

<211>34

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>91

tccgaattcc accatgaagc tgctgccgtc ggtg 34

<210>92

<211>34

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>92

tttgcggccg ctagaggctc agcccatgac acct 34

<210>93

<211>25

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>93

ctgggtcttt ctcttcctcc tgtca 25

<210>94

<211>25

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>94

tgagattgtg atgacccaga ctcca 25

<210>95

<211>24

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>95

ttctcaccca gtctccagca atca 24

<210>96

<211>31

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>96

ttggatccgt cactttatcc tccaagccac a 31

<210>97

<211>29

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>97

ttctcgagga ggctcagccc atgacacct 29

<210>98

<211>29

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>98

ttctcgagcc gaagacatgg gtccctctt 29

<210>99

<211>29

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>99

taggatccaa gagggaccca tgtcttcgg 29

<210>100

<211>66

<212>DNA

<213> Artificial

<220>

<223> PCR primer

<400>100

gatccaagag ggacccatgt cttcggaaat acaaggactt ctgcatccat ggagaatgca 60

aatatc 66

<210>101

<211>66

<212>DNA

<213> Artificial

<220>

<223> chimeric protein

<400>101

tcgagatatt tgcattctcc atggatgcag aagtccttgt atttccgaag acatgggtcc 60

ctcttg 66

<210>102

<211>69

<212>DNA

<213> Artificial

<220>

<223> chimeric protein

<400>102

gatcctgcat ccatggagaa tgcaaatatg tgaaggagct ccgggctccc tcctgcatct 60

gccacccgc 69

<210>103

<211>69

<212>DNA

<213> Artificial

<220>

<223> chimeric protein

<400>103

tcgagcgggt ggcagatgca ggagggagcc cggagctcct tcacatattt gcattctcca 60

tggatgcag 69

<210>104

<211>66

<212>DNA

<213> Artificial

<220>

<223> chimeric protein

<400>104

gatccgctcc ctcctgcatc tgccacccgg gttaccatgg agagaggtgt catgggctga 60

gcctcc 66

<210>105

<211>66

<212>DNA

<213> Artificial

<220>

<223> chimeric protein

<400>105

tcgaggaggc tcagcccatg acacctctct ccatggtaac ccgggtggca gatgcaggag 60

ggagcg 66

Claims (28)

1. An anti-HB-EGF antibody having internalization activity.

2. An anti-HB-EGF antibody conjugated with a cytotoxic substance.

3. The antibody of claim 2, having internalization activity.

4. An anti-HB-EGF antibody having ADCC activity or CDC activity.

5. The antibody of any one of claims 1-4, further having neutralizing activity.

6. An antibody selected from the group consisting of [1] to [13] below:

[1] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 14, SEQ ID NO as CDR 2: 16 and the amino acid sequence of SEQ ID NO: 18, or a heavy chain variable region of the amino acid sequence of seq id no;

[2] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 20, SEQ ID NO as CDR 2: 22 and the amino acid sequence of SEQ ID NO: 24, or a light chain variable region of the amino acid sequence of seq id no;

[3] an antibody comprising the heavy chain of [1] and the light chain of [2 ];

[4] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 26, the amino acid sequence of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 30, or a heavy chain variable region of the amino acid sequence of seq id no;

[5] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 32, the amino acid sequence of SEQ ID NO: 34 and the amino acid sequence of SEQ ID NO: 36, or a light chain variable region of the amino acid sequence of seq id no;

[6] an antibody comprising the heavy chain of [4] and the light chain of [5 ];

[7] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 76, SEQ ID NO as CDR 2: 77 and the amino acid sequence of SEQ ID NO: 78 (HE-39 heavy chain);

[8] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 79, as CDR2, SEQ ID NO: 80 and the amino acid sequence of SEQ ID NO: 81 (HE-39 light chain-1);

[9] comprises a polypeptide having the amino acid sequence of SEQ ID NO as CDR 1: 82, SEQ ID NO as CDR 2: 83 and SEQ ID NO: 84 (HE-39 light chain-2);

[10] an antibody comprising the heavy chain of [7] and the light chain of [8 ];

[11] an antibody comprising the heavy chain of [7] and the light chain of [9 ];

[12] an antibody having an activity equivalent to that of the antibody according to any one of [1] to [11 ]; and

[13] an antibody that binds to the same epitope as the antibody described in any one of [1] to [12 ].

7. A pharmaceutical composition comprising the antibody of any one of claims 1-6.

8. A pharmaceutical composition comprising a cytotoxic agent conjugated to an antibody of any one of claims 1-6.

9. The pharmaceutical composition of claim 7 or 8, which is a cell proliferation inhibitor.

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

11. The pharmaceutical composition of claim 10, wherein the cancer is pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, bladder cancer, brain tumor, or hematological cancer.

12. A method for delivering a cytotoxic substance into a cell using an anti-HB-EGF antibody.

13. A method for inhibiting cell proliferation using a cytotoxic substance bound to an anti-HB-EGF antibody.

14. The method of claim 13, wherein the cell is a cancer cell.

15. The method of any one of claims 12-14, wherein the cytotoxic substance is a chemotherapeutic agent, a radioactive substance, or a toxic peptide.

16. Use of an anti-HB-EGF antibody for transporting a cytotoxic substance into a cell.

17. Use of an anti-HB-EGF antibody having internalization activity for inhibiting cell proliferation.

18. The use of claim 17, wherein the anti-HB-EGF antibody further comprises a neutralizing activity.

19. The use of claim 18, wherein the anti-HB-EGF antibody further comprises antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

20. The use of any one of claims 16-19, wherein the cell is a cancer cell.

21. The use of any one of claims 16-19, wherein a cytotoxic agent is bound to the anti-HB-EGF antibody.

22. A method of preparing a pharmaceutical composition comprising the steps of:

(a) providing an anti-HB-EGF antibody;

(b) determining whether the antibody of (a) has internalizing activity;

(c) selecting an antibody having internalization activity; and

(d) binding a cytotoxic agent to the antibody selected in (c).

23. The method of claim 22, wherein the pharmaceutical composition is an anticancer agent.

24. A method for diagnosing cancer using an anti-HB-EGF antibody.

25. The method of claim 24, comprising using an anti-HB-EGF antibody bound with a labeling substance.

26. The method of claim 24 or 25, wherein the anti-HB-EGF antibody incorporated into the cell is detected.

27. An anti-HB-EGF antibody bound with a labeling substance.

28. The antibody of claim 27, having internalization activity.