WO2012008397A1

WO2012008397A1 - Anti-epha2 antibody and use thereof

Info

Publication number: WO2012008397A1
Application number: PCT/JP2011/065766
Authority: WO
Inventors: 剛滝沢; 達司松岡; 長谷川　純
Original assignee: 第一三共株式会社
Priority date: 2010-07-12
Filing date: 2011-07-11
Publication date: 2012-01-19
Also published as: TW201208701A

Abstract

The present invention provides an antibody or the like, which has cell canceration inhibitory activity and/or tumor cell proliferation inhibitory activity. Provided are: an anti-EPHA2 antibody which comprises a humanized heavy chain derived from an anti-EPHA2 antibody SH 348-1 that is produced by a hybridoma SH 348-1 (FERM BP-10836) and a humanized light chain derived from an anti-EPHA2 antibody SH 357-1 that is produced by a hybridoma SH 357-1 (FERM BP-10837); a functional fragment of the antibody; and a pharmaceutical product or the like, which contains the antibody as an active ingredient.

Description

Anti-EPHA2 antibody and use thereof

The present invention relates to an anti-EPHA2 antibody and a pharmaceutical composition containing the antibody.

EPHA2 is a receptor tyrosine kinase having a single transmembrane structure with a molecular weight of 130 kDa (see, for example, Non-Patent Document 1). EPHA2 has an N-terminal extracellular region with a ligand binding domain and two fibronectin type 3 domains, and a C-terminal intracellular region with a tyrosine kinase domain and a sterile-α-motif (SAM) domain. Existing.

As a ligand for EPHA2, GPI-anchored cell membrane proteins Ephrin-A1 to A5 are known (for example, see Non-patent Document 2). By binding a ligand to EPHA2, the tyrosine kinase domain is activated, tyrosine residues existing in the intracellular region of EPHA2 are phosphorylated, and a signal is transmitted into the cell. In addition, EPHA2 bound to a ligand has been reported to be taken up into cells by endocytosis and finally degraded by the proteasome (see, for example, Non-Patent Document 3).

EPHA2 is clinically highly expressed in many cancers, particularly breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme (for example, Non-Patent Document 4). 5, 6, 7, 8, 9, 10, 11). In esophageal cancer, patients with positive expression of EPHA2 have a significantly high frequency of local lymph node metastasis and the number of lymph node metastases, and the degree of differentiation of cancer is significantly low. Patients with positive expression of EPHA2 survive 5 years. The rate is significantly lower (see, for example, Non-Patent Document 5), non-small cell lung cancer patients with a disease-free survival of less than 5 years have significantly higher expression of EPHA2 than patients with longer than 5 years, EPHA2 expression is significantly higher in patients who have metastasized to the brain (see, for example, Non-Patent Document 9), and liver metastasis, lymphatic vessel invasion, and lymph node metastasis are likely to occur in patients with colorectal cancer who are positive for EPHA2 expression. In addition, it has been reported that there are many EPHA2 expression positive patients in patients with high clinical stage (see, for example, Non-Patent Document 10).

Also, by introducing the EPHA2 gene into cells, non-cancerous cells can acquire cancer traits such as anchorage-independent growth ability, tubular morphogenesis on the extracellular matrix, and in vivo tumor growth ability. It has been reported (for example, see Non-patent Documents 4 and 13) and that the invasiveness of cancer cells to the extracellular matrix is enhanced (see Non-Patent

Documents

12 and 13 for example). Conversely, suppression of EPHA2 expression with siRNA suppresses cancer cell invasion, anchorage-independent growth, and in vivo tumor growth (see, for example, Non-Patent Documents 13 and 14) and the ligand Ephrin -EPHA2 is activated using a fusion protein of A1 and the Fc region of human IgG, and the degradation of EPHA2 by endocytosis induces cancer cell invasion, anchorage-independent growth, and tubular morphogenesis It has been reported that it is suppressed (for example, see Non-Patent

Documents

4, 11, and 12).

On the other hand, it has been reported that EPHA2 is expressed not only in cancer cells but also in blood vessels in or around the tumor (for example, see Non-Patent Document 15). In mice, it has been reported that EPHA2 signal is involved in angiogenesis induced by Ephrin-A1, especially that EPHA2 expressed in vascular endothelial cells is required for the formation and survival of vascular endothelial cells. It has also been reported that a fusion protein of the extracellular region of EPHA2 and the Fc region of human IgG suppresses angiogenesis in vivo and exhibits an antitumor effect (see, for example, Non-Patent Document 16) (for example, Non-patent document 17).

Regarding EPHA2, as in the case of the ligand, an agonist anti-EPHA2 monoclonal antibody having phosphorylation-inducing activity of EPHA2 tyrosine residue and degradation-inducing activity of EPHA2 is used for anchorage-independent growth of breast cancer cell lines and tubular form on the extracellular matrix. It has been reported to inhibit formation (see, for example, Non-Patent Document 14). Furthermore, an agonistic anti-EPHA2 monoclonal antibody that binds to an epitope on EPHA2 exposed in cancer cells as compared to non-cancer cells and has phosphorylation-inducing activity of EPHA2 tyrosine residue and EPHA2 degradation-inducing activity is anti-tumor in vivo. It has been reported that an effect is shown (for example, refer nonpatent literature 23 and patent documents 1). On the other hand, although the anti-EPHA2 monoclonal antibody of Kiewrich et al. Has phosphorylation-inducing activity of EPHA2 tyrosine residue and EPHA2 degradation-inducing activity, it has been reported that it did not exhibit an antitumor effect in vivo (eg, non- (See Patent Document 24).

Patent Document 2 describes LUCA19, SG5, LUCA40, and SPL1, which are anti-EPHA2 monoclonal antibodies obtained by immunizing cancer cells in mice. For these antibodies, LUCA19 and SG5 contain EPHA2 tyrosine residues. It does not affect the phosphorylation of the group, LUCA40 inhibits cancer cell growth in vitro, and LUCA19, SG5 and LUCA40 are internalized in cancer cells in the presence of anti-mouse antibody labeled with sapolin. It is described that it is done. In addition, LUCA40 and SPL1 have been reported to show an antitumor effect in vivo, but it is not clear whether or not they have agonist activity.

Meanwhile, a monoclonal antibody that binds to the extracellular region of EPHA2 and has antitumor activity and a humanized antibody thereof have been reported (Patent Document 4).

International Publication No. WO03 / 094859 Pamphlet International Publication No. WO2006 / 084226 Pamphlet International publication pamphlet WO2003 / 057881 International Publication WO2009 / 028639

One object of the present invention is to provide an antibody against EPHA2. Another object of the present invention is to provide a pharmaceutical composition containing the anti-EPHA2 antibody having a therapeutic effect on cancer. Another object of the present invention is to provide a method for producing the antibody. Another object of the present invention is to provide a method for suppressing tumor growth using the antibody.

The present inventors diligently studied to solve the above-mentioned problems. As a result, the anti-EPHA2 monoclonal antibody described in International Publication Pamphlet WO2009 / 028639, which is a heavy chain derived from SH348-1, and a light chain derived from SH357-1. A humanized antibody (hereinafter referred to as a “swapped antibody”) in which a chain is combined was prepared. While maintaining the growth-inhibitory activity of cancer cells, the humanized antibody derived from SH348-1 and the derived from SH357-1 It was found that the binding activity to the antigen was improved and the ADCC activity was also improved as compared with the humanized antibody, and the present invention was completed.

That is, the present invention
(1) It contains CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. And a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48, a CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50, and an amino acid sequence shown in SEQ ID NO: 52. An antibody comprising a light chain comprising CDRL3 and specifically recognizing a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, or a functional fragment of the antibody,
(2) the antibody or the functional fragment of the antibody according to (1), which specifically recognizes a polypeptide comprising the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing;
(3) It contains CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. And a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48, a CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50, and an amino acid sequence shown in SEQ ID NO: 52. A Kd value for a polypeptide comprising a light chain comprising CDRL3 and comprising an amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.5 nM or less (1) or ( 2) an antibody or a functional fragment thereof,
(4) The antibody or the function of the antibody according to (3), wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.0 nM or less Sex fragment,
(5) The antibody or the function of the antibody according to (3), wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 0.5 nM or less Sex fragment,
(6) The antibody or the functional fragment of the antibody according to any one of (1) to (5), which suppresses phosphorylation of an EPHA2 tyrosine residue induced by an EPHA2 ligand,
(7) Heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 54 and 56 in the sequence listing or the amino acid sequence of any one of the amino acid sequences in which one or several amino acids are deleted, substituted, or added And the amino acid sequence shown in SEQ ID NOs: 66 and 68 in the sequence listing or any one of amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence The antibody or the functional fragment of the antibody according to any one of (1) to (6), comprising a light chain comprising a light chain variable region,
(8) a heavy chain comprising any one of the amino acid sequences shown in SEQ ID NOs: 53 and 55 of the sequence listing or any one of amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence; The amino acid sequence shown in SEQ ID NOs: 65 and 67 in the sequence listing, or a light chain comprising any one of the amino acid sequences in which one or several amino acids are deleted, substituted, or added in the amino acid sequence The antibody or the functional fragment of the antibody according to any one of (1) to (7).
(9) an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the sequence listing, or a functional fragment of the antibody,
(10) an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the sequence listing, or a functional fragment of the antibody,
(11) an antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the sequence listing, or a functional fragment of the antibody,
(12) an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the sequence listing, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the sequence listing, or a functional fragment of the antibody,
(13) The antibody or the functional fragment of the antibody according to any one of (1) to (12), which has the properties shown in the following a) to d):
a) not having the ability to phosphorylate EPHA2 tyrosine residues;
b) has ADCC activity against EPHA2 expressing cells;
c) has CDC activity against EPHA2 expressing cells;
d) Has anti-tumor activity in vivo.
(14) The antibody or the functional fragment of the antibody according to any one of claims 1 to 12, which has the properties shown in the following a) to e):
a) not having the ability to phosphorylate EPHA2 tyrosine residues;
b) Does not show an effect of decreasing the amount of EPHA2 protein;
c) has ADCC activity against EPHA2 expressing cells;
d) has CDC activity against EPHA2 expressing cells;
e) has anti-tumor activity in vivo,
(15) The antibody according to any one of claims 1 to 14, which is an IgG antibody,
(16) The method according to any one of claims 1 to 14, wherein the antibody is any one selected from Fab, F (ab ') 2, Fv, scFV, diabody, linear antibody, and multispecific antibody. Or a functional fragment of the antibody,
(17) A pharmaceutical composition comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody,
(18) A pharmaceutical composition for treating cancer, comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody,
(19) Breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell, comprising at least one of an antibody selected from (1) to (16) or a functional fragment of the antibody A pharmaceutical composition for treating lung cancer, colon cancer and / or glioblastoma multiforme.
(20) A method for suppressing tumor growth of a mammal by administering any one selected from (1) to (18),
(21) The method for inhibiting tumor growth according to (19), wherein the tumor is a tumor expressing EPHA2.
(22) A polynucleotide encoding the antibody according to any one of (1) to (16) or a functional fragment of the antibody,
(23) A host cell transformed with the polynucleotide according to (22),
(24) Production of the antibody or the functional fragment of the antibody, comprising culturing the host cell according to (23) and recovering the antibody or the functional fragment of the antibody from the culture solution. Method,
Consists of.

The present invention succeeded in obtaining an anti-EPHA2 swapped humanized antibody and a functional fragment of the antibody. And it was found that the antibody has excellent antitumor activity.

Furthermore, a pharmaceutical composition containing the antibody as an active ingredient and a pharmaceutical composition for cancer treatment containing the antibody were provided.

1) 357-L4: A diagram showing the amino acid sequence of the variable region of 357-L4. 2) 357-L5: A diagram showing the amino acid sequence of the variable region of 357-L5. 3) The figure which shows the amino acid sequence of the variable region of 357-H4: 357-H4. 4) 357-H5: A diagram showing the amino acid sequence of the variable region of 357-H5. 5) SH348-1_CDRL1: A figure showing the amino acid sequence of CDRL1 of SH348-1. 6) SH348-1_CDRL2: A figure showing the amino acid sequence of CDRL2 of SH348-1. 7) SH348-1_CDRL3: A drawing showing the amino acid sequence of CDRL3 of SH348-1. 8) SH348-1_CDRH1: A figure showing the amino acid sequence of CDR4 of SH348-1. 9) SH348-1_CDRH2: A figure showing the amino acid sequence of CDR4 of SH348-1. 10) SH348-1_CDRH3: A figure showing the amino acid sequence of CDR4 of SH348-1. 11) 348-L4: A drawing showing the amino acid sequence of the variable region of 348-L4. 12) 348-L5: A drawing showing the amino acid sequence of the variable region of 348-L5. 13) 348-H4: A diagram showing the amino acid sequence of the variable region of 348-H4. 14) 348-H5: A diagram showing the amino acid sequence of the variable region of 348-H5. 15) SH357-1_CDR_L1: A diagram showing the amino acid sequence of CDRL1 of SH357-1. 16) SH357-1_CDR_L2: A diagram showing the amino acid sequence of CDRL2 of SH357-1. 17) SH357-1_CDR_L3: A diagram showing the amino acid sequence of CDRL3 of SH357-1. 18) SH357-1_CDR_H1: A drawing showing the amino acid sequence of CDR35 of SH357-1. 19) SH357-1_CDR_H2: A drawing showing the amino acid sequence of CDR35 of SH357-1. 20) SH357-1_CDR_H3: A drawing showing the amino acid sequence of CDR3 of SH357-1. 1) SEQ ID NO: 71: nucleotide sequence of DNA fragment encoding human κ chain secretion signal, human κ chain constant region and human polyA addition signal. 2) SEQ ID NO: 72: A diagram showing a nucleotide sequence of a DNA fragment containing a human IgG1 signal sequence and a DNA sequence encoding an amino acid in the constant region. 3) SEQ ID NO: 73: A diagram showing the nucleotide sequence of DNA containing a gene encoding the hSH348-1-T1L type light chain variable region fused with a secretion signal. 4) SEQ ID NO: 74: A diagram showing the nucleotide sequence of DNA containing a gene encoding the hSH357-1-T1L type light chain variable region fused with a secretion signal. 5) SEQ ID NO: 75: A drawing showing the nucleotide sequence of DNA containing a gene encoding the hSH348-1-T1H type heavy chain variable region. 6) SEQ ID NO: 76 is a view showing the nucleotide sequence of DNA containing a gene encoding the hSH357-1-T1H type heavy chain variable region. 7) SEQ ID NO: 77: hSH348-1-T1L: a drawing showing the nucleotide sequence of hSH348-1-T1L. 8) SEQ ID NO: 78: hSH348-1-T1L: A drawing showing the amino acid sequence of hSH348-1-T1L. 9) A sequence showing the nucleotide sequence of SEQ ID NO: 79: hSH357-1-T1L: hSH357-1-T1L. 10) A sequence showing the amino acid sequence of SEQ ID NO: 80: hSH357-1-T1L: hSH357-1-T1L. 11) The figure which shows the nucleotide sequence of sequence number 81: 348-L4: 348-L4. 12) The figure which shows the amino acid sequence of sequence number 82: 348-L4: 348-L4. 13) The figure which shows the nucleotide sequence of sequence number 83: 357-L4: 357-L4. 14) The amino acid sequence of SEQ ID NO: 84: 357-L4: 357-L4. 15) A drawing showing the nucleotide acid sequence of SEQ ID NO: 85: 348-L5: 348-L5. 16) The figure which shows the amino acid sequence of sequence number 86: 348-L5: 348-L5. 17) The figure which shows the nucleotide sequence of sequence number 87: 357-L5: 357-L5. 18) The figure which shows the amino acid sequence of sequence number 88: 357-L5: 357-L5. 19) The nucleotide sequence of SEQ ID NO: 89: 348-H4: 348-H4. 20) The figure which shows the amino acid sequence of sequence number 90: 348-H4: 348-H4. 21) The nucleotide sequence of SEQ ID NO: 91: 348-H5: 348-H5. 22) The amino acid sequence which shows sequence number 92: 348-H5: 348-H5. 23) The nucleotide sequence of SEQ ID NO: 93: 357-H4: 357-H4. 24) The amino acid sequence of SEQ ID NO: 94: 357-H4. 25) The nucleotide sequence of SEQ ID NO: 95: 357-H5: 357-H5. 26) The amino acid sequence of SEQ ID NO: 96: 357-H5: 357-H5. The figure which shows the measurement result of ADCC activity of each antibody. The horizontal axis indicates antibody concentration, and the vertical axis indicates cell-like discipline. The black dotted line (●) is the control antibody hIgG1, the solid black line (●) is hSH357-1T1, the black square (■) is 357-H4-357-L4, and the white circle (◯) is 348-H4-357- L4, white triangle (Δ) indicates 348-H4-357-L5, and white square (□) indicates 348-H5-357-L5. The figure which shows the antigen-specific binding of each antibody. A) shows the result of confirming the expression level of each protein on the cell membrane using Anti-FLAG mAB. Both proteins indicate that the same level of expression on the cell membrane was observed. B) shows the binding results to each protein for humanized anti-EPHA2 antibody hSH357-1-T1, C) for 348-H4-357-L4, D) 348-H4-357-L5, E) shows the results for 348-H5-357-L5. In B) to E), each antibody binds strongly to cells expressing human EPHA2, whereas pFLAG-GW, which is a negative control, is transfected into cells expressing human EPHA3a and human PTPRS-4. It shows that it showed only the same level of binding as the transfected cells. The figure which shows the result of the in vivo antitumor activity of each antibody. A) shows results for hSH357-1-T1, B) for 348-H4-357-L4, C) for 348-H4-357-L5, and D) for 348-H5-357-L5. The horizontal axis indicates the number of days after transplantation of human breast cancer cell line MDA-MB-231 cells, and the vertical axis indicates the tumor volume.

1. Definitions In the present specification, “cancer” and “tumor” are used interchangeably.

In this specification, the term “gene” includes not only DNA but also mRNA, cDNA and cRNA thereof. Therefore, “EPHA2 gene” in the present invention includes EPHA2 DNA, mRNA, cDNA, and cRNA.

In the present specification, the term “polynucleotide” is used in the same meaning as a nucleic acid, and includes DNA, RNA, probes, oligonucleotides, and primers.

In the present specification, “polypeptide” and “protein” are used without distinction.

In the present specification, “cell” includes cells in an individual animal and cultured cells.

In the present specification, “cell canceration” means that the cell exhibits abnormal growth, such as loss of sensitivity to the contact-inhibiting phenomenon, or anchorage-independent growth, A cell exhibiting such abnormal growth is called a “cancer cell”.

In the present specification, a protein having a function equivalent to the cell carcinogenic activity and / or cell proliferation activity of EPHA2 is also referred to as EPHA2.

As used herein, “swapped antibody” refers to an antibody in which heavy chains and / or light chains are exchanged among a plurality of types of antibodies, and the full lengths of heavy chains and / or light chains are exchanged. In addition to those, only variable regions, those in which only constant regions are exchanged, and those in which only complementarity determining regions are exchanged are included. That is, for example, in the full-length antibody sequence, when the heavy chain of antibody A is AH, the light chain is AL, the heavy chain of antibody B is BH, and the light chain is BL, the combination of heavy chain / light chain is AH / Antibodies that become BL and BH / AL are swapped antibodies. As another example, CDR of the variable region of the heavy chain of antibody A is CDRH1A, CDRH2A, CDRH3A, CDR of the variable region of the light chain is CDRL1A, CDRL2A, CDRL3A, and CDR of the variable region of the heavy chain of antibody B Is CDRH1B, CDRH2B, CDRH3B, and the CDR of the light chain variable region is CDRL1B, CDRL2B, CDRL3B, the combination of CDRs is heavy chain CDRH1A, CDRH2A and CDRH3A, and the light chain is CDRRL1B, CDRRL2B and CDRL3B In addition, an antibody in which the heavy chain is CDRH1B, CDRH2B and CDRH3B, and the light chain is CDRL1A, CDRL2A and CDRL3A is a swapped antibody. In the present specification, unless otherwise specified, “anti-EPHA2 antibody” includes anti-EPHA2 swapped antibodies, and “anti-EPHA2 humanized antibody” also includes anti-EPHA2 humanized swapped antibodies.

In the present specification, the “functional fragment of an antibody” means a partial fragment of an antibody having an antigen-binding activity, and includes Fab, F (ab ′) 2, scFv, and the like. Further, Fab ', which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the functional fragment of the antibody. However, the molecule is not limited to these molecules as long as it has the ability to bind to an antigen. These functional fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes. It is.

In the present specification, “phosphorylation of tyrosine residue” means that a tyrosine residue contained in the amino acid sequence of a peptide is phosphorylated, and whether or not a tyrosine residue is phosphorylated is, for example, The binding of the peptide to an anti-phosphotyrosine antibody (for example, Anti-Phosphotyrosine, recombinant 4G10, HRP-conjugate (Millipore (Upsate) # 16-184)) was examined. In the present specification, the term “EPHA2 tyrosine residue phosphorylation ability” refers to the ability to phosphorylate tyrosine residues in the amino acid sequence of EPHA2, and the antibody is EPHA2 Whether or not it has the ability to phosphorylate tyrosine residues of Eg to after incubation of the antibody and EPHA2, can be determined by whether or not there bind with anti-phosphotyrosine antibody and EPHA2.

In the present specification, “the expression level of EPHA2 protein decreases” means that the amount of EPHA2 protein decreases, and whether or not an antibody has an action of decreasing the amount of EPHA2 protein is, for example, an antibody Can be examined by quantifying the amount of EPHA2 after incubation with EPHA2.

In the present specification, “EPHA2 ligand” refers to a substance that can be a ligand of EPHA2, and specific examples thereof include GPI-anchored cell membrane proteins, Ephrin-A1 to A5 (Annual Review of Neuroscience, 1998, Vol. 21, pp. 309-345).

In the present specification, “cytotoxicity” refers to pathological changes in cells in some form, not just direct trauma, but also DNA cleavage, base dimer formation, chromosomes. This refers to any structural or functional damage to cells, such as cleavage of cells, damage to cell division apparatus, or reduction of various enzyme activities. As used herein, “cytotoxic activity” refers to causing the above-mentioned cytotoxicity.

In the present specification, ADCC is synonymous with antibody-dependent cellular cytotoxicity, and Fcγ receptor-bearing cells are mediated through Fcγ receptor in the Fc part of the antibody bound to the surface antigen of the target cell. The ADCC activity is also referred to as antibody-dependent cytotoxic activity and refers to the activity of the above reaction. ADCC activity can be measured by a method commonly used by those skilled in the art. For example, it can be measured by the method described in Example 8 herein.

As used herein, “CDC” is synonymous with complement-dependent cytotoxicity, and CDC activity refers to an activity that causes complement-dependent cytotoxicity. CDC activity can be measured by methods commonly used by those skilled in the art.

In the present specification, “having anti-tumor activity in vivo” means having an activity of suppressing or reducing the growth of a tumor of an animal individual having a tumor. Whether or not an anti-EPHA2 antibody has “in vivo antitumor activity” can be examined by a method commonly used by those skilled in the art, but can also be examined, for example, by the following method. That is, an anti-EPA2 antibody as a test substance is intraperitoneally injected into a nude mouse (for example, BALB / cAJc1-nu / nu; obtained from Clea Japan Co., Ltd.) into which tumor cells (for example, MDA-MB-231 cells) are transplanted subcutaneously. It is a test substance when the appropriate volume is administered and the change in the tumor volume over time is compared with the control in which the anti-EPA2 antibody was not administered, and the tumor volume is significantly smaller in the anti-EPA2 antibody administration group than in the control. The anti-EPA2 antibody can be determined to have “in vivo antitumor activity”.

It is known that there are three complementarity determining regions (CDRs) in each of the heavy and light chains of an antibody molecule. In the present specification, with regard to the complementarity determining regions of antibodies, the complementarity determining regions of heavy chains are denoted as CDRH1, CDRH2, and CDRH3, and the complementarity determining regions of light chains are denoted as CDRL1, CDRL2, and CDRL3.

As used herein, “epitope” means a partial peptide of EPHA2 having antigenicity and / or immunogenicity in an animal, preferably a mammal, more preferably a mouse or human body. The epitope that is a partial peptide of EPHA2 having antigenicity can be determined by methods well known to those skilled in the art, such as immunoassay, and can be carried out, for example, by the following method. Various partial structures of EPHA2 are produced. In producing the partial structure, a known oligopeptide synthesis technique can be used. For example, after preparing a series of polypeptides sequentially shortened by an appropriate length from the C-terminal or N-terminal of EPHA2, using gene recombination techniques well known to those skilled in the art, the reactivity of antibodies against them is examined, After determining the recognition site, the epitope can be determined by synthesizing shorter peptides and examining their reactivity with those peptides.

As used herein, a mutant antibody refers to an antibody in which one or several amino acids in the amino acid sequence of the antibody are substituted, deleted, or added.

Examples of the properties of antibodies in the present specification include biological, chemical, and physical properties, and more specific examples include biological activity, binding to antigens and epitopes, production, distribution, The stability at the time of storage, thermal stability, etc. can be mentioned.

In the present invention, “hybridize under stringent conditions” means to hybridize at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech) or use a filter on which DNA is fixed. After hybridization at 68 ° C in the presence of 0.7-1.0 M NaCl, 0.1-2 fold SSC solution (1 fold SSC consists of 150 mM NaCl, 15 mM sodium citrate) ) And hybridization under conditions that can be identified by washing at 68 ° C. or equivalent conditions.

2. About EPHA2 (1) EPHA2 gene The nucleotide sequence and amino acid sequence of the EPHA2 gene are registered in GenBank as EPH receptor A2 (accession numbers NM_004431 and NP_004422, respectively). The nucleotide sequence of the open reading frame (ORF) of the EPHA2 gene is described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence thereof is described in SEQ ID NO: 2 in the sequence listing.

EPHA2 includes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of EPHA2, and having a biological activity equivalent to those of these enzymes.
(2) Cancer-specific expression of EPHA2 gene EPHA2 gene is reported to be highly expressed in many cancers, especially breast cancer, esophageal cancer, prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer, and glioblastoma multiforme Has been.

Further, a substance that suppresses the expression level and / or activity of EPHA2 has an activity of suppressing canceration of cells caused by EPHA2 and / or suppressing proliferation of cancer cells.

(3) Preparation of EPHA2 EPHA2, which is an antigen of an anti-EPHA2 antibody, can include a full-length EPHA2 polypeptide or a partial polypeptide thereof, more specifically, a full-length EPHA2 polypeptide, or preferably an EPHA2 cell. An outer region polypeptide (consisting of the amino acid sequence shown in amino acid Nos. 1 to 540 of SEQ ID No. 4 in the sequence listing), more preferably a polypeptide comprising the amino acid sequence shown in amino acid No. 426 to 540 of SEQ ID No. 4 in the sequence listing Peptides, and more preferably, a polypeptide containing the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing, or a derivative in which any amino acid sequence or carrier is added to these. Moreover, the polypeptide which consists of at least 6 continuous partial amino acid sequences of each said polypeptide, or the derivative | guide_body to which arbitrary amino acid sequences and carriers were added to these can be mentioned.

Note that the EPHA2 full-length polypeptide or a partial polypeptide thereof serving as an antigen can be obtained by causing a host cell to produce an EPHA2 gene or a gene of the partial polypeptide thereof by genetic manipulation.

EPHA2 can be directly purified from human tumor tissue or tumor cells, and EPHA2 full-length polypeptide or a partial polypeptide thereof can be synthesized in vitro or produced in a host cell by genetic manipulation. Can be obtained by:

Specifically, in genetic manipulation, EPHA2 or a partial polypeptide thereof is incorporated into a vector capable of expression, and then synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic organisms. Alternatively, the protein can be obtained by expressing EPHA2 or a partial polypeptide thereof by transforming a eukaryotic host cell.

The cDNA of the partial polypeptide of EPHA2, for example, using a cDNA library expressing EPHA2 as a template, and using a primer that specifically amplifies the DNA encoding EPHA2 cDNA or the partial polypeptide (hereinafter referred to as polymerase chain reaction) (Referred to as “PCR”) (see Saiki, R. K., et al. Science (1988) 239, p. 487-489).

Examples of in vitro synthesis of a polypeptide include, but are not limited to, a rapid translation system (RTS) manufactured by Roche Diagnostics.

3. Anti-EPHA2 Antibody (1) Acquisition of Anti-EPHA2 Monoclonal Antibody SH348-1 which is an anti-EPHA2 antibody can be obtained from hybridoma SH348-1. Moreover, SH357-1 which is an anti-EPHA2 antibody can be obtained from the hybridoma SH357-1. Hybridoma SH348-1 and Hybridoma SH357-1 are dated June 8, 2007 to Japan National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (address: 1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki, Japan) The hybridoma SH348-1 is assigned the deposit number FERM BP-10836 under the name SH348-1, and the hybridoma SH357-1 is assigned the deposit number FERM BP-10837 under the name SH357-1.

The nucleotide sequence of the heavy chain of SH348-1 is described in SEQ ID NO: 5 in the sequence listing, and the amino acid sequence is described in SEQ ID NO: 6. The nucleotide sequence of the light chain of SH348-1 is set forth in SEQ ID NO: 7 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 8. The nucleotide sequence of the heavy chain variable region of SH348-1 is set forth in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 10. The nucleotide sequence of the heavy chain constant region of SH348-1 is set forth in SEQ ID NO: 11 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 12. The nucleotide sequence of the light chain variable region of SH348-1 is shown in SEQ ID NO: 13 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 14. The nucleotide sequence of the light chain constant region of SH348-1 is shown in SEQ ID NO: 15 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 16. The nucleotide sequence of CDRH1 of SH348-1 is SEQ ID NO: 17, the amino acid sequence is SEQ ID NO: 18, the nucleotide sequence of CDRH2 is SEQ ID NO: 19, the amino acid sequence is SEQ ID NO: 20, and the nucleotide sequence of CDRH3 is SEQ ID NO: In 21, the amino acid sequence is set forth in SEQ ID NO: 22. The nucleotide sequence of CDRL1 of SH348-1 is SEQ ID NO: 23, the amino acid sequence is SEQ ID NO: 24, the nucleotide sequence of CDRL2 is SEQ ID NO: 25, the amino acid sequence is SEQ ID NO: 26, and the nucleotide sequence of CDRL3 is SEQ ID NO: 27, the amino acid sequence is set forth in SEQ ID NO: 28.

The nucleotide sequence of the heavy chain of SH357-1 is described in SEQ ID NO: 29 of the sequencing method, and the amino acid sequence is described in SEQ ID NO: 30. The nucleotide sequence of the light chain of SH357-1 is set forth in SEQ ID NO: 31 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 32. The nucleotide sequence of the heavy chain variable region of SH357-1 is set forth in SEQ ID NO: 33 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 34. The nucleotide sequence of the heavy chain constant region of SH357-1 is set forth in SEQ ID NO: 35 in the Sequence Listing, and the amino acid sequence is set forth in SEQ ID NO: 36 in the Sequence Listing. The nucleotide sequence of the light chain variable region of SH357-1 is set forth in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 38. The nucleotide sequence of the light chain constant region of SH357-1 is set forth in SEQ ID NO: 39 in the sequence listing, and the amino acid sequence is set forth in SEQ ID NO: 40. The nucleotide sequence of CDR357 of SH357-1 is SEQ ID NO: 41, the amino acid sequence is SEQ ID NO: 42, the nucleotide sequence of CDRH2 is SEQ ID NO: 43, the amino acid sequence is SEQ ID NO: 44, and the nucleotide sequence of CDRH3 is SEQ ID NO: At 45, the amino acid sequence is set forth in SEQ ID NO: 46. The nucleotide sequence of CDRL1 of SH357-1 is SEQ ID NO: 47, the amino acid sequence is SEQ ID NO: 48, the nucleotide sequence of CDRL2 is SEQ ID NO: 49, the amino acid sequence is SEQ ID NO: 50, and the nucleotide sequence of CDRL3 is SEQ ID NO: In 51, the amino acid sequence is set forth in SEQ ID NO: 52.

(2) Acquisition of anti-EPHA2 humanized swapped antibody Examples of anti-EPHA2 humanized swapped antibodies include humanized antibodies having a humanized heavy chain derived from SH348-1 and a humanized light chain derived from SH357-1. it can.

Examples of the anti-EPHA2 humanized swapped antibody include antibodies in which the heavy chain and the light chain are humanized in the above swapped antibody. As a humanized antibody, only a complementarity determining region (CDR) is human. An antibody incorporated in an antibody derived from the antibody (see Nature (1986) 321, p.522-525), an antibody in which some framework amino acid residues in addition to the CDR sequence are grafted to a human antibody by the CDR grafting method (WO90 No. 077861, US Pat. No. 6,972,323).

Specific examples of the anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody include CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and a sequence. From the heavy chain comprising CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing, the CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48 in the sequence listing, and the amino acid sequence shown in SEQ ID NO: 50 in the sequence listing A peptide comprising an amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing, and comprising a light chain comprising CDRL3 comprising the amino acid sequence represented by SEQ ID NO: 52 in the sequence listing Or a functional fragment of the antibody.

Other specific examples of the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody include a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 54 of the Sequence Listing and the amino acid shown in SEQ ID NO: 66 An antibody comprising a light chain comprising a light chain variable region comprising a sequence, a heavy chain comprising a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 54 in the sequence listing, and an amino acid sequence represented by SEQ ID NO: 68 in the sequence listing A light chain comprising a light chain variable region, a heavy chain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 56 of the Sequence Listing, and a light chain comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 66 A heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 56 of the sequence listing and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 68 An antibody comprising a light chain, a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 53 in the sequence listing and an antibody comprising a light chain represented by SEQ ID NO: 65, a heavy chain represented by SEQ ID NO: 53 in the sequence listing and SEQ ID NO: 67 An antibody consisting of the light chain shown in SEQ ID NO: 55, an antibody consisting of the heavy chain shown in SEQ ID NO: 55 and the light chain shown in SEQ ID NO: 65, the heavy chain shown in SEQ ID NO: 55 and the SEQ ID NO: 67 At least one selected from an antibody comprising a light chain and a functional fragment of the antibody. In addition, the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody may comprise an amino acid sequence in which one or several amino acids are deleted, substituted, or added. It is contained in an antibody or a functional fragment of the antibody.

The anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody of the present invention specifically recognizes EPHA2, preferably specifically recognizes the extracellular region of EPHA2, and more preferably, SEQ ID NO: in the sequence listing. 4 specifically, a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540, even more preferably a polypeptide comprising the amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing is specifically recognized.

The anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody of the present invention has a Kd value of 1.5 nM or less, preferably 1 with respect to the polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4. 0.0 nM or less, more preferably 0.5 nM or less.

(3) Production of antibody By means of gene recombination technology, eukaryotic cells are transformed with a cDNA encoding each of the heavy chain and light chain of the above humanized swapped antibody, preferably a vector containing the cDNA, and a gene recombinant human This antibody can be obtained from the culture supernatant by culturing transformed cells that produce a monoclonal antibody.

Here, as the host, for example, eukaryotic cells, preferably CHO cells, mammalian cells such as lymphocytes and myeloma can be used.

When an antibody gene is once isolated and then introduced into an appropriate host to produce an antibody, a combination of an appropriate host and an expression vector can be used.

When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used.

Examples of animal cells include (1) mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650) which are monkey cells, mouse fibroblasts NIH3T3 (ATCC) No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strain (Urlauub, G. and Chasin, LA Proc. Natl. Acad. Sci. U.). S. A. (1980) 77, p. 4126-4220).

In addition, as these hosts, a host modified to express an antibody in which the sugar chain structure is modified and the ADCC activity (antibody-dependent cytotoxic activity) or CDC activity of the antibody is increased can be used. As such a host, among N-glycoside-bonded complex sugar chains that bind to the Fc region of an antibody, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 20% or more. A CHO cell into which a gene encoding an antibody molecule that produces an antibody composition has been introduced can be mentioned (see WO02 / 31140).
When prokaryotic cells are used, for example, Escherichia coli and Bacillus subtilis can be mentioned.

An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro.

The isotype of the antibody is not limited and includes, for example, IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE, and preferably IgG or IgM.

In the present invention, in addition to the above antibody, a functional fragment of the antibody may be used. Examples of functional fragments of antibodies include Fab, F (ab ') 2, scFv and the like. Further, Fab ', which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the functional fragment of the antibody. However, the molecule is not limited to these molecules as long as it has the ability to bind to an antigen. These functional fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes. It is.

A single chain antibody (also referred to as scFv), which is an example of an antibody, is obtained by linking a heavy chain V region and a light chain V region of an antibody with a linker of a polypeptide (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenburg and Moore, Ed. Springer Verlag, New York, p. 269-315 (1994), Nature Biotechnology (2005), 23, p. 1126-1136).

Methods for making single chain antibodies are well known in the art (eg, US Pat. No. 4,946,778, US Pat. No. 5,260,203, US Pat. No. 5,091,513, US (See Japanese Patent No. 5,455,030). In this scFv, the heavy chain V region and the light chain V region are linked via a linker that does not form a conjugate, preferably a polypeptide linker (Huston, JS et al., Proc. Natl. Acad. Sci.U.S.A. (1988), 85, p.5879-5883). The heavy chain V region and the light chain V region in scFv may be derived from the same antibody or different antibodies.

As the peptide linker that links the V regions, for example, any single chain peptide consisting of 12 to 19 residues is used.

The DNA encoding scFv is the DNA encoding the heavy chain or heavy chain V region of the antibody, and the DNA encoding the light chain or light chain V region. Amplification is performed by PCR using a primer pair that defines both ends of the encoding DNA portion as a template, and then the DNA that encodes the peptide linker portion, and both ends thereof are connected to the heavy chain and light chain, respectively. Obtained by combining and amplifying the primer pairs defined in 1.

In addition, once a DNA encoding scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to conventional methods, and by using the host, ScFv can be obtained according to the method.

These antibody fragments can be produced by the host by obtaining and expressing the gene in the same manner as described above.

Furthermore, as an example of an antibody, it may be a multispecific antibody having specificity for at least two different antigens.

Usually, such a molecule binds two antigens (ie, bispecific antibody), but the “multispecific antibody” in the present invention is more than that (for example, three types). It includes an antibody having specificity for the antigens.

The antibody of the present invention may be a full-length multispecific antibody or a fragment of such an antibody (for example, F (ab ') 2 bispecific antibody). Bispecific antibodies can be prepared by combining the heavy and light chains (HL pairs) of two types of antibodies, or by hybridizing hybridomas that produce different monoclonal antibodies to produce a bispecific antibody. It can also be produced by producing cells (Millstein et al., Nature (1983) 305, p. 537-539).

An example of an antibody may be a polyclonal antibody that is a mixture of a plurality of types of anti-EPHA2 antibodies having different amino acid sequences. As an example of the polyclonal antibody, a mixture of plural kinds of antibodies having different CDRs can be mentioned. As such a polyclonal antibody, a mixture of cells producing different antibodies can be cultured, and an antibody purified from the culture can be used (see WO 2004/061104).

As the modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.

Examples of antibodies may further include those that form conjugates with these antibodies and other drugs (Immunoconjugate). Examples of such an antibody include those in which the antibody is bound to a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, p. 1137-1146).

The obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins.

For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns, filters, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Stratesies for Protein Purification and Charcterization: A Laboratoy Course Manual, Daniel R.Marshak et al.eds, Cold Spring Harbor Laboratory Press (1996); Antibodies:. A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory (198 )) It is not intended to be limited thereto.

Chromatography includes affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like.

These chromatography can be performed using liquid phase chromatography such as HPLC and FPLC.

Columns used for affinity chromatography include protein A columns and protein G columns.

For example, as a column using a protein A column, Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like.

It is also possible to purify the antibody using the antigen-immobilized carrier using the binding property to the antigen.

4). Properties of anti-EPHA2 humanized swapped antibody The anti-EPHA2 humanized swapped antibody of the present invention or a functional fragment of the antibody has at least one of the following properties (1) to (5).
(1) It specifically binds to a polypeptide consisting of the amino acid sequence represented by amino acid numbers 426 to 534 of SEQ ID NO: 8 in the sequence listing, and has at least one property selected from the following a) to e) .
a) It does not have the ability to phosphorylate EPHA2 tyrosine residues.
b) Does not show an effect of decreasing the amount of EPHA2 protein.
c) It has ADCC activity against EPHA2-expressing cells.
d) Has CDC activity against EPHA2-expressing cells.
e) Has anti-tumor activity in vivo.
(2) at least any two or more properties selected from the following a) to e), which specifically bind to a polypeptide having the amino acid sequence represented by amino acid numbers 426 to 534 of SEQ ID NO: 8 in the sequence listing Have
a) It does not have the ability to phosphorylate EPHA2 tyrosine residues.
b) Does not show an effect of decreasing the amount of EPHA2 protein.
c) It has ADCC activity against EPHA2-expressing cells.
d) Has CDC activity against EPHA2-expressing cells.
e) Has anti-tumor activity in vivo.
(3) It specifically binds to a polypeptide consisting of the amino acid sequence represented by amino acid numbers 426 to 534 of SEQ ID NO: 8 in the sequence listing, and has the following properties a) to e).
a) It does not have the ability to phosphorylate EPHA2 tyrosine residues.
b) Does not show an effect of decreasing the amount of EPHA2 protein.
c) It has ADCC activity against EPHA2-expressing cells.
d) Has CDC activity against EPHA2-expressing cells.
e) Has anti-tumor activity in vivo.

Here, the phosphorylation ability of EPHA2 tyrosine residues by the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody is, for example, (Example 3) 3) -1-1 to 3) -1- of WO2009 / 028639 It can be measured by the method described in 2. That is, MDA-MB-231 cells are cultured under appropriate conditions, and an anti-EPHA2 humanized swapped antibody or the antibody, as a soluble ligand of EPHA2, Recombinant Mouse Ephrin-A1 / Fc Chimera (hereinafter referred to as “Ephrin-A1 / Fc”). Abbreviated; R & D Systems (# 602-A1-200) diluted to an appropriate concentration with RPMI 1640 was added to MDA-MB-231 cells discarded with the medium, at 37 ° C. under 5% CO ₂ conditions. Incubate for the specified time.

In order to immunoprecipitate EPHA2, first, 8 μg of anti-Eck / EphA2, Clone D7 (hereinafter referred to as “anti-EPHA2 antibody (D7)”) was added to 25 μl of Protein G magnetic beads suspension (NEW ENGLAND Bio-Labs) per sample. Abbreviation: Millipore (Upstate) # 05-480) is added and mixed by inversion at 4 ° C. for 2 hours.

Then, add FBS so that the final concentration is 10%, and mix by inverting at 4 ° C. for 30 minutes. After washing the beads 3 times with 1 × Cell Lysis Buffer containing 1 mM PMSF, 200 μg of the cell lysate supernatant prepared above is added and mixed by inversion at 4 ° C. overnight. The next day, the beads were washed with 1 mM PMSF-containing 1 × Cell Lysis Buffer three times, and then the beads were washed with SDS-sample buffer (56.3 mM Tris-HCl pH 6.8, 1.8% (w / v) SDS, 9% glycerol. , 0.72M 2-mercaptoethanol, 0.045 mg / ml bromophenol blue) and heated at 98 ° C. for 5 minutes, and the protein dissociated from the beads is separated by SDS-PAGE.

The protein is transferred from the gel after separation to a polyvinylidene difluoride membrane (hereinafter abbreviated as “PVDF membrane”; pore size 0.45 μm: manufactured by Millipore). The PVDF membrane after transfer was dissolved in a blocking solution (Block Ace Powder (Dainippon Sumitomo Pharma Co., Ltd., Snow Brand Milk Products)) in 100 ml of ultrapure water, and then Tween 20 and sodium azide were each added at a final concentration of 0.1. % (V / v), 0.02% (w / v) added) to block. First, in order to detect immunoprecipitated EPHA2, the PVDF membrane after blocking is immersed in an anti-EPHA2 antibody solution diluted to 0.25 μg / ml with a blocking solution and shaken at room temperature for 1 hour. The PVDF membrane was washed 3 times with TBST (50 mM Tris-HCl pH 8.0, 138 mM NaCl, 2.7 mM KCl, 0.1% (v / v) Tween 20) for 10 minutes, and then diluted 3000 times with TBST. Immerse in Anti-Mouse Ig, HRP-Linked Whole Ab Sheep (GE Healthcare Bio-Sciences) solution and shake at room temperature for 30 minutes. Further, the PVDF membrane is washed 3 times with TBST for 10 minutes, and then the signal is detected with a film for chemiluminescence using ECL Plus (GE Healthcare Bio-Sciences).

Next, in order to remove the antibody from the PVDF membrane, the PVDF membrane is immersed in a Strip solution (50 mM Tris-HCl pH 6.8, 2% (w / v) SDS, 100 mM 2-mercaptoethanol) at 55 ° C. for 30 minutes. After shaking for 30 minutes, it was immersed in Quench solution (TBST containing 1% (v / v) H ₂ O ₂ , 0.1% (w / v) NaN ₃ ) for 20 minutes at room temperature. Wash 3 times for 10 minutes. In order to detect the phosphorylation state of the EPHA2 tyrosine residue, this PVDF membrane was dissolved in a sodium azide-free blocking solution (1 bag of Block Ace powder was dissolved in 100 ml of ultrapure water, and Tween 20 was added at a final concentration of 0.1%. Anti-Phosphotyrosine, recombinant 4G10 HRP-conjugate (“4G10 antibody” in the figure) diluted with a sodium azide-free blocking solution 10,000 times after blocking. Dipped in Millipore (Upsate) # 16-184) solution and shaken at room temperature for 1 hour. The PVDF membrane is washed 3 times with TBST for 10 minutes, then further washed 3 times with H ₂ O for 5 minutes, and the signal is detected with a film for chemiluminescence using ECL Plus.

The effect of reducing the amount of EPHA2 protein can be measured, for example, by the method described in Example 3) 3) -1-3 of WO2009 / 028639. That is, after the cell lysate supernatant was separated by SDS-PAGE, the protein in the gel was transferred to a PVDF membrane, and Western blotting was performed with an anti-EPHA2 antibody to quantify the amount of protein decrease. It can be measured.

Moreover, although ADCC activity can be measured by the method currently performed normally, it can be measured by the method as described in Example 8 of this specification, for example.

Further, CDC activity can be measured by a method commonly used by those skilled in the art. For example, it can be performed by the method described in Example 3) 3) -3 of WO2009 / 028639. That is, MDA-MB-231, A549, and PC-3 cells suspended in RPMI 1640 containing 10% FBS (with antibiotics) were seeded in a 96-well microplate at 5000 cells / well each, at 37 ° C., 5% CO _2. Incubate overnight under the conditions of The following day, SH348-1, SH357-1, and isotype control antibody (mIgG2a) diluted with 10% FBS-containing RPMI 1640 (with antibiotics) were added to a final concentration of 25 μg / ml after complement addition, and 4 ° C. Leave for 1 hour. Rabbit complement (CEDARLANE # CL3051) diluted to 30% with RPMI 1640 was added thereto to a final concentration of 5%, incubated at 37 ° C. under 5% CO ₂ for 1 hour, and further incubated at room temperature for 30 hours. Let stand for a minute. In order to measure the cell viability, CellTiter-Glo Luminescent Cell Viability Assay (manufactured by Promega) equivalent to the culture solution is added, and the mixture is stirred at room temperature for 10 minutes, and then the amount of luminescence is measured with a plate reader. Cell viability is calculated by the following formula.

Cell viability (%) = (ab) / (cb) × 100
a: Amount of light emitted from the sample well b: Average value of the amount of light emitted from the background (well without cells / antibodies) (n = 8). An equal amount of 10% FBS-containing RPMI 1640 (with antibiotics) is added instead of the cell suspension at the time of cell seeding, and an equal amount of 10% FBS-containing RPMI 1640 (with antibiotics) is added to the antibody dilution at the time of antibody addition. . Otherwise, perform the same operation as the sample well.

C: Average value of the amount of luminescence in wells without antibody added (n = 3). At the time of antibody addition, 10% FBS-containing RPMI 1640 (with antibiotics) is added in an amount equal to that of the antibody diluent. Otherwise, perform the same operation as the sample well.

Moreover, the antitumor activity in vivo can be confirmed, for example, by administering an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody to an experimental animal having a tumor and measuring a change in the volume of the tumor. For example, it can be measured by the method described in Example 10 of the present specification.

5. Pharmaceutical containing EPHA2 humanized swapped antibody Anti-EPHA2 humanized swapped antibody or a functional fragment of said antibody is useful as a pharmaceutical composition, especially for the treatment of cancer, or for immunodiagnosis of such diseases. It is useful as an antibody.

The anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody can be treated as a pharmaceutical as long as it is a cancer expressing EPHA2. Examples of the types of cancer include breast cancer, esophageal cancer, Prostate cancer, gastric cancer, non-small cell lung cancer, colon cancer and glioblastoma multiforme can be preferably mentioned, but are not limited thereto.

The present invention relates to a medicament comprising a therapeutically effective amount of an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Compositions are also provided.

The substance used in the preparation acceptable in the pharmaceutical composition of the present invention is preferably a substance that is non-toxic to a person who is administered the pharmaceutical composition at a dosage or concentration.

The pharmaceutical composition of the present invention changes, maintains, or maintains pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, dissolution rate, sustained release rate, absorption rate, and penetration rate. Or a pharmaceutical substance for the preparation.

Substances for formulation may include, but are not limited to: amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine or lysine, antibacterial agents, ascorbic acid, sodium sulfate or sodium bisulfite Antioxidants such as phosphate, citric acid, borate buffer, hydrogen carbonate, buffer such as tris-hydrochloric acid (Tris-Hcl) solution, filler such as mannitol and glycine, chelate such as ethylenediaminetetraacetic acid (EDTA) Agents, caffeine, polyvinylpyrrolidine, complexing agents such as β-cyclodextrin and hydroxypropyl-β-cyclodextrin, bulking agents such as glucose, mannose or dextrin, monosaccharides, disaccharides and glucose, mannose and dextrin, etc. Carbohydrates, colorants, flavors Agents, diluents, emulsifiers and hydrophilic polymers such as polyvinylpyrrolidine, low molecular weight polypeptides, salt-forming counterions, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorexidine, sorbic acid Preservatives such as hydrogen oxide, solvents such as glycerin, propylene glycol or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspension agents, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Surfactants such as triton, tromethamine, lecithin or cholesterol, stabilization enhancers such as sucrose and sorbitol, sodium chloride, potassium chloride and mannini Elasticity enhancers such Lumpur sorbitol, transport agent, diluent, excipient, and / or pharmaceutically adjuvants.

The addition amount of the substance for these preparations is preferably 0.01 to 100 times, particularly 0.1 to 10 times the weight of the anti-EPHA2 humanized swapped antibody or functional fragment thereof.

A pharmaceutical composition containing an immunoliposome containing an anti-EPHA2 humanized swapped antibody in the liposome or an antibody in which the anti-EPHA2 antibody and the liposome are bound (US Pat. No. 6,214,388) is also included in the present invention.

The composition of a suitable pharmaceutical composition in the preparation can be appropriately determined by those skilled in the art according to the applied disease, the applied administration route, and the like.

Excipients and carriers in the pharmaceutical composition may be liquid or solid.
Appropriate excipients and carriers may be water for injection, physiological saline, artificial cerebrospinal fluid and other substances commonly used for parenteral administration.

Neutral physiological saline or physiological saline containing serum albumin can also be used as a carrier.
Pharmaceutical compositions include, for example, pH 7.0-8.5 Tris buffer, pH 4.0-5.5 acetate buffer, pH 5.0-8.0 citrate buffer, pH 5.0-8.0 histidine. A buffer or a buffer containing sorbitol or other compounds can also be used.

The pharmaceutical composition of the present invention is prepared as a lyophilized product or liquid as an appropriate drug with the purity required for the selected composition.

A pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody can be molded as a lyophilized product using an appropriate excipient such as sucrose.

The pharmaceutical composition can be prepared for parenteral administration or can be prepared for oral digestive tract absorption.

The composition and concentration of the preparation can be determined by the administration method, and the affinity of the anti-EPHA2 humanized swapped antibody or the functional fragment of the antibody contained in the pharmaceutical composition of the present invention for EPHA2, that is, dissociation for EPHA2 The higher the affinity (Kd value) for the constant (Kd value), the smaller the dose to humans, the more effective the drug can be. Therefore, based on this result, the pharmaceutical composition of the present invention for humans The dosage can also be determined.

When the pharmaceutical composition is administered to humans, it is usually sufficient to administer about 0.1 to 100 mg / kg once every 1 to 180 days.

Examples of the form of the pharmaceutical composition include injections containing infusions, suppositories, nasal agents, sublingual agents, and transdermal absorption agents.
The pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody as an active ingredient may be used simultaneously or sequentially with the chemotherapeutic agent. That is, after administering the chemotherapeutic agent, a pharmaceutical composition containing an anti-EPHA2 humanized swapped antibody or a functional fragment of the antibody as an active ingredient may be administered, or after administering the pharmaceutical composition, the chemotherapeutic agent In addition, the pharmaceutical composition and the chemotherapeutic agent may be administered simultaneously. For example, agents that can be used include antimetabolites including thymidylate synthase inhibitors, nucleic acid analogs, platinum cytotoxic agents, topoisomerase inhibitors, or anti-microtubule agents. Examples of thymidylate synthase inhibitors that can be used in the present invention include 5-FU, MTA, and TDX. An example of an antimetabolite that can be used is Tomdex (TDX). Examples of platinum cytotoxic agents that can be used include cisplatin and oxaliplatin. In addition to or in place of the specific agents listed above, chemotherapeutic agents that can be used in the present invention include: alkylating agents; alkyl sulfonates; aziridines; ethyleneimines; methylamelamines; nitrogen mustards; Antimetabolites; Folic acid analogs; Purine analogs; Pyrimidine analogs; Androgens; Anti-adrenal substances; Folic acid supplements; Acegraton; Aldophosphamide glycosides; Aminolevulinic acid; Amsacrine; Vestlabsyl; Bisanthrene; May contain: diazicon; efomithine; ellipticin acetate; ethogluside; gallium nitrate; hydroxyurea; lentinan; ionidamine; Topoisomerase inhibitors may be used as chemotherapeutic agents, and the topoisomerase inhibitors used in certain embodiments are topoisomerase I inhibitors such as camptothecin. A suitable topoisomerase I inhibitor that can be used in the present invention is ilenotecan (CPT-11) or its active metabolite SN-38. CPT-11 acts specifically in the S phase of the cell cycle by stabilizing a reversible covalent reaction intermediate, called a cleavage or cleavage complex, and also arrests the cell cycle in the G2-M phase. Can be induced. A chemotherapeutic agent that can be used in certain embodiments of the invention is a fluoropyrimidine, such as 5-FU. It should be understood that when a particular chemotherapeutic agent is used, analogs including biologically active derivatives and substantial equivalents thereof that retain the anti-tumor activity of a particular agent may be used.

It is possible to suppress the growth of tumors expressing EPHA2 by administering the pharmaceutical composition disclosed herein.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

In the following examples, unless otherwise specified, each operation relating to gene manipulation is from “Molecular Cloning” (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press). (Published in 1989) and the methods described in other experimental documents used by those skilled in the art, or when using commercially available reagents and kits, they were performed according to the instructions for commercial products.

Example 1. Design of humanized antibody a) Design of humanized antibody of SH348-1 a) -i) Molecular modeling of the variable region of SH348-1 The heavy chain variable region of SH348-1 is shown in SEQ ID NO: 10 of the Sequence Listing. . The light chain variable region of SH348-1 is shown in SEQ ID NO: 14 in the sequence listing.

Molecular modeling of the variable region of SH348-1 was performed by a method generally known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). Primary sequence of variable region of human immunoglobulin registered in Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) (three-dimensional structure derived from X-ray crystal structure is available) Was compared to the variable region of SH348-1 determined above. As a result, 2JEL and 1A4J were selected as the sequences with the highest sequence homology to the SH348-1 light and heavy chain variable regions, respectively. The three-dimensional structure of the framework region was created by combining the 2JEL and 1A4J coordinates corresponding to the SH348-1 light and heavy chains to obtain a “framework model”. The CDRs of SH348-1 are described in Thornton et al. (J. Mol. Biol., 263, 800-815, (1996)), CDRL ₁ , CDRL ₂ , CDRL ₃ , CDRH ₁ and CDRH ₂ are in clusters 16A, 7A, 9A, 10A, 10A, respectively. Assigned. CDRH ₃ was classified as e (9) D using the H3 rule (FEBS letter 399, 1-8 (1996)). A representative conformation for each CDR was then incorporated into the framework model.

Finally, in order to obtain a molecular model that may be a variable region of SH348-1 in terms of energy, an energy calculation was performed to eliminate unfavorable interatomic contact. The above procedure was performed using a commercially available protein tertiary structure prediction program Prime and a conformational search program MacroModel (Schrodinger, LLC).

a) -ii) Design of amino acid sequence for humanized SH348-1 The construction of humanized SH348-1 antibody is commonly described as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 1000029-10033 (1989)). By a known method. Acceptor antibodies were selected based on amino acid homology within the framework regions. The sequence of the framework region of SH348-1 was compared with all human frameworks of IgBLAST (Nuc. Acid Res. 36, D25-D30 (2007)). As a result, the light chain was 1006261A for the framework region. Selected as acceptor due to 96% sequence homology. The heavy chain was selected as an acceptor due to 86% sequence homology with AAC09074 for the framework region. The amino acid residues of the framework region for L chain 10060061A and H chain AAC09074 were aligned with the amino acid residues for SH348-1, and the position where the different amino acids were used was identified. The positions of these residues are analyzed using the SH348-1 three-dimensional model constructed above, and the donor residues to be grafted on the acceptor are described in Queen et al. (Proc. Natl. Acad. Sci. USA 86, 1000029-10033 (1989)). The humanized SH348-1 sequence was constructed as described in the examples below by transferring several selected donor residues into the acceptor antibody.

b) Humanization of SH348-1 heavy chain b) -i) hSH348-1-T4H type heavy chain:
Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine) of the SH348-1 heavy chain shown in SEQ ID NO: 6 in the sequence listing ), 43 (lysine), 46 (lysine), 69 (alanine), 73 (glutamic acid), 76 (alanine), 80 (phenylalanine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 88 (asparagine) ), 93 (threonine), 95 (phenylalanine), 114 (threonine), 115 (leucine) are valine, alanine, valine, alanine, serine, valine, arginine, glutamine, glutamic acid, valine, aspartic acid, valine, tyrosine, Leucine, serine, serine, alanine, burr , Tyrosine, leucine, and with designed humanized SH348-1 heavy chain to replace the valine designated as "hSH348-1-T4H-type heavy chain". The amino acid sequence of the hSH348-1-T4H type heavy chain is shown in SEQ ID NO: 53, and the amino acid sequence of the variable region of the hSH348-1-T4H type heavy chain is shown in SEQ ID NO: 54.

b) -ii) hSH348-1-T5H type heavy chain:
Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 43 (lysine) of the SH348-1 heavy chain shown in SEQ ID NO: 6 in the sequence listing ), 76 (alanine), 80 (phenylalanine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 88 (asparagine), 93 (threonine), 114 (threonine), 115 (leucine), respectively, valine, A humanized SH348-1 heavy chain designed with replacement of alanine, valine, alanine, serine, valine, glutamine, valine, tyrosine, leucine, serine, serine, alanine, valine, leucine, valine is replaced with “hSH348-1- It was named “T5H type heavy chain”. The amino acid sequence of the hSH348-1-T5H type heavy chain is shown in SEQ ID NO: 55 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH348-1-T5H type heavy chain is shown in SEQ ID NO: 56.

c) Humanization of SH348-1 light chain c) -i) hSH348-1-T1L type light chain:
The amino acid sequence of the hSH348-1-T1L type light chain described in WO2009 / 028639 (Example 10), 10-1-3-1) is represented by SEQ ID NO: 57 in the Sequence Listing, and the variable of the hSH348-1-T1L type heavy chain. The amino acid sequence of the region is shown in SEQ ID NO: 58.

c) -ii) hSH348-1-T5L type light chain:
Amino acid numbers 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 79 (arginine), 88 (leucine), 105 (of the SH348-1 light chain shown in SEQ ID NO: 8 of the Sequence Listing Glycine), 109 (leucine) and 114 (alanine) are replaced with threonine, proline, glutamic acid, proline, lysine, valine, glutamine, valine and threonine, respectively. 1-T5L type light chain ". The amino acid sequence of the hSH348-1-T5L type light chain is shown in SEQ ID NO: 59 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH348-1-T5L type light chain is shown in SEQ ID NO: 60.

d) Design of humanized antibody of SH357-1 d) -i) Molecular modeling of the variable region of SH357-1 The heavy chain variable region of SH357-1 is set forth in SEQ ID NO: 34 of the Sequence Listing. The light chain variable region of SH357-1 is set forth in SEQ ID NO: 38 in the sequence listing.

In the same manner as in a) -i) above, 2JEL and 1A4J were selected as the sequences having the highest sequence homology to the light and heavy chain variable regions of SH357-1, respectively, and CDRL ₁ , CDRL ₂ , CDRL ₃ , CDRH ₁ and CDRH ₂ were assigned to clusters 16A, 7A, 9A, 10A, 10A, respectively. CDRH ₃ was classified as e (9) D.

A) The sequence of humanized SH357-1 was determined by the same method as in -ii, in which 1006661A was selected as the acceptor for the L chain and AAC09074 was selected as the acceptor for the H chain. As a result, as shown below, one type of humanized sequence for the light chain and two types of humanized sequences for the heavy chain were obtained.

e) Humanization of SH3577-1 heavy chain e) -i) hSH357-1-T4H type heavy chain:
Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine) of the SH357-1 heavy chain shown in SEQ ID NO: 30 of the Sequence Listing ), 43 (lysine), 46 (lysine), 69 (alanine), 73 (glutamic acid), 76 (alanine), 83 (isoleucine), 85 (asparagine), 88 (asparagine), 93 (serine), 95 (phenylalanine) ), 114 (threonine), 115 (leucine) are valine, alanine, valine, alanine, serine, valine, arginine, glutamine, glutamic acid, valine, aspartic acid, valine, leucine, serine, alanine, valine, tyrosine, leucine, Humanization designed with valine replacement The H357-1 heavy chain was named "hSH357-1-T4H-type heavy chain". The amino acid sequence of the hSH357-1-T4H type heavy chain is shown in SEQ ID NO: 61 in the sequence listing, and the amino acid sequence of the variable region of the hSH357-1-T4H type heavy chain is shown in SEQ ID NO: 62.

e) -ii) hSH357-1-T5H type heavy chain:
Amino acid numbers 2 (isoleucine), 9 (proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 43 (lysine) of the SH357-1 heavy chain shown in SEQ ID NO: 30 of the Sequence Listing ), 76 (alanine), 83 (isoleucine), 85 (asparagine), 88 (asparagine), 93 (serine), 114 (threonine), 115 (leucine), respectively, valine, alanine, valine, alanine, serine, valine, The humanized SH357-1 heavy chain designed with replacement with glutamine, valine, leucine, serine, alanine, valine, leucine, valine was named “hSH357-1-T5H type heavy chain”. The amino acid sequence of the hSH357-1-T5H type heavy chain is shown in SEQ ID NO: 63 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH357-1-T5H type heavy chain is shown in SEQ ID NO: 64.

f) Humanization of SH3577-1 light chain f) -i) hSH357-1-T1L type light chain:
The amino acid sequence of the hSH357-1-T1L type light chain described in WO2009 / 028639 (Example 10), 10-2-3-1) is represented by SEQ ID NO: 65 in the sequence listing, and the variable of the hSH357-1-T1L type light chain. The amino acid sequence of the region is shown in SEQ ID NO: 66.

f) -ii) hSH357-1-T5L type light chain:
Amino acid numbers 7 (threonine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 88 (leucine), 105 (SH) light chain shown in SEQ ID NO: 32 of the Sequence Listing Glycine), 109 (leucine) and 114 (alanine) are replaced with serine, threonine, proline, glutamic acid, proline, valine, glutamine, valine, and threonine, respectively, and the humanized SH357-1 light chain designed with “hSH357- 1-T5L type light chain ". The amino acid sequence of the hSH357-1-T5L type light chain is shown in SEQ ID NO: 67 of the Sequence Listing, and the amino acid sequence of the variable region of the hSH357-1-T5L type light chain is shown in SEQ ID NO: 68.

Example 2 Construction of universal expression vectors pEF6KCL and pEF1FCCU 1) Construction of human light chain expression vector pEF6KCL By PCR using plasmid pEF6 / V5-HisB (Invitrogen) as a template with the following primers (Sequence Position 2174) To DNA fragments up to Sma I (Sequence Position 2958) (DNA fragment containing f1 origin of replication and SV40 promoter and origin, hereinafter referred to as “fragment A”).
5′-ccacgcgccctgttagcggcgcattaagc-3 ′ (primer EFF1) (SEQ ID NO: 69)
5′-aaacccggggagtttttgcaaaaggcctag-3 ′ (primer EFsmaR) (SEQ ID NO: 70)
The obtained fragment A overlaps with a DNA fragment (SEQ ID NO: 71, hereinafter referred to as “fragment B”) containing a DNA sequence encoding a human κ chain secretion signal, a human κ chain constant region, and a human polyA addition signal. Binding by PCR. The obtained DNA fragment in which fragment A and fragment B were bound was digested with restriction enzymes KpnI and SmaI, and ligated with plasmid pEF6 / V5-HisB (Invitrogen) digested with restriction enzymes KpnI and SmaI, and downstream of the EF1 promoter. A human light chain expression vector pEF6KCL having a signal sequence, a cloning site, a human κ chain constant region, and a human polyA addition signal sequence was constructed.

2) Construction of pEF1 / KCL A DNA fragment cleaved from the restriction enzyme KpnI and SmaI from pEF6KCL obtained by the above method was ligated with pEF1 / myc-HisB (Invitrogen) digested with KpnI and SmaI, and the plasmid pEF1KCL Built.

3) Construction of human heavy chain expression vector pEF1FCCU A plasmid obtained by digesting a DNA fragment (SEQ ID NO: 72) containing a human IgG1 signal sequence and a DNA sequence encoding the amino acid of the constant region with restriction enzymes NheI and PmeI and digesting with NheI and PmeI A human heavy chain expression vector pEF1FCCU was constructed by binding to pEF1KCL and having a signal sequence, a cloning site, a human heavy chain constant region, and a human polyA addition signal sequence downstream of the EF1 promoter.

Example 3 Construction of antibody expression vector 1) Construction of hSH348-1-T1L and hSH357-1-T1L type light chain expression vector hSH348-1-T1L type light chain variable region fused with the secretion signal shown in SEQ ID NO: 73 of the Sequence Listing And a DNA containing a gene encoding the hSH357-1-T1L type light chain variable region fused with the secretion signal shown in SEQ ID NO: 74 (Invitrogen Artificial Gene Synthesis Service), and was cleaved with restriction enzymes NdeI and BsiWI HSH348-1-T1L and hSH357-1-T1L type light chain expression vectors were constructed by inserting the DNA fragment into a site where a humanized antibody light chain expression general-purpose vector (pEF6KCL) was cleaved with restriction enzymes NdeI and BsiWI. . The obtained expression vectors were named “pEF6KCL / hSH348-1-T1L” and “pEF6KCL / hSH357-1-T1L”, respectively.

2) Construction of hSH348-1-T1H, hSH357-1-T1H type heavy chain expression vector hSH348-1-T1H type heavy chain variable region shown in SEQ ID NO: 75 of the sequence listing and hSH357-1- shown in SEQ ID NO: 76 A DNA containing a gene encoding a T1H type heavy chain variable region was synthesized (Invitrogen Artificial Gene Synthesis Service), and a DNA fragment excised with the restriction enzyme BlpI was used as a humanized antibody H chain expression general-purpose vector (pEF1FCCU) as a restriction enzyme BlpI. The hSH348-1-T1H and hSH357-1-T1H type heavy chain expression vectors were constructed by inserting them into the sites cleaved with. The obtained expression vectors were named “pEF1FCCU / hSH348-1-T1H” and “pEF1FCCU / hSH357-1-T1H”, respectively.

3) Construction of 348-L4 and 357-L4 type light chain expression vectors hSH348-1-T1L represented by amino acid numbers 21 to 137 of SEQ ID NO: 78 and amino acid numbers 21 to 137 of SEQ ID NO: 80, respectively. A DNA comprising a type light chain variable region and a gene encoding a hSH357-1-T1L type light chain variable region (GENEART, artificial gene synthesis service), and a DNA fragment excised with restriction enzymes NdeI and BsiWI, A vector expressing hSH348-1-T1L and hSH357-1-T1L type light chains was constructed by inserting a humanized antibody light chain expression general-purpose vector (pEF6KCL) into a site cleaved with restriction enzymes NdeI and BsiWI. Here, the hSH348-1-T1L type light chain was named “348-L4” and the hSH357-1-T1L type light chain was named “357-L4”. The obtained vector expressing 348-L4 was named “pEF6KCL / 348-L4”, and the vector expressing 357-L4 was named “pEF6KCL / 357-L4”. The nucleotide sequence of 348-L4 is shown in SEQ ID NO: 81 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 82. The nucleotide sequence of 357-L4 is shown in SEQ ID NO: 83, and the amino acid sequence is shown in SEQ ID NO: 84.

Example 4 Preparation of humanized antibody 1) Production of humanized antibody 1.2 × 10 ⁹ log-type freestyle 293F cells (Invitrogen) were seeded in fresh 1.2 L FreeStyle293 expression medium (Invitrogen) at 37 ° C. The cells were cultured with shaking at 90 rpm for 1 hour in an 8% CO ₂ incubator. 3.6 mg of Polyethyleneimine (Polyscience # 24765) dissolved in 20 ml of Opti-Pro SFM medium (Invitrogen), and then an H chain expression vector (0.4 mg) and L chain prepared using PureLink HiPure Plasmid kit (Invitrogen) The expression vector (0.8 mg) was suspended in 20 ml of Opti-Pro SFM medium. 20 ml of an expression vector / Opti-Pro SFM mixture was added to 20 ml of a polyethyleneimine / Opti-Pro SFM mixture, gently stirred, allowed to stand for another 5 minutes, and then added to FreeStyle 293F cells. The culture supernatant obtained by shaking culture at 90 rpm in a 37 ° C., 8% CO ₂ incubator for 7 days was filtered with a Disposable Capsule Filter (Advantec # CCS-045-E1H).

The humanized antibodies of SH348-1 obtained by the combination of pEF6KCL / hSH348-1-T1L and pEF1FCCU / hSH348-1-T1H are “hSH348-1-T1”, pEF6KCL / hSH357-1-T1L and pEF1FCCU / hSH357- The humanized antibody of SH357-1 obtained by combination with 1-T1H was named “hSH357-1-T1”.

2) Purification of humanized antibody The culture supernatant obtained in 1) above was purified in two steps, rProtein A affinity chromatography (4-6 ° C.) and ceramic hydroxylapatite (room temperature). The buffer replacement step after purification of rProtein A affinity chromatography and after purification of ceramic hydroxylapatite was carried out at room temperature. First, 1100-1200 ml of the culture supernatant was applied to MabSelectSuRe (GE Healthcare Bioscience, HiTrap column: volume 1 ml × 2 linked) equilibrated with PBS. After all of the culture solution entered the column, the column was washed with 15-30 ml of PBS. Next, elution was performed with a 2M arginine hydrochloride solution (pH 4.0), and fractions containing the antibody were collected. The fraction was replaced with a 5 mM sodium phosphate / 50 mM MES / 20 mM NaCl / pH 6.5 buffer using a desalting column (manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml × 2 linked). Further, the substituted antibody solution was applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT2-1 Hydroxyapatite Column: volume 2 ml) equilibrated with 5 mM NaPi / 50 mM MES / 20 mM NaCl / pH 6.5 buffer. Applied. Linear gradient elution with sodium chloride was performed and the fractions containing antibody were collected. The fraction was subjected to liquid replacement with CBS (10 mM citrate buffer / 140 mM sodium chloride, pH 6.0) with a desalting column (manufactured by GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml × 2 linked). Finally, it was concentrated with Centrifugal UF Filter Device VASPASP20 (fractionated molecular weight 30K, Sartorius, 4 ° C.), and the IgG concentration was adjusted to 1.0 mg / ml or more to obtain a purified sample.

Example 5 FIG. Construction of humanized antibody expression vector 1) Construction of 348-L5 and 357-L5 type light chain expression vector 348-L5 type light chain variable region represented by amino acid numbers 21 to 137 of SEQ ID NO: 86, and SEQ ID NO: 88 DNA comprising a gene encoding the 357-L5 type light chain variable region represented by amino acid numbers 21 to 137 (GENEART, artificial gene synthesis service), and a DNA fragment cleaved with restriction enzymes NdeI and BsiWI, 348-L5 and 357-L5 type light chain expression vectors were constructed by inserting a humanized antibody light chain expression general-purpose vector (pEF6KCL) into a site cleaved with restriction enzymes NdeI and BsiWI. The obtained expression vectors were named “pEF6KCL / 348-L5” and “pEF6KCL / 357-L5”, respectively.

2) Construction of 348-H4, 348-H5, 357-H4, and 357-H5 type heavy chain expression vectors 348-H4 type heavy chain variable region shown in amino acid numbers 20 to 138 of SEQ ID NO: 90 in the sequence listing, sequence 348-H5 type heavy chain variable region represented by amino acid numbers 20 to 138 of number 92, 357-H4 type heavy chain variable region represented by amino acid numbers 20 to 138 of SEQ ID NO: 94, and amino acid numbers 20 to 20 of SEQ ID NO: 96 A DNA fragment containing a gene encoding the 357-H5 type heavy chain variable region shown in 138 (synthetic gene synthesis service of GENEART) and a DNA fragment cleaved by the restriction enzyme BlpI By inserting the vector (pEF1FCCU) into the site cut with the restriction enzyme BlpI It was constructed 348-H4,348-H5,357-H4, and 357-H5-type heavy chain expression vectors. The obtained expression vectors were named “pEF1FCCU / 348-H4”, “pEF1FCCU / 348-H5”, “pEF1FCCU / 357-H4”, and “pEF1FCCU / 357-H5”, respectively.

The sequence of each antibody is shown in FIG. 1 and FIG.

Example 6 Preparation of humanized antibody 1) Production of humanized antibody Antibody was obtained by the same method as described in Example 4.

The humanized antibody of SH348-1 obtained by the combination of pEF1FCCU / 348-H4 and pEF6KCL / 348-L4 is “348-H4-348-L4”, the combination of pEF1FCCU / 348-H4 and pEF6KCL / 348-L5 The humanized antibody of SH348-1 obtained by the combination of “348-H4-348-L5”, the combination of pEF1FCCU / 348-H5 and pEF6KCL / 348-L4 was obtained by “348-H4-348-L5”. -H5-348-L4 ", the humanized antibody of SH348-1 obtained by the combination of pEF1FCCU / 348-H5 and pEF6KCL / 348-L5," 348-H5-348-L5 ", pEF1FCCU / 357-H4 and In combination with pEF6KCL / 357-L4 The humanized antibody of SH357-1 obtained by the combination of “357-H4-357-L4”, pEF1FCCU / 357-H4 and pEF6KCL / 357-L5 was obtained as the humanized antibody of SH357-1. 357-H4-357-L5 ”, the humanized antibody of SH357-1 obtained by the combination of pEF1FCCU / 357-H5 and pEF6KCL / 357-L4 is“ 357-H5-357-L4 ”, pEF1FCCU / 357-H5 SH357-1 obtained by combining “357-H5-357-L5”, a combination of pEF1FCCU / 348-H4 and pEF6KCL / 357-L4 with a humanized antibody of SH357-1 obtained by the combination of pEF6KCL / 357-L5 with pEF6KCL / 357-L5 From the humanized heavy chain of -1 and the humanized light chain of SH357-1 The antibody consists of a humanized heavy chain of SH348-1 and a humanized light chain of SH357-1 obtained by a combination of “348-H4-357-L4”, pEF1FCCU / 348-H4 and pEF6KCL / 357-L5 An antibody comprising a humanized heavy chain of SH348-1 and a humanized light chain of SH357-1 obtained by a combination of “348-H4-357-L5”, pEF1FCCU / 348-H5 and pEF6KCL / 357-L4 An antibody comprising a humanized heavy chain of SH348-1 and a humanized light chain of SH357-1 obtained by a combination of “348-H5-357-L4”, pEF1FCCU / 348-H5 and pEF6KCL / 357-L5 “348-H5-357-L5”, pEF1FCCU / 357-H4 and pEF6KCL / 348-L4 An antibody comprising a humanized light chain of SH348-1 and a humanized heavy chain of SH357-1 obtained by the combination of “357-H4-348-L4”, pEF1FCCU / 357-H4 and pEF6KCL / 348-L5 An antibody comprising a humanized light chain of SH348-1 and a humanized heavy chain of SH357-1 obtained by the combination is “357-H4-348-L5”, a combination of pEF1FCCU / 357-H5 and pEF6KCL / 348-L4 The antibody consisting of the humanized light chain of SH348-1 and the humanized heavy chain of SH357-1 obtained by the method described above by “357-H5-348-L4”, a combination of pEF1FCCU / 357-H5 and pEF6KCL / 348-L5 The obtained antibody comprising the humanized light chain of SH348-1 and the humanized heavy chain of SH357-1 is designated as “357. It was named H5-348-L5 ".

Example 7 Measurement of Kd value of anti-EPHA2 antibody The dissociation constant of the anti-EPHA2 antibody obtained in Example 6 and Fibronctin type III domain was measured as follows.

His-Fibrinctin type III domain (426-540) (polypeptide having a histidine tag added to the amino terminus of a polypeptide consisting of the amino acid sequence of amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing) was prepared. Using Biacore T100 (GE Healthcare Bioscience Co., Ltd.), the antibody was captured (captured) on an immobilized anti-human IgG (Fc) antibody, and the capture method was performed in which the antigen was measured as an analyte. An anti-human IgG (Fc) antibody (Human antibody capture kit, GE Healthcare Biosciences) was covalently bound to a sensor chip CM5 (BIAcore, Inc.) up to 10,000 RU by the amine coupling method. The reference cell was similarly fixed. HBS-EP (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used as a running buffer. An anti-human IgG (Fc) antibody-immobilized chip was added with an antibody solution of about 20 nM at a flow rate of 10 μL / min for 60 seconds, and then a His-Fibroctin type III domain (426-540) dilution series solution (3. 13-50 nM) was added at a flow rate of 30 μl / min for 120 seconds, followed by monitoring the dissociation phase for 180 seconds. As a regeneration solution, 3M magnesium chloride was added at a flow rate of 10 μl / min for 30 seconds. For the analysis of the data, a one-to-one binding model of analysis software (Biacore T100 Evaluation software, version 2.0.1) was used, and the association rate constant kon, the dissociation rate constant koff and the dissociation constant (Kd; Kd = koff / kon) was calculated.

As a result, in the combination of the humanized heavy chain derived from SH348-1 and the humanized light chain derived from SH357-1, the Kd value is higher than that of the humanized antibody of SH348-1 or the humanized antibody of SH357-1. It was found that the binding activity against the antigen was significantly decreased (Table 1). Humanized antibody h348-1 Td Kd value 8.29 (nM), 348-H4-357-L4 0.37 (nM), 348H4-357-L5 0.37 (nM), 348- H5-357-L4 was 0.32 (nM) and 348-H5-357-L5 was 0.30 (nM), indicating that the binding activity was significantly increased.

Example 8 FIG. Measurement of ADCC activity The prostate cancer cell line PC-3 introduced with the EGFP gene is used as a target cell, and human peripheral blood lymphocytes (BIOPREDIC # PBL003, hereinafter referred to as “human PBL”) are used as effector cells and released into the culture supernatant. The antibody-dependent cytotoxic activity (ADCC activity) was calculated by measuring the amount of fluorescence of EGFP. 96-well microwells of EGFP-expressing PC-3 cells suspended in phenol red-free RPMI 1640 (Invitrogen # 11835-030) medium containing 10% Ultralow IgG FBS (Invitrogen # 16250-078) at 1 × 10 ⁴ cells / well. The cells were seeded and cultured overnight at 37 ° C. and 5% CO ₂ . After removing the medium, humanized anti-EPHA2 antibody or isotype control antibody hIgG1 (Alexis Biochemicals # ALX-804-133) diluted in fresh medium (final concentrations 0.1, 1.0, 10 μg, respectively) / ML), 2 × 10 ⁵ cells / well of human PBL were added, and the cells were cultured overnight under conditions of 37 ° C. and 5% CO ₂ . The culture supernatant was collected and the amount of fluorescence (485/535 nm) in the supernatant was measured. The cell lysis rate due to ADCC activity was calculated by the following formula.
Cell lysis rate (%) = (AB) / (CB) × 100
A: Fluorescence amount of culture supernatant of sample well B: Average value of fluorescence amount of culture supernatant of well without antibody / effector cells.
C: Average value of the fluorescence amount of the culture supernatant of a well in which target cells were lysed with Triton-X100 at a final concentration of 1%.
FIG. 3 shows an average value of three samples, and error bars indicate standard deviation. As a result, 348-H4-357-L4, 348-H4-357-L5, 348-H5-357-L5 were obtained from hSH357-1-T1 and 357-H4-357-L4 at an antibody concentration of 0.1 μg / mL. The ADCC activity of 348-H4-357-L4, 348-H4-357-L5, and 348-H5-357-L5 at an antibody concentration of 0.1 μg / mL is 10 times higher than that. It was shown to be comparable to the ADCC activity of high concentrations of 1.0 μg / mL hSH357-1-T1 and 357-H4-357-L4.

Example 9 Antigen-specific binding In order to verify the binding specificity of the humanized anti-EPHA2 antibody, it is homologous to the amino acid sequence of amino acids 439 to 534 of human EPHA2 (the amino acid sequence shown in amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing) The binding property to highly protein was examined by Cell-ELISA method. The homology search was performed using NCBI BLAST, and the amino acid sequence from human 439 to 534 of human EPHA2 was searched for human protein. Among the hit proteins, the top 2 proteins (EPHA3 isoforma (hereinafter referred to as “EPHA3a” in the figure and in the figure)) and human protein tyrosin phosphate, receptor type, sigma isoform 4 (hereinafter referred to as “PTPRS-4” in the figure). )).
Since these proteins are transmembrane proteins whose N-terminus is an extracellular region, in order to produce a construct expressing a protein with FLAG-Tag added to the N-terminus, pFLAG-myc-CMV19 ( The reading frame B of Gateway Vector Conversion System (Invitrogen # 11828-029) was incorporated into a site obtained by cutting Sigma-Aldrich # E8658) with HindIII and BglII and smoothing to create “pFLAG-GW”. Next, human EPHA2 excluding the signal sequence (amino acid sequence from the start codon to the 23rd amino acid), human EPHA3a excluding the signal sequence (amino acid sequence from the start codon to the 20th), or the signal sequence (from the start codon to the 29th) A recombination reaction using LR Clonase (Invitrogen # 11791-019) between the entry vector in which the gene encoding the amino acid sequence of human PTPRS-4 (excluding the amino acid sequence of the above) was cloned and pFLAG-GW, Expression vectors “pFLAG-EPHA2”, “pFLAG-EPHA3a”, and “pFLAG-PTPRS-4” were prepared.

293αvβ3 cells transfected with pFLAG-GW, pFLAG-EPHA2, pFLAG-EPHA3a, or pFLAG-PTPRS-4 using Lipofectamin 2000 (Invitrogen # 11668-019) were expressed by INTGrin αV and integrin β3 stably in HEK293 cells. Cells) were seeded in a 96-well plate and cultured overnight in DMEM containing 10% FBS under conditions of 37 ° C. and 5% CO ₂ . Discard the medium and humanized anti-EPHA2 antibody (5 μg / mL or 50 μg / mL) diluted in PBS containing 5% FBS, hIgG (5 μg / mL or 50 μg / mL) as a negative control for humanized antibody (Jackson ImmunoResearch Laboratories # 109 -035-098), Monoclonal ANTI-FLAG M2 antibody (0.02 μg / mL or 0.1 μg / mL) (Sigma # F3165: and indicated as “Anti-FLAG mAb” in the figure), Anti-FLAG mAb negative As a control, mIgG1 (0.02 μg / mL or 0.1 μg / mL) (BD Bioscience # 553447) was added and incubated at 4 ° C. for 1 hour. After washing the cells with PBS containing 5% FBS, for detection of humanized anti-EPHA2 antibody and its negative control antibody, Peroxidase AffiniPure Goat Anti-Human IgG Fcγ Fragment Specific (Jackson Immense Immense) was diluted 500 times with PBS containing 5% FBS. 109-035-098) was added 500-fold diluted Goat anti-Mouse IgG Peroxidase Conjugate (Millipore # AP181P) for the detection of Anti-FLAG mAb and its negative control antibody and incubated at 4 ° C. for 1 hour. After washing the cells with PBS containing 5% FBS, color reaction was performed by OPD, and the reaction was stopped with 1M HCl, and the absorbance at 490 nm was measured. FIG. 4 shows an average value of three samples, and error bars indicate standard deviation.

When the expression level of each protein on the cell membrane was confirmed using Anti-FLAG mAb, the same level of expression on the cell membrane was observed for each protein (FIG. 4A). On the other hand, the humanized anti-EPHA2 antibodies hSH357-1-T1, 348-H4-357-L4, 348-H4-357-L5, 348-H5-357-L5 are against human EPHA2 expressing cells. While binding strongly, cells expressing human EPHA3a and human PTPRS-4 showed only the same level of binding as cells transfected with pFLAG-GW, which is a negative control (FIG. 4, BE). ). From the above results, it was shown that these humanized anti-EPHA2 antibodies specifically recognize and bind to human EPHA2.

Example 10 Confirmation of in vivo antitumor activity Human breast cancer cell line MDA-MB-231 cells were trypsinized and detached from the culture flask, suspended in Leibovitz's L-15 containing 10% FBS, and centrifuged to remove the supernatant. did. The cells were washed once with the same medium, then suspended in a BD Matrigel basement membrane matrix (BD Biosciences # 354234), and 6-week-old nude mice (CAnN.Cg-Foxn1 [nu] / CrlCrlj [Foxn1nu / Foxn1nu]: Japan (Charles River) was transplanted subcutaneously between the second and third nipples of the right axilla at 5 × 10 ⁶ cells / animal. Grouping was performed on day 17 with the day of transplantation as day 0, and on days 17, 24, 31 hSH357-1-T1, 348-H4-357-L4, 348-H4-357-L5, or 348-H5 -357-L5 was administered at 0.01 mg / animal or 0.03 mg / animal. The vehicle group was administered with the same amount of vehicle as the antibody. Tumor volume was measured on days 17, 24, 31, and 38. FIG. 5 shows the mean value of 9 tumor volumes per group, and error bars indicate standard errors. Further, statistical analysis was performed by Dunnett's multiple comparison test using values at 38 days (**: P <0.05, **: P <0.01, ***: P <0.001). .

As a result, all humanized anti-EPHA2 antibodies of hSH357-1-T1, 348-H4-357-L4, 348-H4-357-L5, 348-H5-357-L5 were administered at a dose of 0.03 mg / animal. A significant antitumor effect was confirmed, and for hSH357-1-T1, 348-H4-357-L5, 348-H5-357-L5, a significant antitumor effect was observed even at 0.01 mg / animal (FIG. 5). AD).

The anti-EPHA2 antibody of the present invention has antitumor activity, and a pharmaceutical composition containing the anti-EPHA2 antibody can be an anticancer agent.

FERM BP-1083
FERM BP-10837

SEQ ID NO: 3: EPHA2 variant SEQ ID NO: 53: 348-H4
SEQ ID NO: 54: 348-H4 variable region SEQ ID NO: 55: 348-H5
SEQ ID NO: 56: 348-H5 variable region SEQ ID NO: 57: 348-L4
SEQ ID NO: 58: 348-L4 variable region SEQ ID NO: 59: 348-L5
SEQ ID NO: 60: 348-L5 variable region SEQ ID NO: 61: 357-H4
SEQ ID NO: 62: 357-H4 variable region SEQ ID NO: 63: 357-H5
SEQ ID NO: 64: 357-H5 variable region SEQ ID NO: 65: 357-L4
SEQ ID NO: 66: 357-L4 variable region SEQ ID NO: 67: 357-L5
SEQ ID NO: 68: 357-L5 variable region SEQ ID NO: 69: Primer EFF1
Sequence number 70: Primer EfsmaR
SEQ ID NO: 71: human κ signal sequence + κ constant region + poly A
SEQ ID NO: 72: human IgG signal sequence + + constant region SEQ ID NO: 73: signal sequence + hSH348-1-T1L variable region SEQ ID NO: 74: signal sequence + 357-T1L variable region SEQ ID NO: 75: hSH348-1-T1H variable region SEQ ID NO: 76 h357-1-T1H variable region SEQ ID NO: 77: hSH348-T1L
SEQ ID NO: 79: hSH357-T1L
SEQ ID NO: 81: 348-L4
SEQ ID NO: 83: 357-L5
SEQ ID NO: 85: 348-L5
SEQ ID NO: 87: 357-L5
SEQ ID NO: 89: 348-H4
SEQ ID NO: 91: 348-H5
SEQ ID NO: 93: 357-H4
SEQ ID NO: 95: 357-H5

[Supplement under rule 26 11.10.2011]

Claims

A CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, a CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and a CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. Chain, and CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48 in the sequence listing, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50 in the sequence listing, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 52 in the sequence listing. An antibody or a functional fragment of the antibody that specifically recognizes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
The antibody or the functional fragment of the antibody according to claim 1, which specifically recognizes a polypeptide comprising an amino acid sequence represented by amino acid numbers 439 to 534 of SEQ ID NO: 4 in the sequence listing.
A CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, a CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, and a CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. Chain, and CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 48 in the sequence listing, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 50 in the sequence listing, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 52 in the sequence listing. The Kd value for a polypeptide consisting of the light chain and comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.5 nM or less. An antibody or a functional fragment of the antibody.
The antibody or the functional fragment of the antibody according to claim 3, wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 1.0 nM or less.
The antibody or the functional fragment of the antibody according to claim 3, wherein the Kd value for a polypeptide comprising the amino acid sequence represented by amino acid numbers 426 to 540 of SEQ ID NO: 4 in the sequence listing is 0.5 nM or less.
The antibody or the functional fragment of the antibody according to any one of claims 1 to 5, which suppresses phosphorylation of an EPHA2 tyrosine residue induced by an EPHA2 ligand.
A heavy chain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 54 and 56 in the sequence listing or the amino acid sequence of any one of the amino acid sequences in which one or several amino acids are deleted, substituted, or added. Light chain variable consisting of the amino acid sequence shown in SEQ ID NOs: 66 and 68 in the sequence listing or the amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence. The antibody or the functional fragment of the antibody according to any one of claims 1 to 6, comprising a light chain comprising a region.
A heavy chain comprising any one of the amino acid sequences shown in SEQ ID NOs: 53 and 55 in the sequence listing or an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence; The amino acid sequence shown in SEQ ID NOs: 65 and 67 or a light chain comprising any one of the amino acid sequences in which one or several amino acids are deleted, substituted, or added in the amino acid sequence. 8. The antibody or the functional fragment of the antibody according to any one of 1 to 7.
An antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the Sequence Listing and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the Sequence Listing, or a functional fragment of the antibody.
An antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 53 of the Sequence Listing and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the Sequence Listing, or a functional fragment of the antibody.
An antibody consisting of a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the Sequence Listing and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 65 of the Sequence Listing, or a functional fragment of the antibody.
An antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 55 of the Sequence Listing and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 67 of the Sequence Listing, or a functional fragment of the antibody.
The antibody or the functional fragment of the antibody according to any one of claims 1 to 12, which has the properties shown in the following a) to d):
a) not having the ability to phosphorylate EPHA2 tyrosine residues;
b) has ADCC activity against EPHA2 expressing cells;
c) has CDC activity against EPHA2 expressing cells;
d) Has anti-tumor activity in vivo.
The antibody or the functional fragment of the antibody according to any one of claims 1 to 12, which has the properties shown in the following a) to e):
a) not having the ability to phosphorylate EPHA2 tyrosine residues;
b) Does not show an effect of decreasing the amount of EPHA2 protein;
c) has ADCC activity against EPHA2 expressing cells;
d) has CDC activity against EPHA2 expressing cells;
e) Has anti-tumor activity in vivo.
The antibody according to claim 1, which is an IgG antibody.
The method according to any one of claims 1 to 14, wherein the antibody is any one selected from Fab, F (ab ') 2, Fv, scFV, diabody, linear antibody, and multispecific antibody. Or a functional fragment thereof.
A pharmaceutical composition comprising at least one of an antibody selected from claims 1 to 16 or a functional fragment of the antibody.
A pharmaceutical composition for treating cancer comprising at least one of an antibody selected from claims 1 to 16 or a functional fragment of the antibody.
A breast cancer, an esophageal cancer, a prostate cancer, a stomach cancer, a non-small cell lung cancer, a colon cancer, comprising at least one of an antibody selected from claims 1 to 16 or a functional fragment of the antibody, And / or a pharmaceutical composition for the treatment of glioblastoma multiforme.
A method for suppressing the growth of a tumor possessed by a mammal, by administering any one selected from claims 1 to 18.
The method for inhibiting tumor growth according to claim 19, wherein the tumor is a tumor expressing EPHA2.
A polynucleotide encoding the antibody or the functional fragment of the antibody according to any one of claims 1 to 16.
23. A host cell transformed with the polynucleotide of claim 22.
A method for producing the antibody or a functional fragment of the antibody, comprising the steps of culturing the host cell according to claim 23 and recovering the antibody or the functional fragment of the antibody from the culture solution.