WO2016171242A1 - Detection of epha2 - Google Patents

Abstract

The purpose of the present invention is to provide an antibody capable of binding to human EPHA2, a medicinal composition comprising the antibody, a diagnostic composition comprising the antibody, etc.

Description

EPHA2 detection

The present invention introduces a novel antibody, a functional fragment of the antibody, a modified form of the antibody, a nucleotide containing a base sequence encoding the amino acid sequence of the antibody, a vector having the nucleotide inserted therein, the nucleotide or a vector. The present invention relates to a method for producing the antibody, a pharmaceutical composition, a diagnostic or test composition, and the like, which comprise a step of culturing the cell.

EPHA2 is a receptor tyrosine kinase having a single transmembrane structure with a molecular weight of 130 kDa (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, Ephrin-A1 to A5 of GPI-anchored cell surface proteins are known (Non-patent Document 2). Activation of the tyrosine kinase domain of EPHA2 by ligand binding results in autophosphorylation of tyrosine residues present in the intracellular region of EPHA2, and induces intracellular signal transduction. In addition, it has been reported that EPHA2 is taken into cells by endocytosis after binding to a ligand and finally degraded by proteasome (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 (Non-Patent Documents 4 and 5). , 6, 7, 8, 9, 10, 11). Furthermore, in esophageal cancer, a significant correlation has been observed between EPHA2 expression and local lymph node metastasis, the number of lymph node metastases, and the low degree of differentiation of cancer, and the survival rate of EPHA2 positive patients is EPHA2 negative. It has been reported that it is lower than that of patients (Non-patent Document 5). In non-small cell lung cancer, the expression level of EPHA2 is higher in patients less than 5 years compared to patients whose disease-free survival is longer than 5 years, and those who relapse have higher expression levels of EPHA2 than those who do not relapse, In patients who have progressed to brain metastasis, it has been reported that the expression level of EPHA2 is higher than those in patients who have no recurrence or in patients whose metastasis is confined to the contralateral lung (Non-patent Document 9). It has been reported that the expression level of EPHA2 also significantly correlates with liver metastasis, lymphatic vessel invasion and clinical stage in colorectal cancer (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. (Non-Patent Document 4) and cancer cells have been reported to be more invasive to the extracellular matrix (Non-Patent Documents 12 and 13). Conversely, suppression of EPHA2 expression with siRNA suppresses cancer cell invasion, anchorage-independent growth, in vivo tumor growth (Non-Patent Documents 13 and 14), and ligand Ephrin-A1 EPHA2 is activated using a fusion protein of human and human IgG Fc region, and the degradation of EPHA2 by endocytosis suppresses the invasion, anchorage-independent growth, and tubular morphogenesis of cancer cells (Non-Patent Documents 4, 11, and 12) have been reported.

On the other hand, EPHA2 has been reported to be expressed not only in cancer cells but also in blood vessels in or around tumors (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 luminal 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 (Non-patent document 17). .

From the above, it is suggested that EPHA2 may be an excellent therapeutic target for cancer, and some monoclonal antibodies against EPHA2 have actually been acquired and clinical trials are being conducted (Patent Documents 1 to 3).

From these facts, the provision of a method capable of detecting the expression of EPHA2 is useful for testing or diagnosis of diseases related to EPHA2, such as cancer, and EPHA2 expression.

Many monoclonal antibodies that recognize human EPHA2 are known, but there are few that can be applied to immunohistochemical staining, and monoclonal antibodies that can be immunostained by recognizing modified EPHA2 fixed with formalin. As such, only clones D7 and B2D6 (Non-patent Document 18) are known.

International Publication No. WO2003 / 094859 Pamphlet International Publication No. WO2006 / 084226 Pamphlet International Publication No. WO2009 / 028639 Pamphlet

Molecular and Cellular Biology, 1990, Vol. 10, p. 6316-6324 Annual Review of Neuroscience, 1998, Vol. 21, p. 309-345 Molecular Cancer Research, 2002, Volume 1, p. 79-87 Cancer Research, 2001, Vol. 61, p. 2301-2306 International Journal of Cancer, 2003, Vol. 103, p. 657-663 The Prostate, 1999, vol. 41, p. 275-280 American Journal of Pathology, 2003, Volume 163, p. 2271-2276 Cancer Science, 2005, Vol. 96, p. 42-47 Clinical Cancer Research, 2003, Vol. 9, p. 613-618 Oncology Reports, 2004, Volume 11, p. 605-611 Molecular Cancer Research, 2005, Volume 3, p. 541-551 Biochemical and Biophysical Research Communications, 2004, 320, p. 1096-1102 Oncogene, 2004, Vol. 23, p. 1448-1456 Cancer Research, 2002, 62, p. 2840-2847 Oncogene, 2000, Vol. 19, p. 6043-6052 Journal of Cell Science, 2004, Vol. 117, p. 2037-2049 Oncogene, 2002, Vol. 21, p. 7011-7026 Prostate, 1999, 41, p. 275-280

One object of the present invention is to provide an antibody against EPHA2.

Another object of the present invention is to provide a diagnostic or test composition containing an anti-EPHA2 antibody.

The subject of the present invention also includes a nucleotide encoding the amino acid sequence of the antibody, a vector into which the nucleotide has been inserted, a cell into which the nucleotide or vector has been introduced, and a step of culturing the cell. Methods etc. are included.

Furthermore, another object of the present invention is to provide a pharmaceutical composition and a treatment method.

The inventor diligently studied to solve the above problems, found out that a novel anti-EPHA2 antibody was created, and EPHA2 could be detected using the antibody, thereby completing the present invention. That is, the present invention includes the following inventions.
(1) The heavy chain sequence includes a variable region having CDRH1, CDRH2, and CDRH3, the CDRH1 is composed of an amino acid sequence represented by SEQ ID NO: 60, the CDRH2 is composed of an amino acid sequence represented by SEQ ID NO: 61, and the CDRH3 Consists of the amino acid sequence shown in SEQ ID NO: 62;
The light chain sequence includes a variable region having CDRL1, CDRL2, and CDRL3, wherein the CDRL1 is composed of an amino acid sequence represented by SEQ ID NO: 63, the CDRL2 is composed of an amino acid sequence represented by SEQ ID NO: 64, and the CDRL3 is represented by SEQ ID NO: Consisting of the amino acid sequence shown in 65; and specifically binding to EPHA2 shown in SEQ ID NO: 2;
An antibody or an antigen-binding fragment of the antibody,
(2) A heavy chain variable region sequence consisting of amino acid residues 20 to 137 of the amino acid sequence shown in SEQ ID NO: 51 and a light chain variable consisting of amino acid residues 20 to 128 of the amino acid sequence shown in SEQ ID NO: 54 The antibody or antigen-binding fragment of the antibody according to (1), which comprises a region sequence;
(3) A heavy chain variable region sequence consisting of amino acid residues 20 to 137 of the amino acid sequence shown in SEQ ID NO: 51 and a light chain variable consisting of amino acid residues 20 to 128 of the amino acid sequence shown in SEQ ID NO: 54 The antibody or antigen-binding fragment of the antibody according to (1) or (2), comprising a region sequence, wherein the isotype of the heavy chain constant region is IgG1;
(4) The heavy chain sequence includes a variable region having CDRH1, CDRH2, CDRH 3, wherein CDRH1 consists of an amino acid sequence shown in SEQ ID NO: 66, CDRH2 consists of an amino acid sequence shown in SEQ ID NO: 67, CDRH3 consists of the amino acid sequence shown in SEQ ID NO: 68;
The light chain sequence includes a variable region having CDRL1, CDRL2, and CDRL3. The CDRL1 is composed of an amino acid sequence represented by SEQ ID NO: 69, the CDRL2 is composed of an amino acid sequence represented by SEQ ID NO: 70, and the CDRL3 is represented by SEQ ID NO: Consisting of the amino acid sequence shown in 71; and specifically binding to EPHA2 shown in SEQ ID NO: 2;
An antibody or an antigen-binding fragment of the antibody,
(5) A heavy chain variable region sequence consisting of amino acid residues 20 to 138 of the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable consisting of amino acid residues 20 to 133 of the amino acid sequence shown in SEQ ID NO: 59 The antibody or antigen-binding fragment of the antibody according to (4), comprising a region sequence,
(6) A heavy chain variable region sequence consisting of amino acid residues 20 to 138 of the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable consisting of amino acid residues 20 to 133 of the amino acid sequence shown in SEQ ID NO: 59 The antibody or antigen-binding fragment of the antibody according to (4) or (5), comprising a region sequence, wherein the isotype of the heavy chain constant region is IgG2b,
(7) The antigen of the antibody according to any one of (1), (2), (4) or (5) selected from the group consisting of Fab, F (ab ′) 2, Fab ′ and Fv Binding fragment,
(8) The antibody according to any one of (1), (2), (4) or (5), which is scFv,
(9) A composition comprising the antibody according to any one of (1) to (8) or an antigen-binding fragment of the antibody,
(10) A tissue specimen comprising the antibody according to any one of (1) to (8) or an antigen-binding fragment of the antibody, which has been subjected to paraffin embedding and then deparaffinized (hereinafter simply referred to as “specimen”) .) The composition according to (9), which is used for a method of detecting or measuring EPHA2 in
(11) Used in a method for detecting or measuring EPHA2 in a specimen, comprising a step of contacting the test specimen with the antibody or antigen-binding fragment of the antibody according to any one of (1) to (8). The composition according to claim (9) or (10),
(12) When EPHA2 is detected or measured in the test specimen, EPHA2 is detected or measured in the test specimen, or the expression level or expression level of EPHA2 in the test specimen is equal to or higher than a predetermined standard The test specimen is determined to be positive, and EPHA2 is not detected or measured in the test specimen, or the expression level or expression level of EPHA2 in the test specimen is equal to or lower than a predetermined criterion The composition according to (11), comprising a step of determining that the test specimen is negative,
(13) The composition according to any one of (9) to (12), which is used in a test or diagnostic method for an EPHA2-positive disease,
(14) The subject from which the test specimen determined to be positive in the detection or measurement of EPHA2 by the test or diagnostic method for EPHA2-positive disease is an antibody that specifically binds to EPHA2, or an antigen-binding fragment of the antibody The subject from which the test specimen determined to be suitable for the treatment or prevention method of EPHA2-positive disease including the step of administering an antibody and specifically determined to be negative is an antibody that specifically binds to EPHA2, or antigen binding of the antibody The composition according to (13), comprising determining that it is not suitable for a method of treating or preventing an EPHA2-positive disease comprising a step of administering a sex fragment
(15) The composition according to (13) or (14), wherein the EPHA2-positive disease is EPHA2-positive cancer,
(16) A pharmaceutical composition comprising an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody, which is administered to the subject according to any one of (i) to (iii) below:
(I) A subject derived from a test specimen in which EPHA2 is detected or measured using the composition according to any one of (9) to (11) or (13);
(Ii) A subject derived from a test specimen determined to be positive in the detection or measurement of EPHA2 using the composition according to (12);
(Iii) Suitable for treatment or prevention of an EPHA2-positive disease comprising a step of administering an antibody that specifically binds to EPHA2 or an antigen-binding fragment thereof using the composition according to (14) or (15) Subjects determined to be,
(17) A polynucleotide encoding the antibody according to any one of (1) to (8) or an antigen-binding fragment of the antibody,
(18) A vector comprising the polynucleotide according to (17),
(19) A cell comprising the polynucleotide according to (17) or the vector according to (18),
(20) The method for producing the monoclonal antibody or antigen-binding fragment of the antibody according to any one of (1) to (8), comprising the following steps (i) and (ii):
(I) culturing the cell according to (19);
(Ii) recovering a monoclonal antibody or an antigen-binding fragment of the antibody from the culture of step (i),
(21) The antibody or antigen-binding fragment thereof according to any one of (1) to (8), wherein the antibody is a monoclonal antibody;
(22) EPHA2 detection or measurement method comprising the following steps (i) or (i) and (ii):
(I) contacting the test specimen with the antibody according to any one of (1) to (8) or the antigen-binding fragment of the antibody or the composition according to (9);
(Ii) if EPHA2 is detected or measured in the test specimen, or if the expression level or expression level of EPHA2 in the test specimen is equal to or higher than a predetermined criterion, If it is judged positive and EPHA2 is not detected or measured in the test specimen, and the expression level or expression level of EPHA2 in the test specimen is equal to or lower than a predetermined criterion, the test specimen Determining the sample as negative,
(23) A method for identifying an individual to be administered with a pharmaceutical composition comprising an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody, comprising the following steps (i) or (i) and (ii):
(I) contacting the antibody according to any one of (1) to (8) or an antigen-binding fragment of the antibody or the composition according to (9) with a sample derived from an individual;
(Ii) if EPHA2 is detected or measured in a sample from the individual, or if the expression level or level of EPHA2 in the sample from the individual is equal to or higher than a predetermined criterion, Whether or not EPHA2 was detected or measured in the test specimen, EPHA2 was not detected or measured in the sample derived from the individual, and the expression level or expression level of EPHA2 in the sample derived from the individual was determined in advance Determining the individual as negative if equal to or less than the criteria determined in
(24) The method according to (22) or (23), which is used for a test or diagnostic method for EPHA2-positive disease,
(25) The method according to (23), which is used for a method for identifying an individual suffering from or at risk of having an EPHA2-positive disease,
(26) A pharmaceutical composition comprising an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody is administered to the subject according to any one of (i) to (iii) of (16) A method of treating an EPHA2-positive disease, comprising:
(27) A kit for examining or diagnosing an EPHA2-positive disease comprising the antibody according to any one of (1) to (8) or an antigen-binding fragment of the antibody,
and,
(28) The method according to (24) to (26) or the kit according to (27), wherein the EPHA2-positive disease is EPHA2-positive cancer,
Etc.

Expression of Gene Transfected into 293F Cells Gray dotted line and gray solid line indicate the binding of isotype control antibody and anti-FLAG antibody to 293F cells transfected with pFLAG-GW, respectively. The black dotted line and the black solid line indicate the binding of the isotype control antibody and the anti-FLAG antibody to 293F cells transfected with the gene shown in FIG. Evaluation of binding specificity of mouse anti-human EPHA2 antibody A201 by flow cytometry Gray dotted line and gray solid line indicate binding of isotype control antibody and A201 antibody to 293F cells transfected with pFLAG-GW, respectively. The black dotted line and the black solid line indicate the binding of the isotype control antibody and the A201 antibody to 293F cells transfected with the gene shown in FIG. Evaluation of Binding Specificity of Mouse Anti-Human EPHA2 Antibody A205 by Flow Cytometry Gray dotted line and gray solid line indicate the binding of isotype control antibody and A205 antibody to 293F cells transfected with pFLAG-GW, respectively. The black dotted line and the black solid line indicate the binding of the isotype control antibody and the A205 antibody to 293F cells transfected with the gene shown in FIG. 3, respectively. It is a figure which shows the EPHA2 specific binding property of the mouse | mouth anti-human EPHA2 antibodies A201 and A205 which concern on Example 5. FIG. It is an immuno-staining image by the mouse | mouth anti-human EPHA2 antibody A201 of the human pharyngeal laryngeal cancer and stomach cancer concerning Example 5. FIG. 6 is an immunostained image of mouse throat and laryngeal cancer and gastric cancer according to Example 5 with mouse anti-human EPHA2 antibody A205. It is the figure which showed the amino acid sequence (sequence number 51) of the heavy chain of mouse | mouth anti-human EPHA2 antibody A201, and the nucleotide sequence (sequence number 50) which codes this. It is the figure which showed the amino acid sequence (sequence number 54) of the light chain of the mouse | mouth anti-human EPHA2 antibody A201, and the nucleotide sequence (sequence number 53) which codes this. It is the figure which showed the amino acid sequence (sequence number 57) of the heavy chain of mouse | mouth anti-human EPHA2 antibody A205, and the nucleotide sequence (sequence number 56) which codes this. It is the figure which showed the amino acid sequence (sequence number 59) of the light chain of mouse | mouth anti-human EPHA2 antibody A205, and the nucleotide sequence (SEQ ID NO: 58) which codes this. It is the figure which showed the amino acid sequence (sequence number 60 to 65) of CDR which mouse | mouth anti-human EPHA2 antibody A201 has. It is the figure which showed the amino acid sequence (sequence number 66 to 71) of CDR which mouse anti-human EPHA2 antibody A205 has. It is a figure which shows the comparison in the performance in the immunohistochemical dyeing | staining of mouse | mouth anti-human EPHA2 antibodies A201 and A205 which concern on Example 6, and a commercially available anti-EPHA2 antibody. It is an immunostaining image by the mouse | mouth anti-human EPHA2 antibodies A201 and A205 and the commercially available anti-EPHA2 antibody of the EPHA2 non-expression 293α cell, the EPHA2 expression 293α cell, and the EPHA2 endogenous expression MDA-MB-231 cell according to Example 6. FIG. 5 is an immunostained image of mouse anti-human EPHA2 antibodies A201 and A205 of human esophageal cancer according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924). . FIG. 4 is an immunostaining image of mouse anti-EPHA2 antibodies A201 and A205 of human cervical cancer according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924). . FIG. 9 shows immunostained images of mouse colonic cancer mouse anti-EPHA2 antibodies A201 and A205 according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924). FIG. 9 shows immunostaining images of mouse gastric cancer mouse anti-EPHA2 antibodies A201 and A205 according to Example 6, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924).

1. Definitions In the present invention, “gene” means a nucleotide containing a nucleotide sequence encoding a protein amino acid or a complementary strand thereof, for example, a nucleotide containing a nucleotide sequence encoding a protein amino acid or a complementary strand thereof. Certain polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA and the like are included in the meaning of “gene”. Such a gene is a single-stranded, double-stranded, or triple-stranded nucleotide, and an assembly of a DNA strand and an RNA strand, and ribonucleotide (RNA) and deoxyribonucleotide (DNA) are mixed on a single nucleotide strand. Also included within the meaning of “gene” are double-stranded or triple-stranded nucleotides including those and such nucleotide chains. Examples of the “EPHA2 gene” of the present invention include DNA, mRNA, cDNA, cRNA and the like containing a base sequence encoding the amino acid sequence of EPHA2 protein.

In the present invention, “nucleotide” and “nucleic acid” are synonymous, and for example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer and the like are also included in the meaning of “nucleotide”. Such a nucleotide is a nucleotide composed of a single strand, a double strand, or three or more strands, and an assembly of a DNA strand and an RNA strand, and ribonucleotide (RNA) and deoxyribonucleotide (DNA) on a single nucleotide strand. Also included within the meaning of “nucleotide” are those that are intermingled and aggregates of two or more strands containing such nucleotide strands.

In the present invention, “polypeptide”, “peptide” and “protein” are synonymous.

In the present invention, “antigen” is sometimes used to mean “immunogen”.

In the present invention, “cell” includes various cells derived from individual animals, subculture cells, primary culture cells, cell lines, recombinant cells, microorganisms, and the like.

In the present invention, an antibody that recognizes EPHA2 may be referred to as an “anti-EPHA2 antibody”. Such antibodies include chimerized antibodies, humanized antibodies, human antibodies and the like.

In the present invention, the “functional fragment of an antibody” means an antibody fragment that exhibits at least a part of the function exhibited by the original antibody. Examples of the “functional fragment of an antibody” include, but are not limited to, Fab, F (ab ′) 2, scFv, Fab ′, single chain immunoglobulin and the like. Such functional fragments of antibodies are recombinant proteins produced in appropriate host cells using recombinant genes in addition to those obtained by treating full-length antibody protein molecules with enzymes such as papain and pepsin. May be.

In the present invention, the “site” to which the antibody binds, that is, the “site” recognized by the antibody means a partial peptide or a partial higher order structure on the antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site. As a site | part on EPHA2 protein which the anti- EPHA2 antibody of this invention couple | bonds or recognizes, the partial peptide or partial higher order structure etc. on EPHA2 protein can be illustrated.

It is known that there are three complementarity determining regions (CDRs) in each of the heavy and light chains of an antibody molecule. The complementarity-determining region is also called a hypervariable domain, and is located in the variable region of the heavy and light chains of an antibody and has a particularly high primary structure variability. In the primary structure of the polypeptide chain, it is usually separated at three points. In the present invention, for the complementarity determining region of an antibody, the complementarity determining region of the heavy chain is denoted as CDRH1, CDRH2, CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the complementarity determining region of the light chain is defined as the light chain amino acid. CDRL1, CDRL2, and CDRL3 are represented from the amino terminal side of the sequence. These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to be bound.

In the present invention, “antibody variant” has an amino acid sequence in which amino acids are substituted, deleted, added and / or inserted (hereinafter collectively referred to as “mutation”) in the amino acid sequence of the original antibody. And a polypeptide that binds to the EPHA2 protein of the present invention. The number of variant amino acids in such antibody variants is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9 1 to 10, 1 to 12, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, or 1 to 50. Such antibody variants are also included in the “antibody” of the present invention. *

In the present invention, “several” in “1 to several” refers to 3 to 10.

Examples of the activity / property exhibited by the antibody of the present invention include biological activity, physicochemical properties, and the like. Specifically, various biological activities, binding activity to antigens and epitopes, stability during production and storage Property, heat stability, etc. can be raised.

In the present invention, “hybridize under stringent conditions” means hybridization at 65 ° C. in a solution containing 5 × SSC, and then in an aqueous solution containing 2 × SSC-0.1% SDS. 20 minutes at 65 ° C. in an aqueous solution containing 0.5 × SSC-0.1% SDS for 20 minutes at 65 ° C. and 65 ° C. in an aqueous solution containing 0.2 × SSC-0.1% SDS Means to hybridize under conditions of washing for 20 minutes or under equivalent conditions. SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and nx SSC means n-fold concentration of SSC.

In the present invention, “cytotoxicity” refers to causing a pathological change in a cell in some form, and is not limited to direct trauma, but also includes DNA breakage, base dimer formation, chromosome breakage, It means any structural or functional damage of cells, such as damage to cell division apparatus or reduction of various enzyme activities.

In the present invention, “cytotoxic activity” means causing the above cytotoxicity. In the present invention, “antibody-dependent cytotoxic activity” refers to “antibody dependent cellular cytotoxicity (ADCC) activity”, and means an activity in which NK cells damage target cells such as tumor cells via antibodies.

In the present invention, “antibody-dependent cell-mediated phagocytic activity” refers to “antibody dependent cell phagocytosis (ADCP) activity”, which is an activity that monocytes and macrophage cells phagocytose target cells such as tumor cells via antibodies. means. Also referred to as “antibody-dependent phagocytic activity”.

In the present invention, “complement-dependent cytotoxic activity” refers to “complement dependent cytotoxicity (CDC) activity” and means an activity that complements a target cell such as a tumor cell via an antibody.

In the present invention, “cancer” and “tumor” are used interchangeably.

In the present invention, “immunohistochemistry (IHC)” means a histological (histochemical) method for detecting an antigen in a tissue specimen, and is synonymous with “immunoantibody method”. “immunostaining”) is also used interchangeably.

In the present invention, “denatured” EPHA2 means an EPHA2 molecule in a specimen fixed with formalin. EPHA2 molecules in specimens fixed with formalin and then paraffin-treated and deparaffinized are also referred to as “denatured” EPHA2.

In the present invention, “non-denatured” EPHA2 means EPHA2 in a sample not fixed with formalin. EPHA2 molecules in specimens not fixed with formalin are also referred to as “non-denaturing” EPHA2.

2. EPHA2
Is EPHA2 used in the present invention directly purified from human, non-human mammals (eg, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, etc.) or chicken T cells or mast cells? Alternatively, the cell membrane fraction of the above-mentioned cells can be prepared and used, and it can be obtained by synthesizing EPHA2 in vitro or producing it in a host cell by genetic manipulation. Specifically, in genetic manipulation, EPHA2 cDNA 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 prokaryote or eukaryote. The protein can be obtained by expressing EPHA2 by transforming a host cell of an organism.

The nucleotide sequence of human EPHA2 cDNA is registered in GenBank with accession number: NM_004431. Further, the amino acid sequence of human EPHA2 is registered in GenBank with an accession number: NP_004422.

EPHA2 cDNA is, for example, a polymerase chain reaction (hereinafter referred to as “PCR”) using a cDNA library of an organ expressing EPHA2 mRNA as a template and a primer that specifically amplifies EPHA2 cDNA (Saiki, R K., et al., Science, (1988) 239, 487-49).

In addition, a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence encoding human EPHA2 under stringent conditions and encodes a protein having biological activity equivalent to that of EPHA2, is also included in the EPHA2 cDNA. included. Furthermore, a polynucleotide encoding a splicing variant transcribed from the human EPHA2 locus or a protein that hybridizes to this under stringent conditions and also has a biological activity equivalent to that of EPHA2 is also included in the EPHA2 cDNA. included.

In addition, from the amino acid sequence of human EPHA2, or the amino acid sequence obtained by substituting, deleting, or adding 1, 2, or 3, or 4 or 5 amino acids in the amino acid sequence obtained by removing the signal sequence from these sequences Thus, a protein having a biological activity equivalent to that of EPHA2 is also included in EPHA2. Furthermore, an amino acid sequence encoded by a splicing variant transcribed from the human EPHA2 locus, or an amino acid sequence in which 1, 2 or 3 or 4 or 5 amino acids are substituted, deleted or added in the amino acid sequence EPHA2 is also included in the protein having a biological activity equivalent to that of EPHA2.

As used herein, the nucleotide sequence encoding mature human EPHA2 that does not contain a signal peptide is set forth in SEQ ID NO: 1 in the Sequence Listing, and the amino acid sequence of the human EPHA2 is set forth in SEQ ID NO: 2 in the Sequence Listing. Has been.
2. Production of anti-EPHA2 antibody The antibody against EPHA2 of the present invention is obtained by immunizing an animal with any polypeptide selected from the amino acid sequence of EPHA2 or EPHA2 using a conventional method, and collecting and purifying the antibody produced in vivo Can be obtained. The species of EPHA2 serving as an antigen is not limited to humans, and animals can be immunized with EPHA2 derived from animals other than humans such as mice and rats. In this case, an antibody applicable to a human disease can be selected by testing the cross-reactivity between the obtained antibody that binds to heterologous EPHA2 and human EPHA2. In addition, EPHA2 used as an antigen can be obtained by causing a host cell to produce the EPHA2 gene by genetic manipulation. Specifically, a vector capable of expressing the EPHA2 gene is prepared, introduced into a host cell to express the gene, and the expressed EPHA2 may be purified.

The antibody against EPHA2 of the present invention can also be obtained using a DNA immunization method. The DNA immunization method is a technique for inducing immunity to an antigen by introducing an antigen expression plasmid into an individual animal such as a mouse or a rat and expressing the antigen in the individual. Gene transfer methods include direct injection of plasmids into muscle, intravenous injection of liposomes and polyethyleneimine and other introduction reagents, viral vector methods, and gold particles with plasmids attached to them by Gene Gun. There are techniques, such as the Hydrodynamic method in which a large amount of plasmid solution is intravenously injected. As a technique for improving gene expression by intramuscular injection of an expression plasmid, a technique called in vivo electroporation is known (Aihara H, Miyazaki J. et al.). Nat Biotechnol.1998 Sep; 16 (9): 867-70 or Mir LM, Bureau MF, Gehl J, Rangara R, Roue D, Cailaud JM, DelaureD, BranellS.Branellec. S A. 1999 Apr 13; 96 (8): 4262-7.). In this method, the amount of expression is further improved by treating the muscle with hyaluronidase before intramuscular injection of the plasmid (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ. Gene Ther. 2001 Aug; 8 (16) : 1264-70).

Also, known methods (for example, Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980). ), Hybridomas can be established by fusing antibody-producing cells that produce antibodies against EPHA2 and myeloma cells to obtain monoclonal antibodies. Specific examples of such a method are described in International Publication No. WO 09/48072 (published on April 16, 2009) and WO 10/11711 (published on October 14, 2010).

Examples of the mouse anti-human EPHA2 antibody thus established include A201 antibody and A205 antibody. The amino acid sequence of the A201 antibody heavy chain is shown in SEQ ID NO: 51 of the Sequence Listing, and the nucleotide sequence encoding it is shown in SEQ ID NO: 50 of the Sequence Listing. The amino acid sequence of the A201 antibody light chain is shown in SEQ ID NO: 54 of the sequence listing, and the nucleotide sequence encoding it is shown in SEQ ID NO: 53 of the sequence listing. The amino acid sequence of the A205 antibody heavy chain is shown in SEQ ID NO: 57 in the sequence listing, and the nucleotide sequence encoding this is shown in SEQ ID NO: 56 in the sequence listing. The amino acid sequence of the A205 antibody light chain is shown in SEQ ID NO: 59 in the sequence listing, and the nucleotide sequence encoding it is shown in SEQ ID NO: 58 in the sequence listing.

The antibody of the present invention may be an antibody that retains all six CDR sequences derived from A201 and has an activity of binding to EPHA2. That is, the heavy chain variable region of the antibody of the present invention is represented by CDRH1 (DTYVH) consisting of the amino acid sequence shown in SEQ ID NO: 60, CDRH2 (RIDPANANTKYDPKFQG) consisting of the amino acid sequence shown in SEQ ID NO: 61, and SEQ ID NO: 62. It possesses CDRH3 (YGKSAWFY) consisting of an amino acid sequence. The light chain variable region of the antibody comprises CDRL1 (RASQDIGNYLN) consisting of the amino acid sequence shown in SEQ ID NO: 63, CDRL2 (YTSRLHS) consisting of the amino acid sequence shown in SEQ ID NO: 64, and amino acids shown in SEQ ID NO: 65. It has CDRL3 (QQGHSLPPT) consisting of the sequence. The amino acid sequences of these CDRs are also shown in FIG.

The antibody of the present invention may be an antibody that retains all six CDR sequences derived from A205 and has an activity of binding to EPHA2. That is, the heavy chain variable region of the antibody of the present invention is shown in CDRH1 (DYSMN) consisting of the amino acid sequence shown in SEQ ID NO: 66, CDRH2 (WINTYTGEPTYADDFKG) consisting of the amino acid sequence shown in SEQ ID NO: 67, and SEQ ID NO: 68. It possesses CDRH3 (PISLLLRLDY) consisting of an amino acid sequence. In addition, the light chain variable region of the above-mentioned antibody has CDRL1 (RSSQSLVHSNGNTYLH) consisting of the amino acid sequence shown in SEQ ID NO: 69, CDRL2 (KVSNRFS) consisting of the amino acid sequence shown in SEQ ID NO: 70, and amino acids shown in SEQ ID NO: 71. It possesses CDRL3 (SQSTHVPWT) consisting of the sequence. The amino acid sequences of these CDRs are also shown in FIG.
The antibody of the present invention specifically recognizes EPHA2 protein. In other words, preferred antibodies of the present invention specifically bind to the EPHA2 protein.
In certain embodiments, suitable antibodies specifically bind to both undenatured human EPHA2 and denatured human EPHA2 in formalin-fixed specimens. More preferable antibodies include antibodies that specifically bind to both non-denatured human EPHA2 and denatured human EPHA2 in formalin-fixed specimens and that do not specifically bind to other Eph families. However, it is not limited to them.

In addition to the above monoclonal antibody against EPHA2, the antibodies of the present invention include genetically modified antibodies that have been artificially modified for the purpose of reducing heteroantigenicity against humans, such as chimeric antibodies, or humans. Also included are humanized antibodies. These antibodies can be produced using known methods.

Examples of the chimeric antibody include antibodies in which the variable region and the constant region of the antibody are different from each other, for example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is joined to a human-derived constant region (Proc. Natl. Acad). Sci.U.S.A., 81, 6851-6855, (1984)).

The above-described chimeric antibody sequence against EPHA2 can be artificially modified for the purpose of, for example, reducing the heterologous antigenicity to humans, thereby producing a humanized antibody that is a recombinant antibody. The antibody of the present invention includes an antibody obtained by modifying the CDR of the humanized antibody. These antibodies can be produced using known methods.

As humanized antibodies, antibodies (see Nature (1986) 321, p.522-525) in which only complementarity determining regions (CDRs) are incorporated into human-derived antibodies (see Nature (1986) 321, p.522-525), some sequences in addition to CDR sequences Examples of the amino acid residues of the framework include an antibody grafted on a human antibody (International Publication No. WO90 / 07861 pamphlet).

It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced by cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)). It is known that two amino acid residues of glycine and lysine at the chain carboxyl terminal are deleted, and a proline residue located at the carboxyl terminal is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). ). However, deletion and modification of these heavy chain sequences do not affect the antigen-binding ability and effector functions (such as complement activation and antibody-dependent cytotoxicity) of the antibody. Accordingly, the present invention also includes antibodies having such modifications, including deletions in which one or two amino acids have been deleted at the heavy chain carboxyl terminus, and such a deletions that have been amidated (eg, at the carboxyl terminus site). Heavy chain in which a proline residue is amidated). However, as long as the antigen binding ability and the effector function are maintained, the carboxyl-terminal deletion of the heavy chain of the antibody according to the present invention is not limited to the above type. The two heavy chains constituting the antibody according to the present invention may be either one of the full length and the heavy chain selected from the group consisting of the above-mentioned deletion forms, or a combination of any two of them. It may be a thing. The amount ratio of each deletion can be influenced by the type and culture conditions of the cultured mammalian cells that produce the antibody according to the present invention, but the main component of the antibody according to the present invention is a carboxyl in both two heavy chains. A case where one terminal amino acid residue is deleted can be mentioned.

The antibody obtained by the above method can be evaluated for binding to an antigen and a suitable antibody can be selected. An example of another index for comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is a method that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is an indicator of good relative structural stability of proteins. The difference in thermal stability can be compared by measuring the Tm value using DSC and comparing the values. It is known that the storage stability of an antibody shows a certain degree of correlation with the thermal stability of the antibody (Lori Burton, et. Al., Pharmaceutical Development and Technology (2007) 12, p.265-273), and heat. A suitable antibody can be selected using stability as an index. Other indicators for selecting antibodies include high yields in appropriate host cells and low aggregation in aqueous solutions. For example, since the antibody with the highest yield does not always exhibit the highest thermal stability, it is necessary to select the most suitable antibody based on a comprehensive judgment based on the above-described indicators.

Also known is a method of obtaining single chain immunoglobulins by linking the full length sequences of antibody heavy and light chains using an appropriate linker (Lee, HS, et. al., Molecular Immunology (1999) 36, p. 61-71; Shirrmann, T. et. al., mAbs (2010), 2, (1) p. By dimerizing such a single-chain immunoglobulin, it is possible to retain a structure and activity similar to those of an antibody that is originally a tetramer. The antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence. Such antibodies are called single domain antibodies (sdAbs) or nanobodies, and are actually observed in camels or llamas and reported to retain antigen-binding ability. (Muyldemans S. et.al., Protein Eng. (1994) 7 (9), 1129-35, Hamers-Casterman C. et.al., Nature (1993) 363 (6428) 446-8). The above-described antibody can also be interpreted as a kind of antigen-binding fragment of the antibody in the present invention.

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. Specific examples of the antibody gene include a combination of a gene encoding the heavy chain sequence of the antibody described herein and a gene encoding the light chain sequence. When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into separate expression vectors. is there. 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 strains (Urlauub, G. and Chasin, LA Proc. Natl. Acad. Sci. U.). S. A. (1980) 77, p. 4126-4220). Moreover, when using a prokaryotic cell, Escherichia coli and Bacillus subtilis can be mentioned, for example. An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro. In the above culture method, the yield may vary depending on the sequence of the antibody. From the antibodies having equivalent binding activity, those that can be easily produced as pharmaceuticals can be selected using the yield as an index.

There is no limitation as to the isotype of the antibody of the present invention, and examples include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE, preferably IgG or IgM, More preferably, IgG1 or IgG2 can be mentioned.

The antibody of the present invention may be an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof. A fragment of the antibody can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin, or modifying the antibody gene by a genetic engineering technique and expressing it in an appropriate cultured cell. Among such antibody fragments, a fragment that retains all or part of the functions of the full-length antibody molecule can be called an antigen-binding fragment of an antibody. Antibody functions generally include antigen-binding activity, activity that neutralizes antigen activity, activity that enhances antigen activity, antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, and complement-dependence Mention may be made of cellular cytotoxic activity. The function retained by the antigen-binding fragment of the antibody in the present invention is the binding activity to EPHA2.

For example, antibody fragments include Fab, F (ab ′) 2, Fv, or single chain Fv (scFv), diabodies (diabodies), linear antibody in which heavy and light chain Fvs are linked by an appropriate linker. And multispecific antibodies formed from antibody fragments. 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 antibody fragment.

Furthermore, the antibody of the present invention may be a multispecific antibody having specificity for at least two different antigens. Usually, such a molecule binds to two types of antigens (ie, bispecific antibodies), 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 multispecific antibody of the present invention may be a full-length 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).

The antibody of the present invention may be a single chain antibody (also referred to as scFv). A single chain antibody is obtained by linking an antibody heavy chain variable region and a light chain variable region with a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore, edited by Springer Verlag, New). York, p. 269-315 (1994), Nature Biotechnology (2005), 23, p. 1126-1136) In addition, a BiscFv fragment produced by linking two scFvs with a polypeptide linker is used as a bispecific antibody. It can also be used.

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 variable region and the light chain variable 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 variable region and the light chain variable region in scFv may be derived from the same antibody or different antibodies. As the polypeptide linker that links the variable regions, for example, any single chain peptide consisting of 12 to 19 residues is used.

The DNA encoding the scFv is the DNA encoding the heavy chain or heavy chain variable region of the antibody, and the DNA encoding the light chain or light chain variable region. Amplification is performed by PCR using a coding DNA portion as a template and a primer pair defining both ends thereof, and then the DNA encoding the polypeptide 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 a host after obtaining and expressing the gene in the same manner as described above.

The antibody of the present invention may be one that has been increased in quantity and has increased affinity for the antigen. The antibody that multiplies may be one type of antibody or a plurality of antibodies that recognize multiple epitopes of the same antigen. Examples of the method for increasing the amount of antibody include binding of IgG CH3 domain to two scFvs, binding to streptavidin, and introduction of helix-turn-helix motif.

The antibody of the present invention 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.

The antibody of the present invention may be one in which these antibody and another drug form a conjugate (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 column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Stratesies) for Protein Purification and Characterization: A Laboratory 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 ( 988)) it is not intended to be limited thereto.

Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. These chromatography can be performed using liquid chromatography, such as HPLC and FPLC. Examples of the column used for affinity chromatography include a protein A column and a protein G column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F.R. F. (Pharmacia) and the like. It is also possible to purify an antibody using a carrier on which an antigen is immobilized, utilizing the binding property to the antigen.

3. Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising an anti-EPHA2 antibody or a functional fragment thereof or a modified form thereof.

The pharmaceutical composition of the present invention can be used in various diseases (hereinafter referred to as “induced or exacerbated” by EPHA2 signal abnormality or enhancement by EPHA2 or its ligand overexpression or EPHA2 mutation or gene amplification, or by switching of EPHA2 isoforms). , Referred to as “diseases associated with EPHA2”), especially for the treatment or prevention of various cancers.

Causes of the induction or exacerbation of cancer that is the subject of treatment or prevention include single nucleotide substitution (SNP) in the intron of the EPHA2 gene, high expression of EPHA2, a missense mutation that constantly activates EPHA2, and the EPHA2 gene Amplification or overexpression, switching of EPHA2 isoform, etc. can be exemplified.

Examples of such cancer types include lung cancer such as breast cancer, endometrial cancer, ovarian cancer, non-small cell lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colon cancer, esophageal cancer, bladder cancer, uterus. Cervical cancer, hematological cancer, lymphoma, malignant melanoma and the like can be mentioned, and those cancers expressing the EPHA2 protein can be mentioned preferably.

In the present invention, for the treatment or prevention of a disease, the onset of such a disease, preferably the onset of such a disease in an individual expressing the EPHA2 protein, the suppression or inhibition of progression or progression, and the individual suffering from such a disease Examples include, but are not limited to, alleviation of one or more symptoms present, suppression or remission of progression or progression, treatment or prevention of secondary diseases, and the like.

The pharmaceutical composition of the present invention comprises a therapeutically or prophylactically effective amount of an anti-EPHA2 antibody or a functional fragment of the antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Can be contained.

“Therapeutically or prophylactically effective amount” means an amount that exhibits a therapeutic or prophylactic effect for a specific disease, administration form, and administration route, and is synonymous with “pharmacologically effective amount”.

The pharmaceutical composition of the present invention has pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability of the composition or antibody contained therein, solubility, sustained release, absorbability, penetration. Substances for changing, maintaining, and maintaining properties, dosage forms, strength, properties, shapes, etc. (hereinafter referred to as “substances for formulation”) can be included. The substance for the preparation is not particularly limited as long as it is a pharmacologically acceptable substance. For example, non-toxicity or low toxicity is a property that a substance for preparation preferably comprises.

Substances for formulation include, for example, amino acids, antibacterial agents, antioxidants, buffers, fillers, chelating agents, complexing agents, bulking agents, monosaccharides, disaccharides, carbohydrates, coloring agents, flavoring agents, diluents , Emulsifier, hydrophilic polymer, preservative, solvent, sugar alcohol, suspending agent, surfactant, stabilization enhancer, elasticity enhancer, transport agent, diluent, excipient, and / or pharmaceutical adjuvant The amount of these substances to be added is 0.001 to 1000 times, preferably 0.01 to 100 times, more preferably the weight of the anti-EPHA2 antibody or functional fragment or modified product thereof. Is 0.1 to 10 times.

A pharmaceutical composition containing an anti-EPHA2 antibody or a functional fragment thereof or a modified product thereof in an liposome, or an antibody modified product (US Pat. No. 6,214,388, etc.) in which an antibody and a liposome are bound is also present. Included in the pharmaceutical composition of the invention.

The excipient or carrier is usually liquid or solid, and is not particularly limited as long as it is a substance used for water for injection, physiological saline, artificial cerebrospinal fluid, and other preparations for oral administration or parenteral administration. . Examples of the physiological saline include neutral ones and those containing serum albumin.

Examples of the buffer include Tris buffer prepared so that the final pH of the pharmaceutical composition is 7.0 to 8.5, acetate buffer prepared so as to be 4.0 to 5.5, and 5. Examples thereof include a citrate buffer prepared to be 0 to 8.0, a histidine buffer prepared to be 5.0 to 8.0, and the like.

The pharmaceutical composition of the present invention is a solid, liquid, suspension or the like. Freeze-dried preparations can be mentioned. An excipient such as sucrose can be used to mold the lyophilized preparation.

The administration route of the pharmaceutical composition of the present invention may be any of enteral administration, topical administration and parenteral administration. For example, intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration Administration, transdermal administration, intraosseous administration, intraarticular administration and the like can be mentioned.

The composition of such a pharmaceutical composition can be determined according to the administration method, the EPHA2 protein binding affinity of the antibody, and the like. The higher the affinity of the anti-EPHA2 antibody of the present invention or the functional fragment thereof or the modified product thereof for the EPHA2 protein (the lower the KD value), the more effective the drug can be exerted.

The dose of the anti-EPHA2 antibody of the present invention is not limited as long as it is a pharmacologically effective amount, and the species of the individual, the type of disease, the symptom, sex, age, prevalence, EPHA2 protein binding affinity of the antibody or Although it can be appropriately determined depending on the biological activity and other factors, it is usually 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg once every 1 to 180 days, or 1 It can be administered twice or more times a day.

The form of the pharmaceutical composition includes injections (including lyophilized preparations and infusions), suppositories, nasal absorption preparations, transdermal absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosols. Examples thereof include pills, pills, powders, suspensions, emulsions, eye drops, and implantable preparations.

A pharmaceutical composition comprising an anti-EPHA2 antibody or a functional fragment thereof or a modified form thereof as an active ingredient can be administered simultaneously with or separately from other drugs. For example, after administering another pharmaceutical, a pharmaceutical composition containing an anti-EPHA2 antibody or a functional fragment of the antibody as an active ingredient is administered, or after administering such a pharmaceutical composition, another pharmaceutical is administered, or You may administer the said pharmaceutical composition and another pharmaceutical simultaneously. Examples of other medicaments include various anticancer agents such as chemotherapeutic agents and radiotherapy. These are collectively referred to as “the combined use of the antibody of the present invention with another drug”, and a pharmaceutical composition containing an additional drug in addition to the antibody of the present invention, a functional fragment thereof or a modified form thereof is also included in the present invention.

The present invention relates to a method for treating or preventing a disease associated with EPHA2, such as cancer, the use of the antibody of the present invention for preparing a pharmaceutical composition for treating or preventing the disease, and the present invention for treating or preventing the disease. Of the use of antibodies. A therapeutic or prophylactic kit containing the antibody of the present invention is also included in the present invention.

4). Diagnostic Composition A diagnostic or diagnostic composition (hereinafter collectively referred to as “diagnostic composition”) comprising the anti-EPHA2 antibody of the present invention or a functional fragment thereof or a modified form thereof is provided.

The diagnostic composition of the present invention is useful for testing or diagnosis of diseases related to EPHA2, such as cancer, and EPHA2 expression. The examination or diagnosis in the present invention includes, for example, determination or measurement of morbidity risk, determination of the presence or absence of morbidity, measurement of the degree of progression or deterioration, measurement or determination of the effect of drug treatment with a pharmaceutical composition such as an anti-EPHA2 antibody. In addition, measurement or determination of the effect of treatment other than drug treatment, measurement of recurrence risk, determination of the presence or absence of recurrence, and the like are included, but the test or diagnosis is not limited thereto.

The diagnostic composition of the present invention is useful for identification of an individual to which the antibody of the present invention or a functional fragment thereof or a modified product thereof, a composition containing them, or a pharmaceutical composition containing them is administered.

Such a diagnostic composition may contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, a preservative, a developer, a sensitizer, an aggregation inhibitor, and the like.

The present invention relates to a method for testing or diagnosing a disease associated with EPHA2, such as cancer, use of the antibody of the present invention for preparing a diagnostic composition for the disease, use of the antibody of the present invention for testing or diagnosing the disease , Also provide. A test or diagnostic kit containing the antibody of the present invention is also included in the present invention.

A sandwich ELISA is preferable as a test or diagnostic method including the antibody of the present invention, but a normal ELISA method, RIA method, ELISPOT (Enzyme-Linked ImmunoSpot) method, dot blot method, octalony method, CIE (Counterimmunoelectrophoresis) method, CLIA Detection methods using antibodies such as (Chemiluminescent immunoassay) and FCM (Flow Cytometry) can be used. As a labeling method for antibodies, in addition to biotin, labeling methods that can be used for biochemical analysis such as labels for fluorophores such as HRP, alkaline phosphatase, FITC and ALEXA, and radioisotopes can be used. For detection using an enzyme label, TMB (3,3 ′, 5,5′-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indoyl phosphate), ρ-NPP (ρ-nitrophenyl phosphate), OPD Fluorescent substrates such as (O-Phenylenediamine), ABTS (3-Ethylbenzothiazine-6-sulfonic acid), SuperSignal ELISA Pico Chemiluminescent Substrate ^TM (Sermo Fisher Scientific) Substrate In addition, it is possible to use chemiluminescent substrate. For this measurement, in addition to a sample derived from a human or non-human animal, a sample to which an artificial treatment such as a recombinant protein has been added can also be used. Examples of test samples derived from living organisms include, but are not limited to, blood, joint fluid, ascites, lymph, cerebrospinal fluid, tissue homogenate supernatant, tissue section, and the like.

The test or diagnostic sandwich ELISA kit containing the antibody of the present invention may contain an EPHA2 protein standard solution, a coloring reagent, a dilution buffer, a solid phase antibody, a detection antibody, a washing solution, and the like. As a method for measuring the amount of antibody bound to the antigen, an absorption method, a fluorescence method, a luminescence method, an RI (Radioisotope) method or the like is preferably applied. An RI liquid scintillation counter or the like is preferably used.

In addition to the immunohistological test described above, a soluble protein is prepared from cells, tissues or organs or a part thereof in a sample according to a conventional method, and the soluble protein is reacted with a labeled antibody in the soluble protein. It can also be used in Western blotting or dot blotting to confirm the presence or absence of EPHA2.

The present invention provides antibodies useful for immunohistochemistry (IHC) analysis, functional fragments thereof and modifications thereof, and compositions containing them. Such a composition is also included in the “diagnostic composition” of the present invention.

Immunohistochemistry is not particularly limited as long as it is a technique in which a tissue section is reacted with an antibody (primary antibody) that binds to an antigen to detect the primary antibody bound to the antigen.
Tissue sections are preferably paraffin-embedded after formalin fixation. After embedding in paraffin, the sliced tissue section is deparaffinized and then subjected to antigen activation treatment and nonspecific reaction suppression treatment. Examples of the antigen activation treatment method include heat treatment, enzyme treatment with protease, etc., and heat treatment is preferred. As conditions for the heat treatment, a temperature range of 90 to 110 ° C., a pH of 8 to 10 and a treatment time of 20 to 60 minutes are usually preferable. Tris-EDTA buffer (for example, 10 mM Tris buffer containing 1 mM EDTA) or the like can be used for pH adjustment. As the nonspecific reaction suppression treatment, a method of inactivating an endogenous enzyme having a catalytic activity similar to or similar to the enzyme used for color development is usually used. When color is developed by a peroxidase reaction, it is preferable to inhibit endogenous peroxidase existing in the tissue in advance with H ₂ O ₂ or the like. As the H ₂ O ₂ solvent, water, methanol or the like can be used, and the concentration of H ₂ O ₂ is 0.1 to 3%, preferably 0.3 to 3%. Sodium azide can be added to the H ₂ O ₂ solution. A method of blocking with serum or casein can also be used as a nonspecific reaction suppression treatment. Serum and casein can treat tissues before the primary antibody reaction, but can also be included in a solvent that dilutes the primary antibody.
The reaction conditions for the primary antibody are not particularly limited, but the temperature is 4 to 50 ° C, preferably 20 to 37 ° C, more preferably 24 ° C. The reaction time is 5 minutes to 1 day, preferably 10 minutes to 4 hours, more preferably 30 minutes to 1 hour.

For detection of the primary antibody, an antibody that can be visualized and binds to the primary antibody (secondary antibody) can be preferably used. In some cases, the reaction is performed three or more times using an antibody (tertiary antibody) that binds to the secondary antibody itself. The secondary or tertiary antibody can be visualized by binding an enzyme such as peroxidase or alkaline phosphatase to these antibodies, or by adding biotin or the like to these antibodies and binding to the aforementioned enzymes such as streptavidin. And a method of reacting a chromogenic substrate corresponding to those enzymes can be preferably used. Examples of a method for binding an enzyme to a secondary antibody or a tertiary antibody include a method using a reagent in which a large number of the enzyme and secondary antibody are bound to a dextrin polymer or an amino acid polymer (polymer method). In a method of reacting a biotinylated secondary antibody and peroxidase-labeled streptavidin (LSAB method), DAB or the like can be used as a chromogenic substrate. A secondary antibody labeled with a fluorescent dye or the like can also be used. When treated with a fluorescently labeled secondary antibody, positive cells are detected using a fluorescence microscope after the treatment.

In the smear method, the isolated cells are applied to glass or separated by a centrifugal separator, divided into cell components and liquid components, and immunostaining is performed on the cell components. That is, cell components can be applied on a slide glass and fixed with an ethanol solution or a 10% formalin solution, and then immunostaining similar to a tissue section can be performed.

In the freezing embedding method, the excised tissue is rapidly frozen with liquid nitrogen after embedding with an OCT compound and sliced with a cryostat to prepare a slide specimen. After fixing this specimen with 10% formalin or ethanol solution, immunostaining similar to the tissue section can be performed.

The operation related to immunohistochemistry can be performed automatically by programming the reaction solution, reaction conditions, number of washings, etc., and incorporating it into the immune device.

In the case of diagnostic imaging, label the antibody with a pharmaceutically acceptable radionuclide or illuminant, administer the antibody to a subject, take an image using diagnostic imaging techniques such as PET / CT, and then the presence of EPHA2 Can be determined or inspected.

The antibody, functional fragment thereof or modified product thereof contained in the diagnostic composition of the present invention is preferably an antibody that binds to or recognizes EPHA2, that is, an antibody having EPHA2 selectivity, a functional fragment thereof, or a modified product thereof.

Examples of antibodies having human EPHA2 selectivity include heavy chain CDRH1 to CDRH3 of the mouse A201 antibody (amino acid sequence shown in SEQ ID NOs: 60 to 62 (FIG. 11)), and light chain CDRL1 to CDRRL3 ( An antibody comprising a light chain comprising the amino acid sequence shown in SEQ ID NO: 63 to 65 (FIG. 11), a heavy chain variable region of the mouse A201 antibody (amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 51) and light Examples include an antibody containing a chain variable region (amino acid sequence shown at amino acid numbers 20 to 128 of SEQ ID NO: 54), an antibody containing the heavy and light chains of mouse A201 antibody, and the like. Examples of such antibodies include mouse A201 antibody and chimeric A201 antibody, but are not limited thereto.

Antibodies having human EPHA2 selectivity include heavy chain CDRH1 to CDRH3 (amino acid sequence shown in SEQ ID NOs: 66 to 68 (FIG. 12)) of mouse A205 antibody, and light chain CDRL1 to CDRRL3 ( An antibody comprising a light chain comprising the amino acid sequence shown in SEQ ID NOs: 69 to 71 (FIG. 12), a heavy chain variable region of the mouse A205 antibody (amino acid sequence shown in amino acids 20 to 138 of SEQ ID NO: 57), and Examples include an antibody containing a light chain variable region (amino acid sequence shown in amino acid numbers 20 to 133 of SEQ ID NO: 59), an antibody containing the heavy chain and light chain of mouse A205 antibody, and the like. Examples of such antibodies include, but are not limited to, mouse A205 antibody and chimeric A205 antibody.

In one preferred embodiment of the present invention, the diagnostic composition is for EPHA2 detection or measurement.

The present invention provides a method for detecting or measuring human EPHA2 in a test sample.

The diagnostic composition of the present invention can be used for these detection or measurement methods. Such measurement methods and diagnostic compositions are also included in the present invention for diagnosis or testing of human EPHA2-positive cancer.

A method for identifying an individual to which the pharmaceutical composition of the present invention is administered is also encompassed by the present invention. In such an identification method, human EPHA2 in the sample derived from the individual is measured, and human EPHA2 is detected in the sample, or compared with the amount of human EPHA2 detected in the sample derived from a healthy individual. If many human EPHA2s are detected, the individual can be determined as positive.

In the method, the diagnostic composition of the present invention can be used.

Also, in a preferred embodiment of such an identification method, the individual is suffering from or at risk of having cancer.

Furthermore, in one aspect, the pharmaceutical composition of the present invention can be administered to an individual who has been determined to be positive by such an identification method.

5. Reagent The antibody of the present invention or a functional fragment thereof or a modified form thereof is also useful as a reagent. Such reagents are used in the above-described examination or diagnostic, research and other applications.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples. In the following examples, unless otherwise specified, each operation relating to gene manipulation is “Molecular Cloning” (Sambrook, J., Fritsch, EF, and Maniatis, T., Cold Spring Harbor Laboratory Press. (Published in 1989) or when using commercially available reagents and kits, they were used according to the instructions for commercially available products.

Example 1 Production of Mouse Anti-Human EPHA2 Antibody 1) -1 Immunization Fcgr2b KO (BALB / c) Homo mouse female (Immuno-Biological Laboratories) was used for immunization. A mixture of Recombinant Human EPHA2 (R & D SYSTEM; 3035-A2) and Freund's Complete Adjuvant (Wako Pure Chemical Industries) was collected subcutaneously and used for hybridoma production.

1) -2 Hybridoma preparation Spleen cells and mouse myeloma SP2 / 0-Ag14 cells (ATCC: CRL-1581) were cell-fused using PEG4000 (Immuno-Biological Laboratories) or Hybridene Hybrid Production System (Cyto Pulse Sciences). The cells were diluted with ClonaCell-HY Selection Medium D (StemCell Technologies) and cultured. Monoclonal hybridomas were prepared by collecting the emerged hybridoma colonies. Each collected hybridoma colony was cultured, and the resulting hybridoma culture supernatant was used to screen for anti-EPHA2 antibody-producing hybridomas.

(Example 2) Evaluation of antigen-binding ability of mouse anti-human EPHA2 antibody 2) -1 Construction of vector expressing human EPHA2 and protein having sequence similarity to human EPHA2 2) -1-1 Construction of expression vector pFLAG- Reading frame of Gateway Vector Conversion System (Life Technologies, Thermo Fisher Scientific) on blind-ended HindIII and BglII sites of myc-CMV19 (Sigma). This vector was named “pFLAG-GW”.

2) -1-2 Construction of a vector expressing EPHA2 A DNA fragment (SEQ ID NO: 1) encoding human EPHA2 without a signal sequence was used as a primer set 5'-GGGGGACAAGTTTGTACAAAAAAAGCAGGCTCAGCGCAGGGGCCAAGGAAG-3 '(EPHA2-Fw: SEQ ID NO: 3)
5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTCAGATGGGGATCCCCCAC-3 ′ (EPHA2-Re: SEQ ID NO: 4)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 (Life Technologies, Thermo Fisher Scientific) by BP recombination reaction. The gene encoding EPHA2 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHA2. The amino acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2.

2) -1-3 Construction of a vector expressing EPHA3 A DNA fragment (SEQ ID NO: 5) encoding human EPHA3 without a signal sequence was used as a primer set 5′-GGGGGACAAGTTTGTACAAAAAAAGCAGGCTCAGAACTGATCTCCAGCACT-3 ′ (EPHA3-Fw: SEQ ID NO: 7)
5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTTACACGGGAACTGGGGCC-3 ′ (EPHA3-Re: SEQ ID NO: 8)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, the gene encoding EPHA3 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHA3. The amino acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 6.

2) -1-4 Construction of a vector expressing EPHA4 A DNA fragment (SEQ ID NO: 9) encoding human EPHA4 without a signal sequence was used as a primer set 5′-GGGGGACAAGTTTGTACAAAAAAAGCAGGCTTCAGTCACGGGTTCGCGGTA-3 ′ (EPHA4-Fw: SEQ ID NO: 11)
5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTCAGACGGGAACCATTTCTG-3 ′ (EPHA4-Re: SEQ ID NO: 12)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, a gene encoding EPHA4 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHA4. The amino acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 10.

2) -1-5 Construction of a vector that expresses EPHA5 A DNA fragment (SEQ ID NO: 13) encoding human EPHA5 without a signal sequence was used as a primer set 5'-GGGGGACAAGTTTGTACAAAAAAAGCAGCCTTCAGCCCCAGCAACGAAGGTGAATTATTTGG-3 '(EPHA5-Fw: SEQ ID NO: 15)
5'-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTCATACAGGCACCATTCCGTTTACCAG-3 '(EPHA5-Re: SEQ ID NO: 16)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, a gene encoding EPHA5 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHA5. The amino acid sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 14.

2) -1-6 Construction of a vector expressing EPHA6 The DNA fragment of human EPHA6 (SEQ ID NO: 17) was used as a primer set 5′-GGGGGACAAGTTTGTACAAAAAAAGCAGCCTCATGGCCAGGCGAACTGCAG-3 ′ (EPHA6-Fw: SEQ ID NO: 19)
5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTCATACATAGAATCCCTTC-3 ′ (EPHA6-Re: SEQ ID NO: 20)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Next, GeneArt (registered trademark) Site-directed Mutagenesis System (Life Technologies, Thermo Fisher Scientific) and primer set 5′-TCCAACAACACAGTGTGTCTCTGATAC-3 ′ (No.)
5′-CAACACACTACTTGGTTGTTGGATGAGCCCTGCTTTTTG-3 ′ (EPHA6-Mut-Re: SEQ ID NO: 22)
The bases 1 to 24 of the nucleic acid sequence shown in SEQ ID NO: 17 were deleted from this vector. Then, the gene encoding EPHA6 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHA6. The amino acid sequence of SEQ ID NO: 17 is shown in SEQ ID NO: 18.

2) -1-7 Construction of a vector expressing EPHA7 A DNA fragment (SEQ ID NO: 23) encoding human EPHA7 without a signal sequence was used as a primer set 5′-GGGGGACAAGTTTGTACAAAAAACAGCGCTCACAGGCTGGCGAAGGAAGTAC-3 ′ (EPHA7-Fw: SEQ ID NO: 25)
5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTCACCACTGAATGCCAGTTC-3 ′ (EPHA7-Re: SEQ ID NO: 26)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, the gene encoding EPHA7 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHA7. The amino acid sequence of SEQ ID NO: 23 is shown in SEQ ID NO: 24.

2) -1-8 Construction of a vector expressing EPHB1 A DNA fragment (SEQ ID NO: 27) encoding human EPHB1 without a signal sequence was used as a primer set 5′-GGGGGACAAGTTTGTACAAAAAAAGCAGCCTCAATGGGAAGAAACGTTAATGGAC-3 ′ (EPHB1-Fw: SEQ ID NO: 29)
5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTCATGCCATGCCGTTGG-3 ′ (EPHB1-Re: SEQ ID NO: 30)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, the gene encoding EPHB1 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHB1. The amino acid sequence of SEQ ID NO: 27 is shown in SEQ ID NO: 28.

2) -1-9 Construction of a vector that expresses EPHB3 A DNA fragment (SEQ ID NO: 31) encoding human EPHB3 without a signal sequence was used as a primer set 5′-GGGGGACAAGTTTGTACAAAAAAAGGCGCTCACTGGGAAGAGACCCTCATG-3 ′ (EPHB3-Fw: SEQ ID NO: 33)
5′-GGGGACCACTTTGTTACAAGAAAGCTGGGTCTCAGACCTGCACAGGGCAG-3 ′ (EPHB3-Re: SEQ ID NO: 34)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, a gene encoding EPHB3 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHB3. The amino acid sequence of SEQ ID NO: 31 is shown in SEQ ID NO: 32.

2) -1-10 Construction of a vector expressing EPHB4 A DNA fragment (SEQ ID NO: 35) encoding human EPHB4 without a signal sequence was used as a primer set 5'-GGGGGACAAGTTTGTACAAAAAAAGCAGCCTCATTGGAAGAGACCCTGCTG-3 '(EPHB4-Fw: SEQ ID NO: 37)
5'-GGGGACCACTTTGTTACAAGAAAGCTGGGTCTCAGTACTGCGGGGCCG-3 '(EPHB4-Re: SEQ ID NO: 38)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, a gene encoding EPHB4 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-EPHB4. The amino acid sequence of SEQ ID NO: 35 is shown in SEQ ID NO: 36.

2) -1-11 Construction of a vector that expresses PTPRS isoform 2 A DNA fragment (SEQ ID NO: 39) encoding human PTPRS isoform 2 (hereinafter referred to as PTPRS-2) without a signal sequence was used as a primer set 5'-GGGACAAGTTTGTACAAAAAAAGCAGCCTTCGAGT 3 ′ (PTPRS-2 / 4-Fw: SEQ ID NO: 41)
5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTTAGGTTGCATAGTGTCAAAG-3 ′ (PTPRS-2 / 4-Re: SEQ ID NO: 42)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, the gene encoding PTPRS-2 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-PTPRS-2. The amino acid sequence of SEQ ID NO: 39 is shown in SEQ ID NO: 40.

2) -1-12 Construction of a vector expressing PPTRS isoform 4 A DNA fragment (SEQ ID NO: 43) encoding human PTPRS isoform 4 (hereinafter referred to as PTPRS-4) without a signal sequence is shown in SEQ ID NOs: 41 and 42. Amplified by PCR using the prepared primer set. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. Then, the gene encoding PTPRS-4 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-PTPRS-4. The amino acid sequence of SEQ ID NO: 43 is shown in SEQ ID NO: 44.

2) -1-13 Construction of a vector expressing PTPRF isoform 2 A DNA fragment (SEQ ID NO: 45) encoding human PTPRF isoform 2 (hereinafter referred to as PTPRF-2) without a signal sequence was used as a primer set 5′-GGGGGACAAGTTTGTACAAAAAATAGCAGCCTACCTGCTAGCAAACTCTGTCT 3 ′ (PTPRF-2-Fw: SEQ ID NO: 47)
5′-GGGGACCACTTTTGTACAAGAAAGCTGGGTCTTACGTTGCATAGTGGTCAAAGCTG-3 ′ (PTPRF-2-Re: SEQ ID NO: 48)
And was amplified by PCR. This PCR fragment was transferred to the entry vector pDONR221 by BP recombination reaction. The gene encoding PTPRF-2 was transferred from this entry vector to pFLAG-GW by LR recombination reaction. The resulting vector was named pFLAG-PTPRF-2. The amino acid sequence of SEQ ID NO: 45 is shown in SEQ ID NO: 46.

2) -2 IHC screening 2) -2-1 IHC screening using culture supernatants 293α cells (a stable expression cell line derived from HEK293 expressing integrin αv and integrin β3) were added to Lipofectamine 2000 (Life Technologies, Thermo Fisher). (Scientific) was used to transfect pFLAG-EPHA2 (hereinafter referred to as EPHA2-expressing 293α cells). To produce a paraffin block of EPHA2-expressing 293α cells, the cell pellet was fixed in formalin and embedded in paraffin. Untransfected 293α cells and endogenous EPHA2-expressing MDA-MB-231 cells (ATCC: No. HTB-26) were similarly embedded in paraffin after fixation with formalin. Sections are prepared from these blocks and using an automated immunostaining device Ventana Discovery ULTRA and DAB map kit (above, Roche Diagnostics) or Autostainer Link 48 and EnVisionFLEX Mini Kit (DAKO) using the instrument's instructions. IHC screening of the antibody contained in the culture supernatant derived from was performed. First, among the 67 hybridoma-derived culture supernatants obtained in Example 1) -2, 12 antibodies that react strongly with EPHA2-expressing 293α cells and do not react with non-transfected 293α cells were obtained. Selected. Further, 6 cells that react with MDA-MB-231 cells were selected, and 3 cells that strongly reacted with endogenous EPHA2 were selected.

2) -2-2 IHC Screening Using Purified Antibodies Example 2) Three antibodies selected in 2-1 were purified from the culture supernatant of hybridomas producing these antibodies, Example 2) -2-1 In the same manner as human esophageal cancer (CR2), stomach cancer (CQ2), colon cancer (CDA3), cervical cancer (CZA2), lung cancer (CCA4), pharyngeal laryngeal cancer (CH3), malignant melanoma (LM481) and commercially available paraffin sections of normal tissues (MAN, MBN, MCN) (above, SuperBioChip Laboratories) were stained. Among the three, A201 and A205 that responded strongly to tumor cells and had low background staining were selected.

2) -3 Isotype determination of antibodies The isotypes of A201 and A205, which are mouse anti-human EPHA2 antibodies, were determined by the Mouse monoclonal isotyping test kit (AbD Serotec). As a result, it was shown that the isotype of A201 is IgG1, and the isotype of A205 is IgG2b.

2) -4 Preparation of antigen gene expressing cells for flow cytometry analysis Free style 293F cells (Life Technologies, Thermo Fisher Scientific, 293F cells) were prepared at 1 × 10 ⁶ cells / mL, of which 8 mL Was seeded in Erlenmeyer Flask with Vent Cap (125 mL). pFLAG-EPHA2, and pFLAG-EPHA3, pFLAG-EPHA5, pFLAG-EPHA6, pFLAG-EPHA7, pFLAG-EPHB1, pFLAG-EPHAB3, pFLAG-EPHB4, pFLAG-PTPRS-2, which encode proteins having sequence similarity to EPHA2. pFLAG-PTPRS-4, pFLAG-PTPRF-2 and pFLAG-GW as controls were transfected into 293F cells using 293fectin (Life Technologies, Thermo Fisher Scientific), 37 ° C., 125 rpm, 8% CO 2, respectively. Cultured overnight under ₂ conditions. The next day, these 293F cells were collected, washed, and then used for flow cytometry analysis.

2) -5 Evaluation of binding ability of mouse anti-human EPHA2 antibody by flow cytometry In order to evaluate the binding specificity of A201 and A205 to EPHA2, each cell prepared by the method shown in Example 2) -4 was treated with A201, A205, a mouse IgG control antibody (Becton Dickinson), and Anti-FLAG M2 antibody (Sigma-Aldrich) were added and incubated at 4 ° C. for 30 minutes. These cells were washed twice with PBS containing 5% FBS, Anti-Mouse IgG FITC conjugate (MP Bio) diluted 500 times with PBS containing 5% FBS was added, and incubated at 4 ° C. for 20 minutes. These cells were washed twice with PBS containing 5% FBS, then resuspended in PBS containing 5% FBS containing 2 μg / mL 7-Aminoactinomycin D (Life Technologies, Thermo Fisher Scientific), and flow site. Analysis was performed with a meter (FACS Canto II, BD Biosciences). Data analysis was performed with Flowjo. 7-Aminoactinomycin D-positive dead cells were excluded at the gate, and a histogram of FITC fluorescence intensity of live cells was prepared.

In order to confirm whether the transfected gene was expressed in 293F cells, the binding of the Anti-FLAG M2 antibody to the FLAG tag was examined. As a result, the expression of all the transfected genes (EPHA2, EPHA3, EPHA5, EPHA6, EPHA7, EPHB1, EPHB3, EPHB4, PTPRS-2, PTPRS-4, and PTPRF-2) was confirmed (FIG. 1). . A201 showed binding to EPHA2. Although it binds slightly to EPHB1, other molecules with sequence similarities to EPHA2 (EPHA3, EPHA5, EPHA6, EPHA7, EPHB3, EPHB4, PTPRS-2, PTPRS-4, and PTPRF-2) are rare. It was shown not to bind (FIG. 2). A205 binds to EPHA2, while for molecules with sequence similarity to EPHA2 (EPHA3, EPHA5, EPHA6, EPHA7, EPHAB1, EPHAB3, EPHB4, PTPRS-2, PTPRS-4, and PTPRF-2) It was shown not to bind (Figure 3). A205 also bound to cells transfected with the negative control pFLAG-GW, which was presumed to be due to A205 binding to EPHA2 that is endogenously expressed in 293F cells. .

(Example 3) Determination of nucleotide sequence of cDNA encoding mouse anti-human EPHA2 antibody

3) -1 Determination of nucleotide sequence of cDNA encoding heavy chain and light chain fragment of mouse anti-human EPHA2 antibody A201

3) -1-1 Preparation of total RNA from A201-producing hybridoma Total RNA was prepared from A201-producing hybridoma using TRIzol Reagent (Ambion).

3) -1-2 Synthesis of cDNA (5′-RACE-Ready cDNA) cDNA (5′-1 cDNA) using 1 μg of total RNA prepared in Example 3) -1-1 and SMARTER RACE cDNA Amplification Kit (Clontech) -RACE-Ready cDNA) was synthesized.

3) -1-3 Amplification of cDNA encoding heavy chain fragment of A201 by 5′-RACE PCR and determination of nucleotide sequence As a primer for PCR amplification of cDNA encoding heavy chain fragment of A201, UPM (Universal Primer A Mix: attached to SMARTER RACE cDNA Amplification Kit) and 5′-CATCCCCAGGGTCACCATGGAGTTTAGTTGG-3 ′ (mG1VR1: SEQ ID NO: 49) were used. mG1VR1 was designed from the sequence of the constant region of mouse heavy chain (IgG1) in the database.

Using these primers and the cDNA synthesized in Example 3) -1-2 (5'-RACE-Ready cDNA) as a template, cDNA encoding the heavy chain fragment of A201 was amplified by 5'-RACE PCR. This PCR was performed using a touch-down PCR program according to the manual of SMARTER RACE cDNA Amplification Kit using KOD-Plus- (TOYOBO) as Polymerase.

A cDNA encoding a heavy chain fragment amplified by 5'-RACE PCR is purified using MinElute PCR Purification Kit (QIAGEN), then cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and cloned cDNA Was sequenced.

As a sequencing primer,
5'-CATCCCCAGGGTCACCCATGGAGTTTAGTTTG-3 '(mG1VR1: SEQ ID NO: 49) and NUP (Nested Universal Primer A: attached to SMARTER RACE cDNA Amplification Kit) were used.

The sequencing reaction was performed with GeneAmp 9700 (Applied Biosystems), and the DNA sequencing was performed with ABI PRISM 3700 DNA Analyzer (Applied Biosystems) or Applied Biosystems 3730xxl Analyzer (Applied Biosystems).

The nucleotide sequence of the cDNA encoding the determined heavy chain of A201 is shown in SEQ ID NO: 50, and the amino acid sequence is shown in SEQ ID NO: 51. The amino acid sequence consisting of the 1st to 19th amino acid residues of SEQ ID NO: 51 corresponds to the signal sequence of the A201 heavy chain, and the amino acid sequence consisting of the 20th to 137th amino acid residues corresponds to the heavy chain variable region of A201. SEQ ID NO: 50 and SEQ ID NO: 51 are also shown in FIG.

3) -1-4 Amplification of cDNA encoding the light chain fragment of A201 by 5'-RACE PCR and determination of nucleotide sequence As a primer for PCR amplification of the cDNA encoding the light chain fragment of A201, UPM (Universal) Primer A Mix: attached to SMARTER RACE cDNA Amplification Kit) and 5′-AGTCCAACTGTTCAGGAGCCCATTTTGTCG-3 ′ (mKVR2: SEQ ID NO: 52) were used. In addition, mKVR2 was designed from the sequence of the constant region of the mouse light chain in the database.

Using these primers and the cDNA synthesized in Example 3) -1-2 as a template (5'-RACE-Ready cDNA), cDNA encoding the light chain fragment of A201 was amplified by 5'-RACE PCR. This PCR was performed using a touch-down PCR program according to the manual of SMARTER RACE cDNA Amplification Kit using KOD-Plus- (TOYOBO) as Polymerase.

A cDNA encoding a light chain fragment amplified by 5'-RACE PCR is purified using MinElute PCR Purification Kit (QIAGEN), then cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and cloned cDNA Was sequenced.

As a sequencing primer,
5'-AGTCCAACTGTTCAGGACGCCCATTTGTCG-3 '(mKVR2: SEQ ID NO: 52) and NUP (Nested Universal Primer A: attached to SMARTER RACE cDNA Amplification Kit) were used.

The sequencing reaction was performed with GeneAmp 9700 (Applied Biosystems), and the DNA sequencing was performed with ABI PRISM 3700 DNA Analyzer (Applied Biosystems) or Applied Biosystems 3730xxl Analyzer (Applied Biosystems).

The determined nucleotide sequence of cDNA encoding the light chain of A201 is shown in SEQ ID NO: 53 of the Sequence Listing, and the amino acid sequence is shown in SEQ ID NO: 54. The amino acid sequence consisting of the 1st to 19th amino acid residues of SEQ ID NO: 54 corresponds to the signal sequence of the A201 light chain, and the amino acid sequence consisting of the 20th to 128th amino acid residues corresponds to the light chain variable region of A201. SEQ ID NO: 53 and SEQ ID NO: 54 are also shown in FIG.

3) -2 Determination of nucleotide sequence of cDNA encoding heavy and light chain fragments of mouse anti-human EPHA2 antibody A205 3) -2-1 Preparation of total RNA from A205 producing hybridoma Example 3) from A205 producing hybridoma Total RNA was prepared in the same manner as in 1-1.

3) -2-2 Synthesis of cDNA (5′-RACE-Ready cDNA) The cDNA (5′-RACE-Ready cDNA) was prepared using 1 μg of the total RNA prepared in Example 3) -2-1. 3) Synthesis was performed in the same manner as in 1-2.

3) -2-3 Amplification of cDNA encoding heavy chain fragment of A205 by 5′-RACE PCR and determination of nucleotide sequence Example 3) cDNA synthesized in 2-2 (5′-RACE-Ready cDNA) Using the template, the cDNA encoding the heavy chain fragment of A205 was amplified and sequenced in the same manner as in Example 3) -1-3.

However, as primers for amplifying the cDNA of the heavy chain fragment of A205 by PCR, UPM (attached to Universal Primer A Mix: SMARTER RACE cDNA Amplification Kit), and 5′-TGCACCACTGCTGGACAGGGATCCAGAGTTC-3 ′ (mG2bVR) Was used. In addition, mG2bVR2 was designed from the sequence of the constant region of the mouse heavy chain (IgG2b) in the database.

As a sequencing primer,
5′-TGCACACTGCTGGACAGGGGATCCAGAGTTC-3 ′ (mG2bVR2: SEQ ID NO: 55) and NUP (Nested Universal Primer A: attached to SMARTER RACE cDNA Amplification Kit) were used.

The nucleotide sequence of the cDNA encoding the determined heavy chain of A205 is shown in SEQ ID NO: 56 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 57. The amino acid sequence consisting of the 1st to 19th amino acid residues of SEQ ID NO: 57 corresponds to the signal sequence of the A205 heavy chain, and the amino acid sequence consisting of the 20th to 138th amino acid residues corresponds to the heavy chain variable region of A205. SEQ ID NO: 56 and SEQ ID NO: 57 are also shown in FIG.

3) -2-4 Amplification of cDNA encoding light chain fragment of A205 by 5'-RACE PCR and determination of nucleotide sequence Example 3) cDNA synthesized in 2-2 (5'-RACE-Ready cDNA) Using the template, the cDNA encoding the light chain fragment of antibody A205 was amplified and sequenced in the same manner as in Example 3) -1-4.

The nucleotide sequence of the determined cDNA encoding the light chain of A205 is shown in SEQ ID NO: 58 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 59. The amino acid sequence consisting of the 1st to 19th amino acid residues of SEQ ID NO: 59 corresponds to the signal sequence of the A205 light chain, and the amino acid sequence consisting of the 20th to 133rd amino acid residues corresponds to the light chain variable region of A205. SEQ ID NO: 58 and SEQ ID NO: 59 are also shown in FIG.

(Example 4) Production of mouse anti-human EPHA2 antibody Mouse anti-human EPHA2 antibodies A201 and A205 were purified from the hybridoma culture supernatant.

First, A201 and A205-producing hybridomas were grown to a sufficient amount with ClonCell-HY Selection Medium E, and then Ultra Low IgG FBS (Life Technologies, ThermoFisher Scifi) was added to 20% of the world's TiMeMeSriMiMeFriSriMeMriFiSriMiSriFiSriMiSriMiSriMiSriFiSriMiSriFlM. The medium was changed to Fisher Scientific) and cultured for 5 days. The main culture supernatant was collected and sterilized through a 0.45 μm filter.

The antibody was purified from the above hybridoma supernatant by Protein G affinity chromatography (under 4-6 ° C.) in a one-step process. The buffer substitution step after protein G affinity chromatography purification was performed at 4 to 6 ° C. First, the culture supernatant of the hybridoma was applied to a column packed with Protein G (GE Healthcare Bioscience) equilibrated with 0.2 M sodium phosphate buffer (pH 7.0). After all of the culture supernatant liquid entered the column, the column was washed with a sodium phosphate buffer having a column volume of 2 times or more. Next, elution was performed with 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7), and fractions containing the antibody were collected. 1 M Tris-HCl (pH 9.0) was added to the collected fractions to adjust the pH to 7.0 to 7.5, and then 0% was obtained using Amicon Ultra 4 (fractionated molecular weight UF50K, Merck Millipore, 4 to 6 ° C.). 0.02% NaN ₃ / PBS was substituted and concentrated, and the antibody concentration was adjusted to 0.3 mg / mL or more to prepare a purified sample.

(Example 5) Immunohistochemical staining with mouse anti-human EPHA2 antibody FIG. 4 shows the specificity of the mouse anti-EPHA2 antibody in immunohistochemical staining. The Eph family molecules shown in FIG. 4 were transiently expressed in 293F cells, and the pellets of these cells were embedded in paraffin after formalin fixation. Sections prepared from these blocks were subjected to antigen activation treatment with Tris-EDTA solution (DAKO) at 97 ° C for 40 minutes, endogenous peroxidase (HRP) blocking reagent (DAKO) 5 minutes, nonspecific protein adsorption blocking reagent (DAKO) After reacting in order of 30 minutes, mouse anti-EPHA2 antibody A201 or A205 prepared to a concentration of 2.5 μg / mL with an antibody diluent (DAKO) was reacted for 1 hour. Immunohistochemical staining was performed by a conventional method using an HRP-labeled universal secondary antibody (DAKO) and DAB chromogenic substrate (DAKO). Microscopic observation confirmed that both antibodies strongly bound to EPHA2-expressing cells and recognized EPHA2. A201 was also slightly bound to EPHA4-expressing cells, but it was considered to be a level that was not a problem in human tissues because it was a very weak cross reaction with forced expression cells.

FIG. 5 shows the results of immunohistochemical staining with mouse anti-EPHA2 antibody A201. A commercially available human micropharyngeal cancer and gastric cancer tissue microarray (SuperBioChips Laboratories) was subjected to antigen activation treatment with a Tris-EDTA solution (DAKO) at 97 ° C. for 40 minutes, and an endogenous peroxidase (HRP) blocking reagent (DAKO) After reacting in the order of 5 minutes and non-specific protein adsorption blocking reagent (DAKO) for 30 minutes, mouse anti-EPHA2 antibody A201 prepared to a concentration of 5 μg / mL with an antibody diluent (DAKO) was reacted for 1 hour. Immunohistochemical staining was performed by a conventional method using an HRP-labeled universal secondary antibody (DAKO) and DAB chromogenic substrate (DAKO). Reactions other than the antigen activation treatment were performed at room temperature. FIG. 5 is a photomicrograph showing specific staining of part of the cell membrane and cytoplasm of tumor cells of laryngeal squamous cell carcinoma and gastric adenocarcinoma by mouse anti-EPHA2 antibody A201. There is no staining in the tissues and cells of the tumor stroma. Tumor tissue and stromal tissue can be distinguished by morphological differences.

FIG. 6 shows the results of immunohistochemical staining with mouse anti-EPHA2 antibody A205. A commercially available human micropharyngeal cancer and gastric cancer tissue microarray (SuperBioChips Laboratories) was subjected to an antigen activation treatment with a Tris-EDTA solution (DAKO) at 97 ° C. for 40 minutes, and an endogenous HRP blocking reagent (DAKO) 5 minutes. After reacting in order of non-specific protein adsorption blocking reagent (DAKO) for 30 minutes, mouse anti-EPHA2 antibody A205 prepared to a concentration of 15 μg / mL with an antibody diluent (DAKO) was reacted for 1 hour. Immunohistochemical staining was performed by an ordinary method using an anti-mouse linker antibody (DAKO), an HRP-labeled universal secondary antibody (DAKO), and a DAB chromogenic substrate (DAKO). Reactions other than the antigen activation treatment were performed at room temperature. FIG. 6 is a photomicrograph showing specific staining of the cell membrane and part of the cytoplasm of tumor cells of laryngeal squamous cell carcinoma and gastric adenocarcinoma by the mouse anti-EPHA2 antibody A205. There is no staining in the tissues and cells of the tumor stroma. Tumor tissue and stromal tissue can be distinguished by morphological differences.

The same site is stained with mouse anti-EPHA2 antibodies A201 and A205, but A201 is more sensitive than A205, and more tumor cells are stained.

(Example 6) Immunohistochemical staining with mouse anti-human EPHA2 antibody FIGS. 13 and 14 show the staining conditions and staining properties of the anti-EPHA2 antibody in immunohistochemical staining. EPHA2 non-expressing 293α cells, EPHA2 overexpressing 293α cells, and EPHA2 endogenously expressed MDA-MB-231 cell pellets were embedded in paraffin after formalin fixation. Sections prepared from these blocks were subjected to antigen activation treatment with Tris-EDTA solution (DAKO or Roche Diagnostics) at 97 ° C. for 36 to 40 minutes, and endogenous peroxidase (HRP) blocking, non-blocking was performed by a conventional method. After carrying out specific protein adsorption blocking, each anti-EPHA2 antibody prepared to the density | concentration optimized by preliminary examination (FIG. 13) was made to react for 1 hour. For detection, immunohistochemical staining was carried out by the LSAB method or the polymer method using a kit of Roche Diagnostics or DAKO. By microscopic observation, a positive positive reaction was observed in the cell membrane or cytoplasm, and a positive positive reaction was observed when a weak positive reaction was observed. Both A201 and A205 antibodies show strong positive reaction to both EPHA2 overexpressing 293α cells and EPHA2 endogenously expressed MDA-MB-231 cells, and recognize EPHA2 with higher sensitivity than various commercially available EPHA2 antibodies compared. Was confirmed (FIG. 13). Of the various commercially available EPHA2 antibodies examined, the goat anti-EPHA2 polyclonal antibody (AF3035) from R & D Systems was judged to have the highest sensitivity and specificity. FIG. 14 is a photomicrograph of cells. In all antibodies, no positive reaction is observed in EPHA2 non-expressing 293α cells, whereas in EPHA2 overexpressing 293α cells, a strong positive reaction is observed in the cell membrane and cytoplasm. For MDA-MB-231 cells, the mouse anti-EPHA2 antibodies A201 and A205, and R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035) strongly reacted with the cell membrane, Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924) and A weak positive reaction is observed in the cell membrane by the Cell Signaling Technology rabbit anti-EPHA2 monoclonal antibody (6997). In addition, in the mouse anti-EPHA2 antibody (D4) manufactured by Millipore, high non-specific staining was observed throughout the cells.

15 to 18 show the results of immunohistochemical staining with mouse anti-EPHA2 antibodies A201 and A205, R & D Systems goat anti-EPHA2 polyclonal antibody (AF3035), and Santa Cruz rabbit anti-EPHA2 polyclonal antibody (sc-924). A commercially available human tumor tissue microarray (SuperBioChips Laboratories) was subjected to antigen activation treatment with a Tris-EDTA solution (DAKO) at 97 ° C. for 40 minutes, blocked in the same manner as described above, and then diluted with an antibody diluent (DAKO). Each anti-EPHA2 antibody diluted in 1) was reacted for 1 hour. Immunohistochemical staining was performed by a conventional method using an HRP-labeled universal secondary antibody (DAKO) or an HRP-labeled anti-goat IgG antibody (Nichirei Bioscience) or a DAB chromogenic substrate (DAKO). For the A205 antibody, an anti-mouse linker antibody (DAKO) was reacted before the reaction of the HRP-labeled universal secondary antibody. 15 to 18 are micrographs of tumor tissues, which are stained images of human esophageal cancer (FIG. 15), cervical cancer (FIG. 16), colon cancer (FIG. 17), and stomach cancer (FIG. 18). In any cancer, A201 antibody and A205 antibody showed specific staining in the cell membrane and part of cytoplasm of tumor cells, and A201 was more sensitive than A205, and more tumor cells were stained. Yes. There is little or no specific staining with the AF3035 and sc-924 antibodies. The A201 antibody, A205 antibody, and AF3035 antibody show no staining in the tissues and cells of the tumor stroma, but the sc-924 antibody also shows a cross reaction in the non-tumor cells of the tumor stroma. Tumor tissue and stromal tissue can be distinguished by morphological differences.

The use of the antibody provided by the present invention enables examination or diagnosis of various cancers.

SEQ ID NO: 1: Nucleotide sequence encoding human EPHA2 SEQ ID NO: 2: Amino acid sequence of human EPHA2 SEQ ID NO: 3: PCR primer EPHA2-Fw
SEQ ID NO: 4: PCR primer EPHA2-Re
SEQ ID NO: 5: nucleotide sequence encoding human EPHA3 SEQ ID NO: 6: amino acid sequence of human EPHA3 SEQ ID NO: 7: PCR primer EPHA3-Fw
SEQ ID NO: 8: PCR primer EPHA3-Re
SEQ ID NO: 9: nucleotide sequence encoding human EPHA4 SEQ ID NO: 10: amino acid sequence of human EPHA4 SEQ ID NO: 11: PCR primer EPHA4-Fw
SEQ ID NO: 12: PCR primer EPHA4-Re
SEQ ID NO: 13: nucleotide sequence encoding human EPHA5 SEQ ID NO: 14: amino acid sequence of human EPHA5 SEQ ID NO: 15: PCR primer EPHA5-Fw
SEQ ID NO: 16: PCR primer EPHA5-Re
SEQ ID NO: 17: nucleotide sequence encoding human EPHA6 SEQ ID NO: 18: amino acid sequence of human EPHA6 SEQ ID NO: 19: PCR primer EPHA6-Fw
SEQ ID NO: 20: PCR primer EPHA6-Re
SEQ ID NO: 21: primer EPHA6-Mut-Fw
SEQ ID NO: 22: Primer EPHA6-Mut-Re
SEQ ID NO: 23: nucleotide sequence encoding human EPHA7 SEQ ID NO: 24: amino acid sequence of human EPHA7 SEQ ID NO: 25: PCR primer EPHA7-Fw
SEQ ID NO: 26: PCR primer EPHA7-Re
SEQ ID NO: 27: nucleotide sequence encoding human EPHB1 SEQ ID NO: 28: amino acid sequence of human EPHB1 SEQ ID NO: 29: PCR primer EPHB1-Fw
SEQ ID NO: 30: PCR primer EPHB1-Re
SEQ ID NO: 31: nucleotide sequence encoding human EPHB3 SEQ ID NO: 32: amino acid sequence of human EPHB3 SEQ ID NO: 33: PCR primer EPHB3-Fw
SEQ ID NO: 34: PCR primer EPHB3-Re
SEQ ID NO: 35: nucleotide sequence encoding human EPHB4 SEQ ID NO: 36: amino acid sequence of human EPHB4 SEQ ID NO: 37: PCR primer EPHB4-Fw
SEQ ID NO: 38: PCR primer EPHB4-Re
SEQ ID NO: 39: nucleotide sequence encoding human PTPRS-2 SEQ ID NO: 40: amino acid sequence of human PTPRS-2 SEQ ID NO: 41: PCR primer PTPRS-2 / 4-Fw
SEQ ID NO: 42: PCR primer PTPRS-2 / 4-Re
SEQ ID NO: 43: nucleotide sequence encoding human PTPRS-4 SEQ ID NO: 44: amino acid sequence of human PTPRS-4 SEQ ID NO: 45: nucleotide sequence encoding human PTPRF-2 SEQ ID NO: 46: amino acid sequence of human PTPRF-2 47: PCR primer PTPRF-2-Fw
SEQ ID NO: 48: PCR primer PTPRF-2-Re
SEQ ID NO: 49: PCR primer mG1VR1 (for A201 heavy chain variable region)
SEQ ID NO: 50: nucleotide sequence encoding A201 antibody heavy chain SEQ ID NO: 51: amino acid sequence of A201 antibody heavy chain SEQ ID NO: 52: PCR primer mKVR2 (for A201, A205 light chain variable region)
SEQ ID NO: 53: nucleotide sequence encoding A201 antibody light chain SEQ ID NO: 54: amino acid sequence of A201 antibody light chain SEQ ID NO: 55: PCR primer mG2bVR2 (for A205 heavy chain variable region)
SEQ ID NO: 56: nucleotide sequence encoding A205 antibody heavy chain SEQ ID NO: 57: amino acid sequence of A205 antibody heavy chain SEQ ID NO: 58: nucleotide sequence encoding A205 antibody light chain SEQ ID NO: 59: amino acid sequence sequence number of A205 antibody light chain 60: A201 antibody CDRH1 amino acid sequence SEQ ID NO: 61: A201 antibody CDRH2 amino acid sequence SEQ ID NO: 62: A201 antibody CDRH3 amino acid sequence SEQ ID NO: 63: A201 antibody CDRRL1 amino acid sequence SEQ ID NO: 64: A201 antibody CDRRL2 Amino acid sequence of SEQ ID NO: 65: amino acid sequence of CDR1 of antibody A201 SEQ ID NO: 66: amino acid sequence of CDRH1 of antibody A205 SEQ ID NO: 67: amino acid sequence of CDRH2 of antibody A205 SEQ ID NO: 68: amino acid of CDRH3 of antibody A205 SEQ SEQ ID NO 69: A 205 antibodies of CDRL1 amino acid sequence SEQ ID NO 70: A 205 antibodies of CDRL2 amino acid sequence SEQ ID NO 71: A 205 CDRL3 amino acid sequence of the antibody

Claims (28)

The heavy chain sequence includes a variable region having CDRH1, CDRH2, CDRH3, the CDRH1 is composed of the amino acid sequence represented by SEQ ID NO: 60, the CDRH2 is composed of the amino acid sequence represented by SEQ ID NO: 61, and the CDRH3 is represented by SEQ ID NO: Consisting of the amino acid sequence shown in 62;
The light chain sequence includes a variable region having CDRL1, CDRL2, and CDRL3, wherein the CDRL1 is composed of an amino acid sequence represented by SEQ ID NO: 63, the CDRL2 is composed of an amino acid sequence represented by SEQ ID NO: 64, and the CDRL3 is represented by SEQ ID NO: Consisting of the amino acid sequence shown in 65; and specifically binding to EPHA2 shown in SEQ ID NO: 2;
Or an antigen-binding fragment of the antibody. A heavy chain variable region sequence consisting of amino acid residues 20 to 137 of the amino acid sequence shown in SEQ ID NO: 51 and a light chain variable region sequence consisting of amino acid residues 20 to 128 of the amino acid sequence shown in SEQ ID NO: 54 The antibody or antigen-binding fragment of the antibody according to claim 1, which comprises the antibody. A heavy chain variable region sequence consisting of amino acid residues 20 to 137 of the amino acid sequence shown in SEQ ID NO: 51 and a light chain variable region sequence consisting of amino acid residues 20 to 128 of the amino acid sequence shown in SEQ ID NO: 54 The antibody or antigen-binding fragment of the antibody according to claim 1 or 2, wherein the heavy chain constant region isotype is IgG1. The heavy chain sequence includes a variable region having CDRH1, CDRH2, CDRH3, the CDRH1 is composed of an amino acid sequence represented by SEQ ID NO: 66, the CDRH2 is composed of an amino acid sequence represented by SEQ ID NO: 67, and the CDRH3 is represented by SEQ ID NO: Consisting of the amino acid sequence shown in 68;
The light chain sequence includes a variable region having CDRL1, CDRL2, and CDRL3. The CDRL1 is composed of an amino acid sequence represented by SEQ ID NO: 69, the CDRL2 is composed of an amino acid sequence represented by SEQ ID NO: 70, and the CDRL3 is represented by SEQ ID NO: Consisting of the amino acid sequence shown in 71; and specifically binding to EPHA2 shown in SEQ ID NO: 2;
Or an antigen-binding fragment of the antibody. A heavy chain variable region sequence consisting of amino acid residues 20 to 138 of the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable region sequence consisting of amino acid residues 20 to 133 of the amino acid sequence shown in SEQ ID NO: 59; The antibody or antigen-binding fragment of the antibody according to claim 4, wherein the antibody or antigen-binding fragment of the antibody is contained. A heavy chain variable region sequence consisting of amino acid residues 20 to 138 of the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable region sequence consisting of amino acid residues 20 to 133 of the amino acid sequence shown in SEQ ID NO: 59; The antibody or antigen-binding fragment of the antibody according to claim 4 or 5, wherein the heavy chain constant region isotype is IgG2b. 6. The antigen-binding fragment of an antibody according to any one of claims 1, 2, 4 or 5, selected from the group consisting of Fab, F (ab ') 2, Fab' and Fv. 6. Antibody according to any one of claims 1, 2, 4 or 5, characterized in that it is a scFv. A composition comprising the antibody or antigen-binding fragment of the antibody according to any one of claims 1 to 8. EPHA2 in a tissue specimen (hereinafter simply referred to as “specimen”) comprising the antibody according to any one of claims 1 to 8 or an antigen-binding fragment of the antibody, which has been subjected to paraffin embedding and then deparaffinization. The composition according to claim 9, which is used in a method for detecting or measuring the above. 9. A method for detecting or measuring EPHA2 in a specimen, comprising the step of contacting the test specimen with the antibody or antigen-binding fragment of the antibody according to any one of claims 1 to 8. Or the composition according to 10. If EPHA2 is detected or measured in a test sample, or the expression level or expression level of EPHA2 in the test sample is equal to or higher than a predetermined standard, If the test specimen is determined to be positive and EPHA2 is not detected or measured in the test specimen, or the expression level or expression level of EPHA2 in the test specimen is equal to or lower than a predetermined criterion, The composition of Claim 11 including the process of determining a test sample as negative. The composition according to any one of claims 9 to 12, which is used in a test or diagnostic method for an EPHA2-positive disease. A subject from whom a test specimen determined to be positive in the detection or measurement of EPHA2 by EPHA2-positive disease test or diagnostic method, administers an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody The subject from whom the test specimen determined to be suitable for the treatment or prevention method of EPHA2-positive disease including the steps and from which the test sample was determined to be negative is an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody. 14. The composition according to claim 13, comprising determining that the method is not suitable for a method of treating or preventing an EPHA2-positive disease including the step of administering. The composition according to claim 13 or 14, wherein the EPHA2-positive disease is EPHA2-positive cancer. A pharmaceutical composition comprising an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody, which is administered to the subject according to any one of (i) to (iii) below:
(I) a subject derived from a test specimen in which EPHA2 is detected or measured using the composition according to any one of claims 9 to 11 or 13;
(Ii) a subject derived from a test specimen determined to be positive in the detection or measurement of EPHA2 using the composition according to claim 12;
(Iii) The composition according to claim 14 or 15 is suitable for treating or preventing an EPHA2-positive disease comprising a step of administering an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody. Subject determined to be. A polynucleotide encoding the antibody or antigen-binding fragment of the antibody according to any one of claims 1 to 8. A vector comprising the polynucleotide according to claim 17. A cell comprising the polynucleotide according to claim 17 or the vector according to claim 18. The method for producing a monoclonal antibody or an antigen-binding fragment of the antibody according to any one of claims 1 to 8, comprising the following steps (i) and (ii):
(I) a step of culturing the cell according to claim 19;
(Ii) A step of recovering a monoclonal antibody or an antigen-binding fragment of the antibody from the culture of step (i). The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody. EPHA2 detection or measurement method comprising the following steps (i) or (i) and (ii):
(I) contacting the test specimen with the antibody according to any one of claims 1 to 8 or the antigen-binding fragment of the antibody or the composition according to claim 9;
(Ii) if EPHA2 is detected or measured in the test specimen, or if the expression level or expression level of EPHA2 in the test specimen is equal to or higher than a predetermined criterion, If it is judged positive and EPHA2 is not detected or measured in the test specimen, and the expression level or expression level of EPHA2 in the test specimen is equal to or lower than a predetermined criterion, the test specimen The step of judging a specimen as negative. A method for identifying an individual to be administered a pharmaceutical composition comprising an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody, comprising the following steps (i) or (i) and (ii):
(I) a step of bringing the antibody according to any one of claims 1 to 8 or an antigen-binding fragment of the antibody or the composition according to claim 9 into contact with a sample derived from an individual;
(Ii) if EPHA2 is detected or measured in a sample from the individual, or if the expression level or level of EPHA2 in the sample from the individual is equal to or higher than a predetermined criterion, If it is determined positive and EPHA2 is not detected or measured in the sample derived from the individual, and the expression level or expression level of EPHA2 in the sample derived from the individual is equal to or lower than a predetermined criterion, Determining the individual as negative. 24. The method according to claim 22 or 23, which is used in a test or diagnostic method for EPHA2-positive disease. The method according to claim 23, which is used in a method for identifying an individual suffering from or at risk of having an EPHA2-positive disease. A pharmaceutical composition comprising an antibody that specifically binds to EPHA2 or an antigen-binding fragment of the antibody, to a subject according to any one of (i) to (iii) of claim 16. A method for treating an EPHA2-positive disease. A kit for examining or diagnosing an EPHA2-positive disease comprising the antibody or antigen-binding fragment of the antibody according to any one of claims 1 to 8. The method according to claims 24 to 26 or the kit according to claim 27, wherein the EPHA2-positive disease is EPHA2-positive cancer.

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