WO2020158959A1

WO2020158959A1 - Anti-mc16 antibody

Info

Publication number: WO2020158959A1
Application number: PCT/JP2020/004695
Authority: WO
Inventors: 道子福田; 元裕野中
Original assignee: 国立研究開発法人産業技術総合研究所
Priority date: 2019-02-01
Filing date: 2020-01-31
Publication date: 2020-08-06
Also published as: JP7224628B2; JP2020125253A

Abstract

The present invention provides: a monoclonal antibody capable of binding specifically to an N-terminal region of annexin A1; a hybridoma capable of producing the antibody; a reagent for detecting the presence of an N-terminal region of annexin A1, which comprises the antibody; a method for detecting the presence of an N-terminal region of annexin A1 on the surface of a tumor vasculature; and others.

Description

Anti-MC16 antibody

The present invention relates generally to the field of cancer biology, and more specifically to an antibody that specifically binds to the N-terminal region of Annexin A1 and its use.

Most brain tumors, such as meningioma and schwannoma, originating from the meninges and the cranial/spinal nerves are benign brain tumors and can be completely cured if they can be removed by surgery. In contrast, neuroepithelial tumors such as gliomas are basically malignant brain tumors. In particular, for grade 4 glioblastoma, the prognosis is extremely poor (5-year survival rate is about 10%) even if radiation therapy and chemotherapy are performed after excision by craniotomy. The presence of the blood-brain barrier is a major reason why effective chemotherapy has not been implemented for brain tumors.

To date, the present inventors have overwhelmed brain tumors by binding to the N-terminal region of annexin A1 (Non-Patent Document 1), which is known to have the highest specificity among tumor blood vessel-specific marker molecules. Of a target peptide for malignant tumor blood vessels (IF7, dTIT7, etc.) that accumulates at a relatively high concentration and can actively cross the wall of vascular endothelium, and uses the peptide to malignant tumor blood vessel-specific anticancer We have also developed a drug delivery system. Further, since the peptide intravenously injected into a tumor-bearing mouse model targets malignant tumor blood vessels, it is considered that the N-terminal region of annexin A1 is actually expressed on the surface of malignant tumor vascular endothelium.

The recent surge in medical expenses is not just an economic burden for patients, it is tense enough to threaten Japan's financial failure, and expensive medicines are out of reach in countries without overseas universal health insurance. It is becoming a reality, leading to unimaginable medical disparities. Under such circumstances, the delivery system of IF7 and dTIT7 developed by the present inventors and an anticancer agent using them can efficiently cross the blood-brain tumor barrier and exert an excellent therapeutic effect on a brain tumor or the like. Because it is possible and cheap, it is expected to correct the medical disparity on a global scale.

The N-terminal region of annexin A1, which is a receptor for IF7 and dTIT7, needs to be present on the surface of vascular endothelium in order for the anticancer drug bound by IF7 and dTIT7 developed by the present inventors to exert a therapeutic effect. .. Therefore, when a patient suffering from a brain tumor or the like is treated with an anticancer agent to which the IF7 or dTIT7 is bound, the tissue of the tumor or the like is preliminarily considered from the viewpoint of the physical and economic burden of the patient. A diagnostic agent capable of detecting the presence of the N-terminal region of annexin A1 on the surface of vascular endothelium is strongly desired.

An antibody that specifically binds to the N-terminal region of Annexin A1 that can be used for immunostaining is suitable as a diagnostic agent for detecting the presence or absence of the N-terminal region of Annexin A1 in the tumor tissue. To our knowledge, such an antibody is not commercially available and has not been reported in academic papers.

An object of the present invention is to provide a monoclonal antibody that specifically binds to the N-terminal region of annexin A1, a hybridoma that produces the antibody, a reagent for detecting the presence of the N-terminal region of annexin A1 containing the antibody, and a surface of a tumor blood vessel. It is intended to provide a method for detecting the presence of the N-terminal region of annexin A1 in E. coli.

As a result of intensive studies, the present inventors have found a monoclonal antibody that specifically binds to the N-terminal region of annexin A1, and found that the monoclonal antibody can positively stain vascular endothelium in immunostaining in malignant brain tumors. Heading out, the present invention has been completed.

That is, the present invention is as follows.
[1] A monoclonal antibody that specifically binds to the N-terminal region of annexin A1.
[2] The monoclonal antibody according to claim 1, wherein the N-terminal region is a region containing the amino acid sequence represented by SEQ ID NO: 1.
[3] A monoclonal antibody produced by the hybridoma deposited under accession number NITE P-02801.
[4] The hybridoma deposited under the deposit number NITE P-02801.
[5] A reagent for detecting the presence of the N-terminal region of annexin A1, which comprises the monoclonal antibody according to any one of [1] to [3].
[6] Application of cancer treatment using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and the anticancer agent, which comprises the monoclonal antibody according to any one of [1] to [3] A reagent for determining the availability of.
[7] The reagent according to [6], wherein the cancer is solid cancer or liquid cancer.
[8] A method for detecting the presence of an N-terminal region of annexin A1 on the surface of a tumor blood vessel, comprising:
(1) Contacting a sample derived from a subject with a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 according to any one of [1] to [3] (2) In the sample , Detecting the N-terminal region of Annexin A1 on the surface of tumor blood vessels by immunostaining.
[9] A method for assisting selection of an application target of cancer treatment using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent,
(1) Contacting a sample derived from a subject with a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 according to any one of [1] to [3] (2) In the sample , Detecting the N-terminal region of Annexin A1 on the surface of tumor blood vessels by immunostaining.
[10] A method for treating cancer, which comprises using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent,
(1) Contacting a sample derived from a subject with a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 according to any one of [1] to [3] (2) In the sample Detecting the N-terminal region of annexin A1 on the surface of the tumor blood vessel by immunostaining (3) selecting an application target of the treatment based on the detection result (4) selecting annexin A1 A method comprising administering a conjugate of an anticancer drug and a peptide that binds to the N-terminal region.
[11] In any one of [8] to [10], wherein the subject-derived sample is a tissue section prepared using a biopsy sample containing cancer cells collected by biopsy from the subject. The method described.
[12] The method according to any one of [9] to [11], wherein the cancer is solid cancer or liquid cancer.

According to the present invention, a monoclonal antibody that specifically binds to the N-terminal region of annexin A1, a hybridoma producing the antibody, a reagent containing the antibody for detecting the presence of the N-terminal region of annexin A1, a surface of a tumor blood vessel It is possible to provide a method for detecting the presence of the N-terminal region of Annexin A1 in the above.

FIG. 1 shows the results of measuring the binding of anti-MC16 antibody (clone 31-8D) to human annexin A1 by ELISA. FIG. 2 shows the results of detection of binding of anti-MC16 antibody to human annexin A1 and a mutant of human annexin A1 lacking the N-terminal domain by Western blotting. FIG. 3 shows the results of fluorescent immunostaining images of mouse vascular endothelial F2 cells with anti-MC16 antibody. FIG. 4 shows the results of immunostaining of glioblastoma tissue with anti-MC16 antibody (left: immunostaining, right: hematoxylin-eosin staining). FIG. 5 shows the results of immunostaining of various cancer tumor tissues (breast cancer) with anti-MC16 antibody and anti-annexin A1 antibody commercially available from Invitrogen. As the tissue specimen, a multiple cancer tissue micro array from Biomax was used. FIG. 6 shows the results of immunostaining of various cancer tumor tissues (lung cancer) with anti-MC16 antibody and anti-annexin A1 antibody marketed by Invitrogen. As the tissue specimen, a multiple cancer tissue micro array from Biomax was used. FIG. 7 shows the results of immunostaining of various cancer tumor tissues (hepatocellular carcinoma) with anti-MC16 antibody and anti-annexin A1 antibody marketed by Invitrogen. As the tissue specimen, a multiple cancer tissue micro array from Biomax was used. FIG. 8 shows the results of immunostaining of various cancer tumor tissues (ovarian cancer) with anti-MC16 antibody and anti-annexin A1 antibody marketed by Invitrogen. As the tissue specimen, a multiple cancer tissue micro array from Biomax was used.

1. Monoclonal Antibody of the Present Invention The present invention provides a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 (RefSeq No. NP — 000691.1) (hereinafter sometimes referred to as “ANXA1” or “Anxa1”). .. Specifically, the antibody is an antibody that specifically binds to an epitope that is present in the N-terminal region of annexin A1 and that contains the amino acid sequence represented by SEQ ID NO: 1 (MAMVSEFLKQAWFIE). The antibody may belong to any immunoglobulin class of IgG, IgA, IgM, IgD or IgE, but is preferably IgG, more preferably IgG2b. Specific examples of the antibody include an antibody (mouse IgG2b) produced by the monoclonal antibody-producing hybridoma (clone 31-8D) of the present invention described below (hereinafter sometimes referred to as "anti-MC16 antibody").

Since the monoclonal antibody of the present invention specifically binds to the N-terminal region of Annexin A1 expressed on the blood side of neovascular endothelial cells formed in (malignant) tumors, the presence or absence of the N-terminal region of Annexin A1 is checked. Suitable for detecting. Therefore, the monoclonal antibody of the present invention is, for example, a cancer treatment comprising a conjugate containing a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent, and the conjugate and a pharmaceutically acceptable carrier. It can be used for companion diagnosis for predicting whether or not a subject is an application target of the treatment before performing cancer treatment using the composition for treatment and the like.

As used herein, a subject is any animal that expresses annexin A1, particularly a mammal. Examples of mammals include, for example, mice, rats, hamsters, experimental animals such as rodents and rabbits such as guinea pigs, pigs, cows, goats, horses, sheep, mink and other domestic animals, dogs, pets such as cats, humans, and the like. Examples thereof include, but are not limited to, primates such as monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees.

2. Preparation of Monoclonal Antibody of the Present Invention The monoclonal antibody of the present invention and an antibody similar thereto can be prepared by a method known per se (phage display method, hybridoma method). When the antibody is obtained from a hybridoma (fused cell) produced by cell fusion, specifically, for example, first, an immunogenic antigenic peptide is subcutaneously or intramuscularly administered to a mammal such as mouse, rat or hamster. Immunization is carried out by injecting once or several times into a vein, a hood, an abdominal cavity or the like, or by transplanting. Next, antibody-producing cells are isolated from the immunized mammal, the cells are fused with myeloma cells to form a hybridoma, the hybridoma is cloned, and immunogenic used for immunization of the mammal. It is produced by selecting a clone that produces an antibody showing a specific affinity for the antigenic peptide.
Further, as the antibody-producing cells, antibody-producing cells produced by reacting previously isolated spleen cells, lymphocytes, etc. with an immunogenic antigen peptide in a culture medium can also be used. In this case, human-derived antibody-producing cells can also be prepared.

The immunogenic antigen peptide can be used for immunization by diluting it with PBS (Phosphate-Buffered Saline) or physiological saline at an appropriate dilution ratio. The immunogenic antigenic peptide may be administered together with an adjuvant, for example, it may be mixed with complete or incomplete Freund's adjuvant (FCA or FIA) and emulsified. Furthermore, a suitable carrier may be used when immunizing with an immunogenic antigen. Particularly when a peptide having a small molecular weight is used as an immunogenic antigen, it is desirable to immunize the immunogenic antigen peptide by binding it to a carrier protein such as albumin or keyhole limpet hemocyanin.
In Examples described later, a synthetic peptide (SEQ ID NO:2: MAMVSEFLKQAWFIEC) in which cysteine is added to the C-terminal of the amino acid sequence from the N-terminal to the 15th amino acid of human annexin A1 (SEQ ID NO: 1 MAMVSEFLKQAWFIE) (hereinafter, “synthetic peptide”). A mixture of bovine serum albumin-bound substance and complete Freund's adjuvant was used as an immunogenic antigen peptide.

Hybridomas that secrete monoclonal antibodies can be prepared according to the method of Kohler and Milstein (Nature, Vol. 256, pp. 495-497, 1975) and its modifications. That is, the monoclonal antibody of the present invention, splenocytes, lymph node cells, peripheral lymphocytes, myeloma cells, tonsil cells, etc. obtained from the immunized animal as described above, preferably antibody-producing cells contained in splenocytes and It is preferably prepared by culturing a hybridoma obtained by fusion with a mammal such as mouse, rat, guinea pig, hamster, rabbit or human of the same species, more preferably mouse, rat or human myeloma cells (myeloma). It Culturing can be performed in vitro or in vivo in mammals such as mouse, rat, guinea pig, hamster, rabbit, etc., preferably mouse or rat, more preferably in the abdominal cavity of mouse, and the antibody is the culture supernatant or mammalian, respectively. Can be obtained from the ascites of an animal.

Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3/X63-AG8, P3/NSI/1-Ag4-1, P3/X63-Ag8. U1, SP2/0-Ag14, F0 or BW5147, rat-derived myeloma 210RCY3-Ag1.2.3. , Human-derived myeloma U-266AR1, GML500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15.

The screening of the hybridoma clone producing the monoclonal antibody of the present invention is carried out by culturing the hybridoma in, for example, a microtiter plate, and measuring the reactivity of the culture supernatant in the wells in which proliferation is observed against the immunogenic antigenic peptide. For example, the measurement can be performed by ELISA, Western blotting, immunostaining, or the like.

As described in Examples below, a hybridoma that produced an antibody showing high reactivity with the synthetic peptide MC16 was subjected to cell cloning by the limiting dilution method to obtain a monoclonal antibody-producing hybridoma of the present invention (clone 31-8D). Clone 31-8D will be placed on October 24, 2018, under the accession number NITE P-02801, at the Patent Microorganism Depositary Center (2-5-8, Kazusa Kamafoot, Kisarazu City, Chiba Prefecture) under the National Institute for Product Evaluation Technology, National Institute for Product Evaluation Technology. Deposited.

The isolation and purification of the monoclonal antibody is carried out by subjecting the antibody-containing culture supernatant or ascites produced by the above-mentioned method to, for example, ion exchange chromatography, anti-immunoglobulin column or affinity column chromatography such as protein A or protein G column. It can be performed by attaching it to a graph.

The monoclonal antibody of the present invention may be labeled with a detectable label. Examples of the label include fluorescent label, low molecular weight compound label, peptide label, enzyme label and the like. In addition, the monoclonal antibody of the present invention may form a conjugate with other drugs.

The monoclonal antibody of the present invention is not limited to the above-mentioned production method, and may be obtained by any method. For example, a gene cloned from the hybridoma of the present invention, which encodes the monoclonal antibody of the present invention, may be expressed as an antibody by incorporating the gene into a suitable vector and introducing it into a host. Methods for the isolation of antibody-encoding genes, their introduction into vectors, and the transformation of host cells have been established (eg, Vandamme, AM et al. Eur. J. Biochem. (1990). 192, 767-775).

For example, from a hybridoma producing the monoclonal antibody of the present invention, cDNA encoding the variable region (V region) of the antibody can be obtained. For that purpose, first, total RNA is usually extracted from the hybridoma. As a method for extracting mRNA from cells, for example, guanidine ultracentrifugation method (Chirgwin, JM, et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P., et al., Anal. Biochem. (1987) 162, 156-159) and the like can be used. The extracted mRNA can be purified using mRNA Purification Kit (manufactured by GE Healthcare Bioscience). Alternatively, a kit for directly extracting total mRNA from cells, such as QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bioscience), is commercially available. Such a kit can also be used to obtain total mRNA from hybridomas. CDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. The cDNA can be synthesized by AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation). For the synthesis and amplification of cDNA, 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and 5'-RACE method using PCR (Frohman, MA, et al., Proc. Natl. Acad. Sci. USA). (1988) 85, 8998-9002, Belyavsky, A. et al. Nucleic Acids Res. (1989) 17, 2919-2932). Furthermore, in the process of synthesizing such a cDNA, appropriate restriction enzyme sites may be introduced at both ends of the cDNA.

The desired cDNA fragment is purified from the resulting PCR product and then operably linked to vector DNA. After the recombinant vector is prepared in this manner and introduced into Escherichia coli or the like to select a colony, a desired recombinant vector can be prepared from E. coli forming the colony. Whether or not the recombinant vector has the nucleotide sequence of the desired cDNA can be confirmed by a method known per se, such as the dideoxynucleotide chain termination method.

Usually, a monoclonal antibody has a sugar chain having a different structure depending on the type of mammal to be immunized, but the monoclonal antibody in the present invention is not limited by the structural difference of the sugar chain, and a monoclonal antibody derived from any mammal It also includes. Furthermore, for example, a recombinant human monoclonal antibody obtained from a transgenic animal into which a human immunoglobulin gene has been incorporated, or a constant region (Fc) of a monoclonal antibody derived from a certain mammal is recombined with the Fc region of a human-derived monoclonal antibody. Also included in the monoclonal antibody of the present invention are chimeric monoclonal antibodies, and further, chimeric monoclonal antibodies in which the entire region other than the complementarity determining region (CDR) that can directly bind complementarily with an antigen is recombined with the corresponding region of a human-derived monoclonal antibody. To be done.

The antibodies of the present invention include natural antibodies such as the above-mentioned monoclonal antibody (mAb), chimeric antibodies that can be produced by gene recombination technology, humanized antibodies and single chain antibodies, and these antibodies. Fragment of. The antibody fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes Fab, Fab′, F(ab′) ₂ , scAb, scFv or scFv-Fc and the like. ..

These antibody fragments can be obtained by a method known per se, and specifically, the monoclonal antibody of the present invention is treated with an enzyme such as papain or pepsin to produce an antibody fragment, or these antibodies are produced. A gene encoding the fragment may be constructed, introduced into an expression vector, and then expressed in an appropriate host cell. The obtained fragment can be evaluated for reactivity with an antigen, specificity and the like in the same manner as for the monoclonal antibody of the present invention.

Further, those skilled in the art can also prepare a fusion antibody of the antibody or fragment of the present invention with another peptide or protein, or a modified antibody having a modifying agent bound thereto. Other peptides or proteins used for fusion are not particularly limited as long as they do not reduce the binding activity of the antibody, and examples thereof include human serum albumin, various tag peptides, artificial helix motif peptides, maltose binding protein, glutathione S transferase, Examples include various toxins and peptides or proteins capable of promoting multimerization. The modifying agent used for modification is not particularly limited as long as it does not reduce the binding activity of the antibody, and examples thereof include polyethylene glycol, sugar chains, phospholipids, liposomes, and low molecular weight compounds.

3. Reagent of the present invention As described above, since the monoclonal antibody of the present invention specifically binds to the N-terminal region of annexin A1 expressed on the blood side of neovascular endothelial cells formed in (malignant) tumors, it can be targeted to malignant tumors. It is suitable for detecting the presence or absence of the N-terminal region of annexin A1, which is a receptor for therapeutic agents for malignant tumors using active IF7 or dTIT7. Therefore, the present invention also provides a reagent for detecting the presence of the N-terminal region of Annexin A1, which comprises the monoclonal antibody of the present invention.

Further, as described above, the monoclonal antibody of the present invention can be used for companion diagnosis for predicting whether or not a specific cancer treatment is applied. Therefore, the present invention provides a conjugate comprising a peptide that binds to the N-terminal region of annexin A1 and the anticancer agent, which comprises the monoclonal antibody of the present invention, and a composition comprising the conjugate and a pharmaceutically acceptable carrier. Also provided is a reagent for determining the applicability of cancer treatment using the above.

In the present specification, malignant tumor (cancer) may be any type of cancer, and may be solid cancer or liquid cancer. The solid cancer preferably includes a solid cancer that expresses annexin A1 on the cell surface, and thus an angiogenic solid cancer. Examples of solid cancer include brain/nervous system cancer (for example, brain tumor, spinal cord tumor, etc.), head and neck cancer (for example, laryngeal cancer, oral cancer, salivary gland cancer, sinus cancer, thyroid cancer). Cancer, etc.), gastrointestinal cancer (for example, gastric cancer, esophageal cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, etc.), urinary organ or Reproductive organ cancer (eg, renal cancer, renal cell cancer, bladder cancer, prostate cancer, renal pelvis and ureteral cancer, gallbladder cancer, bile duct cancer, testicular cancer, penis cancer, uterine cancer) , Endometrial cancer, uterine sarcoma, cervical cancer, vaginal cancer, vulvar cancer, ovarian cancer, fallopian tube cancer, etc., respiratory cancer (eg lung cancer (small cell lung cancer, non Small cell lung cancer, including metastatic lung cancer), bronchial cancer, etc.), breast cancer, skin cancer (eg, malignant melanoma), bone cancer (eg, osteosarcoma), muscle cancer (eg, Rhabdomyosarcoma) and the like.

Liquid cancers include leukemia, malignant lymphoma, multiple myeloma, myelodysplastic syndrome, etc. Examples of leukemia include acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and the like. Malignant lymphoma is classified into Hodgkin lymphoma and non-Hodgkin lymphoma. As non-Hodgkin lymphoma, B-cell lymphoma, adult T-cell lymphoma, lymphoblastic lymphoma, diffuse large cell lymphoma, Burkitt lymphoma, follicular lymphoma , MALT lymphoma, peripheral T cell lymphoma, mantle cell lymphoma and the like.

As a result of the companion diagnosis using the reagent of the present invention, a peptide that binds to the N-terminal region of annexin A1 administered to a subject, a conjugate of an anticancer agent, or the conjugate and a pharmaceutically acceptable carrier are used. As will be described later, the composition containing the compound can efficiently cross the blood-brain tumor barrier, and therefore a brain tumor can be mentioned as a particularly preferable target.

The brain tumor may be a primary brain tumor or a metastatic brain tumor. The brain tumor may be benign (eg, meningioma, pituitary adenoma, schwannoma, etc.) or malignant brain tumor, preferably malignant brain tumor. Malignant brain tumors include grade 2 brain tumors such as astrocytoma (astrocytoma) and oligodendrogliomas, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic Examples thereof include grade 3 brain tumors such as oligoastrocytoma, and grade 4 brain tumors such as glioblastoma.

The reagent of the present invention may consist only of the monoclonal antibody of the present invention or may contain a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, when the reagent of the present invention is prepared as a liquid agent, various carriers commonly used as a formulation material, for example, diluents, solvents, solubilizers, suspending agents, isotonic agents, etc. Agents, buffers, etc. may be included. Further, a surfactant such as Tween 20 (registered trademark) or Tween 80 (registered trademark) may be added to prevent antibody aggregation. Examples of the diluent include water and physiological saline. Examples of the solvent include water, physiological saline, ethanol and the like. Examples of the solubilizing agent include cyclodextrins. Examples of the suspending agent include gum arabic and carmellose. Examples of the isotonicity agent include inorganic salts such as sodium chloride and potassium chloride, carbohydrates such as glycerin, mannitol and sorbitol. Examples of the buffer include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer and the like. Those skilled in the art can appropriately determine the blending ratio of these carriers.

4. Method of the present invention As described above, the monoclonal antibody of the present invention specifically binds to the N-terminal region of annexin A1 expressed on the blood side of neovascular endothelial cells formed in (malignant) tumors. It is suitable for detecting the presence or absence of the N-terminal region. Therefore, the present invention provides a method for detecting the presence of the N-terminal region of annexin A1 (annexin A1 on the surface of tumor blood vessels) expressed on the blood side of neovascular endothelial cells formed in a tumor using the monoclonal antibody. Also provide.

Specifically, the method of the present invention comprises (1) contacting a sample derived from a subject with a monoclonal antibody of the present invention that specifically binds to the N-terminal region of annexin A1, and (2) in the sample Detecting the N-terminal region of annexin A1 (annexin A1 on the surface of tumor blood vessels) expressed on the blood side of neovascular endothelial cells formed in the tumor by immunostaining.

Further, as described above, the monoclonal antibody of the present invention can be used for companion diagnosis for predicting whether or not a specific cancer treatment is applied. Accordingly, the present invention provides a conjugate comprising a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent using the monoclonal antibody, a composition containing the conjugate and a pharmaceutically acceptable carrier, and the like. The present invention also provides a method for selecting an application target of cancer treatment using the method or a method for assisting the selection. Furthermore, the present invention provides a composition comprising the conjugate selected from a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent, and the conjugate and a pharmaceutically acceptable carrier in the subject selected as described above. It may include actually treating cancer by administering a substance or the like.

The measuring means for detecting the presence of the N-terminal region of Annexin A1 using the monoclonal antibody of the present invention is not particularly limited, but an immunostaining method is preferably used. Specific means using the immunostaining method include immunohistochemical staining (IHC) method, flow cytometry analysis, mass cytometry analysis and the like.

Immunohistochemical staining can be performed according to a method known per se (for example, the revised fourth edition Watanabe-Nakone enzyme antibody method, published by Interdisciplinary Planning Co., Ltd., edited by Hiroshi Nagura, Yoshiyuki Nagamura, Hiroshi Tsutsumi, ISBN4-906514). -73-X C304), preferably the enzyme antibody method (Enzyme labeled antibody method) and the fluorescent antibody method are used.

For example, in the enzyme antibody method, the N-terminal region of annexin A1 is detected with an enzyme-labeled antibody. Labeling may be done directly or indirectly using a secondary antibody. Various improvements have been made to increase the detection sensitivity, and very high sensitivity is achieved with improved methods such as the ABC method using biotin-streptavidin and the tyramide method (TSA method, Tyramide Signal Amplification) using tyramide (tylamide). realizable.

As the labeling enzyme, peroxidase (HRP, horseradish peroxidase) and alkaline phosphatase (AP, alkaline phosphatase) are widely used and can be suitably used in the present invention.

As a general protocol, a cell sample deparaffinized or fixed as necessary is prepared, and if necessary, pretreatment (for example, decolorization or removal treatment of vital dye, blocking treatment) or antigen activation (heat bath, microwave treatment, autoclave) Treatment, protease treatment, etc., and react with primary antibody (for example, 0.5° C. to 24 hours at 4° C. to room temperature). After the reaction is complete, the cell sample is washed and reacted with secondary antibody (for example, 4° C.). After completion of the reaction, the cell sample is washed at room temperature for 5 to 120 minutes). Then, a color-developing substrate such as diaminobenzidine (3,3-diaminobenzidine:DAB) is added to develop the color. For morphological observation with a microscope, counterstaining such as hematoxylin staining may be further performed.

Alternatively, the N-terminal region of annexin A1 may be detected as a fluorescent antibody method by using an antibody labeled with a fluorescent dye instead of the enzyme labeling by the above-mentioned enzyme antibody method. Also in the fluorescent antibody method, labeling may be performed directly or indirectly using a secondary antibody.

 Take an image of the immunostained sample using an optical microscope equipped with an imaging device or a fluorescence microscope (in the case of fluorescence staining), and save it in a computer as a digital image. The digital image may be two-dimensional data or three-dimensional data, but two-dimensional data can be sufficiently analyzed in the present invention. Image processing such as filtering may be performed before the analysis.

Image analysis of immunostained samples can be performed using image analysis software installed in the microscope used and other commercially available software. An example of commercially available software is ScanScope (registered trademark, Aperio Inc.).

Classification of immunostaining intensity can follow standard criteria by pathologists for immunohistochemical staining, such as score 0 (undetected), score +1 (weak), score +2 (moderate), and score +3 (significant). ) May be classified. Further, for example, when the score is +1 or more, a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent, a composition containing the conjugate and a pharmaceutically acceptable carrier, and the like were used. It may be judged that the cancer treatment is applied.

In the present specification, a sample derived from a subject can be contacted with a monoclonal antibody of the present invention that specifically binds to the N-terminal region of Annexin A1 to detect the presence or absence of the N-terminal region of Annexin A1. It is not particularly limited as long as it is one, but is preferably a tissue section prepared by using a biopsy sample containing cancer cells collected by biopsy from a subject, and more preferably a paraffin-embedded tissue. It is a section.

Examples of the peptide that binds to the N-terminal region of annexin A1 used in the method of the present invention (hereinafter sometimes referred to as “peptide used in the method of the present invention”) include, for example, the following (I) to (III Peptide containing any of the amino acid sequences of:
(I) Amino acid sequence of (X1)[D]P[D](X2)[D] (wherein X1 represents W or F, X2 represents S or T),
(II) P[D]T[D](X) _n F[D] amino acid sequence (wherein (X) _n represents n arbitrary amino acids selected independently from each other, and n is Indicates an integer of 0 to 4),
(III) An amino acid sequence which is retro-inverso of the amino acid sequence of (I) or (II) above can be mentioned.

The peptide used in the method of the present invention is, for example, a peptide containing any of the following amino acid sequences (i) to (vii):
(I) the amino acid sequence of T[D]I[D]T[D]W[D]P[D]T[D]M[D],
(Ii) the amino acid sequence of L[D]R[D]F[D]P[D]T[D]V[D]L[D],
(Iii) L[D]L[D]S[D]W[D]P[D]S[D]A[D] amino acid sequence,
(Iv) the amino acid sequence of S[D]P[D]T[D]S[D]L[D]L[D]F[D],
(V) the amino acid sequence of M[D]P[D]T[D]L[D]T[D]F[D]R[D],
(Vi) an amino acid sequence having one or several amino acid insertions, substitutions or deletions, or a combination thereof in the amino acid sequence of any of (i) to (v) above,
(Vii) An amino acid sequence which is retro-inverso of the amino acid sequence of any one of (i) to (vi) above can be mentioned.

In this specification, the amino acid sequence of a chain peptide is described with the N-terminal side on the left side and the C-terminal side on the right side according to the convention of peptide notation. In addition, each amino acid symbol immediately after the symbol [D] in the amino acid sequence represents the D-form of the amino acid, and each amino acid symbol without immediately following the symbol [D] in the amino acid sequence is uncontextual. Unless otherwise indicated, the L form of the amino acid is shown.

The amino acid sequence of (I) above is W[D]P[D]S[D], W[D]P[D]T[D], F[D]P[D]S[D], or F It may be any of [D]P[D]T[D].

In the amino acid sequence of (II) above, the integer n is 0 to 4, preferably 2 to 3. X may be any amino acid selected independently of each other.

The peptide used in the method of the present invention may consist of any of the amino acid sequences of (I) to (III) and (i) to (vii) described above, or the N-terminal side of these sequences. And/or one or more amino acids may be added to the C-terminal side.

In the present specification, the peptide used in the method of the present invention refers to a peptide in which two or more amino acids are bound to each other. The length of the peptide of the present invention is not particularly limited, but for example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , At least 12, at least 13, at least 14, or at least 15 amino acids. The peptides used in the method of the present invention include up to 50, 45, 40, 35, 30, 25, 20, 19, 18, 18 and 17 peptides. , Up to 16, up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, up to 9, up to 8, or up to 7 amino acids Good.

The peptide used in the method of the present invention may be composed of a combination of D-type amino acids and L-type amino acids. Further, the peptide used in the method of the present invention is 37 C.I. F. R. It may or may not contain modified or unusual amino acids, such as those mentioned in 1.821-1.822.

The amino terminus and/or carboxy terminus of the peptide used in the method of the present invention may be modified. Further, the peptide used in the method of the present invention may have various modifications other than at the N-terminus or C-terminus.

The substitution in the amino acid sequence of (vi) above is preferably a conservative amino acid substitution. "Conservative amino acid substitutions" are well known in the art. For example, a conservative amino acid substitution can be defined as a substitution between amino acids with similar side chain properties.

The above (vi) sequence may be included in the above (I) or (II) sequence, in which case the above (vii) sequence is included in the above (III) sequence. Becomes

The retro-inverso isomer of a peptide is one in which the chirality of each amino acid residue is opposite to that of the original peptide (“inverso”) and the direction of the amino acid sequence is reversed (“Retro”). Say. The Retro-inverso isomer is known to exhibit a structure and a function similar to that of the original peptide (eg, Acc. Chem. Res., 1993, 26(5), pp 266-273 and PLoS One. 2013 Dec 2;8(12):e80390).

The peptide used in the method of the present invention may contain two or more sequences selected from the above (I) to (III) and (i) to (vii). The peptide used in the method of the present invention is a tandem repeat of any one of the sequences (I) to (III) and (i) to (vii) (that is, identical sequence portions are directly linked to each other). Structure) may be included. In addition, the peptide used in the method of the present invention may include a structure in which two or more sequences (I) to (III) and (i) to (vii) different from each other are directly linked. As a specific repeat structure, for example, T[D]I[D]T[D]W[D]P[D]T[D]M[D]-T[D]I[D]T[D] E[D]P[D]T[D]M[D], and T[D]I[D]T[D]-T[D]I[D]T[D]. Alternatively, the peptide used in the method of the present invention may constitute a multivalent peptide by a dendrimer.

The peptide used in the method of the present invention may be a free form or a salt form. Salts of the peptides used in the method of the present invention include pharmaceutically acceptable acid addition salts and base addition salts. Examples of the acid addition salt include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, and salts with organic acids such as acetic acid, malic acid, succinic acid, tartaric acid and citric acid. Base addition salts include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, and salts with amines such as ammonium and triethylamine.

The peptide used in the method of the present invention is a region consisting of amino acids 1 to 15 shown in SEQ ID NO: 1 in human annexin A1, or 1 to 15 shown in SEQ ID NO: 3 (MAMVSEFLKQARFLE) in mouse annexin A1. Can bind to a region consisting of the th amino acid. The peptide used in the method of the present invention is preferably less than 10 ⁻⁶ M, and more preferably less than 10 ⁻⁶ M, when the intermolecular interaction with, for example, mouse or human annexin A1 is measured using the QCM (Quartz Crystal Microbalance) method. May have a dissociation constant (Kd value) of less than 10 ⁻⁷ M, even more preferably less than 5×10 ⁻⁸ M. For the above measurement, a human recombinant annexin A1 protein composed of 346 amino acid residues commercially available from ATGen (Seongnam-si, South Korea) can be used.

The peptide used in the method of the present invention can be produced according to a known peptide synthesis method. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. A partial peptide or amino acid that can form the peptide of the present invention is condensed with a residual portion, and when the product has a protecting group, the target peptide is produced by removing the protecting group by a method known per se. You can

The peptide thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, a combination thereof, and the like.
When the peptide obtained by the above method is a free form, the free form can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, when the peptide is obtained as a salt, The salt can be converted into a free form or another salt by a known method or a method analogous thereto.

The anticancer drug used in the method of the present invention refers to a drug intended to suppress the growth of malignant tumor (cancer). The action mechanism of the anticancer agent is not particularly limited. The anticancer agent may be an antimetabolite, an alkylating agent, an anticancer antibiotic, a microtubule inhibitor, a platinum preparation, a topoisomerase inhibitor, a molecular targeting drug and the like. The conjugate of the present invention may contain two or more same or different anticancer agents.

Antimetabolites include, for example, antifolates, dihydropteroate synthase inhibitors, dihydrofolate reductase inhibitors (DHFR inhibitors), pyrimidine metabolism inhibitors, thymidylate synthase inhibitors, purine metabolism inhibitors, IMPDH inhibitors , Ribonucleotide reductase inhibitors, ribonucleotide reductase inhibitors, nucleotide analogs, L-asparaginase and the like. Specific examples of the antimetabolites include enocitabine (san-rabine), capecitabine (xeloda), carmofur (mifurol), cladribine (leustatin), gemcitabine (gemzar), cytarabine (kiloside), cytarabine ocphosphate (staracide), tegafur (tegafur). Athyron, Aftful, Tefseal, Futrafur, Lunacin, etc.), Tegafur Uracil (Yufti), Tegafur Guimeracil Oteracil Potassium (TS-1: TS-1), Doxyfluridine (Furtulon), Nerarabine (Alanony), Hydroxycarbamide (Hydrea) ), fluorouracil (5-FU, carzonal, bennan, lunacol, lunabon), fludarabine (fludara), pemetrexed (alimuta), pentostatin (cophorin), mercaptopurine (leukerin), methotrexate (methotrexate) and the like.

Specific examples of the alkylating agent include cyclophosphamide (endoxane), ifosfamide (ifomide), melphalan (alkeran), busulfan, thiotepa (tespamin), and other nitrogen mustard-based alkylating agents, nimustine (nidoran), ranimustine. (Cimerine), dacarbacin (dacarbacin), procarbacin (procarbacin hydrochloride), temozolomide (temodal), carmustine (giliadel), streptozotocin (zanocer), bendamustine (treaxin), and other nitrosourea-based alkylating agents.

Specific examples of the anticancer antibiotics include actinomycin D (cosmegen), aclarubicin (acracinone), amrubicin (calced), idarubicin (idamycin), epirubicin (epirubicin hydrochloride, famorubicin), zinostatin stimalamer (smanx). ), daunorubicin (daunomycin), doxorubicin (adriacin), pirarubicin (pinorbin, teralubicin), bleomycin (bleo), peplomycin (pepleo), mitomycin C (mitomycin), mitoxantrone (novantron), liposomal doxorubicin (doxyl), etc. Are listed.

Microtubule inhibitors include, for example, vinca alkaloid-based microtubule polymerization inhibitors such as vinblastine (exar), vincristine (oncobin), and vindesine (fordesin), and taxane-based microtubule depolymerization such as paclitaxel (taxol) and docetaxel (taxotere). Examples include inhibitors.

Examples of platinum preparations include oxaliplatin (elprat), carboplatin (carboplatin, carbomerk, paraplatin), cisplatin (ie equol, conaburi, cisplatin, etc.), nedaplatin (acpra), etc.

Examples of topoisomerase inhibitors include type I topoisomerase inhibitors such as camptothecin and its derivatives (eg, irinotecan (campto), nogitecan (hycamtin), SN-38); anthracycline drugs such as doxorubicin (adriacin); etoposide. Examples thereof include type II topoisomerase inhibitors such as epipodophyllotoxin drugs such as (Lasted and bepside) and quinolone drugs such as levofloxacin (clavit) and ciprofloxacin (ciproxan).

Examples of molecularly targeted drugs include regorafenib (stibagga), cetuximab (erbitux), panitumumab (vetivics), ramucirumab (cyramza), gefitinib (iressa), erlotinib (tarseva), afatinib (diotryf), crizotinib (crizotinib). Alectinib (Alesensor), Ceritinib, Lenvatinib (Lenvima), Trastuzumab (Herceptin), Lapatinib (Tykerb), Pertuzumab (Perjeta), Sunitinib (Sutent), Sorafenib (Nexavar), Axitinib (Inraitabi), Panitab (Inraitab) Opdivo), pembrolizumab, ipilimumab (Yarvoy), vemurafenib (Zelboraf), everolimus (Afinitor), temsirolimus (Torisel), rituximab (Rituxan), bevacizumab (Avastin), geldanamycin and the like.

The anti-cancer agent may also be an anti-angiogenic agent. Anti-angiogenic agents can be those that inhibit vascular endothelial growth factor (VEGF) or other angiogenic factors, or their receptors. Specific examples of the anti-angiogenic agent include angiostatin, endostatin, metastatin, anti-VEGF antibody (for example, Avastin), VEGFR-2 inhibitor (for example, SU5416, SU6668) and the like.

The mode of binding between the peptide that binds to the N-terminal region of annexin A1 in the conjugate used in the method of the present invention and one or more anticancer agents is not particularly limited. The bond may be direct, or indirect via a linker or the like. The binding may be covalent, non-covalent, or a combination thereof. One or more anti-cancer agents may be attached, directly or indirectly, at the N-terminus, C-terminus, or other position of the peptide that binds to the N-terminal region of Annexin A1. The linkage between the peptide and the anticancer drug is well known in the art, and in the conjugate of the present invention, the binding may be by any known means.

The composition containing the conjugate used in the method of the present invention and a pharmaceutically acceptable carrier can be provided as a dosage form suitable for oral or parenteral administration. As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, etc. Can be included. These can be prepared according to a method known per se.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrup. Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient which is usually used in the field of formulation.

Note that the above-mentioned composition may contain other active ingredients as long as it does not cause an unfavorable interaction with the above-mentioned conjugate.

The above-mentioned parenteral or oral pharmaceutical composition is conveniently prepared in a unit dosage form suitable for the dose of the active ingredient. Examples of dosage forms of such dosage units include tablets, pills, capsules, injections (ampoules), and suppositories. The content of the peptide or conjugate is usually 1 to 500 mg per dosage unit dosage form, particularly 1 to 100 mg for injections, and preferably 10 to 250 mg for the other dosage forms. ..

The dose of the composition containing the conjugate used in the method of the present invention and a pharmaceutically acceptable carrier varies depending on the purpose of administration, administration subject, target disease, symptom, administration route, etc., and is appropriately set by those skilled in the art. can do.

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

Example 1 (production of mouse monoclonal antibody)
(1) Preparation of immunogen The C-terminal cysteine (Cys) of synthetic peptide MC16 was bound to a carrier protein (BSA) using a bivalent reactive reagent to prepare an immunogen.
(2) Preparation of HRP Label for Screening C-terminal Cys of synthetic peptide MC16 was bound to horseradish-derived peroxidase (HRP) using a divalent reactive reagent to prepare an HRP label for screening.

(3) Immunization The immunogenic antigen prepared in (1) above was mixed with FCA to emulsify, and one Balb/c mouse was calculated and injected with 200 μg intramuscularly in the ridge (immunization). (3 in total). Based on the result of the first antibody activity measurement, the same antigen was further mixed with FIA to emulsify, and 50 μg was subcutaneously injected (boost) to one mouse twice at two-week intervals. Based on the results of antibody activity measurement after two booster immunizations, a final immunization (calculated 100 μg per mouse) was performed on the abdominal cavity of the mice.

(4) Test Blood and Measurement of Antibody Activity After a certain period of time from the initial and booster immunizations, blood was sampled from mice, and antibody activity was measured by ELISA in which a synthetic MC16 peptide was bound to a plastic plate. From the results, mice were selected for cell fusion.
(5) Collection of lymphocytes and antiserum Three days after the final immunization, the spleen was collected, lymphocytes were separated, and then frozen and stored at -80°C. At the same time, blood was collected, serum was separated, and then frozen and stored at -40°C. The obtained antiserum was used as a positive control during screening.

(6) Cell fusion-culture The lymphocytes obtained in (5) above and mouse myeloma were subjected to cell fusion in the presence of 50% polyethylene glycol. The fused cells (hybridomas) were dispersed in HAT medium, dispensed into four 96-well microplates, and cultured in a 37° C. carbon dioxide incubator. 6 days after cell fusion, the medium was replaced with HT medium, and the culture was further continued.

(7) Primary screening-culture 9 days after cell fusion, the culture supernatant was sampled from each well of a 96-well microplate, and the reaction of the culture supernatant with MC16 was measured by an ELISA in which a synthetic MC16 peptide was bound to a plastic plate. Then, the primary screening was performed.
(8) Secondary screening and cryopreservation of cells After transferring to a 24-well microplate and expanding and culturing for 3 days, the culture supernatant is sampled from each well of the microplate, and the secondary screening is performed by the same ELISA as above. Performed, cryopreservation of cells in selected wells was performed.

(9) Antibody activity measurement Two weeks after the second booster, the antibody activity of the antiserum obtained by blood test was measured by the same ELISA as above. As a result, antibody activity against the antigen of interest was confirmed in all immunized mice, and it was judged that this was sufficient for carrying out cell fusion.

(10) Primary screening The same ELISA as described above was carried out, 40 clones showing the desired specificity were selected, and expanded and cultured.
(11) Secondary screening and cryopreservation of cells As a result of performing the same ELISA as above, a large number of positive clones were obtained. Of the positive clones, 18 clones of cells were cryopreserved.

Example 2 (Selection of monoclonal antibody-producing hybridoma (clone 31-8D))
Six highly reactive clones were selected from the clones prepared in Example 1, and the clone (#31-8D, mouse IgG2b) that gave the best results in immunostaining of cultured mouse vascular endothelial cells was selected.

Example 3 (binding assay of antibody (anti-MC16 antibody) produced by clone 31-8D)
Plate assay was performed to confirm the binding of anti-MC16 antibody to ANXA1 protein. Specifically, full-length human ANXA1 protein was produced in insect cells using a baculovirus vector using recombinant DNA, and the produced ANXA1 protein was attached to an ELISA plastic well. A control medium (culture medium for cell culture), a control IgG (mouse IgG2b), and an anti-MC16 antibody purified with a protein A column were reacted, and then HRP-anti-mouse IgG was reacted to determine the enzymatic activity of peroxidase, It was quantified by color development using a substrate for peroxidase. In addition, the specificity of the anti-MC16 antibody was confirmed using Western blotting.

As a result of the above assay, it was confirmed that the antibody (anti-MC16 antibody) produced by clone 31-8D binds to full-length human annexin A1 but does not bind to annexin A1 lacking the N-terminal domain (N-terminal 9 amino acids). , ELISA (Fig. 1) and Western blot (Fig. 2 right).
On the other hand, the rabbit anti-annexin A1 antibody (Invitrogen) bound to both full-length human annexin A1 and annexin A1 lacking the N-terminal domain (N-terminal 9 amino acids) (FIG. 2).

Example 4 (Investigation of fluorescent immunostaining)
Mouse vascular endothelial cells F2 were cultured in a monolayer, an extract of a tumor formed subcutaneously in mice with mouse melanoma B16 cells was added, and the cells were cultured for 2 days. The surface of F2 cells was reacted with an anti-MC16 antibody and then with a fluorescently labeled secondary antibody.
As can be seen from the results of the fluorescent immunostaining image of mouse vascular endothelial F2 cells, it was strongly suggested that the anti-MC16 antibody of the present invention also binds to mouse Anxa1 protein. It was also suggested that the B16 tumor extract may contain an active substance that induces the expression of the N-terminal domain of Annexin A1 on the surface of F2 cells (Fig. 3).

Example 5 (antibody peroxidase staining)
Paraffin-embedded tissue sections of tumor tissue collected from patients with glioblastoma were immunostained by the peroxidase method using anti-MC16 antibody. As a result, it was confirmed that the anti-MC16 antibody positively stained vascular endothelial cells in the tumor (Fig. 4, left). The right of FIG. 4 is a hematoxylin-eosin stain.

Example 6 (antibody peroxidase staining)
Paraffin-embedded tissue sections of prostate cancer tumor tissue were immunostained by the peroxidase method using an anti-MC16 antibody and a rabbit anti-ANXA1 antibody commercially available from Invitrogen. As a result, both antibodies stained tumor blood vessels positively, but it was revealed that the staining result of the anti-MC16 antibody was excellent with a low background (FIGS. 5 to 8).

The present invention is based on Japanese Patent Application No. 2019-017446 filed in Japan (filing date: February 1, 2019), and all the contents are included in the present specification.

Since the monoclonal antibody of the present invention specifically binds to the N-terminal region of Annexin A1, it is possible to detect the presence or absence of the N-terminal region of Annexin A1 on the vascular endothelial surface of a tissue such as a tumor. It can be used for selecting an application target of cancer treatment using a conjugate of an anticancer agent and a peptide that binds to the N-terminal region of annexin A1.

Claims

A monoclonal antibody that specifically binds to the N-terminal region of Annexin A1.
The monoclonal antibody according to claim 1, wherein the N-terminal region is a region containing the amino acid sequence represented by SEQ ID NO: 1.
Monoclonal antibody produced by the hybridoma deposited under accession number NITE P-02801.
Hybridoma that has been entrusted with the consignment number NITE P-02801.
A reagent for detecting the presence of the N-terminal region of annexin A1, which comprises the monoclonal antibody according to any one of claims 1 to 3.
Determining the applicability of cancer treatment using the conjugate of the peptide that binds to the N-terminal region of annexin A1 and the anticancer agent, which comprises the monoclonal antibody according to any one of claims 1 to 3. For reagents.
The reagent according to claim 6, wherein the cancer is solid cancer or liquid cancer.
A method for detecting the presence of an N-terminal region of annexin A1 on the surface of a tumor blood vessel, comprising:
(1) Contacting a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 according to any one of claims 1 to 3 with a sample derived from a subject (2) tumor in the sample A method comprising detecting the N-terminal region of Annexin A1 on the surface of blood vessels by immunostaining.
A method for assisting selection of an application target of cancer treatment using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent,
(1) Contacting a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 according to any one of claims 1 to 3 with a sample derived from a subject (2) tumor in the sample A method comprising detecting the N-terminal region of Annexin A1 on the surface of blood vessels by immunostaining.
The method according to claim 8 or 9, wherein the sample derived from the subject is a tissue section prepared by using a biopsy sample containing cancer cells collected by biopsy from the subject.
The method according to claim 9 or 10, wherein the cancer is solid cancer or liquid cancer.