JP7224628B2 - Anti-MC16 antibody - Google Patents

Description

NPMD NITE P-02801

TECHNICAL FIELD The present invention relates generally to the field of cancer biology, and more particularly to antibodies that specifically bind to the N-terminal region of annexin A1 and uses thereof.

Among brain tumors, most tumors such as meningioma and schwannoma that arise from the meninges and brain/spinal nerves are benign brain tumors, and complete cure is possible if they can be surgically removed. On the other hand, neuroepithelial tumors including glioma are basically malignant brain tumors. Especially for grade 4 glioblastoma, the prognosis is extremely poor (5-year survival rate is about 10%) even if radiotherapy and chemotherapy are performed after excision by open-head surgery. The presence of the blood-brain barrier is cited as a major reason why effective chemotherapy has not been implemented for brain tumors.

So far, the present inventors have found that 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, brain tumors are overwhelmed. Development of a malignant tumor vascular-targeting peptide (IF7, dTIT7, etc.) that can accumulate at a high concentration and actively cross the wall of the vascular endothelium, and use the peptide to develop a malignant tumor vascular-specific anticancer A drug delivery system has also been developed. In addition, since the peptide intravenously injected into a tumor-bearing mouse model targets malignant tumor blood vessels, it is thought that the N-terminal region of annexin A1 is actually expressed on the malignant tumor blood vessel endothelium surface.

The recent surge in medical costs is not only an economic burden on patients, but also a strained situation that threatens to bankrupt the national finances of our country. It is becoming a thing, and it is inviting an unimaginable medical disparity. Under these circumstances, the IF7 and dTIT7 developed by the present inventors and the delivery system for anticancer agents using these can efficiently cross the blood-brain tumor barrier and achieve excellent therapeutic effects on brain tumors and the like. Because it is possible and inexpensive, it is expected to help correct disparities in medical care on a global scale.

In order for the anticancer agents that bind IF7 and dTIT7 developed by the present inventors to exhibit therapeutic effects, the N-terminal region of annexin A1, which is a receptor for IF7 and dTIT7, must be present on the vascular endothelial surface. . Therefore, in patients suffering from brain tumors, etc., when performing treatment using anticancer drugs that bind IF7 or dTIT7, from the viewpoint of the patient's physical and economic burden, the tumor tissue etc. A diagnostic agent capable of detecting the presence of the N-terminal region of annexin A1 on the surface of the vascular endothelium is desired.

As a diagnostic agent for detecting the presence or absence of the N-terminal region of annexin A1 in the tumor tissue, an antibody that specifically binds to the N-terminal region of annexin A1 that can be used for immunostaining is suitable. To the best of our knowledge, such antibodies are not commercially available, nor have they been reported in academic papers or the like.

Oh et al., Nature 429: 629-35, 2004

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

As a result of intensive research, the present inventors 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 of malignant brain tumors. This led to the completion of the present invention.

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 shown in SEQ ID NO:1.
[3] A monoclonal antibody produced by the hybridoma deposited under accession number NITE P-02801.
[4] Hybridoma deposited under accession number NITE P-02801.
[5] A reagent for detecting the presence of the N-terminal region of annexin A1, comprising the monoclonal antibody of any one of [1] to [3].
[6] Application of cancer therapy using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent, including the monoclonal antibody according to any one of [1] to [3] Reagent for judging the propriety of
[7] The reagent according to [6], wherein the cancer is solid cancer or liquid cancer.
[8] A method for detecting the presence of the N-terminal region of annexin A1 on the surface of tumor blood vessels, comprising:
(1) contacting a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 according to any one of [1] to [3] with a sample derived from a subject; , detecting the N-terminal region of annexin A1 on the surface of tumor blood vessels by immunostaining.
[9] A method for assisting selection of targets for cancer therapy using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer drug, comprising:
(1) contacting a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 according to any one of [1] to [3] with a sample derived from a subject; , detecting the N-terminal region of annexin A1 on the surface of tumor blood vessels by immunostaining.
[10] A method for treating cancer using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent, comprising:
(1) contacting a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 according to any one of [1] to [3] with a sample derived from a subject; , detecting the N-terminal region of annexin A1 on the surface of tumor blood vessels by immunostaining; A method comprising administering a conjugate of a peptide that binds to the N-terminal region and an anti-cancer agent.
[11] any one of [8] to [10], wherein the subject-derived sample is a tissue section prepared using a biopsy sample containing cancer cells obtained by biopsy from the subject; described method.
[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, there are provided 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 tumor blood vessel surface. can provide a method for detecting the presence of the N-terminal region of annexin A1 in .

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 detecting the binding of the 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 an anti-MC16 antibody and an anti-annexin A1 antibody commercially available from Invitrogen. A multiple cancer tissue microarray from Biomax was used as a tissue sample. FIG. 6 shows the results of immunostaining of various cancer tumor tissues (lung cancer) with an anti-MC16 antibody and an anti-annexin A1 antibody commercially available from Invitrogen. A multiple cancer tissue microarray from Biomax was used as a tissue sample. FIG. 7 shows the results of immunostaining of various cancer tumor tissues (hepatocellular carcinoma) with an anti-MC16 antibody and an anti-annexin A1 antibody commercially available from Invitrogen. A multiple cancer tissue microarray from Biomax was used as a tissue sample. FIG. 8 shows the results of immunostaining of various cancer tumor tissues (ovarian cancer) with an anti-MC16 antibody and an anti-annexin A1 antibody commercially available from Invitrogen. A multiple cancer tissue microarray from Biomax was used as a tissue sample.

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"). offer. Specifically, the antibody is an antibody that specifically binds to an epitope present in the N-terminal region of annexin A1, including the amino acid sequence (MAMVSEFLKQAWFIE) shown in SEQ ID NO:1. The antibody may belong to any immunoglobulin class of IgG, IgA, IgM, IgD or IgE, preferably IgG, more preferably IgG2b. A specific example of the antibody includes 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 can be detected. suitable for detection. Therefore, the monoclonal antibody of the present invention is, for example, a conjugate containing a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent, and a cancer treatment containing the conjugate and a pharmaceutically acceptable carrier. It can be used for companion diagnosis for predicting whether or not a subject is a candidate for cancer treatment using a composition for cancer treatment.

As used herein, a subject is any animal, particularly a mammal, that expresses annexin A1. Mammals include, for example, rodents such as mice, rats, hamsters and guinea pigs, laboratory animals such as rabbits, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Primates such as monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, and the like can include, but are not limited to, these.

2. Production of the Monoclonal Antibodies of the Present Invention The monoclonal antibodies of the present invention and antibodies similar thereto can be produced by methods 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, an immunogenic antigen peptide is first injected subcutaneously or intramuscularly into mammals such as mice, rats and hamsters. , intravenously, in the footpad, intraperitoneally, etc., by one to several injections or by transplantation. Next, antibody-producing cells are isolated from the immunized mammal, the cells are fused with myeloma cells to form hybridomas, the hybridomas are cloned, and the immunogenic antibody used to immunize the mammal is isolated. It is produced by selecting clones that produce antibodies that exhibit specific affinity to the antigenic peptide.
As antibody-producing cells, antibody-producing cells produced by reacting previously isolated splenocytes, lymphocytes, or the like with an immunogenic antigenic peptide in a culture medium can also be used. In this case, human-derived antibody-producing cells can also be prepared.

An immunogenic antigen peptide can be diluted with PBS (Phosphate-Buffered Saline), physiological saline, or the like at an appropriate dilution ratio and used for immunization. Immunogenic antigenic peptides may also be administered with an adjuvant, for example, mixed with Freund's complete or incomplete adjuvant (FCA or FIA) and emulsified. In addition, suitable carriers may be used when immunizing with immunogenic antigens. In particular, when a peptide with a small molecular weight is used as an immunogenic antigen, it is desirable to combine the immunogenic antigen peptide with a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.
In the examples described later, a synthetic peptide (SEQ ID NO: 2: MAMVSEFLKQAWFIEC) (hereinafter referred to as "synthetic peptide A mixture of bovine serum albumin-bound bovine serum albumin and complete Freund's adjuvant was used as an immunogenic antigen peptide.

Hybridomas secreting monoclonal antibodies can be prepared according to the method of Koehler and Milstein (Nature, Vol. 256, pp. 495-497, 1975) and modifications thereof. That is, the monoclonal antibody of the present invention can be obtained from splenocytes, lymph node cells, peripheral lymphocytes, myeloma cells, tonsil cells, etc. obtained from the animal immunized as described above, preferably antibody-producing cells contained in splenocytes. , preferably allogeneic mammals such as mice, rats, guinea pigs, hamsters, rabbits or humans, more preferably mouse, rat or human myeloma cells (myeloma) obtained by culturing. be. Culturing can be performed in vitro or in vivo in mammals such as mice, rats, guinea pigs, hamsters and rabbits, preferably mice or rats, more preferably intraperitoneally in mice. It can be obtained from animal ascites.

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, CEM-T15, and the like.

Screening for hybridoma clones producing the monoclonal antibody of the present invention is carried out by culturing the hybridomas in, for example, a microtiter plate, and measuring the reactivity of the culture supernatant in wells in which growth is observed with an immunogenic antigenic peptide. For example, it can be measured by ELISA, Western blotting, immunostaining, or the like.

As described in Examples below, a hybridoma that produced an antibody highly reactive to the synthetic peptide MC16 was cloned by limiting dilution to obtain the monoclonal antibody-producing hybridoma of the present invention (clone 31-8D). On December 5, 2018, clone 31-8D was transferred to the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Room 122, Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture) under the accession number NITE P-02801. deposited.

Isolation and purification of monoclonal antibodies are carried out by subjecting the antibody-containing culture supernatant or ascitic fluid produced by the method described above to, for example, ion-exchange chromatography, anti-immunoglobulin column, or affinity column chromatography such as protein A or protein G column. This can be done by subjecting it to graphics.

A monoclonal antibody of the invention may be labeled with a detectable label. Examples of labeling include fluorescence labeling, labeling with low-molecular-weight compounds, labeling with peptides, labeling with enzymes, and the like. Monoclonal antibodies of the present invention may also form conjugates with other agents.

The monoclonal antibody of the present invention may be obtained by any method without being limited to the production method described above. For example, the gene encoding the monoclonal antibody of the present invention, cloned from the hybridoma of the present invention, may be incorporated into an appropriate vector and introduced into a host to express it as an antibody. Methods for isolating genes encoding antibodies, introducing them into vectors, and transforming host cells have already been established (see, for example, Vandamme, A. M. et al. Eur. J. Biochem. (1990) 192, 767). -775).

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

The cDNA fragment of interest is purified from the resulting PCR product and then operably ligated with vector DNA. After the recombinant vector is prepared in this way, introduced into Escherichia coli or the like, and colonies are selected, the desired recombinant vector can be prepared from the colony-forming Escherichia coli. Then, whether or not the recombinant vector has the desired cDNA base sequence can be confirmed by a method known per se, such as the dideoxynucleotide chain termination method.

Monoclonal antibodies usually have sugar chains with different structures depending on the types of mammals to be immunized. It also includes Furthermore, for example, a recombinant human monoclonal antibody obtained from a transgenic animal into which a human immunoglobulin gene has been integrated, or a constant region (Fc) of a mammal-derived monoclonal antibody is recombined with the Fc region of a human-derived monoclonal antibody. The monoclonal antibodies of the present invention also include chimeric monoclonal antibodies, and chimeric monoclonal antibodies in which the entire region other than the complementarity determining region (CDR) capable of complementary and direct binding to an antigen is recombined with the corresponding region of a human-derived monoclonal antibody. be done.

Further, the antibodies of the present invention include natural antibodies such as the above-mentioned monoclonal antibodies (mAb), chimeric antibodies that can be produced using genetic recombination technology, humanized antibodies and single-chain antibodies, as well as these antibodies. contains fragments of Antibody fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes Fab, Fab', F(ab') ₂ , scAb, scFv, scFv-Fc, etc. .

These antibody fragments can be obtained using a method known per se. Specifically, the monoclonal antibodies of the present invention are treated with enzymes such as papain and pepsin to produce antibody fragments, or A gene encoding the fragment may be constructed, introduced into an expression vector, and then expressed in a suitable host cell. The resulting fragment can be evaluated for antigen reactivity, specificity, etc. in the same manner as the monoclonal antibody of the present invention.

In addition, those skilled in the art can produce fusion antibodies from the antibodies or fragments of the present invention and other peptides or proteins, or produce modified antibodies bound with modifiers. Other peptides and proteins used for fusion are not particularly limited as long as they do not reduce the binding activity of the antibody. , various toxins, peptides or proteins capable of promoting multimerization, and the like. Modifiers used for modification are not particularly limited as long as they do not reduce the binding activity of the antibody, and examples thereof include polyethylene glycol, sugar chains, phospholipids, liposomes, low-molecular-weight compounds and the like.

3. Reagent 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, and therefore, It is suitable for detecting the presence or absence of the N-terminal region of annexin A1, which is a receptor for therapeutic drugs for malignant tumors, using IF7 and dTIT7, which have tumor-targeting activity. Accordingly, the invention also provides reagents for detecting the presence of the N-terminal region of annexin A1, including the monoclonal antibodies of the invention.

Also, as described above, the monoclonal antibodies of the present invention can be used as companion diagnostics to predict whether a particular cancer therapy is indicated. Accordingly, the present invention provides a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent, including the monoclonal antibody of the present invention, and a composition comprising the conjugate and a pharmaceutically acceptable carrier. Also provided are reagents for determining the applicability of cancer therapy using, etc.

As used herein, a malignant tumor (cancer) may be any type of cancer, solid or liquid. Solid tumors preferably include solid tumors that express annexin A1 on the cell surface, and therefore angiogenic solid tumors. Solid cancers include, for example, cancers of the brain and nervous system (e.g., brain tumors, spinal cord tumors, etc.), head and neck cancers (e.g., laryngeal cancer, oral cavity cancer, salivary gland cancer, sinus cancer, thyroid cancer, etc.). cancer, etc.), gastrointestinal cancer (e.g., gastric cancer, esophageal cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, etc.), urological or Cancers of the reproductive organs (e.g., kidney cancer, renal cell cancer, bladder cancer, prostate cancer, renal pelvic and ureteral cancer, gallbladder cancer, bile duct cancer, testicular cancer, penile cancer, uterine cancer , endometrial cancer, uterine sarcoma, cervical cancer, vaginal cancer, vulvar cancer, ovarian cancer, fallopian tube cancer, etc.), respiratory system cancer (e.g., lung cancer (small cell lung cancer, non- small cell lung cancer, metastatic lung cancer), bronchial cancer, etc.), breast cancer, skin cancer (e.g., malignant melanoma, etc.), bone cancer (e.g., osteosarcoma, etc.), muscle cancer (e.g., rhabdomyosarcoma, etc.).

Liquid cancers include leukemia, malignant lymphoma, multiple myeloma, and myelodysplastic syndrome. Leukemia includes acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and the like. Malignant lymphoma is classified into Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma includes B-cell lymphoma, adult T-cell lymphoma, lymphoblastic lymphoma, diffuse large cell lymphoma, Burkitt's lymphoma, and follicular lymphoma. , MALT lymphoma, peripheral T-cell lymphoma, and mantle cell lymphoma.

As a result of companion diagnosis using the reagent of the present invention, a conjugate of a peptide that binds to the N-terminal region of annexin A1 administered to a subject and an anticancer agent, or a combination of the conjugate and a pharmaceutically acceptable carrier. A particularly preferred target is brain tumor, since the composition or the like containing it can efficiently cross the blood-brain tumor barrier, as described later.

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

The reagent of the present invention may consist solely of the monoclonal antibody of the present invention, or may contain a pharmaceutically acceptable carrier. When the reagent of the present invention is prepared as a liquid formulation, the pharmaceutically acceptable carrier includes various carriers commonly used as pharmaceutical materials, such as diluents, solvents, solubilizers, suspending agents, and isotonizing agents. agents, buffers, and the like. Furthermore, a surfactant such as Tween20 (registered trademark) or Tween80 (registered trademark) may be added to prevent antibody aggregation. Diluents include water, physiological saline, and the like. Solvents include water, physiological saline, ethanol, and the like. Examples of solubilizing agents include cyclodextrins. Suspending agents include gum arabic, carmellose and the like. Tonicity agents include inorganic salts such as sodium chloride and potassium chloride, and carbohydrates such as glycerin, mannitol and sorbitol. Buffers include phosphate buffers, acetate buffers, borate buffers, carbonate buffers, citrate buffers, Tris buffers and the like. The blending ratio of these carriers can be appropriately determined by those skilled in the art.

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. 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 tumors, using the monoclonal antibody. also provide.

Specifically, the method of the present invention includes (1) contacting a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 of the present invention with a sample derived from a subject, and (2) in the sample, , including detection 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 tumors by immunostaining.

Also, as described above, the monoclonal antibodies of the present invention can be used as companion diagnostics to predict whether a particular cancer therapy is indicated. Therefore, the present invention provides a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent using the monoclonal antibody, a composition comprising the conjugate and a pharmaceutically acceptable carrier, and the like. Also provided are methods for selecting or assisting in the selection of targets for cancer therapy using . Furthermore, the present invention provides a subject selected as described above, a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer agent, and a composition comprising the conjugate and a pharmaceutically acceptable carrier. It may include actually performing cancer treatment by administering a substance or the like.

Specifically, the method of the present invention includes (1) contacting a monoclonal antibody that specifically binds to the N-terminal region of annexin A1 of the present invention with a sample derived from a subject, and (2) in the sample, , including detection 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 tumors by immunostaining.

The measurement 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 immunostaining is preferably used. Specific means using immunostaining include immunohistochemical staining (IHC), flow cytometry analysis, mass cytometry analysis, and the like.

Immunohistochemical staining can be performed according to a method known per se (for example, Watanabe/Nakane Enzyme Antibody Method, Revised 4th Edition, Published by Gakkan Kikaku Co., Ltd., Edited by Hiroshi Nagura, Yoshiyuki Nagamura, Hiroshi Tsutsumi, ISBN4-906514 -73-X C304), preferably, an enzyme antibody method (Enzyme labeled antibody method), a fluorescence antibody method, or the like is used.

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

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

As a general protocol, if necessary, prepare a deparaffinized or fixed cell specimen, and if necessary, pretreat (e.g., decolorization or removal of biological pigments, blocking) or antigen retrieval (warm bath, microwave treatment, autoclave). treatment, protease treatment, etc.), reacting with a primary antibody (e.g., 4°C to room temperature for 0.5 to 24 hours), and after the reaction is completed, the cell specimen is washed and reacted with a secondary antibody (e.g., 4°C to room temperature). for 5 to 120 minutes), and after the reaction is complete, wash the cell specimen. A chromogenic substrate such as 3,3-diaminobenzidine (DAB) is then added to develop color. For morphological observation under a microscope, counterstaining such as hematoxylin staining may be 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 in the enzyme antibody method described above. Also in the immunofluorescence method, labeling may be performed directly or indirectly using a secondary antibody.

Immunostained samples are imaged using an optical microscope equipped with an imaging device or a fluorescence microscope (in the case of fluorescence staining) or the like, and stored in a computer as a digital image. A 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 prior to analysis.

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

Classification of immunostaining intensity can follow standard criteria by immunohistopathologists, e.g., score 0 (undetected), score +1 (weak), score +2 (moderate), and score +3 (marked). ) can be classified as In addition, 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 drug, a composition containing the conjugate and a pharmaceutically acceptable carrier, etc. were used. It may be judged as an application target for cancer treatment.

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

Peptides that bind to the N-terminal region of annexin A1 used in the methods of the present invention (hereinafter sometimes referred to as "peptides used in the methods of the present invention") include, for example, the following (I) to (III) ) containing any of the amino acid sequences of:
(I) (X1)[D]P[D](X2)[D] amino acid sequence (in which X1 represents W or F and X2 represents S or T),
(II) Amino acid sequence of P[D]T[D](X) _nF [D] (in the sequence, (X) _n represents n arbitrary amino acids selected independently from each other, n is Indicates an integer from 0 to 4.),
(III) An amino acid sequence that is a retro-inverso of the amino acid sequence of either (I) or (II) above.

In addition, the peptide used in the method of the present invention is, for example, a peptide containing any one 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) the amino acid sequence of L[D]L[D]S[D]W[D]P[D]S[D]A[D];
(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 in the amino acid sequence of any one of (i) to (v), or a combination thereof;
(vii) An amino acid sequence that is a retro-inverso of the amino acid sequence of any one of (i) to (vi) above.

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 designation. In addition, each amino acid symbol immediately followed by the symbol [D] in the amino acid sequence indicates the D-form of the amino acid, and each amino acid symbol not immediately followed by the symbol [D] in the amino acid sequence is contradicted by the context. Unless otherwise indicated, the L-form of the amino acid is indicated.

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 Any of [D] P[D] T[D] is acceptable.

In the amino acid sequence of (II) above, the integer n is 0-4, preferably 2-3. 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 one of the amino acid sequences (I) to (III) and (i) to (vii) above, or the N-terminal side of these sequences and/or one or more amino acids may be added to the C-terminal side.

As used herein, the peptide used in the method of the present invention refers to a peptide bond of two or more amino acids. Although the length of the peptide of the present invention is not particularly limited, 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 methods of the invention may be up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 19, up to 18, up to 17 , 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.

Peptides used in the methods of the present invention may be composed of a combination of D- and L-amino acids. Also, the peptides used in the methods of the invention may or may not contain modified or unusual amino acids, such as those mentioned in 37 C.F.R. 1.821-1.822.

The amino- and/or carboxy-terminus of peptides used in the methods of the invention may be modified. In addition, peptides used in the methods of the present invention may be modified in various ways other than at the N-terminus or C-terminus.

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

The above sequence (vi) 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.

Retro-inverso isomers of a peptide are those in which the chirality of each amino acid residue is opposite ("inverso") and the orientation of the amino acid sequence is reversed ("retro") relative to the original peptide. say. Retro-inverso isomers are known to exhibit similar structures and functions to the original peptides (for example, 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 (I) to (III) and (i) to (vii) above. The peptide used in the method of the present invention is a tandem repeat of any of the above sequences (I) to (III) and (i) to (vii) (that is, the same sequence portions are directly linked to each other) structure). In addition, the peptide used in the method of the present invention may contain a structure in which two or more different sequences (I) to (III) and (i) to (vii) above 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 peptides used in the methods of the present invention may constitute multivalent peptides via dendrimers.

Peptides used in the methods of the invention may be in free form or in salt form. Salts of peptides used in the methods of the present invention include pharmaceutically acceptable acid addition salts and base addition salts. Acid addition salts include salts with inorganic acids such as hydrochloric, sulfuric, nitric and phosphoric acid, and salts with organic acids such as acetic, malic, succinic, tartaric and citric. Base addition salts include salts with alkali metals such as sodium and potassium, 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 by SEQ ID NO: 1 in human annexin A1, or 1 to 15 shown by SEQ ID NO: 3 (MAMVSEFLKQARFLE) in mouse annexin A1. It can bind to the region consisting of the th amino acid. The peptide used in the method of the present invention preferably has an intermolecular interaction with, for example, mouse or human annexin A1, when measured using the QCM (Quartz Crystal Microbalance) method, preferably less than 10 ⁻⁶ M, more preferably less than 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 measurements, a human recombinant annexin A1 protein consisting of 346 amino acid residues commercially available from ATGen (Seongnam-si, South Korea) can be used.

Peptides used in the method of the present invention can be produced according to known peptide synthesis methods. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. Condensing a partial peptide or amino acid that can constitute the peptide of the present invention with the remaining portion, and if the product has a protecting group, removing the protecting group by a method known per se to produce the desired peptide. can be done.

The peptide thus obtained can be purified and isolated by a known purification method. Here, examples of purification methods include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When the peptide obtained by the above method is a free form, the free form can be converted into a suitable salt by a known method or a method analogous thereto. Conversely, when the peptide is obtained as a salt, , the salt can be converted into the educt or other salt by known methods or analogous methods.

The anticancer drug used in the method of the present invention refers to a drug intended to suppress the growth of malignant tumors (cancer). The mechanism of action of the anticancer agent is not particularly limited. Anticancer agents can be antimetabolites, alkylating agents, anticancer antibiotics, microtubule inhibitors, platinum agents, topoisomerase inhibitors, molecular targeted agents, and the like. A conjugate of the invention may comprise two or more of the same or different anti-cancer agents.

Antimetabolites are, 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 antimetabolites include enocitabine (Sunlavine), capecitabine (Xeloda), carmofur (Miflor), cladribine (leustatin), gemcitabine (Gemzar), cytarabine (kiloside), cytarabine ocphosphate (staracid), tegafur ( atilon, aftofur, tefsil, ftrafur, lunasin, etc.), tegafur-uracil (UFT), tegafur-gimeracil-oteracil-potassium (TS-1: TS-1), doxifluridine (furturon), nerarabine (alanongi), hydroxycarbamide (hydrea) ), fluorouracil (5-FU, calzonal, bennan, lunacol, lunarvone), fludarabine (fludara), pemetrexed (Alimta), pentostatin (cohorin), mercaptopurine (leukeline), methotrexate (methotrexate), and the like.

Specific examples of alkylating agents include nitrogen mustard-based alkylating agents such as cyclophosphamide (endoxan), ifosfamide (ifomide), melphalan (alkeran), busulfan, thiotepa (tespamine), nimustine (nidoran), ranimustine cymerin), dacarbacine (dacarbacine), procarbacine (procarbacine hydrochloride), temozolomide (Temodar), carmustine (Gilliader), streptozotocin (Zanosar), bendamustine (Treakisym) and other nitrosourea alkylating agents.

Specific examples of anticancer antibiotics include actinomycin D (cosmegen), aclarubicin (acracinone), amrubicin (calced), idarubicin (idamycin), epirubicin (epirubicin hydrochloride, famorubicin), dinostatin stimaramer (Sumanx ), daunorubicin (daunomycin), doxorubicin (adriacin), pirarubicin (pinorubine, terarubicin), bleomycin (bleo), peplomycin (pepleo), mitomycin C (mitomycin), mitoxantrone (novantrone), liposomal doxorubicin (doxil), etc. is mentioned.

Microtubule inhibitors include, for example, vinblastine (Exar), vincristine (Oncovin), vinca alkaloid microtubule polymerization inhibitors such as vindesine (Fordesine), taxane microtubule depolymerization such as paclitaxel (Taxol) and docetaxel (Taxotere). inhibitors and the like.

Examples of platinum agents include oxaliplatin (Elplat), carboplatin (Carboplatin, Carbomerc, Paraplatin), cisplatin (IACOL, Conaburi, Cisplatin, etc.), and nedaplatin (Acpla).

Examples of topoisomerase inhibitors include type I topoisomerase inhibitors such as camptothecin and its derivatives (eg, irinotecan (campto), nogitecan (hycamtin), SN-38, etc.); anthracycline drugs such as doxorubicin (adriacin); etoposide; Type II topoisomerase inhibitors such as epipodophyllotoxins such as (Lasted, Bepcid) and quinolone drugs such as levofloxacin (Cravit) and ciprofloxacin (Ciproxan).

Examples of molecular targeted drugs include regorafenib (Stivarga), cetuximab (Erbitux), panitumumab (Vectibix), ramucirumab (Cyramza), gefitinib (Iressa), erlotinib (Tarceva), afatinib (Geotrif), crizotinib (Zarkori), alectinib (Alecensa), ceritinib, lenvatinib (Lenvima), trastuzumab (Herceptin), lapatinib (Tikerb), pertuzumab (Perjeta), sunitinib (Sutent), sorafenib (Nexavar), axitinib (Inlyta), pazopanib (Votrient), nivolumab ( Opdivo), pembrolizumab, ipilimumab (Yervoy), vemurafenib (Zerboraf), everolimus (Afinitor), temsirolimus (Toricel), rituximab (Rituxan), bevacizumab (Avastin), geldanamycin and the like.

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

The mode of binding between the peptide that binds to the N-terminal region of Annexin A1 and the one or more anticancer agents in the conjugate used in the methods of the present invention is not particularly limited. The binding may be direct or indirect via a linker or the like. Binding may be covalent, non-covalent, or a combination thereof. The one or more anticancer 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. Conjugation of peptides and anticancer agents is well known in the art, and in the conjugates of the present invention, the conjugation may be by any known means.

A composition comprising a conjugate used in the methods of the invention and a pharmaceutically acceptable carrier may be provided in a form suitable for oral or parenteral administration. Compositions for parenteral administration include, for example, injections and suppositories, and injections include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. can include 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 and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. agents, emulsions, suspensions and the like. Such compositions may be produced by known methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field.

The composition described above may contain other active ingredients as long as they do not adversely interact with the conjugate when mixed with the conjugate.

The above parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Such dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The content of the peptide or conjugate is usually 1 to 500 mg per dosage unit, preferably 1 to 100 mg for injections, and 10 to 250 mg for other dosage forms. .

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

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

Example 1 (Preparation of mouse monoclonal antibody)
(1) Preparation of immunogen The C-terminal cysteine (Cys) of the synthetic peptide MC16 was bound to a carrier protein (BSA) using a bivalent reactive reagent to prepare an immunogen.
(2) Production of HRP Labeled Product for Screening A HRP labeled product for screening was produced by binding the C-terminal Cys of the synthetic peptide MC16 to horseradish-derived peroxidase (HRP) using a bivalent reactive reagent.

(3) Immunization The antigen with immunogenicity prepared in (1) above is mixed with FCA to emulsify, and 200 μg calculated is injected intramuscularly into the tail region of each Balb/c mouse (immunization). (total 3 animals). Based on the results of the initial antibody activity measurement, the same antigen was further mixed with FIA and emulsified, and 50 μg of the antigen was subcutaneously injected (boost immunization) per mouse twice at 2-week intervals. Based on the results of the antibody activity measurement after the two booster immunizations, a final immunization (100 μg calculated per mouse) was performed in the peritoneal cavity of the mice.

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

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

(7) Primary screening to culture Nine days after cell fusion, the culture supernatant is sampled from each well of a 96-well microplate, and the response of the culture supernatant to MC16 is measured by ELISA using a synthetic MC16 peptide bound to a plastic plate. and a primary screening was performed.
(8) Secondary screening and cryopreservation of cells
After transferring to a 24-well microplate and expanding culture for 3 days, the culture supernatant was sampled from each well of the microplate, secondary screening was performed by the same ELISA as above, and the cells in the selected wells were frozen. Saved.

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

(10) Primary Screening ELISA was performed in the same manner as above, and 40 clones exhibiting the desired specificity were selected and expanded.
(11) Secondary Screening and Cryopreservation of Cells As a result of performing the same ELISA as above, many positive clones were obtained. Cells of 18 clones among the positive clones 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 produced by clone 31-8D (anti-MC16 antibody))
A plate assay was performed to confirm the binding of the anti-MC16 antibody to the 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 ELISA plastic wells. Control medium (culture medium for cell culture), control IgG (mouse IgG2b), and anti-MC16 antibody purified by protein A column were reacted with each other, and then reacted with HRP-anti-mouse IgG to detect peroxidase enzymatic activity. Quantification was performed by color development using a peroxidase substrate. Also, the specificity of the anti-MC16 antibody was confirmed using Western blotting.

As a result of the above assay, 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, a 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 (Study of fluorescent immunostaining)
Mouse vascular endothelial F2 cells were cultured in a monolayer, and an extract of a tumor subcutaneously formed in a mouse with mouse melanoma B16 cells was added and cultured for 2 days. After reacting the surface of F2 cells with an anti-MC16 antibody, they were reacted with a fluorescently labeled secondary antibody.
As can be seen from the results of fluorescent immunostaining images 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 F2 cell surface (Fig. 3).

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

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

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 tissues such as tumors. It can be used to select targets for cancer therapy using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer drug.

Claims (7)

Reagents for detecting the presence of the N-terminal region of annexin A1, including monoclonal antibodies produced by the hybridoma deposited under Accession No. NITE P-02801 . Applicability of cancer therapy using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer drug, including the monoclonal antibody produced by the hybridoma deposited under Accession No. NITE P-02801. Reagent for determination. 3. The reagent according to claim 2 , wherein the cancer is solid cancer or liquid cancer. A method for detecting the presence of the N-terminal region of annexin A1 on the surface of tumor blood vessels, comprising:
(1) contacting a subject-derived sample with a monoclonal antibody produced by the hybridoma deposited under accession number NITE P-02801; , detecting by immunostaining. A method for assisting selection of targets for cancer therapy using a conjugate of a peptide that binds to the N-terminal region of annexin A1 and an anticancer drug, comprising:
(1) contacting a subject-derived sample with a monoclonal antibody produced by the hybridoma deposited under accession number NITE P-02801; , detecting by immunostaining. 6. The method according to claim 4 or 5 , wherein the subject-derived sample is a tissue section prepared using a biopsy sample containing cancer cells biopsied from the subject. 7. The method of claim 5 or 6 , wherein said cancer is solid or liquid cancer.

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