New Anti-C-KIT Antibody

Abstract

The present invention relates to a novel anti-C-KIT antibody or an antibody fragment thereof. The present invention also relates to a composition for preventing or treating angiogenesis-related diseases, or a kit for diagnosing angiogenesis-related diseases, comprising the anti-C-KIT antibody or the antibody fragment thereof.

Description

[Technical field]

[0001] The present invention relates to a novel anti-C-KIT antibody or an antibody fragment thereof. The present invention also relates to a composition for preventing or treating angiogenesis-associated diseases, or a kit for diagnosing angiogenesis-associated diseases, comprising the anti-C-KIT antibody or the antibody fragment thereof. [Background technology]

[0002] Angiogenesis refers to the mechanism by which new blood vessels are generated from existing blood vessels. Abnormal or excessive angiogenesis can cause various diseases. For example, angiogenesis is one of the causes of tumor growth as well as tumor progression from benign to malignant. In addition, excessive formation of angiogenesis has been reported in various diseases such as eye diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma, and rheumatoid arthritis, psoriasis, and chronic inflammation (Cameliet and Jain, Nature, 407:249, 2000 (Non-Patent Document 1)). For this reason, research has been conducted to treat angiogenesis-related diseases using angiogenesis inhibitors, and many angiogenesis promoting and inhibiting factors involved in angiogenesis processes such as growth, migration, and differentiation of vascular endothelial cells have been discovered.

[0003] Angiogenesis inhibitors can be divided into several categories, such as matrix-breakdown inhibitors, endothelial cell inhibitors, and angiogenesis inhibitors, depending on the mechanism of action. Among these, angiogenesis inhibitors include drugs that target VEGFR2, VEGFR1, PDGFR, C-KIT, FLT3, etc. and inhibit their activity, signal transduction, production, etc.

[0004] C-KIT, one of the targets of the angiogenesis inhibitor, belongs to class III receptor tyrosine kinase (RTK) and is also known as a receptor for SCF (Stem Cell Factor). SCF is a cytokine that binds to the C-KIT receptor and has been reported to play an important role in the differentiation and generation of blood cells, sperm, and melanocytes.

[0005] As a result of the interaction between SCF and the ligand-binding domain of C-KIT, the C-KIT protein is phosphorylated and becomes active, and regulates various biological functions such as cell growth, differentiation and proliferation through signal transduction processes such as the PI3K / AKT system, RAS / MAP kinase, etc. In particular, the effect of SCF / C-KIT stimulation in neovascularization has been reported (Angiogenesis in Health, Disease and Malignancy, pp33-36 (Non-Patent Document 2)).

[0006] There are commercially available drugs targeting C-KIT, such as Gleevec (imatinib mesylate) and Sutent (sunitinib malate), which are multi-targeted therapeutic agents that inhibit various kinases, and have been reported to have therapeutic limitations such as numerous side effects, low specificity and bioavailability, antigenicity, and unsuitable pharmacokinetics, etc. Therefore, there is a demand for the development of an effective therapeutic agent specific to C-KIT, without side effects, for diseases associated with angiogenesis caused by activation of C-KIT.

[0007] As a result of extensive efforts to find a therapeutic agent for angiogenesis-related diseases, the inventors have discovered that a specific anti-C-KIT antibody that specifically binds to C-KIT can significantly inhibit neovascularization, and thus has excellent therapeutic properties for angiogenesis-related diseases, thereby completing the present invention. [Prior art documents] [Non-patent literature]

[0008] [Patent Document 1] Cameliet and Jain,Nature,407:249,2000 [Patent Document 2] Angiogenesis in Health,Disease and Malignancy,pp33-36 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide an anti-C-KIT antibody or an antibody fragment thereof that specifically binds to domain II of C-KIT protein and has excellent C-KIT inhibitory activity, and also aims to provide a composition and a diagnostic kit for preventing or treating angiogenesis-related diseases, each comprising the anti-C-KIT antibody or the antibody fragment thereof. [Means for solving the problem]

[0010] According to one aspect, the present invention provides an anti-C-KIT antibody or an antibody fragment thereof that specifically binds to C-KIT.

[0011] According to one embodiment of the present invention, the anti-C-KIT antibody or antibody fragment thereof of the present invention specifically binds to domain II of C-KIT.

[0012] The term "antibody" as used herein means an immunoglobulin molecule immunologically reactive with a specific antigen or a protein molecule that plays the role of a receptor that specifically recognizes an antigen. Thus, in the present invention, "antibody" is used in a broad sense and is interpreted to include all of polyclonal antibodies, monoclonal antibodies, whole antibodies (antibodies consisting of at least two heavy chains and two light chains interconnected by disulfide bonds) and antibody fragments. The whole antibodies include IgA, IgD, IgE, IgM and IgG. In addition, the IgG may include subtypes IgG1, IgG2, IgG3 and IgG4.

[0013] The term "antibody fragment" as used herein refers to an antigen-binding fragment or analog of an antibody that retains at least a portion of the binding specificity of the parent antibody and contains a portion of the antigen-binding region (e.g., one or more CDRs) or variable region of the parent antibody. The antibody fragment may be, for example, a Fab, Fab', F(ab')2, Fv fragment, sc-Fv, unibody, diabody, linear antibody, nanobody, domain antibody, or a multispecific antibody formed from antibody fragments.

[0014] The term "heavy chain" as used herein refers to a full-length heavy chain or a fragment thereof that includes a heavy chain variable region and a heavy chain constant region. Heavy chains include gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types.

[0015] The term "light chain" as used herein refers to a full-length light chain containing a light chain variable region and a light chain constant region, and fragments thereof. Light chains are of kappa (κ) and lambda (λ) types.

[0016] In the present invention, the antibody may be a full-length antibody or an antibody fragment having antigen-binding ability, the heavy chain may be any one of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) types, and the light chain may be kappa (κ) or lambda (λ) type. According to one embodiment of the present invention, the light chain is of the kappa type.

[0017] The term "C-KIT" as used herein belongs to class III receptor tyrosine kinase (RTK) and is also known as an SCF receptor.

[0018] C-KIT, one of the targets of the angiogenesis inhibitors, belongs to class III receptor tyrosine kinase (RTK) and is a receptor for SCF (Stem Cell Factor), which plays an important role in hematopoiesis.

[0019] The term "anti-C-KIT antibody" as used herein means an antibody that specifically binds to C-KIT. Specifically, the anti-C-KIT antibody specifically binds to domain II of C-KIT, thereby inhibiting or neutralizing the activity or activation of C-KIT.

[0020] According to yet another embodiment of the present invention, the anti-C-KIT antibody or antibody fragment thereof of the present invention comprises a light chain variable region including a light chain CDR1 represented by SEQ ID NO:1, a light chain CDR2 represented by SEQ ID NO:2, and a light chain CDR3 represented by SEQ ID NO:3.

[0021] According to yet another embodiment of the present invention, the anti-C-KIT antibody or antibody fragment thereof of the present invention comprises a heavy chain variable region including a heavy chain CDR1 represented by SEQ ID NO: 4, a heavy chain CDR2 represented by SEQ ID NO: 5, and a heavy chain CDR3 represented by SEQ ID NO: 6.

[0022] According to yet another embodiment of the present invention, the light chain variable region of the anti-C-KIT antibody or antibody fragment thereof of the present invention comprises the amino acid sequence of SEQ ID NO:7.

[0023] According to yet another embodiment of the present invention, the heavy chain variable region of the anti-C-KIT antibody or antibody fragment thereof of the present invention comprises the amino acid sequence of SEQ ID NO:8.

[0024] According to yet another embodiment of the present invention, the anti-C-KIT antibody or antibody fragment thereof of the present invention may comprise a light chain comprising the amino acid sequence of SEQ ID NO:25.

[0025] According to yet another embodiment of the present invention, the anti-C-KIT antibody or antibody fragment thereof of the present invention may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:26.

[0026] The term "CDR (Complementarity Determining Region)" as used herein means the amino acid sequence of a hypervariable region that forms an antigen-binding site as a part of the variable region of an antibody produced by B cells and T cells. The amino acid sequences of the heavy chain and the light chain each contain three non-contiguous CDRs, i.e., heavy chain CDR H1 , CDR H2 , CDR H3 and light chain CDR L1 , CDR L2 , CDR L3 CDRs are sites involved in antigen recognition and provide key contact residues when an antibody binds to an antigen or epitope, and thus play a crucial role in the diversity of antigen specificity.

[0027] The antibody or antibody fragment thereof of the present invention comprises a sequence that shows substantial identity to a sequence set forth in the sequence listing, where substantial identity means that the two sequences show 80%, 90%, or 95% or more sequence homology when aligned to maximize correspondence and then analyzed using algorithms commonly used in the art.

[0028] Furthermore, the anti-C-KIT antibody of the present invention may include not only the sequences of the anti-C-KIT antibodies described herein, but also biological equivalents thereof, so long as they are capable of specifically recognizing and binding to C-KIT. For example, the anti-C-KIT antibody may include additional mutations in the sequence to improve the antibody binding affinity and / or biological properties, and may include additional mutations within a range that does not alter the overall activity of the molecule.

[0029] According to another aspect of the present invention, the anti-C-KIT antibody or antibody fragment thereof may comprise a human IgG1-derived constant region. According to one aspect of the present invention, the present invention provides a human anti-C-KIT antibody comprising the light chain variable region, the heavy chain variable region and a human IgG1-derived constant region.

[0030] The term "human antibody" as used herein is understood to mean an antibody having variable regions in which the framework and CDR regions are derived from human immunoglobulin sequences. In the present invention, a human antibody may contain amino acid residues not encoded by human-derived immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The human antibody may be in the form of a full-length antibody or in the form containing a functional fragment of an antibody molecule. Since all components of the human antibody are derived from humans, there is a lower probability of an immune response when administered to humans compared to humanized antibodies or mouse antibodies. Therefore, it has advantageous advantages as a therapeutic antibody for humans.

[0031] Effects related to the inhibitory effect on neovascularization have been confirmed through various examples described below. As a result, the anti-C-KIT antibody or its antibody fragment of the present invention can effectively prevent or treat angiogenesis-related diseases by significantly inhibiting neovascularization.

[0032] In yet another aspect, the present invention provides a nucleic acid encoding an anti-C-KIT antibody or an antibody fragment thereof.

[0033] The nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof according to the present invention may include the nucleotide sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. Here, the nucleotide sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 encode the light chain CDR1 represented by SEQ ID NO: 1, the light chain CDR2 represented by SEQ ID NO: 2, and the light chain CDR3 represented by SEQ ID NO: 3, respectively. The nucleotide sequences of SEQ ID NO: 9 to 11 may be codon-optimized for CHO cells, and the nucleic acid comprising such a codon-optimized nucleotide sequence should be interpreted as being included within the scope of the nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof according to the present invention. For example, the nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof according to the present invention may include SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0034] Alternatively, the nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof according to the present invention may comprise the nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. Here, the nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 encode the heavy chain CDR1 represented by SEQ ID NO: 4, the heavy chain CDR2 represented by SEQ ID NO: 5 and the heavy chain CDR3 represented by SEQ ID NO: 6, respectively. The nucleotide sequences of SEQ ID NO: 12 to 14 may be codon-optimized for CHO cells, and the nucleic acid comprising such a codon-optimized nucleotide sequence should be construed as being within the scope of the nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof according to the present invention. For example, the nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof according to the present invention may comprise SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0035] According to one embodiment of the present invention, a nucleic acid encoding an anti-C-KIT antibody or antibody fragment thereof of the present invention can comprise a light chain variable region-encoding nucleic acid comprising SEQ ID NO:15 or SEQ ID NO:23.

[0036] According to yet another embodiment of the present invention, the nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof of the present invention may comprise a heavy chain variable region-encoding nucleic acid comprising SEQ ID NO:16 or SEQ ID NO:24.

[0037] According to yet another aspect of the present invention, the nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof of the present invention may comprise a light chain-encoding nucleic acid comprising SEQ ID NO:27 or SEQ ID NO:29.

[0038] According to yet another embodiment of the present invention, the nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof of the present invention may comprise a heavy chain-encoding nucleic acid comprising SEQ ID NO:28 or SEQ ID NO:30.

[0039] The term "nucleic acid" as used herein is intended to encompass DNA (gDNA and cDNA) and RNA. Nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogue nucleotides in which the sugar or base moiety is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0040] The nucleic acid molecule encoding the anti-C-KIT antibody or antibody fragment thereof of the present invention comprises a nucleotide sequence that shows substantial identity to the above-mentioned nucleotide sequence, where the substantial identity means that the two sequences show 80%, 90%, or 95% or more sequence homology as determined by aligning the two sequences to maximize their correspondence and analyzing them using algorithms commonly used in the art.

[0041] In yet another aspect, the present invention provides a vector comprising the nucleic acid and a cell transformed with the vector.

[0042] The term "vector" as used herein means any entity capable of gene replication when inserted into a host cell. Such vectors include plasmids, linear nucleic acids, cosmids, RNA vectors, viral vectors, etc., including but not limited to retroviruses, adenoviruses, adeno-associated viruses, etc. The recombinant vector system of the present invention can be constructed through various methods known in the art. In addition, the vector of the present invention can be constructed as a vector for cloning or expression, and can be constructed using prokaryotic or eukaryotic cells as hosts.

[0043] In addition, in the present invention, the cell may be a prokaryotic cell, a eukaryotic cell, or an animal cell. An appropriately selected host cell can be transformed with the vector and used to express and / or secrete the target protein. The host cell may be an immortalized hybridoma cell, an N / SO myeloma cell, a 293 cell, a HuT 78 cell, a CHO cell, a HELA cell, a COS cell, etc., and preferably a CHO cell. However, the host cell is not limited thereto, and any host cell known in the art can be used as the host cell of the present invention.

[0044] In still another aspect, the present invention provides a pharmaceutical composition for preventing or treating angiogenesis-related diseases, comprising an anti-C-KIT antibody or an antibody fragment thereof.

[0045] According to yet another aspect of the present invention, there is provided a method for treating or preventing an angiogenesis-related disease in a subject in need of such treatment or prevention, comprising administering to the subject an anti-C-KIT antibody or an antibody fragment thereof, or a composition comprising the same.

[0046] The term "treatment" as used in the present invention means any treatment in which the symptoms of angiogenesis-related diseases are improved, reversed, cured, etc. by administration of the composition according to the present invention.

[0047] The term "prevention" as used in the present invention means all of the suppression, delay, prevention, etc. of the onset or recurrence of angiogenesis-related diseases by administration of the composition of the present invention.

[0048] The term "angiogenesis-related disease" used in the present invention means a disease caused by angiogenesis. This includes cancer, leukemia, ocular vascular-related disease, rheumatoid arthritis, psoriasis, chronic wound, chronic inflammation, hemangioma, angiofibroma, vascular malformation, arteriosclerosis, vascular adhesion, vasculitis, pyogenic granuloma, blister disease, pulmonary hypertension, asthma, nasal polyp, infectious disease, inflammatory bowel disease, periodontal disease, peritoneal adhesion, endometrium, uterine bleeding, ovarian cyst, osteomyelitis, bone disease, sepsis, and autoimmune disease. Preferably, the disease may be cancer and ocular vascular-related disease, but is not limited thereto.

[0049] The cancer may be bone cancer, lung cancer, head cancer, neck cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, gastric cancer, liver cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, para-anal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, tracheal carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brain stem glioma, pituitary adenoma.

[0050] The ocular vascular related disease may be diabetic retinopathy, macular degeneration, age-related macular degeneration, glaucoma, glaucomatous retinitis pigmentosa, choroidal neovascularization, retinopathy of prematurity, corneal dystrophies and retinal laminar separation.

[0051] The pharmaceutical composition of the present invention may contain the anti-C-KIT antibody or a fragment thereof alone, or may further contain one or more pharma- ceutically acceptable carriers, excipients, or diluents.

[0052] The pharma- ceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose, glycols, etc., and may further include stabilizers and preservatives. Examples of stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Examples of preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharma-ceutically acceptable carriers known in the art may be used (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0053] The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration. For example, the pharmaceutical composition of the present invention can be prepared into an injection dosage form and administered by lightly pricking the skin with a 30-gauge thin needle or by directly applying it to the skin.

[0054] The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the above-mentioned administration route. In the case of an oral administration preparation, the composition of the present invention can be formulated into a powder, granule, tablet, pill, sugar-coated tablet, capsule, liquid, gel, syrup, slurry, suspension, etc., by using a method known in the art. For example, an oral preparation can be obtained by blending an active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing it into a granular mixture to obtain a tablet or sugar-coated tablet. Examples of excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol, starches including corn starch, wheat starch, rice starch, and potato starch, celluloses including methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose, gelatin, polyvinylpyrrolidone, and the like. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, if necessary. The pharmaceutical composition of the present invention may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, and a preservative. In the case of a preparation for parenteral administration, it may be formulated into the form of an injection, a cream, a lotion, an external ointment, an oil, a moisturizer, a gel, an aerosol, or a nasal inhalant by a method known in the art. These formulations are described in a document generally known to all pharmaceutical chemists (Remington's Pharmaceutical Science, 15th Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

[0055] The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have different contents of active ingredients depending on the severity of the disease symptoms. For example, the daily dose of the pharmaceutical composition of the present invention may be 0.0001-100 mg / kg. However, the dosage of the pharmaceutical composition of the present invention may be determined by a person having ordinary skill in the art, taking into consideration various factors such as the age, weight, health condition, sex, severity of the disease, diet, and excretion rate of the patient, as well as the route of administration and number of treatments. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, route of administration, and method of administration, as long as it exhibits the effects of the present invention.

[0056] In addition, the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents. When administered in combination with other therapeutic agents, the composition of the present invention and the other therapeutic agents may be administered simultaneously, separately or sequentially. The other therapeutic agents may be substances already known to have an effect of treating or improving neovascularization-related diseases, and include any anti-cancer therapy other than drug therapy, such as radiation therapy.

[0057] When the pharmaceutical composition of the present invention is administered in combination with other therapeutic agents, the anti-C-KIT antibody and the other therapeutic agent contained in the composition of the present invention may be formulated separately in separate containers or may be formulated together in the same container.

[0058] In yet another aspect, the present invention provides a kit for diagnosing angiogenesis-related diseases, comprising an anti-C-KIT antibody or an antibody fragment thereof.

[0059] The term "biological sample" as used herein includes, but is not limited to, tissues, cells, blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord), and the like.

[0060] The antibody of the present invention can be reacted with a biological sample to diagnose the presence or absence of onset of angiogenesis-related diseases. Specifically, the anti-C-KIT antibody or a functional fragment thereof can be contacted with a biological sample to confirm the presence or absence of formation of an antigen-antibody complex, thereby making a diagnosis. Since the diagnostic kit of the present invention includes an antibody, it can be prepared to be suitable for various immunoassays or immunostaining. The immunoassays or immunostaining include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, radioimmunoassay (RIA), and protein chips.

[0061] Labels for qualitatively or quantitatively measuring the formation of an antigen-antibody complex include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes. Effect of the Invention

[0062] The novel anti-C-KIT antibody or antibody fragment thereof according to the present invention specifically binds to the specific domain II of C-KIT and has strong affinity. As a result, the antibody or antibody fragment thereof according to the present invention has a very excellent effect of significantly inhibiting abnormal or excessive neovascularization, and can effectively prevent or treat angiogenesis-related diseases. In addition, the antibody or antibody fragment thereof according to the present invention has cross-reactivity with mice and rats in addition to humans, and can therefore be effectively used in research on angiogenesis-related diseases. [Brief description of the drawings]

[0063] [Figure 1] 1 is a graph showing the relative levels of the tube formation inhibitory effects of a total of 15 anti-C-KIT monoclonal antibodies when HUVEC cells were treated with SCF, compared to the non-treated control group. [Diagram 2] 1 is a graph showing the relative level of the tube formation inhibitory effect of 2G4 antibody at various concentrations when HUVEC cells were treated with SCF, compared to the untreated control group. [Diagram 3] 1 is a graph showing the inhibitory effect of 2G4 antibody on tube formation at different concentrations when mouse-derived endothelial cells MS-1 were treated with SCF. [Figure 4] The light chain variable region and heavy chain variable region bands of the 2G4 antibody are shown. [Diagram 5] The light chain variable region and heavy chain variable region bands of the 2G4 antibody are shown. [Figure 6] The nucleotide sequence, amino acid sequence and CDR region of the light chain variable region of the 2G4 antibody are shown. [Figure 7] The nucleotide sequence, amino acid sequence and CDR region of the heavy chain variable region of the 2G4 antibody are shown. [Figure 8] 1 shows the results of SDS-PAGE analysis of the 2G4 antibody obtained through cloning, isolation and purification. [Figure 9] 1 is a graph showing the results of SPR analysis to confirm the affinity of 2G4 antibody to C-KIT. [Figure 10] 1 shows the results of an experiment to confirm the C-KIT binding domain of the 2G4 antibody. [Figure 11] The results of comparing the ability of 2G4 antibody and the commercially available Eylea to inhibit abnormal angiogenesis using a mouse oxygen-induced retinopathy model are shown below. [Figure 12] The results of comparing the ability of 2G4 antibody and the commercially available Eylea to inhibit abnormal angiogenesis using a Brown Norway rat macular degeneration model are shown below. [Figure 13] The figure shows the inhibitory effect of 2G4 antibody on AKT phosphorylation induced by SCF in HUVEC cell lines. [Figure 14]The 2G4 antibody was shown to have inhibitory effects on AKT phosphorylation, C-KIT phosphorylation, ERK 1 / 2 phosphorylation and β-catenin in the TF-1 cell line, thereby suppressing leukemia cell proliferation. [Figure 15] 2 shows the ability of 2G4 antibody to inhibit proliferation of HUVEC and TF-1 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples. Terms not specifically defined in the present specification should be understood to have the meanings commonly used in the technical field to which the present invention belongs.

[0065] Example 1. Generation of C-KIT antibody-producing cell lines 1-1. Preparation of immunized mice An emulsion was prepared by mixing 50μg (per mouse) of recombinant C-KIT protein (cat# PKSH030939) purchased from Elabscience with the same volume of Freund's Adjuvant (Sigma, USA). The emulsion thus prepared was injected intraperitoneally into six 7-week-old female humanized NSG mice generated by injection of human CD34+ cells. 50μg of antigen was injected into each mouse in a total volume of 500μl. After one and two weeks, emulsions mixed with antigen and incomplete Freund's Adjuvant (Sigma, USA) were further injected intraperitoneally into the mice.

[0066] 1-2. Confirmation of antibody production Blood was collected from the eyeballs of the mice immunized by the above method, placed in a 1.5 ml microcentrifuge tube, and centrifuged at 13,000 rpm for 10 minutes. Serum was separated and stored at -20°C until an experiment to confirm antibody production was performed. After confirming antibody production by performing an enzyme immunoassay using the antigen protein, an emulsion of incomplete Freund's adjuvant (Sigma, USA) and the antigen was injected into the abdominal cavity of the mice again 3 days before cell fusion.

[0067] 1-3. Hybridoma production After confirming antibody production, the mice were sacrificed, and spleen cells were isolated and fused with myeloma cells P3X63Ag 8.653 (ATCC CRL-1580) to produce hybridomas.

[0068] Specifically, mouse P3X63Ag 8.653 cells were cultured in a culture plate using RPMI 1640 medium supplemented with 10% fetal bovine serum. To perform cell fusion, P3X63Ag 8.653 cells were washed twice with serum-free RPMI 1640 medium (Hyclone, USA) and then cultured at 1 × 10 7 The cell concentration was adjusted to 100%. Mice were sacrificed by cervical dislocation to collect the spleens, which were then placed in mesh containers (Sigma, USA) for cell isolation. A suspension of spleen cells was prepared and the suspension was washed using centrifugation. The spleen cell solution was exposed to Tris-NH4Cl (TRIS 20.6 g / L, NH4Cl 8.3 g / L) to lyse red blood cells. Completely isolated antibody-producing cells were centrifuged at 400 × g for 5 minutes. They were then washed twice with serum-free medium and resuspended in 10 ml medium. Lymphocytes were counted using a hemocytometer to determine lymphocyte count of 1 × 10 8 were mixed with 1 × 10 P3X63Ag 8.653 cells (10:1) in serum-free medium.

[0069] After centrifugation at 400×g for 5 min, 1 ml of the solution was added dropwise using 50% (M / V) polyethylene glycol 1500 (Sigma, USA) warmed to 37°C and mixed for 1 min. The fusion mixture thus prepared was diluted with serum-free RPMI1640 and centrifuged at 400×g for 3 min. The cells were suspended in 35 ml of RPMI1640 selection medium supplemented with 20% fetal bovine serum and HAT (100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine). 100 μl of the suspension was loaded onto a 96-well plate that had been coated with feeder cells (macrophages isolated from the peritoneal cavity using RPMI1640) one day earlier and cultured at 37°C, 5% CO2. After 5 days, the HAT medium was replaced every 2-3 days and the cells were cultured for 14 days. After 14 days, the medium was replaced with RPMI1640 supplemented with 20% fetal bovine serum and HT (medium obtained by removing 0.4 μM aminopterin from HAT) for secondary culture.

[0070] 1-4. Selection and separation of antibody-producing fused cells The supernatant of the culture medium of the fused cells prepared above was collected, and enzyme immunoassay was performed to confirm the production of specific antibodies against the prepared antigen. The culture medium of the fused cells that showed an appropriate concentration of 4 times or more compared to the negative control group was selected and transferred to a 24-well plate for culture. The cells were further diluted to a limiting dilution so that one cell was placed per well in a 96-well plate, and then cultured. The culture medium was then collected, and the C-KIT protein used as the antigen was coated on the 96-well plate at 0.1 μg per well. Then, enzyme immunoassay was performed to select fused cells that produced 15 monoclonal antibodies (1C6, 1H2, 1A6, AFA, 2B3, 2G4, 4G5, 4C4, 4C7, 4D7, 1E1, 2H6, 1G3, 1A3, 1D3).

[0071] Example 2. Selection of C-KIT antibodies 2-1. Tube formation analysis using HUVEC After dispensing 300 μl of Matrigel (Corning, USA) into a 24-well plate, HUVECs (Human Umbilical Vein Endothelial Cells) were added to the Matrigel with SCF (50 ng / ml) or SCF (50 ng / ml) + anti-C-KIT antibody (1 μg / ml). After that, tube formation of HUVECs was observed, and the results are shown in Figure 1.

[0072] In Figure 1, the results of in vitro angiogenesis using HUVEC confirmed that 2G4 had the strongest inhibitory effect on HUVEC tube formation induced by SCF among 15 antibodies, suggesting that the anti-C-KIT antibody designated as 2G4 may be effectively used in the prevention or treatment of angiogenesis-related diseases.

[0073] 2-2. Inhibitory effect of 2G4 antibody on angiogenesis by different concentrations After dispensing 300 μl of Matrigel (Corning, USA) into a 24-well plate, HUVECs were dispensed into the Matrigel containing SCF (50 ng / ml), SCF (50 ng / ml) + 2G4 antibody (0.1 μg / ml), or SCF (50 ng / ml) + 2G4 antibody (1 μg / ml). After that, tube formation of HUVECs was observed, and the results are shown in Figure 2.

[0074] According to the results of tube formation analysis using HUVEC, it was confirmed that the 2G4 antibody inhibited HUVEC tube formation in a concentration-dependent manner, and in particular, the 2G4 antibody had excellent angiogenesis inhibitory ability even at a concentration of 0.1 μg / ml.

[0075] 2-3. Cross-reactivity test in mice To test the cross-reactivity of the 2G4 antibody to mice, the procedure was carried out in the same manner as in Example 2-2 using mouse-derived endothelial cells MS-1.

[0076] As a result, as shown in FIG. 3, it was confirmed that the 2G4 antibody significantly inhibited the angiogenesis of mouse endothelial cells, MS-1, using mouse-derived endothelial cells.

[0077] Example 3. Nucleotide sequence analysis of anti-C-KIT antibody IgG variable region The fusion cells 2G4 clone obtained from Examples 1 and 2 were 5×10 5 Total RNA was isolated from the sarcoma and cDNA was synthesized using random primers (Bioneer, Korea) and reverse transcriptase.

[0078] The kappa light chain domain was amplified from the cDNA using a human IgG library primer set from PROGEN. The amplified nucleic acid was confirmed by agarose gel electrophoresis, and the results are shown in Figure 4. The heavy chain domain was also amplified using a human IgG library primer set from PROGEN, and the results are shown in Figure 5.

[0079] As shown in Figures 4 and 5, a band was found between the kappa light chain domain (414 bp) and the heavy chain domain (483 bp), confirming that a PCR product of the expected size was generated.

[0080] The PCR product was then developed on an agarose gel, the bands were excised, and the agarose gel was melted at 60°C, after which the nucleic acid was purified using a spin column (Qiagen). The nucleic acid thus purified was cloned into a TOPO-TA vector, transformed into E. coli DH5α to obtain colonies, which were then cultured to extract plasmids. PCR was then performed again to obtain four plasmids, and the base sequence of the 2G4 antibody was analyzed.

[0081] Figure 6 shows the amino acid sequence and nucleotide sequence of the light chain variable region of the 2G4 antibody, which correspond to the amino acid sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 15 in the sequence listing attached hereto. Also, in Figure 6, CDR1, CDR2, and CDR3 of the light chain variable region are shown in red order, which correspond to the amino acid sequences of SEQ ID NOs: 1 to 3 and the nucleotide sequences of SEQ ID NOs: 9 to 11 in the sequence listing attached hereto.

[0082] Figure 7 shows the amino acid sequence and nucleotide sequence of the heavy chain variable region of the 2G4 antibody, which correspond to the amino acid sequence of SEQ ID NO: 8 and the nucleotide sequence of SEQ ID NO: 16 in the sequence listing attached hereto. In addition, in Figure 7, CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in red order, which correspond to the amino acid sequences of SEQ ID NOs: 4 to 6 and the nucleotide sequences of SEQ ID NOs: 12 to 14 in the sequence listing attached hereto.

[0083] Example 4. Preparation of anti-C-KIT antibody 4-1. Cloning of fully humanized antibodies The variable region of the 2G4 antibody obtained in Example 3 was grafted to a human Fc amino acid sequence and cloned into the pCHO vector (Life Technology).

[0084] The light chain variable region was fused in frame to the human kappa constant region, and the heavy chain variable region was fused in frame to the human IgG1 constant region. Leader peptide sequences for the secretion of full-length IgG1 antibodies into the medium were added to the two genes, and the genes were synthesized and verified again through sequence analysis. Three clones were selected for CHO cell expression testing. Glycerol stocks for the three clones were prepared, and endotoxin-free plasmids were prepared for CHO cell expression testing.

[0085] 4-2.Isolation and purification of antibodies The obtained plasmid DNA was transfected into CHO-S cells. One week before transfection, CHO-S cells (Invitrogen, 10743-029) were transferred to a monolayer culture in DMEM supplemented serum. One day before transfection, the cells were aliquoted, and the nucleic acid-lipofectamine complex was prepared for the transfection sample and incubated at 37°C in an incubator with 5% CO2 overnight. The cells were cultured for one week while adding medium every 2-3 days. The culture medium was then collected and bound to Protein A / G agarose (Invitrogen) and washed with PBS. The cells were then eluted with 0.1M glycine (pH 2.8) and neutralized with 1M Tris-HCl (pH 8.0). The cells were dialyzed against PBS and stored at -70°C.

[0086] The purified 2G4 antibody was then run on 6% SDS-PAGE under non-reducing and reducing conditions to confirm the purity and size of the antibody, and the results are shown in Figure 8. As shown in Figure 8, a 50 kDa heavy chain and a 25 kDa light chain band were observed in the SDS-PAGE, confirming that the antibody was produced correctly.

[0087] Example 5. Affinity of 2G4 antibody To confirm the C-KIT binding ability of the 2G4 antibody, SPR (Surface Plasmon Resonance) was performed. Using an SR7500DC (Reichert, USA), 20 μg of human C-KIT (eLabScience, PKSH030939), 20 μg of mouse C-KIT (SB, Lot#LC05DE2304), and 20 μg of rat C-KIT (SB, Lot#LC06SE1787), which were used to generate the antibody, were immobilized on a PEG (Reichert, USA) chip. Then, the 2G4 antibody was injected at different concentrations, and the affinity for C-KIT, K D The values ​​were analyzed using the Scrubber2 program. DThe value is calculated by dividing kd by ka, and a lower value indicates a higher binding ability to the target.

[0088] The results are shown in Figure 9. The 2G4 antibody inhibited the K-KIT signaling for human C-KIT. D The value is approximately 2.8237 (±0.9) × 10 -12 M, showing strong affinity. The highest affinity was for humans, followed by mice and rats.

[0089] Example 6. Domain Mapping Deletion mutants of human C-KIT gene (NM_000222) (Q26-P520, D113-P520 △domain I, A207-P520 △domain I-II, K310-P520 △domain I-III) were tagged with FLAG at the c-terminus and transfected into HEK293. They were then secreted into the culture medium and purified using FLAG antibody beads (Sigma-Aldrich). ELISA was then performed.

[0090] As shown in FIG. 10, the 2G4 antibody was unable to recognize C-KIT when domain II was deleted, demonstrating that the specific binding site of the 2G4 antibody to C-KIT is domain II.

[0091] Comparative Example 1. Comparison of in vivo efficacy using mouse models We used the mouse oxygen-induced retinopathy (OIR) model, which is widely used as an animal model of proliferative diabetic retinopathy and retinopathy of prematurity. C57BL / 6 mice were exposed to a 75% hyperoxia environment for 5 days from 7 days after birth, resulting in the formation of abnormal blood vessels.

[0092] Specifically, C57BL / 6 mice were exposed to a 21% oxygen environment from 0 to 7 days after birth, and then to a 75% high oxygen environment from 7 to 12 days after birth. On day 12 after birth, 2G4 antibody (2μg / eye) and Eylea (2μg / eye) were injected intravitreally into the right eye, and the left eye was injected with PBS as a control group. They were then exposed to a 21% oxygen environment again from days 12 to 17 after birth, and sacrificed on day 17 after birth.

[0093] As a result, as shown in FIG. 11, abnormal angiogenesis inhibition was observed in the right eyeball injected with 2G4 antibody and Eylea (2 μg / eye), and the degree of inhibition was confirmed to be at the same level.

[0094] Comparative Example 2. In vivo efficacy comparison using a rat model A macular degeneration model was established using brown Norway rats.

[0095] Specifically, a laser was used to induce CNV (choroidal neovascularization) in the eyes of rats. At the same time, 2G4 antibody (6.28μg / eye) and Eylea (10μg / eye) were injected into the vitreous cavity of the eyes at a dose of 4μl / eye, respectively. The group injected with IgG antibody (10μg / eye) at a dose of 4μl / eye was used as the control group.

[0096] After 14 days, the results of analysis using BS-1 lectin are shown in Figure 12. Abnormal angiogenesis caused by macular degeneration was significantly suppressed in both the Eylea group and the 2G4 antibody group. In particular, the 2G4 antibody showed the same level of efficacy as Eylea despite being administered at a lower concentration, indicating that the 2G4 antibody is more effective than Eylea.

[0097] Example 7. Ability of 2G4 antibody to inhibit SCF / C-KIT signaling SCF / C-KIT signaling is known to induce phosphorylation of AKT. As shown in Figure 13, when HUVECs were treated with SCF, phosphorylation of AKT increased. In contrast, AKT phosphorylation was decreased by the 2G4 antibody.

[0098] Moreover, it can be seen from Figure 14 that AKT phosphorylation by SCF is also inhibited by the 2G4 antibody in the leukemia cell line TF-1. Furthermore, it can be seen from Figure 14 that C-KIT phosphorylation and ERK1 / 2 phosphorylation by SCF are also inhibited.

[0099] β-catenin is known to be an important factor in cell proliferation as an AKT downstream signal. It can be seen from FIG. 14 that the 2G4 antibody inhibits the increase in β-catenin by SCF in a concentration-dependent manner, which means that the 2G4 antibody significantly inhibits the proliferation of the leukemia cell line TF-1. Many cases of leukemia are found to show resistance or tolerance to anticancer drugs due to the high C-KIT mutations, but the antibody according to the present invention can also show preventive or therapeutic effects against leukemia, thus overcoming the limitations of conventional anticancer drugs.

[0100] Example 8.2 Inhibitory effect of G4 antibody on proliferation of HUVEC and TF-1 cells TF-1 and HUVEC were pretreated with 2G4 antibody at different concentrations (0, 0.1, 1, 5, 10μg / ml) for 30 minutes, then treated with 50ng / ml SCF, and cell numbers were counted 36 hours later to compare cell proliferation rates.

[0101] As shown in Figure 15, the SCF-treated group showed an increase of about 26% in the number of TF-1 cells and about 70% in the number of HUVEC cells compared to the negative control group. In contrast, in the 2G4 antibody-treated group, SCF-induced cell proliferation was inhibited in both TF-1 and HUVEC in a concentration-dependent manner. This indicates that the 2G4 antibody has a very excellent ability to inhibit HUVEC and TF-1 cell proliferation.

Claims

1. An anti-C-KIT antibody or an antigen-binding fragment thereof, which specifically binds to domain II of C-KIT, The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a light chain CDR1 represented by SEQ ID NO: 1, a light chain CDR2 represented by SEQ ID NO: 2, and a light chain CDR3 represented by SEQ ID NO: 3; An anti-C-KIT antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR1 represented by SEQ ID NO:4, a heavy chain CDR2 represented by SEQ ID NO:5, and a heavy chain CDR3 represented by SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:7; and The anti-C-KIT antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

3. The anti-C-KIT antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a constant region derived from human IgG1.

4. A nucleic acid encoding the anti-C-KIT antibody or antigen-binding fragment thereof according to claim 1.

5. The nucleic acid is (i) a light chain CDR1 nucleotide sequence represented by SEQ ID NO:9, a light chain CDR2 nucleotide sequence represented by SEQ ID NO:10, and a light chain CDR3 nucleotide sequence represented by SEQ ID NO:11; a heavy chain CDR1 nucleotide sequence represented by SEQ ID NO:12, a heavy chain CDR2 nucleotide sequence represented by SEQ ID NO:13, and a heavy chain CDR3 nucleotide sequence represented by SEQ ID NO:14; (ii) a light chain variable region nucleotide sequence as set forth in SEQ ID NO:15, and a heavy chain variable region nucleotide sequence as set forth in SEQ ID NO:16; (iii) a light chain CDR1 nucleotide sequence represented by SEQ ID NO: 17, a light chain CDR2 nucleotide sequence represented by SEQ ID NO: 18, and a light chain CDR3 nucleotide sequence represented by SEQ ID NO: 19; a heavy chain CDR1 nucleotide sequence represented by SEQ ID NO: 20, a heavy chain CDR2 nucleotide sequence represented by SEQ ID NO: 21, and a heavy chain CDR3 nucleotide sequence represented by SEQ ID NO: 22; or (iv) the light chain variable region nucleotide sequence represented by SEQ ID NO:23, and the heavy chain variable region nucleotide sequence represented by SEQ ID NO:

24.

6. A vector comprising the nucleic acid of claim 4.

7. A cell transformed with the vector according to claim 6.

8. 2. A pharmaceutical composition for preventing or treating angiogenesis, comprising the anti-C-KIT antibody or an antigen-binding fragment thereof according to claim 1, wherein the angiogenesis is angiogenesis associated with a disease selected from the group consisting of cancer, leukemia, ocular vascular disease, rheumatoid arthritis, psoriasis, chronic wounds, chronic inflammation, hemangioma, angiofibroma, vascular malformation, arteriosclerosis, vascular adhesion, vasculitis, pyogenic granuloma, bullous disease, pulmonary hypertension, asthma, nasal polyp, infectious disease, inflammatory bowel disease, periodontal disease, peritoneal adhesion, endometriosis, uterine bleeding, ovarian cyst, osteomyelitis, bone disease, sepsis, and autoimmune disease.

9. 9. The pharmaceutical composition of claim 8, wherein the cancer is selected from the group consisting of bone cancer, lung cancer, head cancer, neck cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, gastric cancer, liver cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, perianal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, tracheal carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brain stem glioma, and pituitary adenoma.

10. 9. The pharmaceutical composition of claim 8, wherein the ocular vascular disease is selected from the group consisting of diabetic retinopathy, macular degeneration, age-related macular degeneration, glaucoma, glaucomatous retinitis pigmentosa, choroidal neovascularization, retinopathy of prematurity, corneal dystrophy and retinal laminar separation.

11. 2. A diagnostic kit for angiogenesis comprising the anti-C-KIT antibody or an antigen-binding fragment thereof according to claim 1, wherein the angiogenesis is angiogenesis associated with a disease selected from the group consisting of cancer, leukemia, ocular vascular disease, rheumatoid arthritis, psoriasis, chronic wound, chronic inflammation, hemangioma, angiofibroma, vascular malformation, arteriosclerosis, vascular adhesion, vasculitis, pyogenic granuloma, bullous disease, pulmonary hypertension, asthma, nasal polyp, infectious disease, inflammatory bowel disease, periodontal disease, peritoneal adhesion, endometriosis, uterine bleeding, ovarian cyst, osteomyelitis, bone disease, sepsis, and autoimmune disease.

Images