NOVEL ANTI-C-KIT ANTIBODY

Abstract

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

Claims

1. An anti-C-KIT antibody or antibody fragment thereof, specifically binding to domain II of C-KIT.

2. The anti-C-KIT antibody or antibody fragment thereof according to claim 1, wherein the antibody comprises a light chain variable region comprising a light chain CDR 1 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; and 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.

3. The anti-C-KIT antibody or antibody fragment thereof according to claim 1, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

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

5. A nucleic acid encoding the anti-C-KIT antibody or antibody fragment thereof of claim 1.

6. The nucleic acid according to claim 5, the nucleic acid comprises (i) SEQ ID NOs: 9 to 14, (ii) SEQ ID NOs: 15 and 16, (iii) SEQ ID NOs: 17 to 22, or (iv) SEQ ID NOs: 23 and 24.

7. A vector comprising the nucleic acid of claim 5.

8. A cell transformed with the vector of claim 7.

9. A method for preventing or treating an angiogenesis-related disease, comprising administering the anti-C-KIT antibody or antibody fragment thereof of claim 1 to a subject in need thereof.

10. The method according to claim 9, the angiogenesis-related disease is selected from a group consisting of cancer, leukemia, ophthalmic vascular diseases, rheumatoid arthritis, psoriasis, chronic wounds, chronic inflammation, hemangioma, hemangiofibroma, vascular malformations, arteriosclerosis, vascular adhesions, vasculitis, pyogenic granuloma, blister diseases, pulmonary hypertension, asthma, nasal polyps, infectious diseases, inflammatory bowel disease, periodontal disease, peritoneal adhesions, endometriosis, uterine bleeding, ovarian cysts, osteomyelitis, osteitis, sepsis and autoimmune diseases.

11. The method according to claim 10, the cancer is selected from a group consisting of bone cancer, lung cancer, brain cancer, neck cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, gastric cancer, liver cancer, pancreatic cancer, skin cancer, intradermal or intraocular melanoma, rectal cancer, anal cancer, colon cancer, uterine cancer, ovarian cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, or 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.

12. The method according to claim 10, the ophthalmic vascular diseases is selected from a group consisting of diabetic retinopathy, macular degeneration, senile macular degeneration, glaucoma, glaucoma-related retinal pigment degeneration, choroidal neovascularization, retinopathy of prematurity, corneal dystrophy and retinoschisis.

13. An angiogenesis-related disease diagnostic kit comprising the anti-C-KIT antibody or antibody fragment thereof of claim 1.

Description

BRIEF DESCRIPTION OF FIGURES

[0061] FIG. 1 is a graph showing the tube formation inhibitory effect of a total of fifteen anti-C-KIT monoclonal antibodies in a relative level compared to the non-treated control group when HUVEC cells were treated with SCF.

[0062] FIG. 2 is a graph showing the tube formation inhibitory effect by concentration of 2G4 antibody in a relative level compared to the non-treated control group when HUVEC cells were treated with SCF.

[0063] FIG. 3 is a graph showing the tube formation inhibitory effect by concentration of 2G4 antibody when mouse-derived endothelial cell MS-1 were treated with SCF.

[0064] FIGS. 4 and 5 show the bands of the light chain variable region and the heavy chain variable region of the 2G4 antibody, respectively.

[0065] FIG. 6 shows the nucleotide sequence, amino acid sequence, and CDR region of the light chain variable region of the 2G4 antibody.

[0066] FIG. 7 shows the nucleotide sequence, amino acid sequence, and CDR region of the heavy chain variable region of the 2G4 antibody.

[0067] FIG. 8 shows the analysis results of SDS-PAGE of 2G4 antibodies obtained through cloning, separation and purification.

[0068] FIG. 9 is a graph showing the analysis results of SPR for confirming the C-KIT affinity of 2G4 antibody.

[0069] FIG. 10 shows the experimental results for confirming the C-KIT binding domain of the 2G4 antibody.

[0070] FIG. 11 shows the results of comparing the ability to inhibit abnormal angiogenesis of 2G4 antibody and commercially available Eylea using a mouse oxygen-induced retinopathy model.

[0071] FIG. 12 shows the results of comparing the ability to inhibit abnormal angiogenesis of 2G4 antibody and commercially available Eylea using a Brown Norwegian rat macular degeneration model.

[0072] FIG. 13 shows the inhibitory ability of 2G4 antibodies to AKT phosphorylation by SCF in HUVEC cell lines.

[0073] FIG. 14 shows the inhibitory ability of 2G4 antibodies to AKT phosphorylation, C-KIT phosphorylation, ERK 1/2 phosphorylation, and β-catenin in TF-1 cell line, thereby showing the inhibition of leukemia cell proliferation.

[0074] FIG. 15 shows the cell proliferation inhibitory ability of HUVEC and TF-1 by the 2G4 antibody.

EXAMPLES

[0075] In the following, exemplary embodiments of the inventive concept will be explained in further detail with reference to examples. However, the following examples are meant to exemplify the present invention, and the scope of the invention is not restricted by these examples. Terms that are not specifically defined in the present specification should be understood as having meanings commonly used in the technical field to which the present invention belongs.

Example 1. C-KIT Antibody Production Cell Line Preparation

[0076] 1-1. Preparation of Immunized Mice

[0077] An emulsion was prepared by mixing 50 μg (based on one mouse) of recombinant C-KIT protein (cat #PKSH030939) purchased from Elabscience with the same volume of complete Freund's Adjuvant (sigma, USA). The prepared emulsion was injected intraperitoneally into six humanized NSG mice prepared by injection of 7-week-old female human CD34+ cells. 50 μg of antigen was injected into each mouse in a total volume of 500 μl. After 1 week and 2 weeks, an emulsion prepared by mixing an incomplete Freund's Adjuvant (sigma, USA) with an antigen was further injected into the intraperitoneal cavity of the mouse, respectively.

[0078] 1-2. Antibody Production Confirmation

[0079] Blood was collected from the eyeballs of mice immunized through 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 is performed. After confirming the antibody production by carrying out an enzyme immunoassay method using an antigenic protein, an emulsion in which an antigen was mixed with an incomplete Freund's Adjuvant (Sigma, USA) was further injected into the intraperitoneal cavity of the mouse 3 days before cell fusion.

[0080] 1-3. Preparation of Hybridomas

[0081] After confirming the antibody production, the mice were sacrificed. The splenocytes were isolated and fused with myeloma cells P3X63Ag 8.653 (ATCC CRL-1580) to prepare hybridomas.

[0082] Specifically, P3X63Ag 8.653 cells of mice were cultured in a culture plate using RPMII640 medium supplemented with 10% fetal bovine serum. To perform cell fusion, P3X63Ag 8.653 cells were washed twice with serum-free RPMI640 medium (Hyclone, USA), and adjusted to a cell concentration of 1×10.sup.7. The mice were sacrificed by cervical dislocation, and the spleen was collected, and then placed in a mesh container (Sigma, USA) to separate cells. After preparing a suspension of splenocytes, the suspension was washed by centrifugation. Red blood cells were lysed by exposing the splenocyte solution to Tris-NH.sub.4Cl (TRIS 20.6 g/L, NH.sub.4Cl 8.3 g/L). Completely isolated antibody-producing cells were centrifuged at 400×g for 5 minutes. After that, it was washed twice in serum-free medium and resuspended in 10 ml medium. Lymphocytes were counted using a hemocytometer, and 1×10.sup.8 lymphocytes were mixed with 1×10 P3X63Ag 8.653 cells (10:1) in serum-free medium.

[0083] After centrifugation at 400×g for 5 minutes, 1 ml of a solution was added dropwise using 50% (MN) polyethylene glycol 1500 (sigma, USA) heated at 37° C. and mixed for 1 minute. The fusion mixture solution thus prepared was diluted with serum-free RPMI1640 and centrifuged at 400×g for 3 minutes. Cells were suspended in 35 ml of RPMI1640 selective 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 coated with feeder cells (macrophages isolated from the peritoneal cavity using RPMI1640) one day before, and cultured at 37° C. 5% CO.sub.2. After 5 days, the HAT medium was changed every 2-3 days, and the cells were cultured for 14 days. After 14 days, the secondary culture was performed by replacing with RPMI1640 medium supplemented with 20% fetal bovine serum and HT (a medium in which 0.4 μM aminopterin was removed from HAT).

[0084] 1-4. Selection and Isolation of Antibody-Producing Fusion Cells

[0085] The supernatant of the previously prepared fusion cells culture medium was collected and subjected to an enzyme immunoassay to determine whether specific antibodies for the prepared antigen were produced or not. A culture medium of fusion cells exhibiting 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. In addition, after culturing by dilution to contain one cell per well in a 96-well plate (limiting dilution), the culture solution was recovered, and the C-KIT protein used as an antigen was coated at 0.1 μg per well on a 96-well plate. After that, enzyme immunoassay is performed to finally select fusion cells producing 15 monoclonal antibodies (1C6, 1H2, 1A6, AFA, 2B3, 2G4, 4G5, 4C4, 4C7, 4D7, 1E1, 2H6, 1G3, 1A3, 1D3).

Example 2. C-KIT Antibody Selection

[0086] 2-1. Tube Formation Analysis Using HUVEC

[0087] After dispensing 300 μl of Matriegel (Corning, USA) into a 24-well plate, HUVECs (Human Umbilical Vein Endothelial Cells) was dispensed into Matrigel with SCF (50 ng/ml) or SCF (50 ng/ml)+anti-C-KIT antibody (1 μg/ml). Thereafter, tube formation of HUVEC was observed, and the results are shown in FIG. 1.

[0088] In FIG. 1, according to the results of in vitro angiogenesis using HUVEC, it was confirmed that 2G4 is the most potent among 15 antibodies to inhibit HUVEC tube formation induced by SCF. This suggests that an anti-C-KIT antibody, referred to as 2G4, can be effectively used in the prevention or treatment of angiogenesis-related diseases.

[0089] 2-2. Angiogenesis Inhibitory Effects Depending on the Concentrations of the 2G4 Antibody

[0090] After dispensing 300 μl of Matrigel (Corning, USA) into a 24-well plate, HUVEC was dispensed into Matrigel with SCF (50 ng/ml), SCF (50 ng/ml)+2G4 antibody (0.1 μg/ml) or SCF (50 ng/ml)+2G4 antibody (I μg/ml). Thereafter, tube formation of HUVEC was observed, and the results are shown in FIG. 2.

[0091] In FIG. 2, 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. In particular, the 2G4 antibody was excellent in the ability to inhibit angiogenesis even at a concentration of 0.1 μg/ml.

[0092] 2-3. Cross-Reaction Test on Mice

[0093] In order to test the cross-reactivity of the 2G4 antibody on mice, it was carried out in the same manner as in Example 2-2 using the mouse-derived endothelial cells MS-1.

[0094] As a result, as shown in FIG. 3, it was confirmed that the 2G4 antibody significantly inhibited angiogenesis of the mouse endothelial cells in the mouse-derived endothelial cells MS-1.

Example 3. Nucleotide Sequence Analysis of Anti-C-KIT Antibody IgG Variable Region

[0095] Total RNA was isolated from the fusion cell 2G4 clone 5×10.sup.5 obtained from Examples 1 and 2. cDNA was synthesized using random primer (bioneer, Korea) and reverse transcriptase. The kappa light chain domain was amplified from the cDNA using PROGEN's human IgG library primer set. The amplified nucleic acid was confirmed by agarose gel electrophoresis, and the results are shown in FIG. 4. Similarly, the heavy chain domain was amplified using PROGEN's human IgG library primer set and the results are shown in FIG. 5.

[0096] As shown in FIGS. 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.

[0097] Thereafter, the PCR product was spread on an agarose gel, the band was cut, the agarose gel was dissolved at 60° C., and then the nucleic acid was purified using a spin column (Qiagen). The purified nucleic acid was cloned into a TOPO-TA vector, transformed into E. coli DH5a to obtain colonies, and then the obtained colonies were cultured to extract plasmids. Subsequently, PCR was performed again to obtain four plasmids, and then the nucleotide sequence of the 2G4 antibody was analyzed.

[0098] FIG. 6 shows the amino acid sequence and nucleotide sequence of the light chain variable region of the 2G4 antibody, which respectively correspond to the amino acid sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 15 in the sequence list attached to the present specification. In addition, CDR1, CDR2, and CDR3 of the light chain variable region in FIG. 6 are indicated in order in red, which correspond to the amino acid sequences of SEQ ID NOs: 1 to 3 and nucleotide sequences of SEQ ID NOs: 9 to 11, respectively, in the sequence list attached to the present specification.

[0099] FIG. 7 shows the amino acid sequence and nucleotide sequence of the heavy chain variable region of the 2G4 antibody, which respectively correspond to the amino acid sequence of SEQ ID NO: 8 and the nucleotide sequence of SEQ ID NO: 16 in the sequence list attached to the present specification. In addition, CDR1. CDR2, and CDR3 of the heavy chain variable region in FIG. 7 are indicated in order in red, which correspond to the amino acid sequences of SEQ ID NOs: 4 to 6 and nucleotide sequences of SEQ ID NOs: 12 to 14, respectively, in the sequence list attached to the present specification.

Example 4. Preparation of Anti-C-KIT Antibody

[0100] 4-1. Fully Humanized Antibody Cloning

[0101] The variable region of the 2G4 antibody obtained in Example 3 was grafted onto a human Fc amino acid sequence, and cloned into a pCHO vector (lifetechnology).

[0102] The light chain variable region was fused in the frame for the human kappa constant region, and the heavy chain variable region was fused in the frame for the human IgG1 constant region.

[0103] A leader peptide sequence for secretion of the whole IgG1 antibody in the medium was added to the two genes to synthesize the gene, and then again verified through sequencing. Three clones were selected for the expression test in CHO cells. Glycerol stocks were prepared for the three clones, and a plasmid without endotoxin was prepared for the expression test in CHO cells.

[0104] 4-2. Isolation and Purification of Antibody

[0105] The plasmid DNA obtained above was transfected into CHO-S cells. One week before transfection, CHO-S cells (Invitrogen, 10743-029) were transferred into monolayer cultures in the presence of DMEM supplemented with serum. After the cells were dispensed 1 day before transfection, a nucleic acid-lipofectamine complex was prepared for the transfection sample, and the cells were incubated overnight at 5% CO.sub.2 and 37° C. in an incubator. The medium was incubated for a week while being added once every 2-3 days. Then, the culture solution was recovered, bonded to Protein A/G agarose (Invitrogen), and washed with PBS. Then, after eluting with 0.1 M glycine (pH 2.8), it was neutralized with 1 M Tris-HCl (pH 8.0). After dialysis with PBS, it was stored at −70° C.

[0106] The separated and purified 2G4 antibody was running on 6% SDS-PAGE under Non-reducing and Reducing conditions to confirm the purity and size of the antibody. The results are shown in FIG. 8. As shown in FIG. 8, a 50 kDa heavy chain and a 25 kDa light chain band were observed as a result of SDS-PAGE, confirming that the antibody was accurately produced.

Example 5. Affinity of 2G4 Antibody

[0107] In order to confirm the C-KIT binding ability of the 2G4 antibody, SPR (Surface Plasmon Resonance) was performed. Using SR7500DC (Reichert, USA), 20 μg of human C-KIT (elabscience, PKSH030939) used for antibody preparation. 20 μg of mouse C-KIT (SB, Lot #LC05DE2304), and rat C-KIT (SB, Lot #LC06SE1787) 20 μg was fixed on a PEG (Reichert, USA) chip. Thereafter, after flowing 2G4 antibody by concentration, the K.sub.D value, which is the affinity for C-KIT, was analyzed using the Scrubber2 program. The K.sub.D value is obtained by dividing kd by ka, and the lower the value means the greater the binding ability to the target.

[0108] The results are shown in FIG. 9. The 2G4 antibody showed a strong affinity for human C-KIT with a K.sub.D value of about 2.8237(±0.9)×10.sup.−12 M. The affinity for humans was highest, followed by mice and rats.

Example 6. Domain Mapping

[0109] The deletion variants (Q26-P520. D113-P520 Δdomain I, A207-P520 Δdomain I-II, K310-P520 Δdomain I-III) of the human C-KIT gene (NM_000222) were tagged with FLAG at the c-terminus and then were transfected with HEK293. Then, after secretion into the culture medium, these were purified using the FLAG antibody beads (Sigma-Aldrich). Then, ELISA was performed.

[0110] As shown in FIG. 10, the 2G4 antibody did not recognize C-KIT when domain II was deleted, and from this, it was proved that the specific binding site for C-KIT of the 2G4 antibody was domain II.

Comparative Example 1. Comparison of In Vivo Efficacy Using a Mouse Model

[0111] As an animal model for proliferative diabetic retinopathy and premature retinopathy, a widely used mouse oxygen-induced retinopathy (OIR) model was used. Abnormal blood vessels are formed when C57BL/6 mice are exposed to a 75% high oxygen environment for 5 days from 7 days after birth.

[0112] C57BL/6 mice were exposed to a 21% oxygen environment from 0 to 7 days after birth, and to a 75% high oxygen environment from 7 to 12 days after birth. On the 12th day after birth, 2G4 antibody (2 μg/eye) and Eylea (2 μg/eye) were injected intravitreally in the right eye, respectively, and PBS was injected into the left eye and compared as a control group. Then, from the 12th to the 17th after birth, they were exposed to an oxygen environment of 21% again, and sacrificed on the 17th day after birth.

[0113] As a result, as shown in FIG. 11, abnormal angiogenesis inhibition was observed in the right eye injected with the 2G4 antibody and Eylea (2 μg/eye), and the degree was confirmed to be at the equivalent level.

Comparative Example 2. Comparison of In Vivo Efficacy Using a Rat Model

[0114] A macular degeneration model was constructed using brown Norway rats. CNV (choroidal neovasculanzation) in the rat's eye was induced by using a laser. At the same time, 2G4 antibody (6.28 μg/eye) and Eylea (10 μg/eye) were injected intravitreally at a dose of 4 μl/eye, respectively. A group injected with an IgG antibody (10 μg/eye) at a dose of 4 μl/eye was used as a control.

[0115] FIG. 12 shows the results of analysis using the BS-1 lectin after 14 days. Abnormal angiogenesis caused by macular degeneration was significantly inhibited in both the Eylea group and the 2G4 antibody group. In particular, the 2G4 antibody showed an equivalent level of efficacy even though the dose concentration was lower than that of Eylea, and it indicates that the 2G4 antibody is more effective than Eylea.

Example 7. SCF/C-KIT Signaling Inhibitory Ability by 2G4 Antibody

[0116] SCF/C-KIT signaling is known to basically induce phosphorylation of AKT. As seen in FIG. 13, it was confirmed that AKT phosphorylation was increased when SCF was treated with HUVEC. On the other hand, it was confirmed that AKT phosphorylation was decreased by the 2G4 antibody.

[0117] In addition, it can be seen from FIG. 14 that AKT phosphorylation by SCF is inhibited by the 2G4 antibody in the leukemia cell line TF-1. Moreover, it can be seen from FIG. 14 that phosphorylation of ERK1/2 and phosphorylation of C-KIT by SCF are also inhibited. β-catenin is an AKT downstream signal, and is known to be an important factor in cell proliferation. It can be seen in FIG. 14 that the 2G4 antibody inhibits the increase of 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. Leukemia has many C-KIT mutations, and thus the resistance or tolerance on anticancer drug is often found. However, the antibody according to the present invention can show a preventive or therapeutic effect against leukemia, and thus it can overcome the limitations of prior anticancer drugs.

Example 8. Proliferation Inhibitory Ability of HUVEC and TF-1 Cell by 2G4 Antibody

[0118] 2G4 antibodies were pretreated on TF-1 and HUVEC for 30 minutes at different concentrations (0, 0.1, 1, 5, 10 μg/ml). Thereafter, 50 ng/ml of SCF was treated, and after 36 hours, the number of cells was measured to compare the cell proliferation rate.

[0119] As shown in FIG. 15, the SCF-treated group increased the number of TF-1 cells by about 26% and the number of HUVEC cells by about 70% compared to the negative control group. On the other hand, in the group treated with 2G4 antibody, cell proliferation by SCF was inhibited in a concentration-dependent manner in both TF-1 and HUVEC. This means that the 2G4 antibody has a very good ability to inhibit the proliferation of HUVEC and TF-1 cells.