KR20190113398A - Anti-Tspan12 antibody or antigen-binding fragment thereof, and use thereof - Google Patents

Abstract

Provided according to one aspect are an antibody or antigen-binding fragment thereof that specifically binds to Tspan12 and a use thereof. The present invention, accordingly, can be used to effectively prevent or treat diseases related to overproduction or activation of Tspan12 protein, specifically diseases related to angiogenesis.

Description

Anti-Tspan12 antibody or antigen-binding fragment thereof, and use thereof

An antibody or antigen-binding fragment thereof that specifically binds to Tspan12 (Tetraspanin12) protein and its use.

The retina receives its blood from the retinal vessels that occupy the interior of the retina and the choroidal tubes that occupy the exterior. Retinal vessel damage occurs in a number of disease processes, including diabetic retinopathy, prematurity retinopathy, and central and branch retinal vein occlusion (ischemic retinopathy). Retinal ischemia from this injury results in undesirable neovascularization. Choroidal neovascularization occurs in numerous other disease processes, including macular degeneration disease. In addition, patients with familial exudative vitreoretinopathy (FEVR) and Nori disease caused by mutations in FZD4 (Frizzled-4), LRP5 (Low-density lipoprotein receptor-related protein 5) or Norrin Incomplete angiogenesis of the retina is known to occur in patients with certain genetic diseases, such as Norrie disease (Berger et al., Nature Genet. 1: 199-203 (1992); Chen et al., Nature Genet. 1 : 204-208 (1992); Robitaille et al., Nature Genet. 32: 326-30 (2002); Toomes et al., Am. J. Hum. Genet. 74: 721-30 (2004)).

Recently, the use of biological agents has gradually increased to treat various retinopathies including diabetic retinopathy and age related macular degeneration diseases. In particular, anti-vascular endothelial growth factor (VEGF) antibodies, which are topically applied by intravitreal injection, are highly effective in reducing abnormal angiogenesis, leading to several drugs, including Aflibercept (Regeneron Pharmaceuticals). This is commercially available. However, the effects of preventing or preventing new blood vessel formation in proliferative diabetic retinopathy are limited, and therefore, the development of therapeutic agents independent of VEGF is required.

Tspan12 (Tetraspanin12) is a protein found in various cell membranes, encoded by the human Tspan12 gene. It belongs to the Tspan (transmembrane 4 superfamily) family and mainly includes four transmembrane domains and two extracellular domains. The Tspan family participates in various cellular signal transduction processes and serves as a signaling platform. In particular, it has been found that Tspan12 protein is selectively expressed in the retinal vascular system and is involved in retinal vascular development by activating β-catenin signaling (Junge HJ et al., Cell. 2009; 139: 299-311).

However, although Tspan12 has been shown to be involved in retinal neovascularization, there has been no attempt to treat in vitro in vivo effects in vascular proliferative retinopathy and to treat vascular proliferative retinopathy.

Therefore, it is necessary to develop new therapeutic agents for vascular proliferative retinopathy using Tspan12.

An antibody or antigen-binding fragment thereof that specifically binds to Tspan12 is provided.

It provides a pharmaceutical composition for the prevention or treatment of diseases associated with the activation or overproduction of Tspan12 protein.

A method of preventing or treating a disease associated with the activation or overproduction of a Tspan12 protein in an individual is provided.

One aspect provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 1 in the extracellular domain 2 (ECD2) of the Tspan12 protein. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 19; A light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 20-37; Or the heavy chain variable region and the light chain variable region.

Tspan12 is a protein encoded by the human Tspan12 gene, also known as NET2 or TM4SF12. The Tspan12 protein may comprise polypeptide fragments of Tspan12, including four transmembrane domains and two extracellular domains. The ECD2 may be an extracellular domain that mainly interacts with other receptors among the two extracellular domains. In addition, the ECD2 may include the amino acid sequence of SEQ ID NO: 1.

The term “epitope” refers to a protein determinant to which an antibody can specifically bind. Epitopes usually consist of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics. Three-dimensional epitopes and non-stereo epitopes are distinguished in that the binding to the former is lost in the presence of a denaturing solvent but not to the latter.

Specific binding to an epitope represented by the amino acid sequence of SEQ ID NO: 1 in ECD2 of the Tspan12 protein may be one having affinity to the Tspan12 protein or fragments thereof.

The antibody or antigen-binding fragment thereof can inhibit the binding of a substance specifically binding to Tspan12 protein and Tspan12 protein, inhibit interaction with FZD4 (Frizzled 4), and reduce beta-catenin activity.

The term “antibody” is used interchangeably with the term “immunoglobulin (Ig)”. Since the present invention relates to an anti-Tspan12 antibody, the term “antibody” used without modification unless otherwise indicated, may mean an anti-Tspan12 antibody that specifically binds to an epitope of Tspan12. A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each of which is linked by a heavy chain and a disulfide bond (SS-bond). The antibody may be an animal derived antibody, mouse-human chimeric antibody, humanized antibody, or human antibody.

The term "antigen-binding fragment" refers to a portion of an antibody that is a fragment thereof for the entire structure of the antibody, including a portion capable of binding to the antigen. For example, antigen-binding fragments are scFv, (scFv) ₂ , Fv, Fab, Fab ', Fv, F (ab') ₂ , diabody, triabody, tetrabody, Bis -scFv, nanobody, or a combination thereof.

As used herein, "antibody" may mean either an antibody or an antigen binding fragment.

The antibody or antigen-binding fragment thereof may include not only sequences of anti-Tspan12 antibodies, but also biological equivalents thereof within a range capable of specifically recognizing Tspan12, and may be modified. For example, further changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. In addition, the antibody or antigen-binding fragment thereof may be modified by conjugation or binding, glycosylation, tagging, or a combination thereof. The antibody may bind to cytokine. The cytokines are BMP (Bone morphogenetic protein), CCL (CC motif ligand), CXC (CXC motif ligand), GDF (Growth differentiation factor), GH (Growth hormone), IL (Interleukin), TNFSF (Tumor necrosis factor superfamily) , OSM (Oncostatin M), Interferon (IFN), or a combination thereof.

The antibody or antigen-binding fragment thereof and the cytokine may bind via (GGGGS) ₅ Linker. Specifically, the (GGGGS) ₅ Linker is the antibody or antigen-binding fragment thereof and the cytoplasm between the N-terminus of the cytokine and the Fc region of the antibody or the C-terminus of the cytokine and the N-terminus of the antibody. Combine Cain.

The term "heavy chain (HC or CH)" refers to a variable region domain VH and three constant region domains CH1, comprising an amino acid sequence having sufficient Variable Region (VR) sequences to confer specificity to the antigen, By both full-length heavy chains and fragments thereof including CH2 and CH3. The heavy chain constant region can be selected from any of the isotypes of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε).

The term “light chain (LC or CL)” refers to a full-length light chain and variable region domain VL comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. It means all fragments. The light chain constant region may be selected from the kappa (κ) and lambda (λ) types.

The heavy chain variable region may include a complementary determining region (H-CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 7; H-CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-11; And H-CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 17.

The light chain variable region comprises L-CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 22; L-CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26; And L-CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 29.

The term “Complementary Determining Region (CDR), ie CDR1, CDR2, and CDR3) refers to a region having binding specificity with an antigen among the variable regions of an antibody. Each variable region is typically a CDR1, CDR2. And three CDR regions identified as CDR3.

The antibody or antigen-binding fragment thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 37.

The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region selected from the group consisting of heavy chain variable regions comprising the amino acid sequences set forth in Table 1 below.

number H-CDR1 H-CDR2 H-CDR3 One SEQ ID NO: 2 SEQ ID NO: 8 SEQ ID NO: 12 2 SEQ ID NO: 3 SEQ ID NO: 9 SEQ ID NO: 13 3 SEQ ID NO: 4 SEQ ID NO: 10 SEQ ID NO: 14 4 SEQ ID NO: 5 SEQ ID NO: 10 SEQ ID NO: 15 5 SEQ ID NO: 6 SEQ ID NO: 9 SEQ ID NO: 16 6 SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 17

For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an H-CDR1 amino acid sequence of SEQ ID NO: 2, an H-CDR2 amino acid sequence of SEQ ID NO: 8, and an H-CDR3 amino acid sequence of SEQ ID NO: 12 Can be.

The antibody or antigen-binding fragment thereof may comprise a light chain variable region selected from the group consisting of light chain variable regions comprising the amino acid sequences set forth in the table below.

number L-CDR1 L-CDR2 L-CDR3 One SEQ ID NO: 18 SEQ ID NO: 23 SEQ ID NO: 27 2 SEQ ID NO: 19 SEQ ID NO: 24 SEQ ID NO: 28 3 SEQ ID NO: 20 SEQ ID NO: 25 SEQ ID NO: 29 4 SEQ ID NO: 21 SEQ ID NO: 26 SEQ ID NO: 30 5 SEQ ID NO: 22 SEQ ID NO: 25 SEQ ID NO: 31 6 SEQ ID NO: 22 SEQ ID NO: 25 SEQ ID NO: 29

For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an L-CDR1 amino acid sequence of SEQ ID NO: 18, an L-CDR2 amino acid sequence of SEQ ID NO: 23, and an L-CDR3 amino acid sequence of SEQ ID NO: 27 Can be.

The antibody or antigen-binding fragment thereof may be bound with angiogenesis inhibitors. The angiogenesis is an increase in permeability due to relaxation of the vascular wall, breakage of vascular endothelial cells, and contraction of cells, causing various proteases (proteinases) to be activated, and the basal membranes to be decomposed and vascular endothelium. Movement and proliferation of cells and the like, growth and completion of loops, and formation of new loops may occur. The angiogenesis inhibitor may include any agent that inhibits the phenomena resulting from the angiogenesis. For example, the cell angiogenesis inhibitor may be interferon (IFN), Interleukin (IL), and anti-vascular endothelial growth factor b (VEGFb). The interferon may be interferon alpha (IFN alpha), interferon beta (IFN beta), interferon gamma (IFN gamma). The interleukin may be interleukin-1a (IL1a), interleukin-13 (IL-13), interleukin-4 (IL4), and interleukin-33 (IL33).

Another aspect provides a pharmaceutical composition for the prevention or treatment of a disease associated with Tspan12 activation or overproduction comprising the antibody or antigen-binding fragment thereof. For example, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating a disease associated with Tspan12 activation or overproduction, including a pharmaceutically effective amount of an anti-Tspan12 antibody or an antigen-binding fragment thereof according to the present invention and a pharmaceutically acceptable carrier. .

The antibody, antigen binding fragment, and Tspan12 are as described above.

As demonstrated in the Examples below, the antibody or antigen-binding fragment thereof can be used in diseases related to angiogenesis because it binds to Tspan12 with high affinity and exhibits a potent inhibitory effect on vascular endothelial cell migration and angiogenesis.

The Tspan12 activation or overproduction refers to both biological and immunological aspects, wherein the term “biological” activity refers to a biological function caused by Tspan12 other than the ability to induce antibody production, and “immunological” activity refers to the activity of Tspan12. Refers to the function of inducing antibody production against antigenic epitopes. Thus, the Tspan12 activation or overproduction may include both the occurrence and increase in beta-catenin signaling and interaction with FZD4. The Tspan12 activation or overproduction can increase vascular endothelial cell migration and vasculature and grow blood vessels.

The term "prophylaxis" means any action that inhibits or delays the progression of a disease associated with Tspan12 activation or hyperplasia by administration to the pharmaceutical composition, such as angiogenesis, and "treatment" means Tspan12 activation or hyperplasia Refers to the inhibition, alleviation or elimination of diseases associated with (eg, neovascularization).

The disease associated with Tspan12 activation or hyperplasia may be a disease associated with angiogenesis. Diseases associated with angiogenesis include, for example, diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic macular edema : DME), pathological myopia, von Hippel-Lindau disease, histoplasma in eyes, central retinal vein occlusion (CRVO), branch retina vein occlusion occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage, and hypertensive retinopathy.

The pharmaceutically acceptable carrier is used in the sense including excipients, diluents or adjuvants. The carrier is, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pi It may be selected from the group consisting of rolidone, water, saline, buffers such as PBS, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anti-coagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, or combinations thereof.

The pharmaceutical composition may be prepared in any formulation according to conventional methods. The composition may, for example, be formulated in an oral dosage form (eg, powder, tablet, capsule, syrup, pill, or granule), or parenteral formulation (eg, injection), preferably It may be formulated into a parenteral formulation. Since proteins or peptides are digested, oral dosage forms should be formulated to coat the active agent or protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device that allows the active substance to migrate to the target cell.

The pharmaceutical composition may further comprise an angiogenesis inhibitor. The angiogenesis inhibitor may further include an anti-VEGF antibody or an anti-Norrin antibody. In addition, the angiogenesis inhibitor includes both a biological inhibitor and a chemical inhibitor, and may be administered sequentially or simultaneously with the pharmaceutical composition.

The term “pharmaceutically effective amount” refers to an amount sufficient to have the effect of prevention or treatment when administered to a subject in need thereof. The effective amount varies depending on factors such as the method of formulation, the mode of administration, the age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response to the response of the patient, and those skilled in the art will select Can be selected accordingly. For example, the daily dosage of the pharmaceutical composition may be in the range of 0.0001-100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg. have. The pharmaceutical composition may be administered once per day, multiple times per day, or once per week, once every two weeks, once every three weeks, or once every four weeks to once a year.

The pharmaceutical composition may be administered orally or parenterally. Preferably, it may be parenteral administration, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intravitreal administration, and the like. In one embodiment, the pharmaceutical composition may be administered in the form of intravitreal injection.

Another aspect provides a method of preventing or treating a disease associated with Tspan12 activation or hyperplasia comprising administering the antibody or antigen-binding fragment thereof to an individual.

Diseases, prevention and treatment associated with the activation or overproduction of the antibody, antigen binding fragment, Tspan12, Tspan12 are as described above.

The subject may be a mammal, for example human, cow, horse, pig, dog, sheep, goat or cat. The subject may be a subject that is likely to develop a disease associated with activation or overproduction of Tspan12, eg, angiogenic disease.

The method may further comprise administering an anti-VEGF antibody to the subject. The anti-VEGF antibody may be administered simultaneously, separately or sequentially with the anti-Tspan12 antibody or antigen binding fragment thereof.

The antibody or antigen-binding fragment thereof, anti-VEGF antibody, or a combination thereof can be administered orally or parenterally. Preferably it may be parenteral administration, in the case of parenteral administration may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intravitreal administration and the like. In one embodiment, it may be administered in an intravitreal injection form.

The preferred dosage of the antibody or antigen-binding fragment thereof, anticancer agent, or a combination thereof depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. The dosage is, for example, in the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg on an adult basis. Can be. The administration can be administered once daily, multiple times daily, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

According to one aspect of the antibody or antigen-binding fragment thereof which specifically binds to Tspan12 and its use, it can be used to effectively prevent or treat diseases related to activation or overproduction of Tspan12 protein, specifically diseases related to angiogenesis. .

1 is a diagram showing ECD2 of Tspan12 and Tspan12.
Figure 2 is a graph showing the degree of binding of anti-Tspan12 antibody and Tspan12 by absorbance.
3 is a graph showing wound closure rate (%) of anti-Tspan12 antibody and control.
4 is a graph showing wound closure rate (%) after EGF stimulation of anti-Tspan12 antibody and control.
5 is a graph showing total branching point and tube length of anti-Tspan12 antibody and control.
Figure 6 is a photograph showing the HUVEC cell staining results of the anti-Tspan12 antibody and the control.
7 is a photograph and graph showing the VO region of anti-Tspan12 antibody and control in vascular development model.
8 is a photograph and graph of vascular bifurcation of anti-Tspan12 antibody and control in vascular development model.
9 is a photograph and graph showing VO and NV regions of anti-Tspan12 antibody and control in the OIR model.
10 is a photograph and graph showing vascular bundle formation of anti-Tspan12 antibody and control in the VLDLR KO model.
Figure 11 is a photograph and graph showing Tspan12 and its mRNA after anti-Tspan12 antibody administration in the OIR model.
12 is a photograph and graph showing beta-catenin protein levels after anti-Tspan12 antibody administration in the OIR model.
Figure 13 is a photograph and graph showing Tspan12 after anti-Tspan12 antibody administration in the VLDLR KO model.
Figure 14 is a photograph showing the signal of the retinal vessels after administration of the anti-Tspan12 antibody and the control.
15 is a graph showing the results of FAC analysis of anti-Tspan12 antibody and the control group.
16 is a graph showing the interaction between Tspan12 and FZD4 with or without anti-Tspan12.
17 is a photograph and graph showing beta-catenin levels of control and anti-Tspan12 after anti-Tspan12 antibody treatment.
18 is a graph showing the levels of BMP4, PLVAP, VEGF, and CLAUDIN5 after anti-Tspan12 antibody treatment.
19 is a graph showing a and b waves after administration of a control, aflibercept, anti-Tspan12 antibody and aflibercept and anti-Tspan12 antibody in the OIR model.
20 is a graph showing VO and NV regions according to dose after administration of aflibercept, anti-Tspan12 antibody, in the OIR model.
FIG. 21 is a photograph and graph showing VO and NV regions after administration of control, aflibercept, anti-Tspan12 antibody and aflibercept and anti-Tspan12 antibody in the OIR model.
22 is a photograph and graph showing wound closure rate (%) of IFNG and control.
FIG. 23 is a graph showing total branching point and tube length of IFNG and control.
FIG. 24 is a photograph and graph showing VO and NV regions of IFNG and control in vascular development mouse disease model.
25 is a graph showing the results of Western blot experiment of IFNG and control in the OIR model.
Figure 26 is a graph showing the a and b waves of IFNG and control in the OIR model.
Figure 27 is a diagram showing the binding method of anti-Tspan12 antibody and IFNG.
FIG. 28 is a photograph and graph showing wound closure rate (%) of control group, IFNG, anti-Tspan12 antibody and the combination of IFNG and anti-Tspan12 antibody.
29 is a graph showing Western blot results of a control group, IFNG, anti-Tspan12 antibody, and a combination of IFNG and anti-Tspan12 antibody.
30 is a photograph and graph showing the VO and NV regions of the control group and the combination of IFNG and anti-Tspan12 antibodies.

Hereinafter, the present invention will be described in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

All data in the following examples are expressed as mean +/− standard error for the mean. Data was tested for normality using the D'Agostino-Pearson omnibus normality test. Once normality was established, t-test or covariance analysis was performed. If normality was not established, nonparametric tests of Mann-Whitney or Kruskal-Wallis were performed.

Example 1. Screening of New Anti-Tspan Antibodies

Since Tspan protein interacts with other receptors mainly through ECD2 in extracellular domains, ECS2 of Tspan12 was used as an antigen for screening antibodies using human binding antibody phage libraries (see FIG. 1). At this time, an antigen peptide having the amino acid sequence of SEQ ID NO: 1 of ECD2 common in humans and mice was synthesized and its N-terminal biotinylation. M13 phage was used for display of the human binding antibody library, and six antibody clones were selected after three biopannings with antibody phage library.

The amino acid sequence of the selected antibody is the same as the nucleotide sequence of SEQ ID NOs: 32 to 37, and the complementarity-determining region (CDR) amino acid sequences (SEQ ID NOs: 2 to 31) of the heavy and light chains of each antibody are shown in Table 3, respectively. And in Table 4.

Antibodies H-CDR1 H-CDR2 H-CDR3 1B5 SEQ ID NO: 2 SEQ ID NO: 8 SEQ ID NO: 12 1C9 SEQ ID NO: 3 SEQ ID NO: 9 SEQ ID NO: 13 1D7 SEQ ID NO: 4 SEQ ID NO: 10 SEQ ID NO: 14 2A3 SEQ ID NO: 5 SEQ ID NO: 10 SEQ ID NO: 15 2D10 SEQ ID NO: 6 SEQ ID NO: 9 SEQ ID NO: 16 3C4 SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 17

Antibodies L-CDR1 L-CDR2 L-CDR3 1B5 SEQ ID NO: 18 SEQ ID NO: 23 SEQ ID NO: 27 1C9 SEQ ID NO: 19 SEQ ID NO: 24 SEQ ID NO: 28 1D7 SEQ ID NO: 20 SEQ ID NO: 25 SEQ ID NO: 29 2A3 SEQ ID NO: 21 SEQ ID NO: 26 SEQ ID NO: 30 2D10 SEQ ID NO: 22 SEQ ID NO: 25 SEQ ID NO: 31 3C4 SEQ ID NO: 22 SEQ ID NO: 25 SEQ ID NO: 29

Example 2. Confirmation of antigen-antibody avidity

In order to measure the K _d value of the antibody selected in Example 1, kinetic binding analysis was performed using biolayer interferometry. Specifically, the biolayer interference method used Octet Red 96 system having a streptavidin dip and a biosensor (# 18-5021, ForteBio). Using this, equilibrium dissociation constants (KD), KD2, Kon (association constant), Kon2, Kdis (dissociation constant), Kdis2, and Full correlation coefficient (R ² ) for each antibody were determined. In Example 1 biotinylated antigen was used as ligand loaded on the biosensor and the antibody was used as analyte in solution. Seven concentrations were tested for each antibody and the results were analyzed with a 2: 1 fitting model, with a total R ² > 0.99.

As a result, as shown in Table 5, all six antibodies had a KD value in the range of nanomolar (nM), it was confirmed that the selected antibodies have a high affinity for ECD2 of Tspan12.

In addition, as shown in FIG. 2, specific binding of six anti-Tspan12 antibodies to ECD2 was confirmed by ELISA.

Example 3 In Vitro Analysis

3-1. Wound Healing Assay

To assess the anti-angiogenic potential of the antibodies selected in Example 1, the antibodies were analyzed in vitro as follows.

Specifically, Human Umbilical Vein Endothelial Cells (# 8000, ScienCell Research Laboratory) was inoculated into 96-well ImageLock tissue culture plates (# 4379, Essen BioScience) for wound healing analysis. Human umbilical vein endothelial cells were cultured in endothelial cell medium (# 1001, ScienCell Research Lab) containing 5% FBS, 1% endothelial cell growth supplement and 1% penicillin / streptomycin. Wound-Maker (# 4493, Essen BioScience) was used to make rejuvenated wounds. Assay plates were analyzed for 24 hours with Incucyte scratch wound cell migration software module # 9600-0012, Essen BioScience. The percent closure was calculated by the formula 100% × (W0-Wn) / W0 (W0: wound width at 0 hours, Wn: wound width at n hours).

As shown in FIG. 3, the 2D10 antibody most strongly inhibited HUVEC cell migration, as the wound closure rate was reduced by 35% compared to the control group treated with PBS. In addition, as shown in Figure 4, when exposed to the 2D10 antibody, even in the presence of EGF stimulation, the wound closure rate was reduced by 40% compared to the control.

3-2. Tube formation assay

For tube formation analysis, HUVECs were applied to Matrigel (# 356231, BD Biocoat) in BD Biocoat Biocoatel (# 356231, BIbidi). Well was continuously observed by the Incucyte Zoom system. Total point number and total tube length were calculated by the Wimasis image analysis platform.

When 2D10 antibody was used in the tube formation assay, both total branch point and tube length were reduced as shown in FIG. 5.

3-3. Confirmation of Tspan12 Specific Binding Ability of Antibodies by Cell Staining

Specific binding to Tspan12 antigen expressed on HUVECs was tested by immunocytochemistry.

Specifically, in knockdown experiments, HUVECs were transfected with 20 nM of siRNA (Dharmacon) along with RNAimax. After 24 hours, cells were seeded on collagen-coated cover slides for immunocytochemical observation. The pool of siRNAs targeting human Tspan12 (Dharmacon) contains four siRNAs containing target sequences such as GGAUAUUGUGGAACGGUGA (SEQ ID NO: 38), CUGUAUAAUUCAGUCGAUU (SEQ ID NO: 39), AGAACUGGCUUGUGGCGUU (SEQ ID NO: 40), and CCUAGAUAUCGGUGGCUUA (SEQ ID NO: 41). It is. Untargeted siRNA was used as a control (WT control) and 2D10 antibody or commercially available anti-Tspan12 antibody was incubated with HUVEC (WT control) and HUVEC treated with siRNA targeting Tspan12 (Tspan12 KD group).

As shown in FIG. 6, the 2D10 antibody stained the surface of HUVEC in a pattern similar to a commercial anti-Tspan12 antibody in a WT control sample, but staining was strongly reduced in the Tspan12 KD group. This means that the 2D10 antibody recognizes Tspan12 on the endothelial cell surface.

Example 4. In Vivo Assay

Experiments using C57BL / 6 and transgenic mice in the present invention complied with the NIH Guide for the Care and Use of Laboratory Animals.

The animal studies performed were reviewed and approved by the Institutional Animal Care and Use Committee of the Scripps Research Institute. The effect of 2D10 antibody was shown in wild-type (WT) C57BL / 6 mice and Very low density lipoprotein receptor knockout (VLDLR KO) mice in an oxygen-induced retinopathy (OIR) model. B6; 129S7-Vldlrtm1 Her / J, The Jackson Laboratory) was observed during the development of physiological retinal vessels.

Intravitreal injection was performed using a 33-guage Hamilton syringe. Mice received 2D10 antibody, Appliverscept (Eylea, Regeneron Pharmaceuticals), or a combination thereof in one eye and 100 ng of rhIgG1 (# 110-HG-100, R & D Systems) in the opposite eye as a control. . AngioTool software (National Cancer Institute) was used to quantify the bifurcation during physiological development.

In the vascular development model, wild-type C57Bl / 6 mice were injected intravitreally with 100 ng of 2D10 antibody at 33 days after birth (P2) or 7 days (P7) using a 33-guage Hamilton syringe. In the opposite eye, 100 ng of rhIgG1 (# 110-HG-100, R & D Systems) was injected in the same way. Mice were euthanized and eyes were obtained at P6 or P11.

In the OIR model, mice between 7 and 12 days after birth were transferred indoors after 75% oxygen exposure. At P12 of the OIR model, mice were injected intravitreally with various concentrations of 2D10 antibody and Aflibercept (Aflibercept, Regeneron Pharmaceuticals) and 100 ng of rhIgG1 control.

In the VLDLR KO experiment, mice at day 15 postnatally (B6; 129S7-Vldlrtm1Her / J, The Jackson Laboratory) were injected intravitreally with 100ng of 2D10 antibody or rhIgG1 control. The retina was obtained at day 22 to quantify the bundle formation of the retina. Retinal neovascular proliferation values of VLDLR KO mice were quantified using Image J Cell Counter Plug-In (National Institutes of Health).

Vasoobilteration (VO) and neovascularization (NV) regions were quantified according to existing techniques in OIR P17 (Connor KM, et al., 2009; 4: 1565-1573; Banin E, et al., Invest Ophthalmol Vis Sci. 2006; 47: 2125-2134). VO and NV structures (Rescue) were determined by standardizing the NV region of the treated retina, mean NV region of the control eye treated with IgG1, and VO region for mean VO.

As a result of the above example, FIG. 7 shows a slight but significant decrease in the vascularized site on day 6 when 2D10 antibody was injected on day 2 postnatal day (P2) compared to the control group. Likewise, FIG. 8 shows a significant decrease in vascular bifurcation when injected with 2D10 antibody after formation of superficial vascular nerves at day 7 of birth (P8). That is, 2D10 antibody means that it maintains the anti-angiogenic potential in vivo.

In addition, as shown in FIG. 9, in the OIR model, the retina is characterized by changes in the central VO region (yellow) and the NV region derived from the surface plexus (red). The VO area was significantly reduced. Thus, 2D10 antibodies pathologically inhibit NV and promote capillary regrowth and function as vascular modulators rather than anti-angiogenic agents in vivo.

In the VLDLR KO model, the retina develops neovascular changes originating from the deep and middle vascular plexus, which grows into the subretinal space. As shown in FIG. 10, injection of 2D10 antibody at P15 significantly reduced vascular bundle formation while pathologically inhibiting NV.

It was observed that vascular proliferative retinopathy and Tspan12 and beta-catenin signal transduction are endogenously activated in the model of this example.

As shown in FIG. 11, mRNAs of Tspan12 (P12 6h) and Tspan12 protein (OIR P13) were significantly increased in the OIR model. Similarly, as shown in FIG. 12, the protein level of beta-catenin also increased significantly in P13 compared to P12. The results indicate that Tspan12 and beta-catenin signaling pathways are associated with the acute onset of retinal hypoxia, which pathways may play an important role in the early stages of retinal neovascularization.

As shown in FIG. 13, Tspan12 protein was significantly increased around P21 and P32 in the VLDLR KO model. This means that it plays a role in later stages in the pathway VLDLR KO model.

To confirm that the 2D10 antibody was localized in the vascular layer in vivo, the 2D10 antibody was injected into the vitreous body and then detected with an anti-human IgG1 (Fc) secondary antibody in the entire retinal mount.

As a result, as shown in FIG. 14, after 1 hour, the retinal vessels were clearly classified that the fluorescence signal was increased along the target vessel layer only in the eyes injected with the 2D10 antibody. In addition, after 6 hours, the beta-catenin levels of the retina were significantly reduced in the OIR and VLDLR KO models compared to the control group treated with IgG1.

Example 5. Immunofluorescence Assay

Eyes were obtained and fixed in 4% paraformaldehyde for 40 minutes. The entire retinal mount was dissected and stained with Isolectin GS-IB4 (# I21412, Thermo Fisher Scientific) at 4 ° C. for 1 day. To detect 2D10 antibodies in retinal tissue 1 hour after intravitreal injection, the whole retinal mount was further stained with goat anti-human IgG Fc (# SA5-10135, Thermo Fisher Scientific) cross-sorbed with secondary antibody.

HUVEC was fixed in 100% methanol for 5 minutes at 4 ° C., blocked for 1 hour in 10% BSA, and commercially available anti-Tspan12 antibody (# LS-B9720, Lifespan Biosciences) or 2D10 antibody and anti beta-catenin antibody (# 9562, Cell Signaling) and incubated for 1 day at 4 ° C. Alexa Fluor 488 chicken anti-rabbit IgG (# A-21441, Thermo Fisher Scientific) was used as secondary antibody.

For cryosection, the entire eye was frozen and only central retinal sections including the optic nerve were taken. Retinal sections were fixed in cold ethanol for 10 minutes, permeabilized with 0.1% Triton and blocked with 10% BSA. Primary antibody isolectin GS-IB4 (# I21412, Thermo Fisher Scientific), 2D10 antibody, commercial anti-Tspan12 antibody (# LS-B9720, Lifespan Biosciences), anti-beta-catenin antibody (# 9562, Cell Signaling), mouse Anti-rhodosin antibody (#MAB 5356, EMD Millipore) and rabbit anti-Red / Green opsin antibody (AB5405, EMD Millipore) were incubated at 4 ° C. for 1 day. To maximize the comparison between samples, representative images of the samples were taken only at the central retina by the optic nerve head.

Example 6 Western Blot, Quantitative Real-Time PCR and PCR Array Analysis

Collect samples in a single retina or HUVEC, wash them with PBS, then dissolve buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 10% glycerol, 1% Triton X-100 )). The lysates were then centrifuged at 15,000 rpm at 4 ° C. to remove aggregates, and the soluble protein was denatured with Laemmli sample buffer at 95 ° C. for 5 minutes and then separated by SDS-PAGE. Protein was transferred to nitrocellulose membrane using iBlot and blocked in PBST containing 5% BSA for 1 hour. Commercially available anti-Tspan12 antibody (# LS-B9720, Lifespan Biosciences) and anti beta-catenin antibody (# 9562, Cell Signaling) were used as primary antibodies and incubated at 4 ° C. for 1 day. After washing the membrane several times with PBST, the blots were incubated with HRP-conjugated anti-human or anti-rabbit antibodies for 1 hour. The membrane was washed with PBST and developed with ECL. In the western blot experiment two independent experiments were performed.

For quantitative real-time PCR analysis, mouse actin (assay ID Mm02619580_g1), mouse Tspan12 (Mm00557112_m1), mouse plasma envelope vesicle-associated protein (Mm00453379_m1), human ACTIN (Hs01060665_g1), human bone morphogenesis protein 4 (Hs00610344_m1), human plasma Taqman gene expression assay (Thermo Fisher Scientific) of outer membrane vesicle-associated protein (Hs00229941_m1), human VEGF / A (Hs00900055_m1) and human CLAUDIN5 (Hs00533949_s1) was used. For PCR array analysis, a mouse angiogenesis PCR array (# PAMM-024Z, Qiagen) was performed using RT2 SYBR quantitative real-time PCR Mastermix (# 330522, Qiagen). Data analysis was performed using RT2 Profiler PCR Array Data Analysis 3.5 version (Qiagen).

Example 7. FAC Analysis

To confirm the binding capacity of 2D10 antibody to Tspan12 of the selected antibodies, untreated HEK-293 cells (WT control) and cells overexpressing Tspan12 (Tspan12 OE) were observed as 2D10 antibodies.

Specifically, 293 cells (# CRL-1573, ATCC) were cultured in DMEM medium (# 10569-010, Gibco) containing 10% FBS and 1% penicillin / streptomycin. In total, 293 cells (# CRL-1573, ATCC) were transfected with Tspan12 (# RC206943, OriGene) for 48 hours. After transfection, cells were stained with 2D10 antibody, commercial anti-Tspan12 antibody (# LS-B9720, Lifespan Biosciences) or IgG1 isotype control antibody (# 110-HG-100, R & D Systems). For nuclear staining, Hoechst was used. Stained cells were sorted with an LSRII flow cytometer (Becton Dickinson).

In this analysis, as shown in FIG. 15, the fluorescence signal was stronger in the Tspan12 OE group.

Example 8 Coimmunoprecipitation Method

Coimmunoprecipitation tests examined whether 2D10 antibodies blocked the formation of receptor complexes between Tspan12 and FZD4. Confluent 293 cells were transfected with Lipo2000 (Invitrogen) for 48 hours in a 75 cm2 flask with Tspan12 antibody (RC206943, OriGene) and FZD4 (RG217286, OriGene). Cells were washed with PBS and incubated for 1 hour in the presence or absence of 2D10 (1 μg / mL) at 4 ° C.

As a control, flag-Tspan11 (RC216497, OriGene) was transfected. After washing three times with PBS, cells were incubated with DTSSP (Pierce) in PBS for 1 hour. Cell lysates were incubated with antiflag antibody-protein A / G beads for 1 day at 4 ° C. After thorough washing with lysis buffer (150 mM NaCl, 50 mM Tris / HCl [pH 8.0], 1 mM EGTA, 1% NP-40, 0.1% N-dodecyl-β-D-maltoside, 3 mM MgCl2 and protease inhibitor mixture) , The beads were eluted. Eluates were analyzed by Western blot.

Antibodies were used as anti-flag antibodies (# A00187-200, Genscript) and anti-FZD4 (# MAB194-SP, R & D Systems).

Tspan12 has been shown to affect vascular development by regulating standard Wnt and beta-catenin signaling pathways (Junge HJ et al., Cell. 2009; 139: 299-311).

In Example 8, as shown in Figure 16, the interaction between Tspan12 and FZD4 was significantly reduced after incubation with 2D10 antibody. As shown in Figure 17, after treatment with 2D10 antibody in the immunochemical and Western blot analysis of the above example, beta-catenin protein levels significantly decreased.

In addition, as shown in FIG. 18, 2D10 treatment significantly reduced the expression level of bone morphogenetic protein 4: BMP4, but plasma lemma vesicle-associated protein (PLVAP) levels significantly increased as expected. The expression level of vascular endothelial growth factor (VEGF) or CLAUDIN5 was not affected.

Example 9 Ganzfeld ERG

Ganzfeld ERG mice were dark-adapted for 1 day and subsequent procedures were performed under dim light. Mice were anesthetized by intraperitoneal injection of ketamine (60 mg / kg) and xylazine (10 mg / kg). The pupils were expanded through 0.25% hydroxyamphetamine and 1% trophamide. Ganzfeld ERG was recorded on the corneal surface. Silver needle electrodes were attached to the forehead and tail, and stimulated with Ganzfeld dome (Espion E2 with Colordome, Dignosys). Rod and mixed rod / cone responses were recorded using white flashes of increasing intensity (1 × 10 −5 to 50 cd · s / m 2) in the dark-adapted (dark-adapted) state. Light-adaptation (light acclimatization) was performed on a background of 30 cd · s / m 2. Cone responses to flickering stimuli at 1-Hz (0.63-20 cd · s / m2) and 30-Hz (3.98, 10, 20 cd · s / m2) were recorded.

Retinal function was examined with ERG at P32 after intravitreal injection in OIR model P12 to determine side effects through administration of anti-VEGF antibody and 2D10 antibody.

As shown in FIG. 19, the visual acuity treated with IgG1 in the OIR model significantly decreased the b-wave amplitude and photometric response compared to the control group with normal oxygen concentration. The appliverscept dose to humans (2 μg) further reduced b and a wave amplitudes in mice compared to IgG1 treated controls. However, co-administration of low doses of appliverscept (20 ng) and low doses of 2D10 antibody (10 ng) did not have a negative effect on ERG compared to the OIR IgG1 control.

Example 10. Confirmation of Effect on Combination Therapy

In the combination treatment experiment, mice were injected intravitreally with 10 ng of antibody 2D10, 20 ng of aflibercept and 2D10, and 10 ng of 2D10 and 20 ng of aflibercept and tested in an OIR model according to the method of Example 4.

As a result, as shown in FIG. 20, in the OIR model, the 2D10 antibody reduced the VO and NV regions most significantly between 0.2 ng and 75 ng, while the VO improved by 45% and NV by 68%, while the applivercept was 2 μg. The highest dose was found at, with 37% improvement in VO and 80% improvement in NV. In addition, as shown in FIG. 21, appliverscept (20 ng) causes a slight decrease in VO (38%) and NV (26%) regions, whereas 2D10 antibody (10 ng) shows 40% VO and NV regions, respectively. , 41%. Furthermore, co-administration of appliverscept (20 ng) and 2D10 antibody (10 ng) increased the structural effects on the VO and NV regions by 64% and 75%, respectively.

Example 11 Binding of Anti-Tspan12 Antibodies to Interferon gamma (IFNG)

11-1. In vitro effects of IFNG

According to the method of Example 3, the wound healing analysis for IFNG, as shown in Figure 22, when exposed to IFNG, 50% and 40% reduction in wound closure compared to the control and aflibercept .

When IFNG was used in the tube formation assay, both total branch point and tube length were reduced as shown in FIG. 23.

In Western blot experiments, treatment with IFNG significantly reduced pSTAT1 (phosphorylated signal transducer and activator of transcription 1) protein levels.

11-2. In vivo effects of IFNG

According to the method of Example 4, in vivo analysis of IFNG was performed using PBS (Phosphate buffer saline) as a control in three models. As a result, as shown in Fig. 24, the reduction of the vascularized site was not particularly seen, but significantly reduced both the VO and NV regions.

As shown in FIG. 25, when IFNG was treated in the OIR model, the protein of pSTAT1 increased significantly more than 10-fold when confirming the sample results from four independent mouse retinas.

As shown in FIG. 26, the visual acuity treated with PBS in the OIR model showed a significant decrease in dark-adapted b-wave amplitude and photometric response compared to the control group with normal oxygen concentration, but this was not significantly different from that of IFNG. Had no negative effect on ERG compared to the control.

11-3. Binding of IFNG with Anti-Tspan12 Antibodies

As shown in FIG. 27 using genetic recombination techniques, between the N-terminus of the human IFNG gene and the Fc region of the anti-Tspan12 antibody or the C-terminus of the human IFNG gene and the N-terminus of the anti-Tspan12 antibody (GGGGS) ₅ Linker was mediated.

According to the method of the above embodiment through the above combination, in vitro experiments were performed. As a result of wound healing analysis for the combination of anti-Tspan12 antibody and IFNG, the wound closure rate was reduced by 18% and 25% compared to IFNG alone and anti-Tspan12 antibody alone as shown in FIG. 28.

 In Western blot experiments, when the anti-Tspan12 antibody and IFNG were combined, 80% reduction in Erk phosphorylation and 90% reduction in beta-catenin, as shown in FIG. 29.

Similarly, as a result of in vivo experiments, as shown in Example 4, when the anti-Tspan12 antibody and IFNG were combined, as shown in FIG. 30, both VO and NV regions were significantly reduced compared to the control group. Therefore, it can be seen that the combination of anti-Tspan12 antibody and IFNG maintains anti-vascular potential in vivo and significantly reduces vascular bundle formation.

<110> Daegu Gyeongbuk Institute of Science and Technology <120> Anti-Tspan12 antibody or antigen-binding fragments,          use <130> PN121617 <160> 41 <170> KoPatentIn 3.0 <210> 1 <211> 48 <212> PRT <213> Homo sapiens <400> 1 Cys Cys Gly Val Val Tyr Phe Thr Asp Trp Leu Glu Met Thr Glu Met   1 5 10 15 Asp Trp Pro Pro Asp Ser Cys Cys Val Arg Glu Phe Pro Gly Cys Ser              20 25 30 Lys Gln Ala His Gln Glu Asp Leu Ser Asp Leu Tyr Gln Glu Gly Cys          35 40 45 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 <400> 2 Gly Phe Pro Phe Ser Ser Tyr Gly   1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 <400> 3 Gly Tyr Thr Phe Thr Asn Asn Tyr   1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 <400> 4 Gly Gly Asn Phe Arg Lys Asn Ser   1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 <400> 5 Gly Tyr Thr Phe Thr Ser Tyr Tyr   1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 <400> 6 Gly Tyr Thr Phe Thr Gly Tyr Tyr   1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 <400> 7 Gly Phe Thr Phe Ser His Phe Gly   1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 <400> 8 Ile Ser Arg Ser Ser Ser Tyr Ile   1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 <400> 9 Ile Asn Pro Asn Ser Gly Gly Thr   1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 <400> 10 Ile Asn Pro Ser Gly Gly Ser Thr   1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 <400> 11 Ile Ser Ser Ser Ser Ser Tyr Ile   1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 <400> 12 Ala Arg Gly Arg Ser Phe Asp Leu   1 5 <210> 13 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 <400> 13 Ala Arg Asp Pro Phe Arg Arg Gly Leu Arg Gly Gly Ile Ala Ala Ala   1 5 10 15 Phe Asp Tyr             <210> 14 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 <400> 14 Ala Arg Ala Arg Gly Tyr Cys Thr Asn Gly Val Cys Tyr Thr Gly Pro   1 5 10 15 Phe Asp Tyr             <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 <400> 15 Ala Arg Asp Arg Gly Ser Arg Gly Leu Gly Tyr   1 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 <400> 16 Ala Arg Asp Arg Arg Gly Met Phe Asp Pro   1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3 <400> 17 Ala Arg Gly Met Ala Arg Ala Phe Asp Ile   1 5 10 <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 <400> 18 Gln Gly Ile Ser Ser Tyr   1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 <400> 19 Ser Ser Asp Val Gly Gly Tyr Asn Tyr   1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 <400> 20 Ser Gly Ile Asn Val Gly Ala Tyr Arg   1 5 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 <400> 21 Ser Ser Asn Ile Gly Ser His Phe   1 5 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 <400> 22 Ser Gly Ile Asn Val Gly Ala Tyr Arg   1 5 <210> 23 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 <400> 23 Ala Ala Ser   One <210> 24 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 <400> 24 Glu val ser   One <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 <400> 25 Tyr Lys Ser Asp Ser Asp Lys   1 5 <210> 26 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 <400> 26 Gly asn ser   One <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 <400> 27 Gln Gln Ser His Gly Phe Pro Pro Thr   1 5 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 <400> 28 Ala Ser Tyr Arg Ser Gly Ser Thr Trp Val   1 5 10 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 <400> 29 Ala Ile Trp His Ser Ser Ala Trp Val   1 5 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 <400> 30 Gln Ser Tyr Asp Ser Ser Leu Ser Gly Tyr Val   1 5 10 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3 <400> 31 Ala Ile Trp His Ser Ser Ala Trp Met   1 5 <210> 32 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Antibody 1B5 <400> 32 Met Ala Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro   1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser              20 25 30 Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu          35 40 45 Trp Val Ala Ser Ile Ser Arg Ser Ser Ser Tyr Ile Tyr Tyr Thr Asp      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp His Asn Ser  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Gly Arg Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu             100 105 110 Val Thr Val Ser Ser Arg Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly         115 120 125 Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala     130 135 140 Ser Val Gly Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile 145 150 155 160 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys                 165 170 175 Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg             180 185 190 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser         195 200 205 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His Gly     210 215 220 Phe Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 225 230 235 <210> 33 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> Antibody 1C9 <400> 33 Met Ala Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Arg Pro   1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr              20 25 30 Asn Asn Tyr Ile His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu          35 40 45 Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln      50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr  65 70 75 80 Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Pro Phe Arg Arg Gly Leu Arg Gly Gly Ile Ala             100 105 110 Ala Ala Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala Ser     130 135 140 Val Pro Gly Ser Pro Gly Gln Ser Val Thr Ile Ser Cys Thr Gly Thr 145 150 155 160 Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His                 165 170 175 Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Glu Val Ser Lys Arg Pro             180 185 190 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala         195 200 205 Ser Leu Thr Val Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr     210 215 220 Cys Ala Ser Tyr Arg Ser Gly Ser Thr Trp Val Phe Gly Gly Gly Thr 225 230 235 240 Lys Leu Thr Val Leu Gly                 245 <210> 34 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> Antibody 1D7 <400> 34 Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro   1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Asn Phe Arg              20 25 30 Lys Asn Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu          35 40 45 Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln      50 55 60 Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr  65 70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Ala Arg Gly Tyr Cys Thr Asn Gly Val Cys Tyr Thr             100 105 110 Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Leu Thr Gln Pro Ser Ser     130 135 140 Leu Ser Ala Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg 145 150 155 160 Ser Gly Ile Asn Val Gly Ala Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys                 165 170 175 Pro Gly Ser Pro Pro Gln Phe Leu Leu Arg Tyr Lys Ser Asp Ser Asp             180 185 190 Lys Gln Gln Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Asp         195 200 205 Ala Ser Ala Asn Ala Gly Ile Leu Leu Ile Ser Gly Leu Arg Ser Glu     210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Ala Ile Trp His Ser Ser Ala Trp Val 225 230 235 240 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly                 245 250 <210> 35 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Antibody 2A3 <400> 35 Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro   1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr              20 25 30 Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu          35 40 45 Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln      50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr  65 70 75 80 Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Arg Gly Ser Arg Gly Leu Gly Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Ser Gln         115 120 125 Ala Val Leu Thr Gln Pro Ser Ser Val Ser Ala Ala Pro Gly Gln Lys     130 135 140 Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser His Phe 145 150 155 160 Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile                 165 170 175 Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Phe Gly             180 185 190 Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala         195 200 205 Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu Ser     210 215 220 Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly 225 230 235 <210> 36 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Antibody 2D10 <400> 36 Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro   1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr              20 25 30 Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu          35 40 45 Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln      50 55 60 Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr  65 70 75 80 Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Arg Arg Gly Met Phe Asp Pro Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Ser Gln Ala         115 120 125 Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala Ser Val     130 135 140 Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Ala Tyr Arg 145 150 155 160 Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Phe Leu Leu                 165 170 175 Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val Pro Ser             180 185 190 Arg Phe Ser Gly Ser Arg Asp Ala Ser Ala Asn Ala Gly Ile Leu Leu         195 200 205 Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ile     210 215 220 Trp His Ser Ser Ala Trp Met Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu gly         <210> 37 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Antibody 3C4 <400> 37 Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro   1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser              20 25 30 His Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu          35 40 45 Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Gly Met Ala Arg Ala Phe Asp Ile Trp Gly Gln Gly             100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Ser Gln Ala         115 120 125 Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala Ser Val     130 135 140 Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Ala Tyr Arg 145 150 155 160 Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Phe Leu Leu                 165 170 175 Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val Pro Ser             180 185 190 Arg Phe Ser Gly Ser Arg Asp Ala Ser Ala Asn Ala Gly Ile Leu Leu         195 200 205 Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ile     210 215 220 Trp His Ser Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val 225 230 235 240 Leu gly         <210> 38 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SiRNA targeting Tspan12 <400> 38 ggauauugug gaacgguga 19 <210> 39 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SiRNA targeting Tspan12 <400> 39 cuguauaauu cagucgauu 19 <210> 40 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SiRNA targeting Tspan12 <400> 40 agaacuggcu uguggcguu 19 <210> 41 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SiRNA targeting Tspan12 <400> 41 ccuagauauc gguggcuua 19

Claims (18)

An antibody or antigen-binding fragment thereof that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 1 in ECD2 (Extracellular domain 2) of Tspan12 (Tetraspanin12) protein. The method according to claim 1, wherein the antibody or antigen-binding fragment thereof is a heavy chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to 17;
A light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 31;
Or an antibody or antigen-binding fragment thereof comprising the heavy chain variable region and the light chain variable region. The method according to claim 2, wherein the heavy chain variable region is
H-CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-7;
H-CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-11; And
An antibody or antigen-binding fragment thereof comprising H-CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-17. The method according to claim 2, wherein the light chain variable region is
L-CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 22;
L-CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26; And
An antibody or antigen-binding fragment thereof comprising L-CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-31. The antigen binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-37. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of heavy chain variable regions comprising the amino acid sequences set forth in Table 6 below:

The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of light chain variable regions comprising the amino acid sequences set forth in Table 7 below.

The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is bound to an angiogenesis inhibitor. The antibody or antigen-binding fragment thereof of claim 8, wherein the angiogenesis inhibitor is interferon (IFN), Interleukin (IL), and anti-vascular endothelial growth factor b (VEGFb). . The antibody or antigen-binding fragment thereof of claim 9, wherein the interferon is interferon alpha, interferon beta, interferon gamma. A pharmaceutical composition for preventing or treating a disease associated with Tspan12 activation or hyperplasia comprising the antibody of claim 1 or an antigen-binding fragment thereof. The pharmaceutical composition of claim 11, wherein the disease associated with Tspan12 activation or hyperplasia is a disease associated with angiogenesis. The disease of claim 12, wherein the disease associated with angiogenesis is diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic macular edema edema (DME), pathological myopia, von Hippel-Lindau disease, histoplasma of the eye, central retinal vein occlusion (CRVO), branch retina central vein occlusion (branch retina) vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage, and hypertensive retinopathy The pharmaceutical composition of claim 11, further comprising an angiogenesis inhibitor. The pharmaceutical composition of claim 14, wherein the angiogenesis inhibitor is an anti-VEGF antibody or an anti-Norrin antibody. A method of preventing or treating a disease associated with Tspan12 activation or hyperplasia, comprising administering to a subject an antibody of claim 1 or an antigen-binding fragment thereof. The method of claim 16, further comprising administering to the subject an angiogenesis inhibitor. The method of claim 16, wherein the angiogenesis inhibitor is administered simultaneously, separately, or sequentially with the antibody of any one of claims 1-10 or an antigen-binding fragment thereof.

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