WO2018124582A1 - Composition for preventing and treating eye diseases including anti-ang2 antibody - Google Patents

Abstract

The present invention relates to a composition which is for preventing and treating eye diseases and includes an anti-angiopoietin-2 (Ang2) antibody and, more specifically, to a novel use of an anti-Ang2 antibody as an agent for preventing or treating eye diseases, wherein the anti-Ang2 antibody is specifically coupled to Ang2, is coupled to a Tie2 receptor along with Ang2, and has improved affinity. The composition according to the present invention can be usefully used to develop a treatment agent for macular degeneration, diabetic retinopathy, and glaucoma.

Description

Composition for the prevention and treatment of eye diseases comprising an anti-ang2 antibody

The present invention relates to a composition for the prevention and treatment of eye diseases comprising an angiopoietin-2 (Anggiopoietin-2; Ang2) antibody or an antigen-binding fragment thereof, and more specifically, specifically binds to Ang2, And a novel use as an agent for preventing and treating ocular diseases, which binds to the Tie2 receptor and has improved affinity.

Many ocular diseases, which cause millions of blindness worldwide each year, are a major cause of angiogenesis. In particular, representative examples of various eye diseases resulting from abnormal blood vessels include age-related macular degeneration, diabetic retinopathy, and glaucoma.

Age-related macular degeneration is a disease name that refers to the breakdown or destruction of the macula accompanying aging and occurs mainly in people over 50 years of age. This disease is caused by the accumulation of small waste products called drusen, which exude leaks from non-exudatives (dry type), which causes the macular atrophy to progress and loss of visual function, and abnormal neovascularization resulting from the accumulation of hypoxia and oxidative stress in the retina and choroid. As the macular edema is induced and this process is repeated, the visual and retinal pigment epithelial cells of the macula are permanently denatured and divided into exudatives (wet type) that cause vision loss. Particularly, in case of wet macular degeneration, the incidence of blindness is high, and the prevalence rate in Korea is estimated to reach 37 per 10,000 people. The cause of macular degeneration is not yet known, but the risk factors are age, and other environmental factors of interest include smoking, hypertension, obesity, genetic predisposition, excessive UV exposure, and low blood antioxidant levels. have. Currently, the treatment of wet macular degeneration mainly consists of antibody injection treatment for vascular endothelial growth factor, and other treatments such as laser photocoagulation, photodynamic therapy, and vitrectomy are known, but there is no complete treatment yet. to be.

Diabetic retinopathy is a diabetic retinopathy caused by diabetic peripheral circulatory disorders, which is a complication of the eye that causes a reduction in visual acuity due to disorders in the microcirculation of the retina. In early diabetic retinopathy, it can cause no symptoms or only mild vision problems, but can eventually lead to blindness. Diabetic retinopathy can develop in anyone with type 1 diabetes or type 2 diabetes.

Glaucoma (glaucoma) is a chronic optic nerve disease, a severe intractable disease that causes progressive degeneration of the optic nerve, progressive loss of retinal ganglion cells and vision loss due to visual field defects. Risk factors for glaucoma include age, race, sex, and high blood pressure, but increased intraocular pressure is known as the most important cause of glaucoma, especially primary open angle glaucoma. Glaucoma is thought to be due to increased intraocular pressure due to increased resistance of the aqueous humoral outflow pathways through the Schlemm's canal and trabecular meshwork in the eye, but the molecular biological mechanisms that cause problems It has not been clear so far. For the treatment of glaucoma, there are drugs that reduce the production of waterproofing, or methods using laser trabeculoplasty, or surgically creating a new waterproofing release route. Existing non-surgical treatments resolve the abnormality of the waterproofing pathway. The effect is limited because it is not possible, and in case of surgical treatment, the newly created waterproof discharge path is frequently blocked due to normal tissue healing reaction, and thus there is no fundamental treatment yet.

Angiopoietin-2 (Angiopoietin-2; Ang2) is an antagonistic ligand of receptor Tie2 present in vascular endothelial cells. Angiopoietin-1, an agonist of Tie2. Inhibits signaling by Tie2 by competing for Ang1) and Tie2 binding. Ang1, a ligand that activates the Tie2 receptor, acts as a major regulator of vascular stability by maintaining the barrier function of vascular endothelial cells. In the overexpression or inflammatory state of vascular endothelial growth factor, vascular endothelial cells are activated, and vascular permeability is increased. At this time, Ang1 induces stabilization of vascular endothelial cells by promoting junctional integrity of vascular endothelial cells and decreases vascular permeability, whereas increased Ang2 in activated vascular endothelial cells competes with Ang1 Inhibits endothelial cell stabilization. Therefore, Ang2 inhibits Ang1-Tie2 binding and signal transduction through maintaining stability of vascular endothelial cells, thereby promoting angiogenesis through dynamic rearrangement of blood vessels. Overexpression of Ang2 has been reported in various solid and hematological cancers, and overexpression of Ang2 has also been reported in various diseases such as sepsis, bacterial infections, lung damage and kidney damage.

Conventionally, Korean Patent Publication No. 10-2015-0014077 specifically binds to angiopoietin-2 (Ang2), an angiogenesis inducer, binds to Tie2 receptor with Ang2, and humanizes it. And / or an anti-Ang2 antibody with improved affinity was identified and its use as an anticancer agent using this anti-Ang2 antibody (Korean Patent Publication No. 10-2015-0032075) and its use in sepsis (Korean Patent Publication No. 10- 2015-0028087) was confirmed. However, the therapeutic use of anti-ang2 antibodies in eye diseases has not been proved experimentally.

Therefore, the present inventors have tried to develop a therapeutic agent for various eye diseases resulting from abnormal blood vessels, and as a result, anti-ang2 antibody that specifically binds to Ang2 and binds to Tie2 receptor with Ang2 is a blood vessel leak and Inhibition of choroidal neovascularization, improvement of choroidal perfusion, improvement of vascular leakage and inhibition of macrophage infiltration in diabetic retinopathy animal model, by confirming the effect of improving Schlemm's tube homeostasis and IOP in glaucoma model, To complete.

Prior art literature

Republic of Korea Patent Publication No. 10-2015-0136031

Republic of Korea Patent Publication No. 10-2015-0014077

Republic of Korea Patent Publication No. 10-2015-0032075

Republic of Korea Patent Publication No. 10-2015-0028087

Summary of the Invention

The present invention relates to a novel use of an anti-ang2 antibody, and an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of eye diseases comprising an anti-ang2 antibody or antigen-binding fragment thereof.

The present invention also provides a method for treating ocular disease comprising administering to a patient a pharmaceutical composition comprising an anti-ang2 antibody or antigen-binding fragment thereof, and an ocular disease of the pharmaceutical composition comprising the anti-ang2 antibody or antigen-binding fragment thereof. It is aimed at providing a therapeutic or prophylactic use.

The present invention provides a pharmaceutical composition for preventing or treating ocular disease, comprising an anti-Ang2 antibody or an antigen-binding fragment thereof, in order to achieve the above object.

The present invention also provides a pharmaceutical composition, wherein the ocular disease is selected from the group consisting of macular degeneration, diabetic retinopathy and glaucoma.

The present invention also provides a pharmaceutical composition wherein the glaucoma is primary open angle glaucoma.

The present invention also said anti Ang2 antibody,

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (CDR-H3) A heavy chain complementarity determining region (CDR) comprising at least one selected from the group consisting of C), or a heavy chain variable region comprising the at least one heavy chain complementarity determining region; And

A polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3) Provided is a pharmaceutical composition comprising a light chain complementarity determining region comprising one or more selected from the group consisting of or a light chain variable region comprising the one or more light chain complementarity determining regions.

The present invention also said anti Ang2 antibody,

It provides a pharmaceutical composition comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

The present invention also provides a method for preventing or treating ocular disease, comprising administering to a patient a pharmaceutical composition comprising an anti Ang2 antibody or antigen-binding fragment thereof.

The present invention also provides a method of preventing or treating primary open angle glaucoma comprising administering to a patient a pharmaceutical composition comprising an anti Ang2 antibody or antigen binding fragment thereof.

The invention also provides the use of the treatment or prevention of ocular disease in a pharmaceutical composition comprising an anti Ang2 antibody or antigen binding fragment thereof.

The invention also provides for the use of the primary open angle glaucoma treatment or prevention of a pharmaceutical composition comprising an anti Ang2 antibody or antigen binding fragment thereof.

Figure 1 shows the inhibitory effect of vascular leakage and choroidal neovascularization in the prevention and treatment of anti-ang2 antibody administration in wet macular degeneration model.

Figure 2 shows the improvement of choroidal perfusion of anti-ang2 antibody administration in wet macular degeneration model.

Figure 3 shows the establishment and verification of the diabetic retinopathy model. Specifically, we established a diabetic retinopathy model by infusion of vascular endothelial growth factor (VEGF-A) and perivascular cell loss (DTAiΔPC) (A). Significantly increased blood leakage was observed in manufactured diabetic retinopathy model mice. Observed (BC).

Figure 4 shows the effects of anti-ang2 antibody administration in the improvement of blood vessel leakage and macrophage infiltration in diabetic retinopathy model.

5 shows the process of establishing and verifying a glaucoma model. Specifically, a glaucoma model that inhibits Ang-Tie2 signal was established (A), and it was confirmed that intraocular pressure increased significantly in the prepared glaucoma model mouse, which suppressed the formation of Schlemm's canal and maintained homeostasis.

Figure 6 shows the effect of anti-Ang2 antibody administration of Schlemm's tube homeostasis and lowering IOP in glaucoma model.

Detailed description of the invention and preferred Embodiment

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

In one embodiment of the present invention, the anti-ang2 antibody or antigen-binding fragment thereof that specifically binds to Ang2 and binds to the Tie2 receptor together with Ang2 reduces the vascular leakage, choroidal neovascularization, and choroidal perfusion in macular degeneration. In the animal model of diabetic retinopathy, blood vessel leakage was improved and macrophage infiltration was inhibited.

Therefore, in one aspect, the present invention relates to a composition for preventing or treating ocular disease, including an anti Ang2 antibody or an antigen-binding fragment thereof.

In another aspect, the present invention relates to a method for preventing or treating ocular disease, comprising administering to a patient a pharmaceutical composition comprising an anti Ang2 antibody or antigen-binding fragment thereof.

In another aspect, the present invention relates to the use of the prophylaxis or treatment of ocular diseases of the pharmaceutical composition comprising an anti Ang2 antibody or antigen-binding fragment thereof.

Anti-ang2 antibody of the present invention is an antibody targeting the angiogenesis inducer Ang2, by binding specifically to Ang2 inhibits the function of Ang2 to inhibit neovascularization and reduce blood vessel density in cancer tissues, In combination with Ang2, Tie2 binds to induce activation of Tie2, thereby structurally and / or functionally normalizing blood vessels. The anti-Ang2 antibody binds to the Tie2 receptor along with Ang2 and activates the Tie2 receptor with downregulation of Ang2 in diseases related to abnormal activation and dysfunction of blood vessels such as cancer, sepsis, and eye diseases. It has the effect of treating a disease by inducing functional normalization.

Specifically, the anti Ang2 antibody of the present invention may specifically recognize Ang2 and bind to both Ang2 and Tie2 receptors. In addition, the anti Ang2 antibody may be to induce the activation of the Tie2 receptor. Such activation of the Tie2 receptor is one selected from the group consisting of increased phosphorylation of the Tie2 receptor and / or proteins associated with its underlying signaling pathways, such as Akt (NM_005163), eNOS (NM_000603), 42/44 (NM_002745), and the like. It can be induced by phosphorylation of more than one species. In addition, the anti Ang2 antibody may be to induce internalization of the Tie2 receptor into the cell. That is, while the anti-Ang2 antibody specifically binds to Ang2, unlike the conventional anti-Ang2 antibody, it does not inhibit the binding of Ang2 to the Tie2 receptor, thereby binding to the Tie2 receptor together with Ang2 to form a complex, Tie2 It may be to induce activation of the receptor.

In the present invention, the Ang2 protein, which acts as an antigen of the anti-Ang2 antibody, is closely related to neovascularization and is a soluble ligand present in blood, and is widely used for angiogenesis, metastasis, cancer cell invasion, and the like. It works. The Ang2 may be derived from mammals such as humans, primates such as monkeys, rodents such as mice, rats, and the like, for example, human Ang2 (NCBI Accession No. O15123, etc.), monkey Ang2 (NCBI Accession No. Q8MIK6, etc.). ), Mouse Ang2 (NCBI Accession No. O35608, etc.), rat Ang2 (NCBI Accession No. O35462, etc.) and the like, but is not limited thereto.

The Tie2 receptor (TEK tyrosine kinase) of the present invention is an Ang1 receptor, expressed in various mammalian endothelial cells such as mice (NM_013690; NP_038718), rats, humans (NM_000459; NP_000450), and various downstream signals. It is involved in downstream signaling.

Anti Ang2 antibody of the present invention binds Ang2 specifically but does not inhibit the binding of Ang2 and Tie2 receptor, and the antibody complexed with Ang2 and Tie2 receptor (antibody / Ang2 / Tie2), dimerization of antibody ), Thereby effectively clustering the Tie2 receptor complexed thereto, thereby inducing the activation of the Tie2 receptor and its downstream signaling. Due to this mechanism of action, the antibody of the present invention binds to Ang2, induces intracellular migration and degradation, thereby inhibiting Ang2 function and lowers circulating Ang2 levels, and binds to Tie2 receptors with Ang2 to bind Tie2 like Ang1. By having a dual function that activates the receptors to induce stabilization of vascular endothelial cells, it can be useful in the treatment of diseases caused by Ang2 overexpression as well as decreased stabilization of vascular endothelial cells, ie increased vascular permeability. have.

The anti-Ang2 antibody may be exposed to all or a portion of a human Ang2 (hAng2; SEQ ID NO: 9; Accession # O15123) from 417 to 434 amino acids in loop 1 SEQ ID NO: 9 (eg, exposed to the outside in a loop). One or more amino acid residues selected from the group consisting of the selected amino acid residue sites) or 2-20, 2-15, non-contiguous, comprising at least one amino acid residue exposed to the outside of loop 1 in SEQ ID NO: 9, An amino acid sequence region comprising 2 to 10 or 2 to 5 amino acids may be recognized as an epitope or specifically bound to this region. Table 1 shows the Ang2 sequence of SEQ ID NO.

The anti Ang2 antibody is a contiguous or noncontiguous 2 comprising amino acid residues in combination with Q418, P419, Q418 and P419, located in loop 1 of SEQ ID NO: 9, or in combination with Q418, P419, Q418 and P419 in SEQ ID NO: 9. An amino acid sequence region comprising from 20 to 2, 2 to 15, 2 to 10, or 2 to 5 amino acids may be recognized as an epitope or specifically bound to this site. Preferably, the anti-Ang2 antibody may recognize amino acid residues of Q418 and P419 of SEQ ID NO: 9 as epitopes or specifically bind to this portion.

Q418, P419, or an amino acid site including the specific binding site of the anti-Ang2 antibody is an exposed amino acid residue located at loop 1 of the three-dimensional structure of Ang2, and is directly involved in binding between Ang2 and Tie2 receptors. It is considered to be a site controlling this. In the site where the anti-Ang2 antibody specifically binds, Q418, P419, or an amino acid site including the same, “non-contiguous amino acids” are adjacent to each other on the secondary or tertiary structure of the protein, but are not contiguous on the amino acid sequence. It may mean an amino acid that does not. Thus, “contiguous or non-contiguous amino acid residues” herein may mean contiguous amino acid residues on the primary, secondary or tertiary structure of a protein.

The anti Ang2 antibodies include not only complete antibody forms, but also antigen binding fragments of the antibody molecules.

A complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), Gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types. The basic four-chain antibody unit is a heterodimeric glycoprotein consisting of two identical light chains (L) and two identical heavy chains (H). The light chain has a variable region (VL) at the N-terminus, followed by a constant region at its other end. The heavy chain has a variable region (VH) at the N-terminus, followed by three constant regions (CH) for the α and γ chains and four CH regions for the μ and ε isotypes, respectively. Variables indicate that certain portions of the variable region differ greatly in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for that particular antigen. Variability is concentrated in three segments called hypervariable regions (HVRs), or CDRs, in both the light and heavy chain variable regions. The more highly conserved portions of the variable regions are called framework regions (FRs). The heavy and light chain variable regions have the structures FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus.

As used herein, the term “heavy chain” refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 And fragments thereof.

In addition, the term "light chain" as used herein refers to a full-length light chain and fragment thereof 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 the antigen. All means.

The antigen binding fragment or antibody fragment of an antibody means the fragment which has antigen binding function, and includes Fab, F (ab ') 2, Fv, etc.

"Fv" fragments are antibody fragments containing complete antibody recognition and binding sites. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly and covalently associated, for example, with scFv.

"Fab" fragments contain the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain. F (ab ') 2 antibody fragments generally comprise a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH domain and the VL domain that allows the scFv to form the desired structure for antigen binding.

In the present invention, the anti Ang2 antibody,

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (CDR-H3) A heavy chain complementarity determining region (CDR) comprising at least one selected from the group consisting of C), or a heavy chain variable region comprising the heavy chain complementarity determining region;

A polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3) A light chain complementarity determining region comprising at least one selected from the group consisting of C), or a light chain variable region comprising the light chain complementarity determining region;

A combination of the at least one heavy chain complementarity determining site and at least one light chain complementarity determining site; Or a combination of the heavy chain variable region and the light chain variable region.

Preferably, the anti Ang2 antibody,

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (CDR-H3) A heavy chain complementarity determining site comprising: or a heavy chain variable region comprising the heavy chain complementarity determining site; And

A polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3) ) May include light chain complementarity determining regions, or light chain variable regions comprising the light chain complementarity determining regions.

Specifically, the heavy chain complementarity determining site and the light chain complementarity determining site of the anti Ang2 antibody may be those having the amino acid sequences shown in Table 2 below.

In one embodiment, the heavy chain variable region of the antibody may comprise the amino acid sequence of SEQ ID NO: 7 in Table 3 below, the light chain of the antibody may include the amino acid sequence of SEQ ID NO: 8 in Table 3 below .

Therefore, the anti-Ang2 antibody may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or a combination of the heavy chain variable region and the light chain variable region. . For example, the anti Ang2 antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

Antibodies in the present invention include all animal-derived antibodies, chimeric antibodies, humanized antibodies and human antibodies. Animal-derived antibodies that are produced by immunizing a desired antigen with an immunized animal are generally capable of immunorejection upon administration to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such rejection. Chimeric antibodies are obtained by replacing the constant region of an animal-derived antibody causing an anti-isotype reaction with the constant region of a human antibody using genetic engineering methods. Chimeric antibodies have been significantly improved in anti-isotype responses compared to animal derived antibodies, but still contain adverse effects on potential anti-idiotypic responses due to the presence of animal derived amino acids in the variable region. Doing. Humanized antibodies have been developed to ameliorate these side effects. It is produced by implanting a region of complementarity determining regions (CDRs) that play an important role in binding of antigens in the variable regions of chimeric antibodies to the human antibody framework.

The most important aspect of CDR grafting technology for producing humanized antibody is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. structure analysis and molecular modeling techniques. However, even when the CDR region of an animal-derived antibody is implanted into an optimized human antibody skeleton, there are many amino acids that are located in the skeleton of an animal-derived antibody and affect antigen binding. As such, the application of additional antibody engineering techniques to restore antigen binding capacity is essential.

According to one embodiment, the antibody may be a mouse derived antibody, mouse-human chimeric antibody, humanized antibody, or human antibody.

Since the anti-ang2 antibody or antigen-binding fragment thereof of the present invention has a function of inhibiting abnormal angiogenesis by inhibiting the function of Ang2, eye diseases accompanying vascular abnormalities such as macular degeneration, diabetic retinopathy, and glaucoma It can be used for prevention and / or treatment.

"Prevention" means any action that inhibits or delays the progression of an ocular disease by administration of a composition according to the present invention, and "treatment" means inhibiting, alleviating or eliminating the development of ocular disease.

As used herein, the term “macular degeneration” is a condition in which new blood vessels grow abnormally and the macula is damaged to affect vision. Macular degeneration occurs mainly in people over 50 years of age and is divided into non-exudative (dry type) or exudative (wet type). In particular, wet macular degeneration can lead to blindness, the cause of which is unknown, but the risk factor is age, and environmental factors include smoking, hypertension, obesity, genetic predisposition, and excessive UV exposure. And low blood antioxidant levels.

As used herein, the term "diabetic retinopathy" is a peripheral circulatory disorder caused by diabetes, which is a complication of the eye that causes a decrease in vision due to a disorder in the microcirculation of the retina, which initially causes no symptoms or only mild vision. It can cause problems, but ultimately blindness. Diabetic retinopathy can develop in anyone with type 1 diabetes or type 2 diabetes.

As used herein, the term "glaucoma" is a chronic optic nerve lesion, a severe refractory disease that causes progressive degeneration of the optic nerve, progressive loss of retinal ganglionic cells, and vision loss due to visual field defects. Risk factors for glaucoma include age, race, sex, and hypertension, but increased intraocular pressure is known as the most important cause of glaucoma, especially primary open angle glaucoma. Accordingly, the present invention can be used to prevent and / or treat primary open angle glaucoma.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, which is commonly used in the formulation of drugs, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, Calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, minerals It may be one or more selected from the group consisting of oils, but is not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, which are conventionally used for preparing pharmaceutical compositions.

The pharmaceutical composition, or pharmaceutically effective amount of the antibody, may be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration. When orally administered, the protein or peptide is digested so that the oral composition can be formulated to coat the active agent or protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.

The content of anti Ang2 antibody in the pharmaceutical composition varies depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, interval of administration, route of administration, rate of excretion and reaction sensitivity of the patient. Can be prescribed. For example, the daily dosage of the anti Ang2 antibody may range from 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg, but is not limited thereto. The daily dosage may be formulated into one formulation in unit dosage form, may be formulated in appropriate quantities or may be prepared within a multi-dose container. The "pharmaceutical effective amount" may refer to the amount or dosage of an active ingredient that may exhibit a desired pharmacological effect, and may include a formulation method, a mode of administration, a patient's age, weight, sex, morbidity, food, and time of administration. It can be determined variously by factors such as the administration interval, the route of administration, the rate of excretion, and the reaction sensitivity.

The pharmaceutical compositions may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media or may be formulated in the form of extracts, powders, powders, granules, tablets or capsules, etc. It may further include a topic.

In particular, the pharmaceutical composition comprising the anti Ang2 antibody or antigen-binding fragment thereof comprises an antibody and thus may be formulated into an immune liposome. Liposomes comprising the antibody can be prepared according to methods well known in the art. The immune liposomes can be prepared by reverse phase evaporation as a lipid composition comprising phosphatidylcholine, cholesterol and polyethyleneglycol-derivatized phosphatidylethanolamine. For example, Fab 'fragments of antibodies can be conjugated to liposomes via disulfide-replacement reactions.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Effect of Anti Ang2 Antibody on Macular Degeneration Model

1. Wet macular degeneration model manufacturing

To prepare a wet macular degeneration model, the pupils of an 8-week-old mouse were dilated by the use of a Shandong agent, and then the laser was irradiated to four locations for each mouse eye while observing the retina using a slit lamp microscope. Induced. At this time, only the lesions located at the location where the Bruch's membrane was found to be ruptured were included in the analysis, and bleeding was excluded from the analysis.

2. Inhibitory effect of vascular leakage and choroidal neovascularization

To determine whether anti-ang2 antibodies comprising the heavy chain CDRs of SEQ ID NOs: 1 to 3 and the light chain CDRs of SEQ ID NOs: 4 to 6 listed in Table 2 are effective in inhibiting vascular leakage and choroidal neovascularization, The evaluation was divided into categories. In order to compare the therapeutic effect with the anti-vascular endothelial growth factor antibody, the most widely used drug, experiments were conducted in three groups (placebo-administered group, anti-ang2 antibody-administered group and anti-vascular endothelial growth factor-administered group). Proceeded.

As a result, in both the prevention and treatment sectors, the anti-Ang2 antibody-treated group had vascular leakage (prevention: 63.1%; treatment: 66.4%) and the volume of choroidal neovascularization (prevention: 61.3%; treatment: 50.1). %) Was significantly reduced, and compared with the anti-vascular endothelial growth factor antibody administration group showed a similar degree of vascular leakage inhibition and choroidal neovascular volume reduction effect (Fig. 1).

3. Improvement of choroidal perfusion

Anti-vascular endothelial growth factor treatment, which has been widely used for the treatment of wet macular degeneration, requires only periodic repetitive injection treatment for a long period of time because it temporarily reduces the volume of CNV and suppresses vascular leakage. As a side effect of the anti-vascular endothelial growth factor treatment, the retinal and choroidal hypoxia and oxidative stress intensify the retinal neovascularization. In addition, there is a fatal limitation that can lead to permanent vision loss by inducing degeneration of retinal pigment epithelium and optic nerve cells.

To determine whether the anti-Ang2 antibody can overcome these limitations, time series of angiography using light coherence tomography showed that the choroids at the periphery of the laser-induced choroidal neovascularization in the anti-vascular endothelial growth factor antibody-administered group. The size of the avascular site increased (35.9%, 28.3%, and 47.5%) during the follow-up period, while the anti-ang2 antibody-treated group gradually decreased (27.3%, 43.1%, and 45.5%). 2).

Therefore, anti-Ang2 antibody has similar inhibitory effect of vascular leakage and choroidal neovascularization compared with conventional anti-vascular endothelial growth factor treatment, resulting in an immediate therapeutic effect as well as reducing the size of choroidal avascular region. By reducing hypoxia and oxidative stress, it has been shown to fundamentally inhibit the recurrence of choroidal neovascularization and not to induce side effects such as degeneration of retinal pigment epithelium and optic nerve cells.

Example 2: Effect of Anti Ang2 Antibody in Diabetic Retinopathy Model

1. Preparation of Diabetic Retinopathy Model

The first vascular changes in diabetic retinopathy are known to be thickening of the retinal capillary basement membrane and loss of perivascular cells. As these changes occur and over time, extensive capillary non-perfusion occurs and excessive regeneration of vascular endothelial growth factors from the ischemic retina results in retinal neovascularization causing complications such as bleeding and inflammation. The diabetic retinopathy model was newly prepared in the mouse, considering that the early changes in diabetic retinopathy caused loss of perivascular cells and that excessively produced vascular endothelial growth factor was important for the progression of diabetic retinopathy.

First, all cells expressing platelet-derived growth factor receptor (PDGF-receptor-beta), which are known to be specifically expressed in perivascular cells using genetically engineered mice, are produced by externally injected tamoxifen to produce and kill diphtheria toxin. It was. When vascular endothelial growth factor was injected into the eye of the mammary gland engineered mouse and compared with the retina of the control eyeball, it was observed that the leakage of blood vessels was significantly increased in the genetically engineered mouse (FIG. 3). This produced an animal model based on the mechanism of development of diabetic retinopathy and showing an vascular disease similar to diabetic retinopathy.

2. Improvement of vascular leakage and inhibition of macrophage invasion

Diabetic retinopathy model mice show vascular leakage and macrophage infiltration. Intraperitoneal administration of an anti-Ang2 antibody comprising the heavy chain CDRs of SEQ ID NOS: 1 to 3 and the light chain CDRs of SEQ ID NOs: 4 to 6 described in Table 2 results in diabetic retinopathy model mice. Tran blood vessel leakage was reduced, macrophage infiltration was confirmed to decrease (Fig. 4).

Example 3: Effect of Anti Ang2 Antibody on Glaucoma Model

1. Manufacture of Glaucoma Model

 Glaucoma is known to occur due to an increase in intraocular pressure due to an increase in the resistance of the waterproof outflow path through the Schlemm's canal and trabecular meshwork in the eye. Given that Ang-Tie2 signaling regulates angiogenesis and remodeling of blood vessels, Ubiquitin-Cre; Ang1 & Ang2 double floxed mouse inhibits Ang-Tie2 signaling by inhibiting Ang1 and Ang2 expression overall. Was prepared. The prepared glaucoma model mice were found to significantly increase intraocular pressure by inhibiting formation of Schlemm's canal and maintaining homeostasis (FIG. 5).

2. Schlem tube homeostasis improvement effect and intraocular pressure drop effect

In a glaucoma model mouse and a wild-type mouse, an anti-Ang2 antibody comprising the heavy chain CDRs of SEQ ID NOS: 1 to 3 and the light chain CDRs of SEQ ID NOs: 4 to 6 shown in Table 2 having a Tie2 activation action is released from the mouse together with recombinant Ang2. After injection into the body cavity, changes in intraocular pressure, regions of the Schlemm's canal and expression levels of the transcription factors Prox1 and Tie2 receptors were compared.

As a result, no significant change was observed in wild-type mice even after anti-ang2 antibody injection. On the other hand, in the glaucoma model mouse, a significant intraocular pressure-lowering effect was observed, the region of Schlemm's canal was significantly increased, and the expression of Prox1 and Tie2 was also increased (FIG. 6).

The present invention provides a therapeutic use of an anti-ang2 antibody that inhibits Ang2 and simultaneously activates a Tie2 receptor to promote downstream signal transduction, thereby making it useful for developing macular degeneration, diabetic retinopathy, and glaucoma therapeutic agents. have.

Electronic file attached.

Claims (7)

1. A pharmaceutical composition for preventing or treating ocular disease, comprising an angiopoietin-2 (Anggiopoietin-2; Ang2) antibody or an antigen-binding fragment thereof.
2. The pharmaceutical composition of claim 1, wherein the ocular disease is selected from the group consisting of macular degeneration, diabetic retinopathy, and glaucoma.
3. The pharmaceutical composition of claim 2, wherein the glaucoma is primary open angle glaucoma.
4. The method of claim 1,

   The anti Ang2 antibody,

   A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (CDR-H3) A heavy chain complementarity determining region (CDR) comprising at least one selected from the group consisting of C), or a heavy chain variable region comprising the at least one heavy chain complementarity determining region; And

   A polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3) Pharmaceutical composition, characterized in that it comprises a light chain complementarity determining region comprising one or more selected from the group consisting of; or a light chain variable region comprising the one or more light chain complementarity determining regions.
5. The method of claim 1,

   The anti Ang2 antibody,

   A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
6. A method of treating ocular disease comprising administering to a patient a pharmaceutical composition comprising an angiopoietin-2 (Ang2) antibody.
7. Use for the treatment or prophylaxis of ocular disease of a pharmaceutical composition comprising an angiopoietin-2 (Ang2) antibody.

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