Methods and compositions for the treatment of angiogenesis disorders using anti-VEGF agents

Abstract

To provide methods and compositions for treating angiogenic disorders by using anti-VEGF agents.SOLUTION: Provided herein is a method and a composition for treating an angiogenic disorder by using an anti-VEGF agent. The anti-VEGF agent comprises a VEGF binding domain and has an ability to bind to the vitreous. Provided are exemplary embodiments of Fc-IgG fusion proteins comprising VEGF binding domains with strong heparin-binding characteristics, strong inhibition of mitogenic activity, and improved pharmacokinetics, namely longer half-lives of the anti-VEGF agents and consequently less frequent dosing.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 622,382, filed on January 26, 2018, which is hereby incorporated herein by reference in its entirety. Sequence Listing

[0002] This application includes a sequence listing electronically submitted in ASCII format, which is hereby incorporated herein by reference in its entirety. The name of the above ASCII copy created on January 25, 2019 is 24978 - 0470_SL.txt, and the size is 50,960 bytes.

Background Art

[0003] Blood vessels carry nutrients to tissues and organs and remove catabolites. Therefore, the development of new blood vessel supply or angiogenesis plays an important role in maintaining homeostasis. 1 However, uncontrolled growth of blood vessels can promote or facilitate a number of disease processes, including tumors and intraocular vascular disorders. 1 Although several angiogenic factors have been initially identified and characterized (e.g., EGF, TGF - α, TGF - β, aFGF, bFGF, angiogenin), 2 studies conducted over the past 30 years have established the important role of VEGF - A (hereinafter, VEGF) in normal and pathological angiogenesis. 3 4 VEGF is a member of a gene family that also includes PlGF, VEGF - B, VEGF - C, and VEGF - D. Three related receptor tyrosine kinases (RTKs) have been reported to bind to the VEGF ligands: VEGFR - 1, VEGFR - 2, and VEGFR - 3. 5 PlGF and VEGF - B selectively interact with VEGFR - 1, and VEGF binds to both VEGFR - 1 and VEGFR - 2. VEGFR - 3, the third member of this RTK family 6It binds to VEGF-C and VEGF-D, which are involved in lymphangiogenesis. Each member of this RTK class has seven immunoglobulin (Ig)-like domains in its extracellular portion 7 . VEGFR-2 is considered the main signaling receptor for VEGF 5 . However, VEGFR-1 binds to VEGF with a substantially higher binding affinity than VEGFR-2 7 .

[0004] VEGF inhibitors have become the standard treatment for multiple tumors and have revolutionized the treatment of intraocular neovascular disorders such as neovascular age-related macular degeneration (AMD), proliferative diabetic retinopathy, and retinal vein occlusion, which are the main causes of severe vision loss and legal blindness 8 4 . Currently, three anti-VEGF drugs, bevacizumab, ranibizumab, and aflibercept, are widely used in the United States for ophthalmic indications 4 . Bevacizumab is a full-length IgG antibody that targets VEGF 9 . Bevacizumab has not been developed for ophthalmic indications but is widely used outside the indication due to its low cost. Ranibizumab is an affinity-matured anti-VEGF Fab 10 . Aflibercept is an IgG-Fc fusion protein 11 12 and has elements from VEGFR-1 and VEGFR-2 that bind to VEGF, PIGF, and VEGF-B 13 . Conbercept is a soluble VEGF receptor structurally related to aflibercept and is widely used in China for the treatment of intraocular neovascularization 14 . Millions of patients worldwide are being treated with these drugs. Importantly, after 5 years of treatment with ranibizumab or bevacizumab, approximately half of patients with neovascular AMD show good vision, i.e., vision of 20 / 40 or better, an outcome that would not have been achievable before the availability of anti-VEGF agents 15 .

[0005] However, in actual clinical settings, many patients receive fewer anti-VEGF injections than in clinical trials, and it has been hypothesized that this may correlate with poorer visual outcomes. 16 In fact, the need for relatively frequent intravitreal injections has hindered patient compliance and ultimately hindered the benefits of the therapy, especially in some countries. 16 Therefore, there is a need to develop drugs that have a longer duration of action when injected into the eye, thereby reducing the frequency of injections, and many approaches have been attempted to achieve this. 17 , 1 8 Aflibercept (EYLEA) was approved based on clinical trials showing that a 2 mg dose administered every 8 weeks could be comparable in efficacy to monthly ranibizumab (0.5 mg). However, despite predictions that switching to aflibercept would reduce the number of intravitreal injections, recent studies suggest this is not the case. 19 Therefore, there remains an unmet medical need for intravitreal anti-VEGF agents with improved half-lives.

[0006] In 1996, Davis-Smyth et al. 20 (See also U.S. Patent No. 5,952,199) reported that domain (D)2 of VEGFR-1 is a critical binding element for VEGF and PIGF. Deletion of D2 completely disrupted the binding. By replacing D2 of VEGFR-3 with VEGFR-1 D2, VEGFR-3 was conferred with the ligand specificity of VEGFR-1. 20 Subsequent research demonstrated the interaction between D2 and VEGF (or PlGF) using X-ray crystallography. 21-23 .

[0007] The initial research led to the design of a construct with complete VEGF-binding properties, including the first three Ig-like D (Flt-1-3-IgG) of VEGFR-1 fused to Fc-IgG. 20 Flt-1-3-IgG demonstrated potent ability to neutralize VEGF in vitro and in vivo.24-27 However, the systemic half-life of this molecule was hindered by the presence of D3, which has high heparin affinity due to the presence of a cluster of basic residues and thus facilitates binding to HSPG in various tissues. (Holash et al., 2002) 13 (U.S. Patent No. 7,070,959) described an IgG fusion construct consisting of VEGFR-1 D2 (binding element) and VEGFR2 D3, which has a very weak heparin affinity compared to VEGFR-1 D3. This molecule, now known as aflibercept (marketed as EYLEA), has been reported to have a longer half-life compared to Flt-(1-3-IgG) after systemic administration. 13 For example, it is clearly advantageous for the treatment of ophthalmic indications. [Overview of the project]

[0008] The present invention provides compositions and methods for inhibiting angiogenesis and treating VEGF-related conditions, including but not limited to age-related macular degeneration, proliferative diabetic retinopathy, retinal vein occlusion, choroidal neovascularization secondary to myopia, retinopathy of prematurity, diabetic macular edema, and polypoid choroidal vasculopathy, which involve administering an anti-VEGF agent that inhibits VEGF activity and simultaneously possesses potent heparin-binding properties, thereby providing superior pharmacokinetics, i.e., an anti-VEGF agent with a longer half-life after intravitreal administration.

[0009] In embodiments, the present invention provides compositions and methods for treating conditions in which topical direct administration of anti-VEGF agents is beneficial, such as treating and preventing endothelial cell proliferation or angiogenesis, and for treating solid tumors, such as intracranial administration in glioblastoma, for example.

[0010] In embodiments, the present invention provides an anti-VEGF agent, which is an Fc-IgG construct fusing a domain having VEGF-binding properties and a domain that binds to heparin proteoglycans. In embodiments, the present invention provides an anti-VEGF agent, which is an Fc-IgG construct having the ability to bind to heparin and containing one or more domains having VEGF-binding properties. In embodiments, the present invention provides an anti-VEGF agent, which is a fusion protein having improved efficacy in binding to VEGF and heparin. In embodiments, the present invention provides an anti-VEGF agent, which is a fusion protein with very low endotoxin levels.

[0011] In embodiments, the present invention provides an anti-VEGF agent, which is an IgG chimeric protein containing elements of the VEGF receptor. In embodiments, the present invention provides an IgG chimeric protein, which comprises one or more fragments of seven immunoglobulin (Ig)-like domains in the extracellular portion of the VEGF tyrosine kinase receptor. In embodiments, the present invention provides an IgG chimeric protein, which comprises one or more extracellular domain fragments of VEGFR-1 fused with Fc-IgG. In embodiments, the present invention provides an IgG chimeric protein comprising at least one VEGF-binding domain VEGFR-1 domain 2 and at least one additional VEGFR-1 domain 1 or 3, but not domain 4. In embodiments, the present invention provides an IgG chimeric protein, which comprises one or more extracellular domain fragments of VEGFR-2 fused with Fc-IgG. In embodiments, the present invention provides an IgG chimeric protein, which comprises one or more extracellular domain fragments of VEGFR-1 and VEGFR-2 fused with Fc-IgG.

[0012] In embodiments, the present invention provides an anti-VEGF agent comprising a VEGF-binding moiety operably linked to Fc-IgG, wherein the VEGF-binding moiety comprises at least one VEGF-binding domain which is the IgG-like domain 2 of VEGFR-1, and the anti-VEGF agent has superior VEGF-stimulated mitotic inhibitory ability compared to aflibercept. In embodiments, the present invention provides that the anti-VEGF agent has superior vitreous binding ability compared to aflibercept. In embodiments, the present invention provides that the anti-VEGF agent has superior vitreous-bound VEGF-stimulated endothelial cell proliferation inhibitory ability compared to aflibercept. In embodiments, the present invention provides that the agent has an increased half-life in vivo compared to aflibercept.

[0013] In embodiments, the present invention relates to a VEGF binding moiety that is essentially the IgG-like domains 1, 2, and 3 of VEGFR-1. 1-2-3 The invention provides that the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 1. In the embodiment, the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 1.

[0014] In embodiments, the present invention relates to a VEGF binding moiety that is essentially the IgG-like domains 2 and 3 of VEGFR-1. 2-3 The invention provides that the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 3. In the embodiment, the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 3.

[0015] In embodiments, the present invention relates to a VEGF binding moiety that is essentially the IgG-like domains 1, 2, 3, and 3(V) of VEGFR-1. 1-2-3-3 The invention provides that the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 5. In the embodiment, the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 5.

[0016] In embodiments, the present invention relates to a VEGF binding moiety that is essentially the IgG-like domains 2, 3, and 3(V) of VEGFR-1. 2-3-3 The invention provides that the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 7. In the embodiment, the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 7.

[0017] In embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of an anti-VEGF agent as defined in the claims and a pharmaceutically acceptable excipient. In embodiments, the present invention provides a method for treating a VEGF-related disorder in a subject requiring treatment, comprising administering a therapeutically effective dose of a defined anti-VEGF agent to the subject. The anti-VEGF agent can be injected directly into affected tissue or organs, such as the eyes.

[0018] In embodiments, the present invention provides a method for treating an eye disease, in which the anti-VEGF agent is administered topically to the eye in a dosage corresponding to a molar ratio of 2:1 with respect to VEGF. In embodiments, the present invention provides a method for treating an eye disease, in which the anti-VEGF agent is administered by intravitreal injection. In embodiments, the present invention provides a method for treating an eye disease, in which the anti-VEGF agent is administered every 4 to 6 weeks, and in other embodiments, the treatment is continued for at least one year.

[0019] According to one embodiment, the present invention provides a method for treating an eye disease comprising topically administering a therapeutically effective amount of an anti-VEGF agent to the eye, the treatment being effective for occult, minimally classic, and predominantly classic exudative macular degeneration, and the agent being a fusion protein.

[0020] In embodiments, the present invention can be used to treat a wide variety of VEGF-related diseases, including neovascular age-related macular degeneration, choroidal neovascularization secondary to myopia, proliferative diabetic retinopathy, diabetic macular edema, retinal vascular occlusion such as retinal vein occlusion, eye tumors, von Hippel-Lindau syndrome, retinopathy of prematurity, polypoid choroidal vasculopathy, colorectal cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, schwannomas, gliomas, ependimomas, and neoplastic or non-neoplastic disorders that benefit from anti-VEGF therapy.

[0021] In another aspect, the present invention provides a pharmaceutical formulation containing an anti-VEGF agent in a pharmaceutically acceptable carrier formulation for topical administration to the eyes or other areas.

[0022] In embodiments, the present invention discloses a novel construct that strongly neutralizes the activity of VEGF while simultaneously possessing strong heparin-binding properties. [Brief explanation of the drawing]

[0023] A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description and accompanying drawings illustrating exemplary embodiments in which the principles of this disclosure are utilized. [Figure 1] Schematic diagrams of exemplary constructed fusion proteins using various Ig-like extracellular domains of VEGFR-1(V) fused to Fc-IgG(Fc) are shown. The following constructs are shown: V1-2-3-Fc; V2-3-Fc; V1-2-3-3-Fc; V2-3-3-Fc; V1-2-3-4-Fc; V2-3-4-Fc; V1-2-4-Fc, and V2-4-Fc. [Figure 2] This document outlines strategies for plasmid construction and expression. [Figure 3] The amino acid and nucleic acid sequences of construct V1-2-3 are shown. Sequence IDs are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. [Figure 4] The amino acid and nucleic acid sequences of constructs V2-3 are shown. These are Sequence ID No. 3 and Sequence ID No. 4, respectively. [Figure 5] The amino acid sequence and nucleic acid sequence of construct V1-2-3-3 are shown. Sequence IDs are SEQ ID NOs. 5 and 6, respectively. [Figure 6] The amino acid sequence and nucleic acid sequence of construct V2-3-3 are shown. Sequence IDs 7 and 8, respectively. [Figure 7] The amino acid and nucleic acid sequences of construct V1-2-3-3-4 are shown. Sequence IDs 9 and 10, respectively. [Figure 8] The amino acid and nucleic acid sequences of construct V2-3-4 are shown. Sequence IDs 11 and 12, respectively. [Figure 9] The amino acid and nucleic acid sequences of constructs V2-4 are shown. Sequence IDs 13 and 14, respectively. [Figure 10] This shows the expression of the VEGFR-1 construct in 293 cells. [Figure 11] Silver-stained PAGE gels of 200 ng of each VEGFR-1 Fc fusion protein under reducing and non-reducing conditions are shown, compared to EYLEA. [Figure 12] This study demonstrates the inhibitory effect of VEGF receptor chimeric proteins on VEGF-stimulated endothelial cell proliferation. [Figure 13] This shows the competition among biotinylated VEGF (100 ng / ml) for binding to the VEGFR1 soluble receptor. [Figure 14] This shows the VEGFR-1 soluble receptor that binds to biotinylated VEGF and bovine vitreous humor. [Figure 15] This demonstrates that bovine vitreous binding V1-2-3-3 is biologically active. [Figure 16] This study demonstrates the effects of control IgG, EYLEA, or VEGFR-1 Fc fusion protein on choroidal neovascularization (CNV) regions. Each protein was injected intravitreously into mice at a dose of 2.5 mg one day prior to laser treatment. EYLEA was also tested at 25 mg. Asterisks indicate statistical significance (Student's t-test) compared to the appropriate IgG control group (**p<0.01, *p<0.05). [Figure 17A] This shows the effect of EYLEA, V1, 2, 3, 3, or control IgG on the CNV area after a single intravitreal administration (2.5 mg) 1, 7, or 14 days prior to laser treatment. An asterisk indicates a statistically significant difference (p<0.05, Student's t-test) compared to the IgG control group. [Figure 17B] Figure 17B shows representative CD31 immunofluorescence images of the group shown in Figure 17A. [Modes for carrying out the invention]

[0024] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0025] The aspects and embodiments of the present invention described herein are understood to include aspects and embodiments consisting of and / or essentially consisting of. Other objects, advantages, and features of the present invention will become apparent from the following specification in conjunction with the accompanying drawings.

[0026] When describing elements of the present invention or its preferred embodiments, the articles "a," "an," "the," and "said" are intended to indicate that one or more of the elements exist. The terms "equip," "include," and "have" are intended to be comprehensive and mean that additional elements other than those listed may exist.

[0027] As used herein, “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” “containing,” “characterizes,” or any other variation thereof, are intended to encompass a non-exclusive inclusion of the enumerated components, subject to any limitations expressly indicated elsewhere. For example, “including” a list of elements (e.g., components, features, or steps), a fusion protein, pharmaceutical composition, and / or method may include other elements (or components or steps) that are not expressly listed or specific to the fusion protein, pharmaceutical composition, and / or method, but are not necessarily limited to those elements (or components or steps).

[0028] As used herein, the transitional phrases “consists of” and “consisting of” exclude any elements, steps, or components not specified. For example, “consists of” or “consisting of” as used in a claim limits the claim to the components, materials, or steps specifically enumerated in the claim, with the exception of impurities that are normally associated (i.e., impurities within a given component). If the phrase “consists of” or “consisting of” appears in a clause of the claim text rather than immediately following the preamble, the phrase “consists of” or “consisting of” limits only the elements (or components or steps) described in that clause, and does not exclude other elements (or components) as a whole from the claim.

[0029] As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define a fusion protein, pharmaceutical composition, and / or method that includes materials, steps, features, components, or elements in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not substantially affect the basic and novel characteristics of the claimed invention. The term “consisting essentially of” occupies a compromise between “equipped with” and “consisting of.”

[0030] As used herein, the term “pharmaceutical composition” refers to a composition comprising one or more therapeutic agents or drugs as described below, and one or more pharmaceutically acceptable excipients, carriers, or vehicles.

[0031] As used herein, the term “pharmaceutically acceptable excipient, carrier, or vehicle” includes any acceptable material and / or any one or more additives known in the art. As used herein, the terms “excipient,” “carrier,” or “vehicle” refer to a material suitable for drug administration by various conventional routes of administration known in the art. Excipients, carriers, and vehicles as useful herein include any such material known in the art that is non-toxic and does not interact in a harmful manner with other components of the composition, and generally refer to excipients, diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or vehicles to which an activator or drug is administered together. Such carriers may be sterile liquids such as water and oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, including those of petroleum, animal, plant, or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity such as sodium chloride or dextrose can also be carriers. Methods for preparing compositions in combination with carriers are known to those skilled in the art. In some embodiments, the term “pharmaceutically acceptable carrier” is intended to include all kinds of solvents, dispersions, coatings, isotonic agents, and absorption retarders that are suitable for pharmacopoeias. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0032] As used herein, the term “therapeutic effective dose” means an amount of a therapeutic agent or drug that, when administered to a particular subject, has a desired therapeutic effect, taking into account the nature and severity of the condition in question, for example, an amount that cures, prevents, inhibits, or at least partially halts or partially prevents the target condition. In some embodiments, the term “therapeutic effective dose” or “effective dose” means an amount of a therapeutic agent or drug that, when administered alone or in combination with additional therapeutic agents or drugs to cells, tissues, organs, or subjects, is effective in preventing or relieving eye diseases and cancers, including but not limited to age-related macular degeneration, proliferative diabetic retinopathy, retinal vein occlusion, choroidal neovascularization secondary to myopia; retinopathy of prematurity, diabetic macular edema, polypoid choroidal vasculopathy, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, and prostate cancer. A therapeutic effective dose further means an amount of a therapeutic agent or drug sufficient to result in remission of a symptom, for example, treatment, cure, prevention, or remission of the associated medical condition, or an increase in the rate of treatment, cure, prevention, or remission of such a condition. When applied to individual active ingredients administered alone, the therapeutic dose refers to that ingredient alone. When applied to combinations, the therapeutic dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered together, sequentially, or simultaneously.

[0033] As used herein, the terms “treating,” “treatment,” or “alleviating” refer to therapeutic actions whose purpose is to slow down (reduce) a target pathological condition or disorder, if they do not cure the condition or prevent its recurrence. A subject is successfully “treated” if, after receiving a therapeutic dose of a therapeutic drug or medication, the subject exhibits an observable and / or measurable reduction or absence of one or more signs and symptoms of a particular condition. The reduction of signs or symptoms of a condition may also be felt by the subject. A subject is also considered treated if it experiences a stable condition. In some embodiments, treatment with a therapeutic drug or medication is effective in keeping the subject asymptomatic for three months, preferably six months, more preferably one year, and even more preferably two years or more after treatment. These parameters for evaluating the success of treatment and improvement of condition are readily measurable by routine procedures well known to those skilled in the art.

[0034] As used herein, “preventive” treatment means delaying the onset of a condition or its symptoms, suppressing any symptoms that may appear, or reducing the risk of the onset or recurrence of a condition or its symptoms. “Cure” treatment includes reducing the severity of an existing symptom or condition, or preventing its worsening.

[0035] As used herein, the terms “therapeutic,” “anti-VEGF agent,” “fusion protein,” “chimeric protein,” or “recombinant protein” include a first polypeptide operably linked to a second polypeptide; “therapeutic,” “anti-VEGF agent,” “fusion protein,” “chimeric protein,” or “recombinant protein” inhibits VEGF activity. A chimeric protein may optionally include a third, fourth, fifth, or other polypeptide operably linked to the first or second polypeptide. A chimeric protein may include two or more different polypeptides. A chimeric protein may include multiple copies of the same polypeptide. A chimeric protein may also include one or more mutations in one or more polypeptides. Methods for producing chimeric proteins are well known in the art. In some embodiments, the terms “therapeutic,” “fusion protein,” “chimeric protein,” or “recombinant protein” refer to any construct that is expressed or synthesized, including, but not limited to, peptides or proteins operably linked to one or more Ig-like domains or domain fragments of VEGFR-1 and / or VEGFR-2 having Fc-IgG.

[0036] The term "Ig-like domain" refers to Ig-like domains 1-7 of VEGFR-1 and VEGFR-2. The term "Ig-like domain fragment" refers to a portion of a full-length domain, generally including heparin and / or its VEGF-binding or variable region. Examples of domain fragments include segments comprising at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% of the full-length domain, and containing an amino acid sequence with 100% sequence identity and its variations. As encompassed by this disclosure, changes in the amino acid sequence of the fusion protein are intended, provided that the changes in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. This includes certain intermediate percentages of sequence identity, such as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. In particular, conservative amino acid substitutions are intended. Conservative substitutions are substitutions that occur within a family of amino acids related to the side chain. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) non-charged amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.Other families of amino acids include (i) the aliphatic hydroxyl family serine and threonine, (ii) the amide-containing family asparagine and glutamine, (iii) the aliphatic family alanine, valine, leucine, and isoleucine, and (iv) the aromatic family phenylalanine, tryptophan, and tyrosine. For example, it is reasonable to expect that single substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, and threonine with serine, or analogous substitutions of amino acids with structurally related amino acids, will not significantly affect the binding or properties of the resulting molecule, especially if the substitution does not involve amino acids within the framework site. Whether an amino acid change results in a functional fusion protein can be easily determined by assaying the specific activity of the fusion protein derivative. Fragments or analogs of fusion proteins can be readily prepared by those skilled in the art. The preferred amino and carboxyl terms of the fragments or analogs are located near the boundaries of the functional domain.

[0037] As used herein, “isolated” or “purified” fusion protein is the dominant species in which the fusion protein resides (i.e., in molar terms, more abundant than any other individual species in the composition), and preferably a substantially purified fraction means a composition containing at least about 50% (in molar terms) of all the macromolecular species in which the fusion protein resides. Generally, a purified composition constitutes more than about 80%, more preferably about 85%, 90%, 95%, and more than 99% of all the macromolecular species present in the composition. Most preferably, the fusion protein is purified to the point of being essentially homogeneous (contaminant species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species.

[0038] Values ​​or ranges may be expressed herein as “approximately,” “approximately” one particular value, and / or “approximately” another particular value. Where such values ​​or ranges are expressed, other embodiments disclosed include the enumerated particular values, from one particular value, and / or other particular values. Similarly, where values ​​are expressed as approximations by using the antecedent “approximately,” it will be understood that particular values ​​form another embodiment. It will be further understood that there are several values ​​disclosed herein, and each value is disclosed herein not only as the value itself, but also as “approximately” its particular value. In embodiments, “approximately” may be used to mean, for example, within 10% of the enumerated values, within 5% of the enumerated values, or within 2% of the enumerated values.

[0039] In one embodiment, the present invention discloses a composition comprising a therapeutic agent comprising one or more heparin-binding domains of VEGFR-1 or VEGFR-2 and one or more VEGF-binding domains, thereby inhibiting the binding of VEGF to its homologous receptor.

[0040] In embodiments, the present invention provides an anti-VEGF agent comprising a VEGF-binding moiety operably linked to Fc-IgG, wherein the VEGF-binding moiety comprises at least one VEGF-binding domain which is the IgG-like domain 2 of VEGFR-1, and the anti-VEGF agent has superior VEGF-stimulated mitotic inhibitory ability compared to aflibercept. In embodiments, the present invention provides that the anti-VEGF agent has superior vitreous binding ability compared to aflibercept. In embodiments, the present invention provides that the anti-VEGF agent has superior vitreous-bound VEGF-stimulated endothelial cell proliferation inhibitory ability compared to aflibercept. In embodiments, the present invention provides that the agent has an increased half-life in vivo compared to aflibercept.

[0041] In embodiments, the present invention relates to a VEGF binding moiety that is essentially the IgG-like domains 1, 2, and 3 of VEGFR-1. 1-2-3 The invention provides that the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 1. In the embodiment, the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 1.

[0042] In embodiments, the present invention relates to a VEGF binding moiety that is essentially the IgG-like domains 2 and 3 of VEGFR-1. 2-3 The invention provides that the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 3. In the embodiment, the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 3.

[0043] In embodiments, the present invention relates to a VEGF binding moiety that is essentially the IgG-like domains 1, 2, 3, and 3(V) of VEGFR-1. 1-2-3-3 The invention provides that the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 5. In the embodiment, the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 5.

[0044] In embodiments, the present invention relates to a VEGF binding moiety that is essentially the IgG-like domains 2, 3, and 3(V) of VEGFR-1. 2-3-3 The invention provides that the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 7. In the embodiment, the anti-VEGF agent comprises the amino acid sequence defined in SEQ ID NO: 7.

[0045] In embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of an anti-VEGF agent as defined in the claims and a pharmaceutically acceptable excipient. In embodiments, the present invention provides a method for treating a VEGF-related disorder in a subject requiring treatment, comprising administering a therapeutically effective dose of a defined anti-VEGF agent to the subject. The anti-VEGF agent can be injected directly into affected tissue or organs, such as the eyes.

[0046] In embodiments, the present invention can be used to treat a wide variety of VEGF-related diseases, including neovascular age-related macular degeneration, choroidal neovascularization secondary to myopia, proliferative diabetic retinopathy, diabetic macular edema, retinal vascular occlusion such as retinal vein occlusion, ocular tumors, von Hippel-Lindau syndrome, retinopathy of prematurity, polypoid choroidal vasculopathy, colorectal cancer, lung cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, schwannomas, gliomas, ependymomas, and neoplastic or non-neoplastic disorders that benefit from anti-VEGF therapy.

[0047] In some embodiments, the therapeutic agent is an administerable dosage form comprising the therapeutic agent and additional excipients, carriers, adjuvants, solvents, or diluents.

[0048] In some embodiments, the present invention discloses pharmaceutical compositions suitable for treating and / or prophylactically treating a subject, wherein the therapeutic agent is contained in an amount effective to achieve its intended purpose.

[0049] In some embodiments, the therapeutic agents or compositions disclosed herein are administered by injection. In certain embodiments, the composition or therapeutic agent is injected directly into the affected organ or tissue. In some embodiments, the therapeutic agent can be administered topically, for example, by patching or direct application to the affected organ or tissue, or by iontophoresis. The therapeutic agent may be provided as a sustained-release composition, such as those described in U.S. Patents 5,672,659 and 5,595,760. The use of immediate-release or sustained-release compositions depends on the nature of the condition being treated. If the condition consists of an acute or over-acute disorder, treatment with an immediate-release type is preferred over a sustained-release composition. Alternatively, for certain prophylactic or long-term treatments, a sustained-release composition may be appropriate.

[0050] Therapeutic agents may also be delivered using implants, including but not limited to intraocular implants. Such implants may be biodegradable and / or biocompatible, or they may be non-biodegradable. The implants may be permeable or impermeable to the active agent. The specific implant for delivering the therapeutic agent depends on both the affected tissue or organ, as well as the nature of the condition being treated. The use of such implants is well known in the art.

[0051] The inhibitors described in the present invention may be formulated into nanoparticles or other drug formulations to provide precise delivery to specific tissues and to provide controlled-release therapy.

[0052] The inhibitors described in this application can be delivered not only as purified recombinant proteins but also by gene therapy approaches. VEGF inhibitors can be delivered subretinal or intravitreous delivery using recombinant adeno-associated vectors (rAAV) or other suitable vectors. 43,44 .

[0053] In a related aspect, the present invention provides a method for treating VEGF-related or neovascularization in a subject, the method comprising administering to the subject (a) an effective amount of a fusion protein that binds to heparin and can reduce or prevent the development of undesirable neovascularization. The fusion protein may be combined with other anti-VEGF agents, including, but not limited to, antibodies or antibody fragments specific to VEGF, antibodies specific to the VEGF receptor, compounds that inhibit, regulate, and / or modulate tyrosine kinase signaling, VEGF polypeptides, oligonucleotides that inhibit VEGF expression at the nucleic acid level, such as antisense RNA, and various organic compounds and other agents having anti-angiogenic activity.

[0054] This invention relates to heparin binding mediated by VEGFR1's D3 (or other Ig-like domain). 28 However, while it is disadvantageous for systemic administration, it offers significant advantages for intravitreous (or other local) administration. In fact, it has the ability to bind to HGPSG, a major component of the extracellular matrix. 29 This promotes accumulation in the vitreous humor and penetration into the retina. 30 This invention provides a series of VEGFR-1 Fc fusion constructs having different abilities to interact with HSPG. This allows for the selection of VEGF inhibitors with different durations / half-lives in the eye that are useful under different clinical conditions.

[0055] Features and other details of the present invention are described more specifically herein and are shown in the following examples which describe preferred technical and experimental results. The examples are provided for illustrative purposes only and should not be construed as limiting the present invention. [Examples]

[0056] Accordingly, in embodiments, the present invention discloses a novel anti-VEGF agent that improves upon existing anti-VEGF agents, including aflibercept, by its high biological potency combined with potent heparin-binding properties. Heparin binding predicts a longer half-life and a resulting reduction in administration frequency.

[0057] This invention relates to heparin binding mediated by VEGFR1 D3 (or other Ig-like domains). 28 However, while it is disadvantageous for systemic administration, it offers significant advantages for intravitreous (or other local) administration. In fact, it has the ability to bind to HGPSG, a major component of the extracellular matrix. 29 This can promote accumulation in the vitreous humor and penetration into the retina. 30 The present invention provides a series of VEGFR-1 Fc fusion constructs having different capabilities for interacting with HSPG.

[0058] Figure 1 provides a schematic diagram of the construct used here. Figure 2 illustrates the vector and cloning strategy used. Figures 3–9 show the nucleic acid and amino acid sequences of the generated construct.

[0059] The examples show that the expression levels of most constructs are low. 1-2-3-4 , V 1-2-3-3 , V 2-3-4 , and V 1-2-4 It was hardly detected at all in the conditioned medium. Previous studies have shown that heparin (VEGF) 189 or VEGF 206 The VEGF isoform, which has high affinity for VEGF, was not detected in the conditioned medium of the transfected cells, indicating that it was tightly bound to the cell surface or extracellular matrix. 31 32 However, they can be released in a soluble form by the addition of heparin or heparinase, and the binding site consists of HSPG. 31 32This example demonstrates that the addition of heparin may also affect the levels of recombinant VEGFR-1 fusion protein. Indeed, when heparin was added to the culture medium of transfected cells in a 6-well plate, the concentration of recombinant protein in the medium increased in a dose-dependent manner (Figure 10).

[0060] When attempting to purify recombinant proteins, conventional protein A (PA) affinity chromatography alone yielded the expected major mass band, but there were numerous smaller bands likely reflecting the interaction of recombinant proteins with host cell-derived HSPGs and other molecules. A protocol was developed to remove such impurities, as described in the methods. Washing at high pH (e.g., 9.2) in the presence of 1.2 M NaCl released numerous contaminants when the proteins bound to PA. The subsequent anion exchange chromatography using Hi-Trap Q was highly effective in removing most contaminants and aggregates while the purified protein remained in a flow-through state. The LPS level of the final purified preparation was <0.1 EU / mg (range 0.02-0.08), indicating a very low level of compatibility with preclinical studies. 33 As shown in Figure 11, the purity of the recombinant protein, as evaluated by silver-stained SDS / PAGE gel, was >95%, which was equivalent to the purity of EYLEA, an FDA-approved drug.

[0061] Recombinant proteins were tested for their ability to inhibit VEGF (10 ng / ml)-induced mitosis in bovine choroidal endothelial cells. The recombinant proteins showed inhibitory effects, and IC 50 The values ​​were in the range of approximately 1 nM, but V1-2-4 and V2-4 were not as potent (Figure 12). Interestingly, even at the highest concentration tested, EYLEA inhibited less than 80% of VEGF-stimulated proliferation (Figure 12). In contrast, this VEGFR-1 construct (V 1-2-4 and V 2-4Except for [specific component], VEGF-induced proliferation was completely blocked (Figure 12). Binding assays demonstrated the interaction between the soluble VEGF receptor and biotinylated VEGF, and VEGF's ability to replace binding (Figures 13, 14).

[0062] To further define the treatment-related interactions, we attempted to evaluate whether the recombinant protein bound to the bovine vitreous humor in vitro. As illustrated in Figure 14, EYLEA or control IgG bound little to no, while our protein showed significant binding. The strongest binding agents were V1-2-3-3, V2-3-3, and V1-2-3-4, followed by V1-2-3. V2-3 bound to EYLEA (or control IgG) and V 1-2-3-3 It showed intermediate binding characteristics with AVASTIN, a monoclonal antibody commonly used in the treatment of intraocular neovascularization. 9 They hardly or never bonded.

[0063] To determine whether VEGFR-1 FC fusions bound to the vitreous humor were biologically active, plates were coated with bovine vitreous humor. Addition of V1-2-3-3, rather than EYLEA or control IgG, inhibited the ability of exogenously added VEGF to stimulate endothelial cell proliferation (Figure 15).

[0064] Recombinant proteins were tested using a mouse CNV assay and compared to control IgG or EYLEA. Each protein was injected intravitreously at a dose of 2.5 μg one day prior to laser treatment. EYLEA was also tested at 25 μg. 1-2-3 , V 2-3 , V 1-2-3-3 , and V 2-3-3 The degree of inhibition was equivalent to or greater than that achieved with 25 μg of EYLEA. However, none of the constructs containing D4 showed significant inhibition under the conditions tested (Figure 16).

[0065] To determine whether heparin binding provides a permanent therapeutic effect after a single dose, V 1-2-3-3EYLEA or control IgG was injected intravitreously (2.5 μg) 1, 7, or 14 days before laser-induced injury. As shown in Figure 17, EYLEA produced significant inhibition only when administered 1 day before injury. However, V 1-2-3-3 It also produced significant inhibition when administered 7 or 14 days prior to injury.

[0066] Several novel VEGFR-1-Fc constructs evaluated in various in vitro and in vivo assays are disclosed. A multi-step protocol was used to purify the recombinant protein. This was largely determined by the relatively low expression levels in transient expression 293 cells, which required the addition of heparin to the culture medium to improve release. However, the need to use heparin may be partially or completely eliminated by different host cells (e.g., HSPG or its variants with different compositions) or by higher expression levels in amplified stable cell lines.

[0067] All constructs except V2-4 potently neutralize VEGF activity and simultaneously possess strong heparin-binding properties, which predict a long half-life after intravitreous administration. This example demonstrates that these proteins bind to bovine vitreous humor. The strongest binding agents were V1-2-3-3, V2-3-3, V1-2-3-4, followed by V1-2-3. V2-3 was significant but showed low vitreous binding. Control IgG, EYLEA, or AVASTIN, on the other hand, showed minimal binding. We selected V1-2-3-3, one of the strongest vitreous binding agents, to test the hypothesis that a VEGFR1 Fc construct bound to the vitreous matrix may be biologically active. As shown in Figure 15, when V1-2-3-3 was added to a plate coated with bovine vitreous humor, rather than EYLEA or control IgG, the ability of exogenously added VEGF to stimulate endothelial cell proliferation was inhibited.

[0068] Next, recombinant proteins were tested for their ability to inhibit laser-induced neovascularization in a mouse CNV model. EYLEA was used as a positive control and human IgG1 as a negative control. Relatively low doses were selected for the in vivo study, as they are optimal for clarifying differences in potency between various constructs. It has also been reported that relatively high doses of antibodies of IgG1 isotypes may have off-target inhibitory effects mediated by FcgRI(CD64) and c-Cbl when injected intravitreously. 34 The dosage used should avoid such off-target effects and allow for the detection of true specific effects.

[0069] As shown in Figure 16, EYLEA resulted in approximately 30% inhibition at a dose of 2.5 μg and approximately 50% inhibition at 25 μg. These findings are largely consistent with published literature. Saishin et al. reported that intravitreal injection of approximately 5 μg of aflibercept inhibited the CNV region in mice by approximately 30%. 35 In fact, a dose of 40 μg is commonly used to achieve the maximum effect in mouse CNV models. 36 An unexpected finding in our study was the high potency of some of our V1-2-3, V2-3, V1-2-3-3, and V2-3-3 constructs. Administration of 2.5 μg of these constructs one day prior to injury was consistent with or exceeded the inhibitory levels achieved with 25 μg of EYLEA. The finding that V1-2-3-3, rather than EYLEA, had a significant effect in preventing CNV when administered 7 or 14 days prior to injury (Figure 17) demonstrates the persistence and therapeutic value of the effect.

[0070] Unexpected findings include these molecules (V 2-4Despite the fact that (excluding) demonstrated the ability to block VEGF-stimulated mitosis in vitro, none of the D4-containing constructs (V1-2-3-4, V2-3-4, V1-2-4, V2-4) produced significant inhibition in vivo (at least at the tested doses). However, all of these constructs tended to form polymers or aggregates, as assessed by SDS / PAGE gel under non-reducing conditions (Figure 11) or size exclusion chromatography (not shown). Early studies 37 D4 (along with D7) was identified as a requirement for VEGFR-1 dimerization, but such an effect is known to be ligand-dependent. Crystal structure studies revealed the D4 loop responsible for such isomorphic interactions. 23 It is thought that forced dimerization imposed by high concentrations and / or Fc constructs may lead to ligand-independent interactions and aggregation. In any case, given the potential for inflammation and immunogenicity, aggregates are not a desirable drug. 38 , 3 9 Therefore, an embodiment of the present invention is the identification of a structure having VEGFR-1 D2 / D3 rather than D4.

[0071] It is worth noting that well-characterized Fc mutations, well known to those skilled in the art, which reduce effector function, may be useful additions to the present invention to minimize antibody-dependent cytotoxicity (ADCC) and interaction with C1q and the initiation of the complement cascade. 40 .

[0072] In conclusion, aflibercept was designed to eliminate the heparin-binding heparin domain to improve the systemic half-life for oncological indications. The construct described in this study is instead designed to promote binding and retention in the vitreous humor to ensure a more sustained and therapeutically relevant interaction.

[0073] method VEGFR-1 ECD- For the construction of Fc expression plasmids, nucleic acid fragments encoding signal peptides and various extracellular Ig-like domains (VEGRF-1 (gene ID: 2321) 1-4) 20 However, these were synthesized by GenScript USA Inc. The various extracellular Ig-like domain constructs are as follows: V123 contains D1, 2, and 3; V23 contains D1 and D2; V1234 contains D1, 2, 3, and 4; V1233 contains D1, 2, 3, and 3; V234 contains D2, 3, and 4; V124 contains D1, 2, and 4; V24 contains D2 and 4; F7 contains ECD2, 2, and 3; and F8 contains ECD2 and 3. The synthesized fragments were inserted into the pFUSE-hIgG1-Fc vector (InvivoGen, #pfuse-hgifc1) at the EcoRI and BgIII sites to generate plasmids containing various Flt1 ECDs. Next, using the PrineSTAR Mutagenesis Basal Kit (Takara, R046A), the interval amino acids R and S (BgIII site) between the ECD and Fc fragments were removed to generate plasmids (F1-F8) expressing a fusion protein of Flt1 ECD containing 227 amino acids human IgG1-Fc.

[0074] Preparation of transfection and conditioned medium Following the manufacturer's instructions, a conditioned medium for purification was prepared using the Expi293 expression system (Life Technologies, A14524). Briefly, Expi293F® cells (ThermoFisher) were incubated in a humidified atmosphere of 8% CO2 for 37 minutes. oCells were cultured in suspension in Expi293® expression medium at 13C. When the cell density reached 2.5 million / ml, the plasmid DNA and ExpiFectamine® 293 reagent were mixed, incubated for 5 minutes, and added to the cells. The final concentrations of the DNA and transfected reagent were 1 μg and 2.7 μl / ml, respectively. Five hours after transfection, 100 μg / ml of heparin (Sigma, H3149) and a protease inhibitor cocktail 1:400 (Sigma, P1860) were added to the cells. Sixteen hours after transfection, enhancer reagents 1 and 2 were added. Ninety-six hours after transfection, the conditioned medium was collected. Aliquots were tested for Fc fusion protein concentration using a human Fc ELISA Kit (Syd Labs, EK000095-HUFC-2) according to the manufacturer's instructions. Add the protease inhibitor to the bulk (1:500) and continue until use -80 o Saved in C.

[0075] Recombinant protein purification A pyrogenic reagent was used. Before use, the column and instrument (Akta Explorer System) were disinfected by exposure to 0.5 N NaOH for approximately 45 minutes. The conditioned medium from the transfected cells was prepared in PBS and 0.01% polysorbate (PS) 20. PS 20 was added to the buffer at every step. After centrifugation at 20,000 xg for 30 minutes, the supernatant was subjected to protein A (PA) affinity chromatography using Hi-Trap MabSelect SuRe (5 ml, GE Healthcare). After loading, the column was washed with 20 mM diethanolamine, pH 9.2, and 1.2 M NaCl, then eluted with 0.1 M citrate, pH 3.0, and immediately neutralized. Next, the PA elution pool was diluted with 20 mM diethanolamine, pH 9.2, and applied to a Hi-Trap Q (5 ml, GE Healthcare) anion exchange column. The bound substance was eluted with a NaCl gradient. The flow-through containing the purified recombinant protein was immediately adjusted to 20 mM Tris, pH 6.8, and concentrated by binding to heparin-Sepharose (Hi-Trap®-HS). After washing with 0.2–0.45 M NaCl (construct-dependent), the recombinant VEGFR1 fusion protein was eluted with 1 M NaCl. The final polishing step consisted of size exclusion chromatography (SEC) using, for example, Superdex 200 Increase, 10 / 300 GL, or Hi-Load 16 / 600 Superdex 200 pg, GE Healthcare. Finally, the protein was buffer-exchanged by dialysis to 10 mM Tris, pH 6.8, 10 mM histidine, 1% threalose, 40 mM NaCl, and 0.01% PS20. To determine endotoxin levels, the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript, L00350) was used according to the manufacturer's protocol.

[0076] Cell proliferation assay The bovine endothelial cell proliferation assay was performed essentially as previously described.41 Bovine choroidal endothelial cells (BCEC) in the logarithmic growth phase (passage < 10) were trypsin-treated, resuspended, and seeded at a density of 1000 cells / well in 200 μl volumes into 96-well (uncoated) plates of low-glucose DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics. rhVEGF 165 Peprotech was added at a concentration of 10 ng / ml. Aflibercept (EYLEA) was purchased from a pharmacy. Inhibitors were added to the cells at various concentrations, as shown in the figure, before ligand addition. After 5 or 6 days, the cells were incubated with Alamar Blue for 4 hours. Fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm.

[0077] Solid-phase VEGFR-1 variant binding assay Costar 96-well EIA / RIA strip wells (#2592, Corning Incorporated, Kennebunk, ME) were coated with purified VEGF receptor protein (250 ng / well) in coating buffer (Biolegend, San Diego, CA, #421701) overnight at 4°C. After one wash with ELISA washing buffer (R&D systems 895003), the strips were incubated with 2% BSA (Sigma, A6003) in PBS at room temperature (RT) for 1 hour to block nonspecific binding sites. The strips were then washed three times, followed by incubation at 37°C for 2 hours with biotinylated human VEGF. 165 (G&P Biosciences, Santa Clara, CA) was added to the assay diluent (Biolegend, #421203) either alone or in combination with various concentrations of non-biotinylated human VEGF165 (R&D systems). After three washes, biotinylated human VEGF bound to VEGFR1 was added. 165The substance was detected by incubation with HRP streptavidin (1:1000, Biolegend, #405210) at room temperature for 1 hour. After washing the strips five times, they were incubated with TMB high-sensitivity substrate solution (Biolegend, #421501) for 30 minutes, and absorbance at 450 nm was measured after adding an equal volume of stop solution (Biolegend, #77316). All experiments were performed in two wells and repeated at least twice.

[0078] In vitro binding of bovine vitreous humor Bovine vitreous humor (InVision BioResource, Seattle, WA) was thawed at 4°C. The material was first diluted 1:1 with PBS, filtered through a 0.22 μm filter, divided into equal portions, and stored at -80°C. The total protein concentration of the bovine vitreous material was measured by Pierce BCA protein assay. Costar 96-well EIA / RIA strip wells were coated with bovine vitreous humor (1 μg / well) for 4 hours at room temperature, followed by one wash with ELISA wash buffer. Nonspecific binding sites were blocked by adding 2% BSA to PBS for 1 hour at room temperature, followed by three washes with 0.01% PBS-T. 50 μl of VEGFR1 or control protein was added to each well overnight at 4°C. The following day, the plates were washed three times with 0.01% PBS-T, followed by incubation with 100 μl of AP conjugate goat anti-human Fc (1:2000, Invitrogen, #A18832) at room temperature for 1 hour. After washing the plates five more times with 0.01% PBS-T, 50 μl of one-step PNPP substrate (Thermo Scientific, Rockford, IL, #37621) was added and incubated at room temperature for 15–30 minutes. OD was measured at 405 nm.

[0079] The effect of vitreous-bound VEGFR1 on VEGF-stimulated endothelial cell proliferation in Costar 96-well EIA / RIA strip wells was investigated. The plates were first UV-sterilized for 1 hour, then coated with 1 μg / well of bovine vitreous diluted in PBS for 4 hours at room temperature. The plates were washed once with PBS and 4... oAt 1°C, the plates were blocked with 2% BSA and washed twice with PBS in a biosafety hood. Equal volumes of soluble receptor or control IgG were added to the plates and diluted in PBS O / N at 4°C (50 μl / well). The plates were then washed once with PBS, followed by one wash with assay medium containing 10% BCS. 100 μl of medium was added to each well, followed by 5 ng / ml of VEGF, or PBS alone as a control without VEGF. The plates were incubated with VEGF or PBS for 1 hour, followed by the addition of 100 μl of BCEC cell suspension (ultimately 2500 cells / well). After 48 hours, growth was measured by adding Alamar Blue.

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Claims

1. A composition for the treatment of VEGF-related eye diseases in subjects requiring treatment, comprising an anti-vascular endothelial growth factor (anti-VEGF) agent, wherein the anti-VEGF agent consists of amino acid residues 27-459 of SEQ ID NO: 3, and the VEGF-related eye disease is choroidal neovascularization secondary to myopia, proliferative diabetic retinopathy, diabetic macular edema, retinal vascular occlusion, ocular tumor, von Hippel-Lindau syndrome, retinopathy of prematurity, or polypoid choroidal vasculopathy.

2. The composition according to claim 1, wherein the VEGF-related eye disease is proliferative diabetic retinopathy.

3. The composition according to claim 1, wherein the VEGF-related eye disease is diabetic macular edema.

4. The composition according to claim 1, wherein the VEGF-related eye disease is retinopathy of prematurity.

5. The composition according to claim 1, wherein the VEGF-related eye disease is an eye tumor.

6. The composition according to claim 1, wherein the VEGF-related eye disease is retinal vascular occlusion.

7. The composition according to claim 6, wherein the retinal vascular occlusion is retinal vein occlusion.

8. The composition according to any one of claims 1 to 7, wherein the anti-VEGF agent has a superior ability to inhibit vitreous-bound VEGF-stimulated endothelial cell proliferation compared to aflibercept.

9. The composition according to any one of claims 1 to 7, wherein the anti-VEGF agent has a longer duration of action when injected into the eye compared to aflibercept.

10. The composition according to any one of claims 1 to 6, wherein the anti-VEGF agent is formulated for intravitreal injection.

11. The composition according to any one of claims 1 to 6, wherein the anti-VEGF agent is administered topically to the eye in a dosage corresponding to a molar ratio of 2:1 with respect to VEGF.

12. The composition according to any one of claims 1 to 7, wherein an anti-VEGF agent is administered to the subject every 4 to 6 weeks.

13. The composition according to any one of claims 1 to 7, wherein the anti-VEGF agent is administered to the subject for at least one year.

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