VEGFR fusion protein pharmaceutical composition

Abstract

The present invention relates to biologics that inhibit angiogenesis. In particular, the present invention relates to fusion proteins that inhibit the integrin activation pathway and one other angiogenic factor activation pathway, as well as pharmaceutical compositions of such fusion proteins and methods of making and using the same.

Description

Technical Field

[0001] Cross-reference of related applications This application is an international application claiming the priority of U.S. Non-Provisional Patent Application No. 17 / 663,260, filed on May 13, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to electronically submitted sequence listings The content of the electronic sequence listing (065781_WO1.xml; size: 66,200 bytes; created on August 25, 2022) is incorporated herein by reference in its entirety.

[0003] The present invention relates to a fusion protein pharmaceutical composition that inhibits the angiogenesis factor activation pathway. In particular, the present invention relates to fusion proteins that inhibit the angiogenesis factor activation pathway, compositions of these fusion proteins, and methods for their manufacture and use.

Background Art

[0004] Angiogenesis is the process of growing new blood vessels from existing vascular systems. It plays a crucial role in several physiological processes, including embryonic development and tissue and wound repair (Folkman J et al. Angiogenic Factors. Science 1987;235:442-7). The physiological processes of angiogenesis are well-characterized and include proteolysis of the extracellular matrix, proliferation, adhesion, migration and aggregation of endothelial cells into tubular channels, recruitment and differentiation of parietal cells and pericytes, and production of the extracellular matrix (Carmeliet P et al. Nature. 2011;473:298-307). Pathological angiogenesis can occur in tumorigenesis, ocular disorders (e.g., diabetic retinopathy, diabetic macular edema, retinal / choroidal neovascularization, exudative age-related macular degeneration, and neovascular glaucoma), arthritis, psoriasis, fibrous diseases, inflammatory diseases, atherosclerosis, and arteriosclerosis (Polverini P J. Crit Rev Oral Biol Med. 1995;6(3):230-47, Perrotta P et al. Vascular Pharmacology. 2019;112:72-78).

[0005] Pathological neovascularization is often more heterogeneous and disordered, exhibiting tortuous vascular organization, hypoxic voids of varying sizes, heterogeneous and incomplete vascular walls and linings, and ineffective perfusion (Jain R K., Nat Med. 2003;9(6):685-93). These distinct characteristics of neovascularization in the disease present challenges to the therapeutic targeting of angiogenesis. Anti-VEGF therapies, such as the off-label use of LUCENTIS® (ranibizumab), EYLEA® (aflibercept), or AVASTIN® (bevacizumab), can generally stabilize or improve visual function, but subretinal scarring (fibrosis) can occur in approximately half of all treated eyes within two years of anti-VEGF treatment and has been identified as one cause of poor outcomes (Daniel E et al. Ophthalmology. 2014;121(3):656-66). Many of the key players in subretinal fibrosis are likely growth factors and matrix cell proteins involved in the fibrotic process (cell proliferation, migration, and ECM remodeling) (Patsenker E et al. Hepatology. 2009 Nov;50(5):1501-1511, Xu J et al. Biochim Biophys Acta. 2014 Nov;1842(11):2106-2119). Despite its complexity, anti-angiogenic drug development remains a very interesting area as our knowledge of the angiogenic process increases.

[0006] Currently, many important roles in the angiogenesis process have been identified, with the vascular endothelial growth factor (VEGF) family playing a dominant role. The human VEGF family consists of six members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF). Furthermore, multiple isoforms of VEGF-A, VEGF-B, and PIGF are generated by alternative RNA splicing (Sullivan et al. MAbs. 2002;2(2):165-75). VEGF-A is a major factor involved in angiogenesis and binds to both VEGFR-1 and VEGFR-2. Strategies to inhibit angiogenesis by interfering with VEGF-A signaling have established successful therapeutic approaches for certain cancers as well as for retinal angiogenesis and ischemic diseases. (Major et al.J Pharmacol Exp Ther.1997;283(1):402-10;Willet et al.Nat.Med.2004;10:145-7;Papadopoulos et al.Angiogenesis.2012;15(2):171-85;Aiello et al.PNAS.1995;92:10457-61).

[0007] Platelet-derived growth factor (PDGF), transformed growth factor beta (TGF-β), epidermal growth factor (EGF), nerve growth factor (NGF), hypoxia-inducible factor (HIF), basic fibroblast growth factor or fibroblast growth factor (bFGF or FGF-2), connective tissue growth factor (CTGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), insulin-like growth factor (IGF), hepatocyte growth factor / scatter factor (HGF / SF), tumor necrosis factor alpha (TNF-α), stromal cell-derived factor-1 (SDF-1), interleuk Other growth factors, cytokines, and chemokines, including chemotactic substances such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-20 (IL-20), interleukin-23 (IL-23), CC motif ligands (CCL28, CCL21) and CXC motif ligands (CXCL1, CXCL5), macrophage migration inhibitors (MIFs), and immune cell surface proteins such as differentiation antigens (CDs). These factors have been reported to be overexpressed and play an important role in angiogenesis-related diseases (Elshabrawy et al. Angiogenesis. 2015;18:433-448; Somanath PR et al, Cell Biochem Biophys. 2009;53(2):53-64, Eliceiri B P., Circ Res. 2001 Dec 7;89(12):1104-10). By targeting these factors to reduce downstream pathway activation, angiogenesis-related diseases can be reduced.

[0008] Integrins, a family of cell surface receptors, have also been found to be overexpressed on the surface of endothelial cells and are thought to promote the growth and survival of newly formed blood vessels during angiogenesis. Integrins are heterodimeric cell surface receptors that interact with extracellular matrix proteins and are important for many biological processes. Integrin expression in various cell types is involved in tumor progression, and their ability to crosstalk with growth factor receptors and directly interact with several growth factors has made them attractive therapeutic targets. (Staunton DE et al. Adv Immunol. 2006;91:111-57; Avraamides, CJ et al. Nat Rev Cancer. 2008;8:604-617, Somanath PR et al. Cell Biochem Biophys. 2009;53(2):53-64) In particular, integrin αvβ3 is upregulated in both tumor cells and angiogenic endothelial cells and is important for tumor cell migration, angiogenesis, and dysregulated cellular signaling. Therefore, integrin αvβ3 antagonists have been studied intensively for their anti-angiogenic and antitumor properties (Desgrosellier JS et al. Nat Rev Cancer. 2010;10:9-22).

[0009] Disintegrins are proteins found in the venom of viper family snakes and primarily inhibit the function of β1 and β3-related integrins. They were initially identified as inhibitors of integrin αIIbβ3, and subsequently shown to bind to other integrins with high affinity, blocking the interaction between integrins and RGD-containing proteins. They contain 47 to 84 amino acids with approximately 4 to 7 disulfide bonds and share the same RGD motif (McLane MA, et al. Proc Soc Exp Biol Med. 1998; 219: 109-119; Niewiarowski S et al. Semin Hematol 1994; 31: 289-300; Calvete JJ, Curr Pharm Des. 2005; 11: 829-835; Blobel CP et al. Curr Opin Cell Biol. 1992; 4: 760-765). The conserved RGD sequence of the disintegrin family plays a crucial role in integrin recognition. Disintegrins have been found to interact with 8 out of 24 integrins and inhibit integrin-mediated cell proliferation, adhesion, migration, and angiogenesis (McLane MA, et al. Front Biosci. 2008;13:6617-6637; Swenson S, et al. Curr Pharm Des. 2007;13:2860-2871). Animal studies have shown that disintegrins target angiogenic endothelium and metastatic tumors, indicating their potential use in cancer therapy. The specific binding of RGD-containing proteins to integrins is functional in both conformation and local arrangement surrounding the RGD motif. Numerous studies have shown that residues adjacent to the RGD motif in RGD-containing proteins influence their binding specificity and affinity to integrins (Scarborough RM et al. J Biol Chem. 1993;268:1058-1065; Rahman S et al. Biochem J. 1998;335:247-257). [Prior art documents] [Non-patent literature]

[0010] [Non-licensed document 1] Folkman J et al.Angiogenic Factors.Science 1987;235:442-7 [Non-licensed document 2] Carmeliet P et al.Nature.2011;473:298-307 [Non-licensed document 3] Polverini P J.Crit Rev Oral Biol Med.1995;6(3):230-47

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Summary of the Invention

Problems to be Solved by the Invention

[0011] Angiogenesis is a complex biological process involving various growth factors and signal transduction receptors, and targeting a single molecule in the signal transduction cascade may not provide an effective clinical treatment for unregulated angiogenesis in diseases such as cancer. Therefore, there is an increasing need to develop innovative therapeutic agents that can bind cooperatively to several important angiogenic factors in order to effectively inhibit angiogenesis and disease progression. Pharmaceutical formulations of fusion proteins and methods of using such formulations are provided herein.

Means for Solving the Problems

[0012] In one general aspect, the present disclosure provides a) 0 .5 mg / mL to 1 a fusion protein at a concentration of 20 mg / mL, and b) 1 % to 1 a polyol or alcohol selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, benzyl alcohol, polyvinyl alcohol, polyethylene glycol (PEG) 400 to 12000 at a concentration of 0% w / v, and c) 1 0 mM to5 A buffer selected from the group consisting of sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and trisodium citrate dihydrate, at a concentration of 0 mM. d) 0 .01~ 4 A surfactant at a concentration of %w / v, A pharmaceutical preparation containing, The formulation 5 The present invention relates to a pharmaceutical formulation having a pH of 0.5 to 7.5, and optionally further comprising a polysaccharide selected from the group consisting of sodium carboxymethylcellulose, microcrystalline cellulose, or sodium hyaluronate.

[0013] According to embodiments of the present disclosure, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188, and is preferably polysorbate 20.

[0014] According to the embodiments of this application, the surfactant is 0 The concentration is 0.03%.

[0015] According to the embodiments of this disclosure, the fusion protein is 1 mg / mL~ 9 0 mg / mL, preferably 2 0 mg / mL~ 8 The concentration is 0 mg / mL, and more preferably the fusion protein is 4 The concentration is 0 mg / mL.

[0016] According to embodiments of this application, the polyol is 2 5mM~ 2 50 mM, preferably 1 This is trehalose at a concentration of 90 mM.

[0017] According to the embodiments of this disclosure, the buffering agent is 1 0mM~ 4 0 mM, preferably 2 0mM~ 3Histidine at a concentration of 0 mM, more preferably histidine 2 The concentration is 5 mM.

[0018] According to embodiments of this disclosure, the fusion protein is arranged in the following order from the N-terminus to the C-terminus: a) Extracellular domain of vascular endothelial growth factor receptor (VEGFR), b) The Fc domain of human immunoglobulin G, and c) Integrin-binding protein or its fragment Includes.

[0019] According to the embodiments of this application, the fusion protein comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0020] According to the embodiments of this application, pH is 5 .5~ 7 The pH is 0, preferably pH is 6 It is 0.

[0021] According to embodiments of this application, the formulation is stable for at least 24 months at -70°C, -20°C and / or 5°C.

[0022] According to embodiments of this application, the formulation retains its protein purity and potency for at least nine months at -70°C, -20°C and / or 2-8°C, preferably 2-8°C.

[0023] According to the embodiments of this application, the formulation is 1 It also contains salts at concentrations ranging from 0 mM to 50 mM.

[0024] According to the embodiments of this application, the formulation is 1 It further contains at least one amino acid in a concentration of 0 mM to 50 mM.

[0025] According to embodiments of this application, the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

[0026] According to embodiments of the present disclosure, the amino acid is selected from the group consisting of arginine, methionine, proline, histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

[0027] According to embodiments of this application, the pharmaceutical formulation can be used in a method for treating eye diseases.

[0028] According to embodiments of this disclosure, eye diseases include neovascularization or ischemic uveitis, retinal vasculitis, retinal pigment striata, retinitis pigmentosa, corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy without DME (DR, non-proliferative and proliferative DR), and diabetic retina with DME. The following conditions are selected: macular edema (DR, non-proliferative and proliferative DR), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA), and / or vitreomacular traction (VMT).

[0029] According to embodiments of this disclosure, the formulation is 0 0.03-10 mg / eye, preferably 3 It is administered at a dose of 0.0-6.0 mg / eye, and more preferably the formulation 4 It is administered at a dose of mg / eye.

[0030] According to the embodiments of this application, the formulation is per eye 4 It is administered in doses of mg.

[0031] Another general aspect of this disclosure is: a) Fusion protein at a concentration of 40 mg / mL, b) 25 mM histidine, c) 190 mM trehalose, sucrose, or mannitol d) 0.03% polysorbate 20 or polysorbate 80 A pharmaceutical preparation containing, The formulation 6 This pertains to pharmaceutical formulations with a pH of 0.0.

[0032] This specification is considered sufficient to enable those skilled in the art to carry out the invention. In addition to those shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and are included in the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0033] The above and other purposes, aspects, features, and advantages of the exemplary embodiments can be better understood by referring to the following description in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0034] [Figure 1]Graphs show the mean percentage change from baseline in vascular leakage area after single intravitreal injection of fusion protein 1 at 0.6, 1.0, and 1.9 mg / eye in a laser-induced choroidal angiogenesis model in rhesus monkeys. Data are presented as mean ± SEM. All doses were administered as a single intravitreal dose on day 0. Lucentis® (0.5 mg) was used as a system compatibility and positive control. All spots analyzed were grade III / IV. % Vascular leakage = (baseline leakage area - treatment leakage area) ÷ (baseline leakage area) × 100%; statistical analysis was performed by Mann-Whitney U test to compare the treatment group and vehicle group*, p<0.05. In the 1.9 mg group, data from one monkey with severe ocular inflammation was removed, which affected statistical significance (p=0.057). n=4 eyes / group. [Figure 2] Graphs of mean retinal thickness assessment by optical coherence tomography after single intravitreal injection of fusion protein 1 at 0.6, 1.0, and 1.9 mg / eye in a laser-induced choroidal neovascularization model in rhesus monkeys. Data are presented as mean ± SEM. All doses were administered as a single intravitreal dose on day 0. Lucentis® (0.5 mg) was used as a system compatibility and positive control. All spots analyzed were grade III / IV. Retinal thickness % = (baseline retinal thickness - treated retinal thickness) ÷ (baseline retinal thickness - pre-study retinal thickness) × 100%; statistical analysis was performed by the Mann-Whitney U test to compare the treatment group and vehicle group, *, p<0.05. In the 1.9 mg group, data from one monkey with severe ocular inflammation was removed, which affected statistical significance (p=0.057). n=4 eyes / group. [Figure 3]This graph shows Masson's trichrome staining of grade III / IV lesions in a laser-induced choroidal neovascularization model in rhesus monkeys treated as shown. Data are presented as mean ± SEM. Enucleation was performed on day 29. Selected grade 4 lesion spots were analyzed by Masson's trichrome staining. Student's t-test analysis was performed to compare the treatment group with the vehicle group (*, p<0.05). n=4 eyes / group. [Figure 4] The graphs show pulmonary hydroxyproline levels in a veromycin-induced C57BL / 6 mouse pulmonary fibrosis model after the treatment shown (n=8 animals for each treatment group and n=4 for the sham control group). *p<0.05, treatment vs. sham control, unpaired Student's t-test; *p<0.05, treatment vs. vehicle, one-way ANOVA and Dunnett's test. In the fusion protein 1 group, data from one animal was unavailable due to premature death of unknown cause on day 3. [Figure 5A] This is a plot of the diffusion coefficient of fusion protein 1 in phosphate buffer pH 7.0. The diffusion coefficient was determined by dynamic light scattering (DLS). [Figure 5B] This is a plot of the diffusion coefficient of fusion protein 1 in phosphate buffer pH 6.5. The diffusion coefficient was determined by dynamic light scattering (DLS). [Figure 5C] This is a plot of the diffusion coefficient of fusion protein 1 in histidine buffer pH 6.5. The diffusion coefficient was determined by dynamic light scattering (DLS). [Figure 5D] This is a plot of the diffusion coefficient of fusion protein 1 in histidine buffer pH 6.0. The diffusion coefficient was determined by dynamic light scattering (DLS). [Figure 5E] This is a plot of the diffusion coefficient of fusion protein 1 in citrate buffer pH 6.0. The diffusion coefficient was determined by dynamic light scattering (DLS). [Figure 5F] This is a plot of the diffusion coefficient of fusion protein 1 in citrate buffer pH 5.5. The diffusion coefficient was determined by dynamic light scattering (DLS). [Figure 6]This graph shows turbidity measurements analyzed at two wavelengths for both 40 and 80 mg / L fusion protein 1. Figure 6A shows a wavelength of 660 nm, and Figure 6B shows a wavelength of 320 nm. NaPi = sodium phosphate, Cit = citrate, and His = histidine. [Figure 7AB] This is a non-reducing SDS-PAGE of fusion protein 1 using a Coomassie blue stained gel. Figures 7A and 7B show 40 or 80 mg / mL of fusion protein 1 incubated in citrate buffer at 4°C or 40°C (lanes 2 and 3), for 4 days (lanes 4 and 5), 7 days (lanes 6 and 7), and 14 days (lanes 8 and 9). [Figure 7CD] This is a non-reducing SDS-PAGE of fusion protein 1 using a Coomassie blue stained gel. Figures 7C and 7D show 40 or 80 mg / mL of fusion protein 1, incubated in histidine buffer at 4°C or 40°C (lanes 2 and 3), for 4 days (lanes 4 and 5), 7 days (lanes 6 and 7), and 14 days (lanes 8 and 9). [Figure 7E] This is a non-reducing SDS-PAGE of fusion protein 1 using a Coomassie blue stained gel. Figure 7E shows 40 or 80 mg / mL of fusion protein 1 incubated in histidine or citrate buffer at 4°C or 40°C for 28 days. NaPi = sodium phosphate, Cit = citrate, and His = histidine. 3 μg of protein was loaded into each sample well. [Figure 8AB] Figures 8A and 8B show reduced SDS-PAGE gels stained Coomassie blue with fusion protein 1. Each gel contains 40 or 80 mg / mL of fusion protein 1 incubated in citrate buffer at 4°C or 40°C (lanes 2 and 3), for 4 days (lanes 4 and 5), 7 days (lanes 6 and 7), and 14 days (lanes 8 and 9). [Figure 8CD]This is a reduced SDS-PAGE gel stained with Coomassie blue for fusion protein 1. Figures 8C and 8D show 40 or 80 mg / mL of fusion protein 1 incubated in histidine buffer at 4°C or 40°C (lanes 2 and 3), for 4 days (lanes 4 and 5), 7 days (lanes 6 and 7), and 14 days (lanes 8 and 9). [Figure 8E] Figure 8E shows a reduced SDS-PAGE gel stained with Coomassie blue containing fusion protein 1. The gel contains 40 or 80 mg / mL of fusion protein 1 incubated in histidine or citrate buffer at 4°C or 40°C for 28 days. (NaPi = sodium phosphate, Cit = citrate, and His = histidine.) Each sample well was loaded with 3 μg of protein. [Figure 9] This graph shows the thermal stability of fusion protein 1 at pH 6.0 at a concentration of 1 mg / mL, determined by differential scanning calorimetry (DSC), after formulation in 25 mM histidine buffer, 190 mM trehalose, and 0.03% PS2O. [Figure 10] This graph shows the effects of a single dose of fusion protein 1 at the indicated concentrations in Dutch belt rabbits on inhibiting human VEGF-A165-induced retinal vascular permeability. [Modes for carrying out the invention]

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0036] As used herein, the singular forms "a," "an," and "the" include multiple referents unless otherwise explicitly indicated by the context. Thus, for example, a reference to "joint domain" includes multiple joint domains and their equivalents known to those skilled in the art.

[0037] As used herein, the terms “polypeptide” and “protein” may be used interchangeably to refer to the amino acid sequence of a natural protein, or to a long chain of peptides having an amino acid sequence with one or more mutations, such as the deletion, addition, and / or substitution of one or more amino acid residues.

[0038] A "fusion protein" refers to a protein that has two or more parts that are covalently bonded to each other, with each part originating from a different protein.

[0039] The present invention provides a pharmaceutical formulation comprising a fusion protein comprising an integrin-binding peptide selected from the group consisting of integrins (see U.S. Patent No. 7,943,728 and PCT application number PCT / US15 / 46322, which are incorporated by reference in their entirety, respectively, for descriptions of their amino acid sequences), anti-integrin αvβx antibodies (see U.S. Patents No. 6,160,099 and 8,350,010, which are incorporated by reference in their entirety, respectively), anti-integrin α5β1 antibodies, fibronectin targeting integrin isoform αvβx or α5β1 (see U.S. Patent No. 2015 / 0218251, which is incorporated by reference in its entirety, respectively), an integrin-binding peptide, an angiogenic factor, and another protein-binding peptide targeting the Fc domain, wherein x is 1, 3, 5, 6, or 8.

[0040] As used herein, the term “antibody” is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains, two heavy chains, and two light chains interconnected by disulfide bonds. The full-length heavy chain contains a variable region domain VH, as well as three constant region domains CH1, CH2, and CH3. The VH domain is at the amino terminus of the polypeptide, and the CH3 domain is at the carboxy terminus. The full-length light chain contains a variable region domain VL and a constant region domain CL. The antigen-binding fragment (Fab) consists of one light chain and the CH1 and variable region of one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab' fragment consists of one light chain and one heavy chain containing more of the constant region between the CH1 and CH2 domains, and as a result can form an interchain disulfide bond between the two heavy chains to form a diabody. The variable fragment (Fv) region contains variable regions from both the heavy and light chains but lacks a constant region. A single-chain fragment (scFv) is an Fv molecule in which the heavy chain and light chain variable regions are linked by a flexible linker to form a single polypeptide chain that forms an antigen-binding region. Single-chain antibodies are discussed in detail in International Publication No. 88 / 01649 and U.S. Patents 4,946,778 and 5,260,203. As used herein, the term “antibody” includes immunoglobulin molecules having two full-length L chains and two full-length H chains, as well as their fragments such as antigen-binding fragments (Fab), Fv regions, and scFv.

[0041] The term "Fc domain" refers to a molecule or sequence containing the sequence of the non-antigen-binding portion of an antibody, whether in monomeric or polymeric form. The original immunoglobulin source of Fc is preferably of human origin and can be any isotype, e.g., IgG, IgA, IgM, IgE, or IgD. Full-length Fc consists of the following Ig heavy chain regions: flexible hinge regions between CH1 and CH2, and between CH2 and CH3, where the two chains are typically linked by disulfide bonds within the flexible hinge regions.

[0042] The present invention provides a fusion protein comprising a disintegrin and an integrin-binding peptide comprising its integrin-binding fragment, and another protein-binding peptide comprising the extracellular domain and Fc domain of a VEGF receptor, wherein the fusion protein comprises at least one mutation on or adjacent to the RGD motif. According to embodiments of the present invention, the disintegrin and its integrin-binding fragment have an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6 and SEQ ID NOs: 7, or an amino acid sequence having at least 85% sequence identity with SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6 and SEQ ID NOs: 7.

[0043] As used herein, “disintegrin” refers to a class of cysteine-rich proteins or polypeptides that are potent soluble ligands for integrins. The RGD motif is a conserved tripeptide (Arg-Gly-Asp) in most monomeric disintegrins and is located in the integrin-binding loop. The disintegrins described herein are isolated from snake venom or derived from wild-type forms and have at least one mutation on or adjacent to the RGD motif that selectively binds to or targets various integrin isoforms. As used herein, the term “adjacent to the RGD motif” means any mutation occurring at any amino acid residue within 15 to 20 amino acids of the RGD motif in a given peptide, polypeptide, or protein sequence.

[0044] Other amino acid sequence variants of disintegrin are also being considered. For example, the binding affinity and / or other biological properties of disintegrin can be improved by modifying the amino acid sequence that codes for the protein. Disintegrin variants can be prepared by introducing appropriate modifications to the nucleic acid sequence that codes for the protein, or by introducing modifications through peptide synthesis. Such modifications include mutations such as deletions, insertions, and / or substitutions from the nucleic acid or amino acid sequence of disintegrin. Any combination of deletions, insertions, and substitutions can be used to arrive at the final amino acid construct of disintegrin, provided that the final construct has desired characteristics, such as binding to integrin superfamily members and / or inhibition of the integrin activation pathway.

[0045] Substantial modification of the biological properties of a protein or polypeptide is achieved by selecting substitutions that have a significantly different effect on (a) the structure of the polypeptide backbone in the region of substitution, e.g., sheet or helical structure, (b) the molecular charge or hydrophobicity at the target site, or (c) the maintenance of most of the side chain.

[0046] A useful method for identifying specific residues or regions of a fusion protein that are preferred sites for mutagenesis is known as "alanine scanning mutagenesis," as described by Cunningham BC et al. Science. 1989;244:1081-1085. For example, to influence the interaction between an amino acid and a target binding partner, a group of residues or target residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with a neutral amino acid (most preferably glycine, alanine, or leucine) or a reverse-charged amino acid (positively charged to negatively charged or vice versa). The amino acid position that exhibits functional sensitivity to the substitution is then purified by introducing further or other variants into the substitution site or the substitution site. Thus, while the site to introduce amino acid sequence mutations is predetermined, the nature of the mutation itself does not need to be predetermined. For example, random mutagenesis can be performed at a target codon or region to analyze the performance of a mutation at a given site, and the expressed fusion polypeptide variant is screened for desired activity. For example, to improve its stability, cysteine ​​bonds(s) can be added to the fusion protein or protein component.

[0047] Accordingly, disintegrin variants that may be components of any fusion protein disclosed herein are provided. In some embodiments, the disintegrin comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence of a disintegrin selected from the group consisting of rhodostomin (SEQ ID NO: 1), triflavin (SEQ ID NO: 3), exstatin (SEQ ID NO: 4), trimucrine (SEQ ID NO: 5), elegantin (SEQ ID NO: 6), and triglamin (SEQ ID NO: 7). In some embodiments, the disintegrin comprises an amino acid sequence having at least one mutation on or adjacent to the RGD motif of rhodostomin (SEQ ID NO: 1), triflavin (SEQ ID NO: 3), exstatin (SEQ ID NO: 4), trimucrine (SEQ ID NO: 5), elegantin (SEQ ID NO: 6), or triglamin (SEQ ID NO: 7). In some embodiments, the disintegrin comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence of a disintegrin variant (SEQ ID NO: 2). The Xaa in SEQ ID NO: 2 represents various positions that can be modified by insertion, substitution, or deletion to produce amino acid sequence variants different from the wild type of disintegrin. For example, a rhodostomin variant can be created by substituting the Xaa at position 50 in SEQ ID NO: 2, which corresponds to glycine (Gly) in the RGD motif of wild-type rhodostomin (SEQ ID NO: 1), with a naturally occurring amino acid other than glycine. In other examples, one or more Xaa in SEQ ID NO: 2 can also be substituted with naturally occurring amino acids other than those initially found at the corresponding positions in wild-type rhodostomin (SEQ ID NO: 1) to generate a variety of rhodostomin variants. Furthermore, it should be noted that disintegrin variants are not limited to containing only a single mutation in any Xaa in SEQ ID NO: 2, and multiple mutations occurring at several positions of Xaa in SEQ ID NO: 2 or at corresponding positions in other consensus sequences of the disintegrin (e.g., SEQ ID NOs: 3-7) may also be included within the scope of the present invention.

[0048] Rhodostomin variants are described in U.S. Patent No. 7,943,728 and PCT Application No. PCT / US15 / 46322, the sequences of which are incorporated herein by reference. For example, PCT / US15 / 46322 describes a disintegrin variant comprising at least one of a mutant RGD loop having an amino acid sequence selected from the group consisting of SEQ ID NOs. 24 to 26, a mutant linker having an amino acid sequence selected from the group consisting of SEQ ID NOs. 29 to 41, and a mutant C-terminus having an amino acid sequence selected from the group consisting of SEQ ID NOs. 42 to 47. More preferably, the disintegrin variant comprises the mutant RGD loop, mutant linker, and mutant C-terminus described herein.

[0049] For example, mutants of rhodostomin or disintegrin having one or more modifications in addition to the RGD motif in the linker region or C-terminus showed the ability to selectively bind to αvβ3, αvβ5, αvβ6, α5β1, or αIIbβ3. For example, rhodostomin variants having mutations in the linker region (39X40X41X42X43X) in which SRAGK (SEQ ID NO: 50) is replaced by the amino acids KKKRT (SEQ ID NO: 51), KKART (SEQ ID NO: 52), MKKGT (SEQ ID NO: 53), IEEGT (SEQ ID NO: 54), LKEGT (SEQ ID NO: 55), AKKRT (SEQ ID NO: 56), KAKRT (SEQ ID NO: 57), KKART (SEQ ID NO: 58), KKKAT (SEQ ID NO: 59), KKKRA (SEQ ID NO: 60), KAKRA (SEQ ID NO: 61), or SKAGT (SEQ ID NO: 62) showed the highest effect on integrins in the following order: α vβ3 (approximately 14 times) >α5β1 (approx. 5 times) > αIIbβ3 (approx. 2 times) .

[0050] Rhodostomin variants with mutations in the C-terminal region (66X67X68X69X70X), in which RYH is replaced by the amino acids RYH (SEQ ID NO: 63), RNGL (SEQ ID NO: 64), RGLYG (SEQ ID NO: 65), RGLY (SEQ ID NO: 66), RDLYG (SEQ ID NO: 67), RDLY (SEQ ID NO: 68), RNGLYG (SEQ ID NO: 69), or RNPWNG (SEQ ID NO: 70), had the greatest effect on integrins in the following order: αIIbβ3 (approx. 13x) > αvβ5 (approx. 8x) = αvβ6 (approx. 8x) > αvβ3 (approx. 4x) > α5β1 (approx. 2x). Table 1 shows the sequences of SEQ ID NOs. 24-49 and their corresponding positions in SEQ ID NO: 1.

[0051] [Table 1]

[0052] While disintegrin variants have been discussed primarily with reference to the above amino acid sequences, polypeptide or nucleotide sequences encoding snake venom, such as arborabrin, applegin, basilisin, batroxostatin, vitistatin, celeberin, cerastin, clotroxin, durisin, flavoridine, flavostatin, halisin, halistin, jaralasin, jarastatin, quistrin, rachesin, rutosin, morosin, salmonsin, saxatirin, tergeminin, trimestatin, trimutase, usulistatin, viridian, and their variants having at least one mutation on or adjacent to the RGD motif, may also be included in the scope of the present invention.

[0053] While not bound by theory, in this specification, integrins are intended to inhibit the integrin activation pathway by binding to integrin superfamily members and blocking their interaction with polyvalent integrin receptors. In some aspects, disintegrins bind to integrin superfamily members, including but not limited to integrin isoforms αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, and / or αIIbβ3.

[0054] According to the present invention, other protein-binding peptides of the fusion protein may be receptor proteins that bind to targets selected from the group consisting of tumor antigens, TNF receptor superfamily members, Hedgehog family members, receptor tyrosine kinases, proteoglycan-related molecules, TGF-beta superfamily members, Wnt-related molecules, and angiogenesis targets.

[0055] According to some embodiments of the present invention, other protein-binding peptides may specifically bind to angiogenic targets, including, but are not limited to, angiopoietin (ANG), ephrin (Eph), fibroblast growth factor (FGF), neuropilin (NRP), plasminogen activator, platelet-derived growth factor (PDGF), tumor growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), vascular endothelial cadherin (VE-cadherin), tumor necrosis factor alpha (TNF-α), insulin-like growth factor (IGF-1), and their receptors. Accordingly, according to embodiments of the present invention, other protein-binding peptides may include the extracellular portion of receptor proteins that bind to and antagonize angiogenic targets. In other embodiments, other protein-binding peptides may bind to the extracellular portion of angiogenic factor receptors.

[0056] In some embodiments, other protein-binding peptides may be anti-VEGF antibodies that bind to VEGF ligands (see International Publication No. 2015 / 200905 for a description of its amino acid sequence, which is incorporated herein by reference in its entirety) or anti-VEGFR1 or anti-VEGFR2 antibodies that bind to VEGF receptors (see U.S. Patent No. 5,874,542 for a description of its amino acid sequence, which is incorporated herein by reference in its entirety). In other embodiments, other protein-binding peptides may also be anti-PDGF antibodies that bind to PDGF ligands (see U.S. Patent No. 5,094,941 for a description of its amino acid sequence, which is incorporated herein by reference in its entirety) or anti-PDGFRβ antibodies that bind to PDGF receptors (see U.S. Patent No. 9,265,827 for a description of its amino acid sequence, which is incorporated herein by reference in its entirety for any purpose).

[0057] In certain embodiments, the other protein-binding peptide binds to the same VEGF as one of the VEGF receptors (VEGFRs): VEGFR1, VEGFR2, and VEGFR3. In some embodiments, the other protein-binding peptide comprises at least one extracellular portion of any of the VEGFRs described herein. For example, the other protein-binding peptide comprises at least one extracellular portion of VEGFR1 or one extracellular portion of VEGFR2. In another example, the other protein-binding peptide comprises one extracellular portion of VEGFR1, such as Ig-like domain 2 (D2), and one extracellular portion of VEGFR2, such as Ig-like domain 3 (D3). In some embodiments, the other protein-binding peptide comprises one extracellular portion of VEGFR1, such as the amino acid sequence of SEQ ID NO: 8, and one extracellular portion of VEGFR2, such as the amino acid sequence of SEQ ID NO: 9. In some embodiments, the other protein-binding peptide comprises a fusion of the extracellular portions of VEGFR1 and VEGFR2, which includes the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 10.

[0058] In other embodiments, the other protein-binding peptide binds to the same PDGF as either the PDGF receptor (PDGFR): PDGFRα and PDGFRβ. In some embodiments, the other protein-binding peptide comprises at least one extracellular portion of any of the PDGFRs described herein. For example, the other protein-binding peptide comprises at least one extracellular portion of PDGFRα or one extracellular portion of PDGFRβ. In another example, the other protein-binding peptide comprises one extracellular portion of PDGFRβ, such as Ig-like domains 1-3. In some embodiments, the other protein-binding peptide comprises an extracellular portion of PDGFR comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 11.

[0059] In other embodiments, the present invention also provides a fusion protein comprising the amino acid sequence of SEQ ID NO: 1 having at least one mutation on or adjacent to the RGD motif, the amino acid sequence of SEQ ID NO: 2 having at least 85% sequence identity with SEQ ID NO: 1, a human or humanized constant subregion including immunoglobulin CH2 and CH3 domains, and an integrin-binding peptide comprising other protein-binding peptides having Ig-like D2 of VEGFR1 and Ig-like D3 of VEGFR2. In further embodiments of the fusion protein, the integrin-binding peptide has at least 85% sequence identity with SEQ ID NO: 2.

[0060] The term “percent (%) sequence identity” with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after the sequences have been aligned to achieve maximum percent sequence identity and gaps have been introduced as necessary, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the identity percentage of an amino acid sequence or nucleic acid sequence can be achieved in various ways within the scope of the skill of the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al. eds., 1987), and including BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) Software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. For the purposes of this specification, the % amino acid sequence identity of a given amino acid sequence A to or with respect to a given amino acid sequence B is calculated as follows: fraction X / Y multiplied by 100, where X is the number of amino acid residues that are identical matches according to the sequence alignment program in the alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A and B will not be equal to the % amino acid sequence identity of B and A.

[0061] The present invention provides a dimeric fusion protein comprising two fusion proteins, each fusion protein comprising any fusion protein disclosed herein. In one embodiment, the dimeric fusion protein comprises two identical fusion proteins. In another embodiment, the dimeric fusion protein may comprise two different fusion proteins. The fusion proteins disclosed herein may form a polymer of two or more identical fusion proteins, or may form a heterogeneous fusion protein via a polymerizing domain comprising a constant partial region of a human antibody or humanized antibody. In some embodiments, the constant partial region of a human antibody or humanized antibody is selected from the group consisting of IgG Fc regions, IgA Fc regions, IgM Fc regions, IgD Fc regions, and IgE Fc regions. In further embodiments, the constant partial region of a human antibody or humanized antibody is selected from the group consisting of IgG1 Fc regions, IgG2 Fc regions, IgG3 Fc regions, and IgG4 Fc regions. In some embodiments, the partial region comprises the CH2 and CH3 regions of IgG1, IgG2, IgG3, or IgG4. Amino acid sequences encoding immunoglobulins, including the Fc region, are well known in this art.

[0062] The components of a fusion protein may be linked directly to one another, or they may be linked via linkers. Generally, the term “linker” refers to one or more molecules, such as nucleic acids, amino acids, or non-peptide moieties, that can be inserted between one or more component domains. For example, linkers can be used to provide a desired site of interest between components for ease of manipulation. Linkers can also enhance the expression of the fusion protein from a host cell, reduce steric hindrance, and allow its components to assume their optimal tertiary structure and / or interact appropriately with their target molecules. Linker sequences may consist of one or more amino acids naturally linked to the receptor components, or they may be additive sequences used to enhance the expression of the fusion protein, provide a specific site of interest, enable component domains to form an optimal tertiary structure, and / or enhance the interaction between the components and their target molecules.

[0063] Preferably, the linker enhances the flexibility of the fusion protein components without interfering with the structure of each functional component within the fusion protein. In some embodiments, the linker portion is a peptide linker having a length of 2 to 100 amino acids. Exemplary linkers include linear peptides having at least two amino acid residues, such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly(G)n, and Gly-Ser(GS) linkers. GS linkers described herein include, but are not limited to, (GS)n, (GSGSG)n, (G2S)n, G2S2G, (G2SG)n, (G3S)n, (G4S)n, (GGSGG)nGn, and GSG4SG4SG, where n is one or more. An example of a (G)n linker is the G9 linker. Suitable linear peptides include polyglycine, polyserine, polyproline, polyalanine, and oligopeptides consisting of alanyl and / or selinyl and / or prolinyl and / or glycyl amino acid residues. Linker portions may be used to link any of the components of the fusion protein disclosed herein. In some embodiments, a linker is used between the extracellular portion of the receptor protein and the constant subregion of the immunoglobulin. In other embodiments, a linker is used between a disintegrin or a variant thereof and the constant subregion of the immunoglobulin. In certain embodiments, the fusion protein includes a linker between the extracellular portion of the receptor protein and a disintegrin or a variant thereof, and a linker between a disintegrin or a variant thereof and the constant subregion of the immunoglobulin. As implemented in the present invention, the fusion protein may contain at least one linker, but may contain four or fewer linkers.

[0064] The fusion proteins described herein may or may not include a signal peptide that functions to secrete the fusion protein from a host cell. The nucleic acid sequence encoding the signal peptide can be operably ligated to the nucleic acid sequence encoding the protein of interest. In some embodiments, the fusion protein includes a signal peptide. In some embodiments, the fusion protein does not include a signal peptide.

[0065] Furthermore, the fusion protein described in the present invention may include modified forms of protein-binding peptides. For example, the fusion protein component may have post-translational modifications including glycosylation, sialylation, acetylation, and phosphorylation to any protein-binding peptide.

[0066] While embodiments are generally described with reference to two protein-binding peptides contained in the fusion protein, the present invention also envisions a fusion protein incorporating three or more protein-binding peptides to provide any further effects or synergistic effects in terms of inhibiting the process of angiogenesis. For example, there may be additional protein-binding peptides that bind to other angiogenic targets or act as angiogenic factor antagonists linked to the two existing protein-binding peptides.

[0067] Fusion proteins of the pharmaceutical formulations disclosed herein can be purified and identified using commonly known methods such as fractionation on immunoaffinity or ion exchange columns; ethanol precipitation; reversed-phase HPLC; chromatography on cation exchange resins such as silica or DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using Sephadex G-75, for example; ligand affinity using hydrophobic affinity resins, appropriate binding partners immobilized on a matrix; centrifugation; enzyme-linked immunosorbent assay (ELISA); BIACore; Western blot assay; amino acid and nucleic acid sequencing; and biological activity. In some embodiments, the fusion protein is expressed in host cells and purified from host cells using a combination of one or more standard purification techniques, including but not limited to protein A affinity chromatography, protein G affinity chromatography, buffer exchange, size exclusion chromatography, ultrafiltration, and dialysis. Thus, the recovered fusion protein is substantially pure. In further embodiments, the recovered fusion protein has a purity of at least 90%, 95%, 96%, 97%, 98%, or 99%.

[0068] The fusion proteins or fusion protein components disclosed herein can be characterized or evaluated for biological activities including, but not limited to, affinity for target binding partners, competitive binding, inhibitory activity, inhibition of cell proliferation, inhibition of tumor growth, and inhibition of angiogenesis. In some embodiments, the fusion proteins or fusion protein components disclosed herein can be evaluated for biological activity in vitro and in vivo. Many methods for evaluating binding affinity are known in the art and can be used to identify the binding affinity of a fusion protein or fusion protein component to a binding partner by titration. Binding kinetics are determined by the dissociation constant (K). D ) or maximum half-dose effective concentration (EC 50 It can be expressed as a steady-state equilibrium coupling constant, which is expressed as a value.

[0069] In certain embodiments, the fusion protein has an EC of 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM or less for inhibition of activity (e.g., inhibition of angiogenic factor activity and / or integrin activity). 50 It has. In any embodiment of this specification, the fusion protein has K for binding to its binding partner (angiogenic factor and / or integrin). D but 1 0 mM, 500 μM, 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM, 50 pM, 10 pM, or less than 5 pM, and including any value between these numbers.

[0070] In certain embodiments, the isoelectric point (pI) of the fusion protein is 4 It is 0 to 9.0. In certain embodiments, the isoelectric point (pI) of the fusion protein is 4 0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 9.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any value in between. Preferably, the isoelectric point (pI) of the fusion protein is 8.17.

[0071] The present invention also provides a pharmaceutical composition comprising a fusion protein comprising disintegrin, an anti-integrin αvβx antibody, an anti-integrin α5β1 antibody, an integrin-binding peptide selected from the group consisting of fibronectin targeting integrin αvβx or α5β1 and their integrin-binding fragments, another protein-binding peptide targeting angiogenic factors, and an Fc domain, where x is 1, 3, 5, 6, or 8. The composition of the present invention comprises a therapeutically effective amount of the fusion protein.

[0072] The term "therapeutic dose" means the amount of therapeutically active compound required to induce a desired biological or clinical effect. According to embodiments of the present invention, the "therapeutic dose" is an amount sufficient to produce a beneficial or desired outcome, including clinical results. The therapeutic dose can be administered in one or more doses. With respect to a disease condition, the therapeutic dose is an amount sufficient to improve, stabilize, or delay the onset of the disease. According to specific embodiments of the present invention, the therapeutic dose is the amount of fusion protein required to treat or prevent disorders characterized by abnormal angiogenesis, such as diseases characterized by angiogenesis, vascular permeability, edema, inflammation, retinopathy, fibrosis, or cancer.

[0073] The term "potency" refers to the ability of a fusion protein to function as intended at the clinical dose administered.

[0074] In this specification, the term “pharmaceutical preparation” refers to a preparation comprising a pharmaceutically acceptable carrier used for administering a VEGF receptor fusion protein (e.g., aflibercept or convercept) to a target for therapeutic / pharmaceutical purposes.

[0075] In this specification, the term "purity" refers to the absence of contamination in the fusion protein. Contaminants as referred to herein include protein species other than the intended fusion protein molecule, resulting from the compound manufacturing process and / or the degradation of the manufactured protein compound as impurities.

[0076] In some embodiments, the pharmaceutical composition comprising the fusion protein is 0 .5~ 1 00 mg / mL, preferably 4 0~ 8 0 mg / mL, for example 4 0, 50, 60, 70, or 80 mg / mL, most preferably 4 0± 4 The fusion protein is formulated in a buffer at a protein concentration of mg / mL. In other preferred embodiments, the fusion protein is 4The formulation is prepared in a buffer solution with a protein concentration exceeding 0 mg / mL.

[0077] In a particular method of implementation, the buffer is 5 0.5~7.0, more 6 0~6.5, even more 6 It is a buffer having a pH of 0.0. In a particular embodiment, the buffer is 6 0.5 to 8, more 7 ~7.5, even more comfortable 7 It is a phosphate buffer with a pH of 0.2. 5 Sodium phosphate in a concentration of mM to 20 mM, for example, 5 mM, 10 mM, 15 mM, or 20 mM, more preferably. 1 0 mM sodium phosphate; 2 0-60 mM sodium chloride, comfortable 4 0 mM sodium chloride; 1 ~10% weight / volume (w / v) sucrose, more comfortably 5 %w / v sucrose; and 0 Surfactants in a w / v concentration of 0.01 to 0.05%, more preferably 0 Contains 0.03% w / v polysorbate 20.

[0078] In certain embodiments, the pharmaceutical formulation contains a polyol or alcohol selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, benzyl alcohol, polyvinyl alcohol, and polyethylene glycol (PEG) 400 to 12000. 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w / v, etc. 1 %~ 1 Contains at a concentration of 0% w / v.

[0079] In a preferred embodiment, the polyol is 2 5mM~ 2 50 mM, for example 2Trehalose at concentrations of 5 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, or 250 mM. In other preferred embodiments, the polyol is 1 00mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, or 200mM, etc. 1 Trehalose at a concentration of 00-200 mM. In other preferred embodiments, the polyol is 1 This is trehalose at a concentration of 90 mM.

[0080] In certain embodiments, the pharmaceutical formulation includes a buffer. In certain embodiments, the buffer is 1 0mM~ 5 0mM, for example 1 The buffer is selected from the group consisting of sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and trisodium citrate dihydrate at concentrations of 0 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In a preferred embodiment, the buffer is 1 0mM~ 4 0mM, for example 1 The concentration is 0 mM, 20 mM, 30 mM, or 40 mM. In another preferred embodiment, the buffer is 2 0mM to 30mM, for example 2 The concentrations are 0 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. In other preferred embodiments, the buffer is 2 The concentration is 5 mM.

[0081] In certain embodiments, the pharmaceutical formulation further comprises a polysaccharide selected from the group consisting of sodium carboxymethylcellulose, microcrystalline cellulose, or sodium hyaluronate.

[0082] In a preferred embodiment, the buffering agent is 1 0mM~ 4 0mM, for example 1Histidine at concentrations of 0 mM, 20 mM, 30 mM, or 40 mM. In other preferred embodiments, histidine is 2 0mM~ 3 0mM, for example 2 The concentrations are 0 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. In other preferred embodiments, histidine is 2 The concentration is 5 mM.

[0083] In certain embodiments, the pharmaceutical formulation includes a surfactant. In preferred embodiments, the surfactant is 0 0.01%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, or 4.0%, etc. 0 .01~ 4 The concentration is %w / v. In other preferred embodiments, the surfactant is 0 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 1.0% w / v, etc. 0 0.01%~ 1 The concentration is 0.0% w / v. In other preferred embodiments, the surfactant is 0 The concentration is 0.03% w / v.

[0084] In preferred embodiments, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188, and is preferably polysorbate 20. In preferred embodiments, polysorbate 20 is 0 The concentration is 0.03% w / v.

[0085] In some embodiments, the fusion protein is in a pharmaceutical formulation that is stable at -70°C to 5°C for at least two years, such as -70°C, -60°C, -50°C, -40°C, -30°C, -20°C, -10°C, 0°C, or 5°C. In some embodiments, the fusion protein is in a pharmaceutical formulation that is stable at -70°C, -20°C, and / or 5°C for at least six months, for example, six months, seven months, eight months, nine months, ten months, eleven months, or longer. In preferred embodiments, the fusion protein is in a pharmaceutical formulation that is stable at -70°C, -20°C, and / or 5°C for at least one year, two years, three years, four years, five years, six years, seven years, eight years, nine years, or ten years or more. In other preferred embodiments, the fusion protein is in a pharmaceutical formulation that is stable at -70°C, -20°C, and / or 5°C for at least two years.

[0086] In some embodiments, the formulation retains protein purity and potency for at least 6 months at -70°C to 25°C, for example -70°C, -60°C, -50°C, -40°C, -30°C, -20°C, -10°C, 0°C, or 5°C or 25°C. In some embodiments, the formulation retains protein purity and potency for at least 6 months at -70°C, -20°C, and / or 5°C, such as 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years or more.

[0087] In some embodiments, the formulation further comprises a salt. In some embodiments, the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

[0088] In certain embodiments, the formulation further comprises at least one amino acid. In some embodiments, the amino acid is selected from the group consisting of arginine, histidine, methionine, proline, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

[0089] In some embodiments, the pharmaceutical formulation comprises a fusion protein at concentrations of 40 mg / mL, 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20, and the formulation 6 It is at a pH of 0.0.

[0090] The present invention also provides a method for producing any formulation described herein, comprising the step of combining the components of the formulation into a single composition. Such a method may include the step of adding the resulting formulation to a vial or injection device. The method may further include a sterile filtration step. Any composition that is a product of such a method also forms part of the present invention. For example, embodiments herein also include a method for preparing a formulation by combining a histidine-based buffer with a surfactant (such as polysorbate 20), a fusion protein, trehalose, and optionally one or more additional components, as discussed herein.

[0091] The present invention also relates to the use of compositions according to the present invention for treating or preventing integrin-related diseases in individuals or subjects. “Individual” or “subject” is a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, a method for treating or preventing one or more aspects or symptoms of a disease includes administering an effective amount of a composition containing a fusion protein to an individual.

[0092] The methods described herein can be used to treat a variety of diseases, including but not limited to inflammatory diseases, ocular diseases, autoimmune diseases, or cancer. In some embodiments, the diseases to be treated include rheumatoid arthritis, inflammatory arthritis, osteoarthritis, cancer, histo / organ fibrosis, retinitis pigmentosa, uveitis (anterior or posterior uveitis, etc.), and ocular diseases characterized by neovascularization or ischemia (corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, angioretinopathy, diabetic retinopathy without DME (DR, non-proliferative This includes, but is not limited to, diabetic retinopathy (DR, non-proliferative and proliferative DR), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA) and / or vitreomacular traction (VMT), etc.

[0093] The compositions described herein can be administered to an individual via any route, including but not limited to intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intratracheal, subcutaneous, intrathecal, percutaneous, transpleural, topical, mucosal, gastrointestinal, intra-articular, intrasacral, intraventricular, intracranial, intraurethral, ​​intrahepatic, and intratumoral. In some embodiments, the compositions are administered systemically (e.g., by intravenous injection). In some embodiments, the compositions are administered topically (e.g., by intraarterial or intraocular injection).

[0094] In some embodiments, the composition is administered directly to the eye or ocular tissue. In some embodiments, the composition is administered topically to the eye, for example, as eye drops. In some embodiments, the composition is administered by injection into the eye or tissues related to the eye. The composition can be administered, for example, by intraocular injection, periocular injection, subretinal injection, intravitreous injection, suprachoroidal injection, transseptal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjunctival injection, sub-Tenon's capsule injection, retrobulbar injection, peribulbar injection, or posterior parascleral delivery. These methods are known in the art. The composition can be administered, for example, to the vitreous humor, aqueous humor, sclera, conjunctiva, the area between the sclera and conjunctiva, retinal choroidal tissue, macula, or other areas within or near the eye of an individual.

[0095] In some embodiments, the pharmaceutical composition is 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg per eye, etc. 0 It is administered to the eye in doses of 0.03 to 10 mg. In preferred embodiments, the pharmaceutical composition is administered per eye in doses of 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 5.5 mg, or 6 mg, etc. 3 It is delivered to the eye in doses of 0.0 to 6.0 mg. In other preferred embodiments, the pharmaceutical composition is delivered per eye 4 It is delivered to the eye in mg doses.

[0096] The optimal effective amount of the composition can be determined empirically and depends on the type and severity of the disease, the route of administration, the progression of the disease and the patient's health status, the mass and body area of ​​the individual. Such determination is within the scope of the art of the art. The composition containing the fusion protein may also be administered six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every six months, once every nine months, or once a year.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to describe the methods and / or materials cited in those publications.

[0098] Embodiment This application includes, but is not limited to, embodiments numbered as follows:

[0099] Embodiment 1 is a) 0 0.5 mg / mL~ 1 A fusion protein at a concentration of 20 mg / mL, b) 1 %~ 1 A polyol or alcohol selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, benzyl alcohol, polyvinyl alcohol, and polyethylene glycol (PEG) 400-12000, at a concentration of 0% w / v, c) 1 0mM~ 5 A buffer selected from the group consisting of sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and trisodium citrate dihydrate, at a concentration of 0 mM. d) 0 .01~ 4 A surfactant at a concentration of %w / v, A pharmaceutical preparation containing, The formulation 5 The formulation has a pH of 0.5 to 7.5 and optionally further contains a polysaccharide selected from the group consisting of sodium carboxymethylcellulose, microcrystalline cellulose, or sodium hyaluronate.

[0100] Embodiment 2 is the pharmaceutical formulation according to Embodiment 1, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188, and is preferably polysorbate 20.

[0101] Embodiment 3 is the pharmaceutical formulation according to Embodiment 2, wherein the polysorbate is polysorbate 20.

[0102] Embodiment 4 is a surfactant 0 The pharmaceutical preparation is one of the embodiments 1 to 3, with a concentration of 0.03% w / v.

[0103] Embodiment 5 is a fusion protein 1 mg / mL~ 9 0 mg / mL, preferably 2 0 mg / mL~ 8 The concentration is 0 mg / mL, and more preferably the fusion protein is 4 The pharmaceutical preparation is one of the embodiments described in any one of Embodiments 1 to 4, with a concentration of 0 mg / mL.

[0104] Embodiment 6 is a fusion protein 4 This is the pharmaceutical formulation described in Embodiment 5, with a concentration of 0 mg / mL.

[0105] Embodiment 7 is a polyol 2 5mM~ 2 50 mM, preferably 1 The pharmaceutical preparation is a trehalose preparation with a concentration of 90 mM, as described in any one of Embodiments 1 to 6.

[0106] Embodiment 8 is a case in which trehalose 1 This is the pharmaceutical formulation described in Embodiment 7, with a concentration of 90 mM.

[0107] Embodiment 9 is a buffering material, 1 0mM~ 4 0 mM, preferably 20mM~ 3 Histidine at a concentration of 0 mM, more preferably histidine 2 The pharmaceutical preparation is one of the embodiments 1 to 8, with a concentration of 5 mM.

[0108] Embodiment 10 is a configuration in which histidine 2 This is the pharmaceutical formulation described in Embodiment 9, with a concentration of 5 mM.

[0109] Embodiment 11 is a fusion protein in the following order from the N-terminus to the C-terminus: a) Extracellular domain of vascular endothelial growth factor receptor (VEGFR), b) The Fc domain of human immunoglobulin G, and c) Integrin-binding protein or its fragment A pharmaceutical formulation according to any one of Embodiments 1 to 10, including the above.

[0110] Embodiment 12 is a pharmaceutical formulation according to any one of Embodiments 1 to 11, wherein the fusion protein includes SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0111] Embodiment 13 has a pH 5 .5~ 7 0, preferably pH 6 The pharmaceutical preparation is .0, as described in any one of Embodiments 1 to 12.

[0112] Embodiment 14 has a pH of 6 The pharmaceutical formulation described in Embodiment 13 is 0.0.

[0113] Embodiment 15 is a pharmaceutical formulation according to any one of Embodiments 1 to 14, which is stable for at least 24 months at -70°C, -20°C and / or 2 to 8°C.

[0114] Embodiment 16 is a pharmaceutical formulation according to any one of Embodiments 1 to 15, which retains protein purity and potency for at least 6 months at -70°C, -20°C and / or 2 to 8°C, preferably 2 to 8°C.

[0115] Embodiment 17 is, 1 The pharmaceutical preparation according to any one of Embodiments 1 to 16 further comprises a salt with a concentration of 0 mM to 50 mM.

[0116] Embodiment 18 is a pharmaceutical formulation according to Embodiment 17 in which the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

[0117] Embodiment 19 is, 1 The pharmaceutical formulation according to any one of Embodiments 1 to 18 further comprises at least one amino acid in a concentration of 0 mM to 50 mM.

[0118] Embodiment 20 is the pharmaceutical formulation according to Embodiment 19, wherein the amino acid is selected from the group consisting of arginine, methionine, proline, histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

[0119] Embodiment 21 is a pharmaceutical formulation according to any one of Embodiments 1 to 20, used in a method for treating an eye disease.

[0120] Embodiment 22 describes eye diseases such as neovascularization or ischemic uveitis, retinal vasculitis, retinal pigment striata, retinitis pigmentosa, corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy without DME (DR, non-proliferative and proliferative DR), and diabetic retinopathy with DME (DR, non-proliferative and The pharmaceutical formulation described in Embodiment 21 is selected from proliferative DR, diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA), and / or vitreomacular traction (VMT).

[0121] Embodiment 23 is a formulation in which, 0 0.03-10 mg / eye, preferably 3 It is administered at a dose of 0.0 to 6.0 mg / eye, and more preferably the formulation is 4 This is a pharmaceutical preparation according to any one of Embodiments 1 to 22, administered at a dose of mg / eye.

[0122] Embodiment 24 is a formulation that is applied to the eye. 4 This is the pharmaceutical preparation described in Embodiment 23, administered in a dose of mg.

[0123] Embodiment 25 is, a) Fusion protein at a concentration of 40 mg / mL, b) 25 mM histidine, c) 190 mM trehalose, sucrose, or mannitol d) 0.03% polysorbate 20 or polysorbate 80 A pharmaceutical preparation containing, The formulation 6 It is a pharmaceutical product with a pH of 0.0.

[0124] Embodiment 26 is an embodiment in which the fusion protein is arranged in the following order from the N-terminus to the C-terminus: a) Extracellular domain of vascular endothelial growth factor receptor (VEGFR), b) Fc domain of human immunoglobulin G, c) Integrin-binding protein or its fragment This is a pharmaceutical formulation according to Embodiment 25, which includes [the specified element].

[0125] Embodiment 27 is the pharmaceutical formulation according to Embodiment 26, wherein the fusion protein comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0126] Embodiment 28 is a pharmaceutical formulation according to any one of Embodiments 25 to 27, which is stable for at least 24 months at -70°C, -20°C and / or 5°C.

[0127] Embodiment 29 is a pharmaceutical formulation according to any one of Embodiments 25 to 28, which retains protein purity and potency for at least 6 months at -70°C, -20°C and / or 2 to 8°C, preferably 2 to 8°C.

[0128] Embodiment 30 is, 1 The pharmaceutical preparation according to any one of embodiments 25 to 29 further comprises a salt with a concentration of 0 mM to 50 mM.

[0129] Embodiment 31 is a pharmaceutical formulation according to Embodiment 30 in which the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

[0130] Embodiment 32 contains at least one amino acid 1 The pharmaceutical preparation is described in any one of embodiments 25 to 31, further containing the substance at a concentration of 0 mM to 50 mM.

[0131] Embodiment 33 is a pharmaceutical formulation according to Embodiment 32, wherein the amino acid is selected from the group consisting of arginine, methionine, proline, histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

[0132] Embodiment 34 is a pharmaceutical formulation according to any one of Embodiments 25 to 33, used in a method for treating an eye disease.

[0133] Embodiment 35 describes eye diseases such as neovascularization or ischemic uveitis, retinal vasculitis, retinal pigment striata, retinitis pigmentosa, corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy without DME (DR, non-proliferative and proliferative DR), and diabetic retinopathy with DME (DR, non-proliferative and The pharmaceutical formulation described in Embodiment 34 is selected from proliferative DR, diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA), and / or vitreomacular traction (VMT).

[0134] Embodiment 36 is a formulation in which, 0 0.03-10 mg / eye, preferably 3 It is administered at a dose of 0.0 to 6.0 mg / eye, and more preferably the formulation is 4 A pharmaceutical preparation according to any one of embodiments 25 to 35, administered at a dose of mg / eye.

[0135] Embodiment 37 is a formulation that is applied to the eye. 4 This is the pharmaceutical preparation described in Embodiment 36, administered in a dose of mg.

[0136] Embodiment 38 is a method for treating a target eye disease, a) 0 0.5 mg / mL~ 1 A fusion protein at a concentration of 20 mg / mL, b) 1 %~ 1 A polyol or alcohol selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, benzyl alcohol, polyvinyl alcohol, PEG400-12000, etc., at a concentration of 0% w / v. c) 1 0mM~ 5 A buffer selected from the group consisting of sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and trisodium citrate dihydrate, at a concentration of 0 mM. d) 0 .01~ 4 A surfactant at a concentration of %w / v, This includes administering to pharmaceutical preparations, pH 5 The method involves a ratio of 0.5 to 7.5, and in some cases, the formulation further comprises a polysaccharide selected from the group consisting of sodium carboxymethylcellulose, microcrystalline cellulose, or sodium hyaluronate.

[0137] Embodiment 39 is the method of Embodiment 38, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188, and is preferably polysorbate 20.

[0138] Embodiment 40 is the method of Embodiment 39, wherein the surfactant is polysorbate 20.

[0139] Embodiment 41 is a surfactant 0 The method according to any one of embodiments 38 to 40, with a concentration of 0.03% w / v.

[0140] Embodiment 42 is a fusion protein 1 mg / mL~ 90 mg / mL, preferably 4 0 mg / mL~ 8 The concentration is 0 mg / mL, and more preferably the fusion protein is 4 This method is according to any one of embodiments 38 to 41, with a concentration of 0 mg / mL.

[0141] Embodiment 43 is a fusion protein 4 This is the method according to Embodiment 42, with a concentration of 0 mg / mL.

[0142] Embodiment 44 is a polyol 1 50mM~ 2 30 mM, preferably 1 The method according to any one of embodiments 38 to 43, wherein the trehalose is at a concentration of 90 mM.

[0143] Embodiment 45 is a case in which trehalose 1 This is the method according to Embodiment 44, with a concentration of 90 mM.

[0144] Embodiment 46 is a buffering agent 2 0mM~ 4 0 mM, preferably 2 0mM~ 3 Histidine at a concentration of 0 mM, more preferably histidine 2 This method is according to any one of embodiments 38 to 45, with a concentration of 5 mM.

[0145] Embodiment 47 is a configuration in which histidine 2 This is the method described in Embodiment 46, where the concentration is 5 mM.

[0146] Embodiment 48 is a fusion protein in the following order from the N-terminus to the C-terminus: a) Extracellular domain of vascular endothelial growth factor receptor (VEGFR), b) The Fc domain of human immunoglobulin G, and c) Integrin-binding protein or its fragment This is a method according to any one of embodiments 38 to 47, including the above.

[0147] Embodiment 49 is the method according to any one of Embodiments 38 to 48, wherein the fusion protein includes SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0148] Embodiment 50 has a pH 6 .0~ 6 5. Preferably, pH 6 The method is according to any one of embodiments 38 to 49, wherein the value is 0.

[0149] Embodiment 51 has a pH of 6 This is the method of embodiment 50, which is 0.0.

[0150] Embodiment 52 is a method according to any one of Embodiments 38 to 51, wherein the formulation is stable at -70°C, -20°C and / or 2 to 8°C for at least 24 months.

[0151] Embodiment 53 is a method according to any one of Embodiments 38 to 52, wherein the formulation retains its protein purity and potency for at least 6 months at -70°C, -20°C and / or 2 to 8°C, preferably 2 to 8°C.

[0152] Embodiment 54 is a formulation 1 The method according to any one of embodiments 38 to 53, further comprising a salt at a concentration of 0 mM to 50 mM.

[0153] Embodiment 55 is the method described in Embodiment 54, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

[0154] Embodiment 56 is a formulation 1The method according to any one of embodiments 38 to 55, further comprising at least one amino acid at a concentration of 0 mM to 50 mM.

[0155] Embodiment 57 is the method according to Embodiment 56, wherein the amino acid is selected from the group consisting of arginine, methionine, proline, histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

[0156] Embodiment 58 describes eye diseases such as neovascularization or ischemic uveitis, retinal vasculitis, retinal pigment striata, retinitis pigmentosa, corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy without DME (DR, non-proliferative and proliferative DR), and diabetic retinopathy with DME (DR, non- The method is according to any one of embodiments 38 to 57, selected from proliferative and proliferative DR, diabetic macular edema (DME), exudative and non-exudative age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA) and / or vitreomacular traction (VMT).

[0157] Embodiment 59 is a formulation 0 0.03-10 mg / eye, preferably 3 It is administered at a dose of 0.0-6.0 mg / eye, and more preferably the formulation 4 The method according to any one of embodiments 38 to 58, administered at a dose of mg / eye.

[0158] Embodiment 60 is a formulation 4 This is the method according to Embodiment 59, administered at a dose of mg / eye.

[0159] Embodiment 61 is a method for treating a target eye disease, comprising a) a fusion protein at a concentration of 40 mg / mL, b) 25 mM histidine, c) 190 mM trehalose, sucrose or mannitol, d) 0.03% polysorbate 20 or polysorbate 80 administering a pharmaceutical preparation containing the above to the subject, <***********887>where the preparation has 6 a pH of 7.0.

[0160] Embodiment 62 is the method according to Embodiment 61, wherein the fusion protein has the following order from the N-terminus to the C-terminus: a) the extracellular domain of vascular endothelial growth factor receptor (VEGFR), b) the Fc domain of human immunoglobulin G, c) an integrin-binding protein or a fragment thereof

[0161] Embodiment 63 is the method according to Embodiment 62, wherein the fusion protein contains SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

[0162] Embodiment 64 is the method according to any one of Embodiments 61 - 63, wherein the preparation is stable at -70°C, -20°C and / or 5°C for at least 24 months.

[0163] Embodiment 65 is the method according to any one of Embodiments 61 - 64, wherein the preparation retains protein purity and potency after at least 6 months at -70°C, -20°C and / or 2 - 8°C, preferably 2 - 8°C.

[0164] Embodiment 66 is the method according to any one of Embodiments 61 - 65, wherein the preparation further contains a salt at a concentration of about 10 mM - 50 mM.

[0165] Embodiment 66 is that the preparation 1 ​The method according to any one of embodiments 61 to 65, further comprising a salt with a concentration of 0 mM to 50 mM.

[0166] Embodiment 68 is a formulation 1 The method according to any one of embodiments 61 to 67, further comprising at least one amino acid at a concentration of 0 mM to 50 mM.

[0167] Embodiment 69 is the method according to Embodiment 68, wherein the amino acid is selected from the group consisting of arginine, methionine, proline, histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

[0168] Embodiment 70 describes eye diseases such as neovascularization or ischemic uveitis, retinal vasculitis, retinal pigment striata, retinitis pigmentosa, corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy without DME (DR, non-proliferative and proliferative DR), and diabetic retinopathy with DME (DR, non-proliferative and proliferative D). R) The method according to any one of embodiments 61 to 69, selected from diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA) and / or vitreomacular traction (VMT).

[0169] Embodiment 71 is a formulation 0 0.03-10 mg / eye, preferably 3 It is administered at a dose of 0.0-6.0 mg / eye, and more preferably the formulation 4 The method according to any one of embodiments 61 to 70, administered at a dose of mg / eye.

[0170] Embodiment 72 is a formulation 4 This is the method of Embodiment 71, in which the dose is administered as mg / eye. [Examples]

[0171] The following examples are provided to illustrate the present invention, but not to limit it. Those skilled in the art will recognize that the following procedures can be modified using methods known to those skilled in the art.

[0172] Example 1: Evaluation of fusion protein 1 in a laser-induced choroidal angiogenesis model in rhesus monkeys. Fusion protein 1 (an IgG1 Fc fusion protein with multiple targets) was evaluated in a monkey model of choroidal neovascularization (CNV) in the eye.

[0173] In the CNV model, rhesus monkeys (n=4 eyes / group) were subjected to laser burns on the retina using photocoagulation to induce choroidal neovascularization for 21 days (-21 to day 0). On day 0, the monkeys were injected into the eyes of a vehicle, 0.5 mg / eye of Lucentis® (0.5 mg / eye), or fusion protein 1 (0.6 mg, 1.0 mg, 1.9 mg / eye) by a single intravitreal (IVT) injection. Efficacy endpoints were evaluated in all groups at baseline before administration (-2 days) and at 14 and 28 days after injection.

[0174] In eyes treated with 0.5 mg of Lucentis®, the mean leakage area score decreased by 91% and 92% at days 14 and 28, respectively, compared to baseline (Figure 1). Mean retinal thickness also decreased by 117% and 120%, respectively, compared to baseline (Figure 2). Lucentis® showed a statistically significant improvement compared to eyes treated with vehicle controls.

[0175] All three dose groups of fusion protein 1 showed recovery of laser-induced retinal thickness and leakage area compared to baseline and pre-study values. The mean leakage area score decreased by 87%–89% and 79%–90% on days 14 and 28, respectively (Figure 1). Mean retinal thickness also decreased by 98%–121% and 104%–129%, respectively (Figure 2). Fusion protein 1 showed statistically significant improvements at doses of 0.6 mg and 1.0 mg compared to eyes treated with vehicle control on days 14 and 28. No dose-dependent effect of fusion protein 1 was observed, as all doses showed similar efficacy.

[0176] Histological examination of the eye using Masson staining for collagen (a precursor of fibrosis) showed a dose-dependent decrease in collagen thickness (ocular fibrosis) for fusion protein 1, but not for the vehicle or Lucentis® treatment groups (Figure 3).

[0177] In conclusion, single IVT injections of fusion protein 1 at doses of 0.6, 1.0, and 1.9 mg / eye effectively inhibited vascular leakage, retinal thickness, and fibrosis in a laser-induced CNV model in rhesus monkeys. Based on the vitreous volume ratio between humans (4 mL) and monkeys (2 mL), these doses are equivalent to human equivalent doses of 1.2 mg, 2 mg, and 4 mg / eye. This supports the proposed doses of fusion protein 1 in the treatment of retinal diseases such as diabetic macular edema (DME) and neovascular age-related macular degeneration (nAMD). Furthermore, the data suggest that fusion protein 1 has an additive effect that benefits patients compared to conventional anti-VEGF monotherapy.

[0178] Example 2: Evaluation of fusion protein 1 in a bleomycin-induced C57BL / 6 mouse pulmonary fibrosis model. The potential antifibrotic activity of fusion protein 1, administered intravenously (IV) at 39 mg / kg, was evaluated in a mouse model of bleomycin-induced pulmonary fibrosis. Mice were challenged with 1.5 U / kg of bleomycin using a PENNCENTURY® intrapulmonary aerosolizer on day 1. Mice were intravenously administered fusion protein 1, nintedanib ethanesulfonate (nintedanib), or vehicle (formulation buffer) daily from day 1 to day 21 and sacrificed on day 22. Total protein was precipitated from the collected lung tissue using 50% trichloroacetic acid for 20 minutes on ice. The samples were centrifuged, and the pellet was mixed with 1 mL of 12N HCl and baked at 110 °C for 14 - 18 hours until the sample was carbonized and dried. The samples were resuspended in 2 mL of deionized water by incubating at room temperature for 72 hours while applying intermittent vortexing. Starting from 0.5 mg / mL, serial dilutions of trans-4-hydroxy-L-proline standard (source: Sigma, USA) were prepared. 200 μL of vortexed sample (or standard) was added to 500 μL of 1.4% chloramine T in 0.5 M sodium acetate / 10% isopropanol (source: Fisher Sci, USA) and incubated at room temperature for 20 minutes. Next, 500 μL of Ehrlich's solution (1.0 M p-dimethylaminobenzaldehyde in 70% isopropanol / 30% perchloric acid) (source: Fisher Sci, USA) was added, mixed, and incubated at 65 °C for 15 minutes. After the samples reached room temperature, the optical density of each sample and standard was measured at 550 nm, and the concentration of lung hydroxyproline (μg / lung) was calculated from the hydroxyproline standard curve. Proline is a major component of collagen and is one of the markers used to evaluate fibrosis formation.

[0179] Lung hydroxyproline was significantly increased (p < 0.05) in the vehicle control group (Figure 4), suggesting the success of inducing lung fibrosis. Multiple administrations of fusion protein 1 significantly inhibited hydroxyproline levels (p < 0.05) compared to the vehicle control, showing an additive effect on these markers after fusion protein 1 injection (Figure 4). Continuous oral exposure to nintedanib had a mild effect on the decrease in hydroxyproline levels (Figure 4).

[0180] Therefore, fusion protein 1 administered intravenously once daily at 39 mg / kg for 21 days was associated with a significant decrease in pulmonary hydroxyproline, a major pulmonary fibrosis marker in mice with bleomycin-induced pulmonary fibrosis.

[0181] Example 3: Preformulation of Fusion Protein 1 for Intravitreal Injection We conducted pre-formulation studies of fusion protein 1 and developed an appropriate liquid formulation of fusion protein 1. The objectives of this research were as follows: 1. To evaluate the compatibility and colloidal stability of fusion protein 1 at various concentrations (40 mg / mL and 80 mg / mL) with selected buffer solutions. 2. To evaluate changes in quality characteristics under accelerated conditions (40°C, 25°C, 2-8°C) after short-term storage. 3. Selection of excipients: tonics, NaCl, polyols (i.e., trehalose, sucrose, mannitol, and sorbitol), and other stabilizers such as methionine and arginine. 4. Evaluate suitability through a Design of Experiment (DoE) plan using the selected buffer and potential stabilizer. 5. Evaluate the effectiveness of the cryoprotectant.

[0182] Formulation development work for the active pharmaceutical ingredient was initiated using a DoE approach to identify appropriate buffers, polyols, surfactants, and other stabilizers. During formulation development, fusion protein 1 was tested at two concentrations: 40 mg / mL and 80 mg / mL. Thermal stress, as well as agitation and freeze / thaw stress, were used during formulation development. Table 2 summarizes the components tested in the following studies and their associated functions.

[0183] [Table 2]

[0184] Colloidal stability and compatibility of fusion protein 1 in various buffers and pH levels. Solubility and diffusion coefficient (D) are commonly used to evaluate protein-protein interactions and also to demonstrate protein colloidal stability. Colloidal stability is an important parameter for representing the long-term integrity of the molecule after dispersion and its resistance to sedimentation / precipitation in solution. In addition, this method can rapidly capture the compatibility of the buffer with the molecule. As a result, fusion protein 1 was subjected to three commonly used ophthalmic buffers: sodium phosphate (NaPi), histidine (His), and citrate buffer (Cit), and the pH could be buffered (Table 3). The attributes of fusion protein 1 were analyzed by high-performance liquid chromatography (SEC-HPLC) (Table 4), dynamic light scattering (DLS) (Table 5), and turbidity (Figure 6).

[0185] [Table 3]

[0186] The diffusion coefficients of fusion protein 1 in phosphate buffer and citrate buffer showed a negative correlation with increasing concentration, suggesting the possibility of increased protein-protein interactions (Figures 5A, 5B, 5E, and 5F). In contrast, repulsive protein-protein interactions of fusion protein 1 in both 6.5 and 6.0 histidine buffers were observed with a positive correlation between the diffusion coefficient and fusion protein 1 at various concentrations (Figures 5C and 5D). Therefore, the repulsive intermolecular interactions of fusion protein 1 in histidine buffer suggest favorable colloidal stability of fusion protein 1.

[0187] As the concentration increased beyond 40 mg / mL to 60 and 80 mg / mL for fusion protein 1, a downward diffusion coefficient was detected for all formulations, even the histidine-buffered formulations. In the case of the histidine-buffered formulations, this suggests that pre-aggregation may occur when the concentration increases above 40 mg / mL. Based on the SEC-HPLC results, the protein formulated at 80 mg / mL showed lower purity in the buffer and larger high molecular weight aggregates for all buffers and pH levels, except for histidine buffer pH 6.5 (Table 4). Compared with the 40 mg / mL sample, an increase in subvisible particles (100-1,000 nm, by DLS) was detected in all 80 mg / mL formulations (Table 5). There was no significant difference in turbidity between the 40 mg / mL and 80 mg / mL samples (Figures 6A and 6B). In summary, DLS particle size analysis showed an increase in subvisible aggregates for all buffers as the protein concentration increased from 40 mg / mL to 80 mg / mL. A slight increase in HMW by SEC-HPLC was detected for all formulations except histidine buffer (pH 6.5), and no significant increase in turbidity was observed for any of the formulations.

[0188] [Table 4]

[0189] [Table 5]

[0190] Buffer selection and evaluation of one attribute of the fusion protein. The feasibility and accelerated stability of both 40 and 80 mg / mL fusion protein 1 formulations in 25 mM citrate or histidine buffer at pH 6.0 were tested. The amount of non-aggregated (dimeric) fusion protein 1 in citrate and histidine buffers stored at 2–8°C showed no significant change during storage for both 40 and 80 mg / mL concentrations (Table 6). However, under accelerated conditions at 40°C, the main peak of 80 mg / mL fusion protein 1 decreased by approximately 70% in the citrate buffer after 28 days (Table 7). In comparison, the fusion protein 1 content decreased by approximately 50% when formulated in histidine buffer. Furthermore, the protein concentration of 80 mg / mL fusion protein 1 decreased by 29% in the citrate buffer compared to no decrease in the histidine buffer. Therefore, fusion protein 1 in histidine buffer is more stable at pH 6.0 than in citrate buffer.

[0191] [Table 6]

[0192] [Table 7]

[0193] Furthermore, fusion protein 1 in citrate buffer showed a greater decrease in main band intensity at approximately 250 kDa in non-reducing SDS-PAGE (Figures 7A-7E) after incubation at 40°C (Figures 8A-8E). The proteolysis profile of fusion protein 1 showed an increase in bands of product degradation fragments and high molecular weight aggregates over time. Analysis by reducing SDS-PAGE showed a decrease in main band intensity and an increase in fragment bands at approximately 75 kDa (Figures 8A-8E). Overall, investigations of protein purity and integrity indicated that fusion protein 1 exhibited better stability in 25 mM histidine buffer at pH 6.0 than in citrate buffer.

[0194] We also evaluated accelerated conditions involving short-term storage for freeze / thawing to investigate changes in the quality attributes of the fusion protein 1 sample. The sample was incubated at 40°C for 4 days. A single freeze-thaw cycle at -70°C and room temperature (RT) demonstrated that fusion protein 1 was stable without polyol protection (Table 8).

[0195] [Table 8]

[0196] Screening of excipients To investigate the suitability of excipients, a pilot study was conducted to evaluate the change in main peak purity of one formulated fusion protein sample by SEC-HPLC. Based on the above study, 25 mM histidine buffer pH 6.0 was selected for further excipient screening tests. The three excipients included were polyols, salts, and amino acids. The evaluation was designed to have 11 conditions and was tested under accelerated conditions at 40°C for 4 days (Table 9).

[0197] [Table 9]

[0198] When each polyol (Table 10, Tests #1-4), a combination of trehalose and NaCl (Test #6), and a combination of sucrose and methionine (Test #11) were combined, the main peak purity was 90% or higher.

[0199] [Table 10]

[0200] The remaining polyol combinations with NaCl showed a slight increase in the percentage of aggregates compared to the tests without NaCl (Table 10, Tests #6, 7, 8, 9). The arginine and sucrose combination, after storage under accelerated conditions, showed a decrease in purity to 73.88% and yielded over 16% aggregates (Table 10, Test #10). As a result, trehalose, sucrose, and mannitol were selected for further experimental design.

[0201] Optimization of candidate formulations using DoE To investigate the significance arising from candidate stabilizers and to clarify the cross-effects between NaCl and individual polyols, selected polyols and various concentrations of NaCl were tested via a planned DoE, as shown in Table 11. Accelerated test conditions and analyses are summarized in Table 12. According to SEC-HPLC results (Table 13), 25 mM His buffer pH 6.0 containing 200 mM polyol / sugar (particularly trehalose and sucrose) provided the best protection of fusion protein 1 against thermal stress, maintaining the high purity of fusion protein 1.

[0202] [Table 11]

[0203] [Table 12]

[0204] After storing 80 mg / mL fusion protein 1 samples at 40°C for 4 and 7 days (Table 12), fusion protein 1 stabilized with 200 mM polyol was stabilized against heat stress in 25 mM histidine buffer at pH 6.0, maintaining a relative purity >80% and providing the best obtained purity of fusion protein 1 (Tests #4 and #6 in Table 13). All samples containing polyol and 50 mM NaCl (Tests #2 and #5) showed lower purity than samples containing polyol only (Tests #4 and #6). This effect was also observed in samples containing 25 mM polyol but no NaCl (Tests #7 and #9) compared to samples containing 25 mM polyol and 50 mM NaCl (Tests #10 and #11). No difference was distinguishable between these combinations at 4°C and 25°C (data not shown).

[0205] [Table 13]

[0206] Higher histidine buffers containing 200 mM polyol maintained the protein's quality attributes, but the gravimetric osmolality had to be maintained within the ophthalmic physiological range of 280–310 mOsm / kg. Therefore, 300 mOsm / kg was set as the target DoE statistical calculation. The component compositions selected to formulate fusion protein 1 at 80 mg / mL were 25 mM histidine buffer at pH 6.0, 190 mM trehalose, sucrose, or a combination of trehalose and sucrose.

[0207] Evaluation of potential excipient compositions in formulas To evaluate the suitability of appropriate components in combined formulations, fusion protein 1 at 80 mg / mL was tested in further studies for use in four candidate formulations. In these studies, the properties of fusion protein 1 were evaluated after treatment with 0.03% polysorbate 20 (PS20) or polysorbate 80 (PS80) in combination with trehalose or sucrose, under accelerated conditions for 1 week and / or 1 month, various freeze-thaw cycles between -20°C and room temperature (RT), and after stirring at 24 and 48 hours (Tables 14A-14B). Short-term suitability of containers was tested in parallel.

[0208] [Table 14A]

[0209] [Table 14B]

[0210] The four candidate formulation compositions showed similar observations regarding changes in attributes in the heat stress test. Fusion protein 1 at a concentration of 80 mg / mL was sensitive to a temperature of 40°C, causing a decrease in the main peak to less than 95% and more than 5% aggregation by 7 days. This was not observed even after storage at 4°C for at least one month (Tables 15 and 16). Furthermore, the relative potency of VEGF and / or integrin αvβ3 binding compared to the reference was within the target range (70–130%), showing only slight changes in VEGF binding and no significant changes in αvβ3 binding after storage under accelerated conditions.

[0211] In particular, size exclusion chromatography studies showed that formulations containing 25 mM histidine, 190 mM trehalose, and 0.03% PS2O, pH 6.0, enabled better stability of fusion protein 1 at both 4°C and 40°C compared to other formulations.

[0212] [Table 15]

[0213] [Table 16]

[0214] The 80 mg / mL fusion protein 1 formulation in the candidate formulations was frozen at -20°C for approximately 23 hours, thawed at 25°C for at least 1 hour, and tested for quality attributes after 3 and 6 cycles. SEC-HPLC analysis did not detect any significant changes in the percentage main peaks of fusion protein 1 and its aggregates (Table 17). The remaining protein was over 95%, and aggregates were less than 5%. Furthermore, the potency of fusion protein 1 binding to VEGF or integrin αvβ3 compared to the reference was reduced to less than 70% in candidates #3 and #4 (Table 17).

[0215] [Table 17]

[0216] The stirring test examined protein stability to simulate potential handling and transport using fusion protein 1 in liquid form. Conditions were accelerated by shaking the vial at 220 rpm for 24 and 48 hours at 25°C. Processing protein samples under such rigorous conditions affected protein purity and binding to the primary target compared to samples exposed under steady-state conditions and at baseline (Table 18).

[0217] [Table 18]

[0218] In addition to important quality attributes such as quantity, purity, and potency, the formation and distribution of subvisible particles after stress conditions were investigated. Dynamic light scattering (DLS) analysis classified the observed subvisible particles by size, polydispersity %, and the proportion of particles of different sizes. The results showed that progress to obtaining concentrated fusion protein 1 up to the desired concentration of 80 mg / mL was not adequately monitored, and therefore, not only was the main fusion protein 1 molecule with a radius of approximately 5 nm detected, but aggregates could also be seen as larger particles, i.e., 10–100 nm or 100–1000 nm at baseline (D0). It was not possible to conclude which candidate formulation buffer performed best by DLS.

[0219] Furthermore, non-reduced and reduced SDS-PAGE analyses were unable to distinguish changes in the integrity of fusion protein 1 after treatment under stress conditions. The osmotic pressures of these tested candidate formulations (Table 19) showed that the other formulations were in a similar range, with the exception of candidate #4, which showed a lower osmotic pressure.

[0220] [Table 19]

[0221] conclusion Based on the results, the composition of Formulation 1, which contains 190 mM trehalose and 25 mM His buffer containing 0.03% PS20, pH 6.0, was specified to be tested as a pilot formulation for a 12-month stability test.

[0222] Example 4 Formulation of Fusion Protein 1 for Intravitreal Injection Stability studies were conducted to develop the final formulation of fusion protein 1.

[0223] Active pharmaceutical ingredient bulk concentration test To select a suitable concentration of fusion protein 1 to support the final bulk API throughout the manufacturing process, both 40 and 80 mg / mL of fusion protein 1 were formulated into the main formulation components (25 mM histidine, 190 mM trehalose, and 0.03% PS20, pH 6.0). Formulations containing both concentrations of fusion protein 1 were tested under short-term accelerated conditions to evaluate key quality attributes of any changes by SEC-HPLC, DLS, and SDS-PAGE analysis.

[0224] Protein content varied between the 40 mg / mL and 80 mg / mL samples during short-term storage at 40°C for 7 days (Table 20). Slightly more aggregates were observed in the 80 mg / mL sample. Results for samples stored at 40°C showed that fusion protein 1 formed lower high molecular weight aggregates in the 40 mg / mL sample compared to the 80 mg / mL sample by days 4 and 7. Test #2 protein sample at 40 mg / mL containing NaCl showed >5% aggregates. Freeze / thaw cycle tests did not show any significant change in the percentage or purity of aggregates for both the 40 and 80 mg / mL samples (data not shown).

[0225] [Table 20]

[0226] Using DLS, subvisible particles and their distribution were analyzed, and all protein samples contained particles in the size range of 10 nm to 100 nm and / or 100 to 1000 nm at baseline, and it was not possible to distinguish any changes after stress testing.

[0227] Pilot formulation stability test Based on the above studies, the selected formulation composition for fusion protein 1 that maintained the most consistent purity based on SEC-HPLC was as follows: 40 mg / mL of fusion protein 1 at pH 6.0, 25 mM histidine, 190 mM trehalose, and 0.03% PS2O. To verify and evaluate compatibility with container systems and to collect initial stability, 40 mg / mL of fusion protein 1 was prepared using this formula, filled into type I borosilicate glass vials (with rubber stoppers and flip-cap seals), and incubated under three conditions: -25°C to -15°C, 2 to 8°C, and 25°C / 60% relative humidity (RH) (Table 21).

[0228] [Table 21]

[0229] The results of the study showed that fusion protein 1 at 40 mg / mL was stable when stored at -20°C for at least 12 months (Table 22). After 6 months under storage conditions at 4°C, there was a slight decrease in purity and an increase in aggregates (Table 23). For accelerated storage at 25°C, SEC-HPLC detected an increase in aggregate percentage over 6 months, but the main peak remained above 95%, and molecular specific potency and integrity (i.e., capillary electrophoresis sodium dodecyl sulfate (CE-SDS) data) were maintained with minimal change (Table 24).

[0230] [Table 22]

[0231] [Table 23]

[0232] [Table 24]

[0233] According to DLS analysis, when the test samples were stored at -20°C and 4°C for at least 12 months, no subvisible particles were detected, except for one sample stored at 4°C that had experienced one freeze / thaw cycle at the 12-month mark and formed a trace amount (2%) of larger particles (radius 2208 nm). Larger particles (radii 2195 and 2264 nm) were detected in samples stored at 25°C for 3 and 6 months.

[0234] A preliminary sample stored at 4°C was analyzed to confirm the presence of subvisible particles observed after 12 months. This sample was also stored for 12 months and subjected to freeze / thaw cycles. Subvisible particles were observed in the tested preliminary sample. Therefore, fusion protein 1 is sensitive to freeze / thaw stress after 12 months of storage at 4°C. As a result, fusion protein 1 can be stored at 4°C for at least 12 months without freeze / thaw cycles. However, for storage of fusion protein 1 longer than 6 months, storage at -20°C is recommended.

[0235] The viscosity of the formulation was determined using a viscometer. The target formulation (40 mg / mL fusion protein 1 in 25 mM histidine buffer containing the selected component) had a low viscosity of 5.402 cP (centipoise) (Table 25). The selected formulation exhibits favorable thermal properties for the protein, as shown by the representative differential scanning calorimetry (DSC) thermogram in Figure 9. The deconvoluted thermogram shows four thermal transition peaks with Tm values ​​of 61.47°C, 66.51°C, and 81.55°C–84.21°C.

[0236] [Table 25]

[0237] conclusion In light of the results of this study, fusion protein 1, formulated at 40 mg / mL in 25 mM histidine, 190 mM trehalose, and 0.03% PS20, pH 6.0, was compatible with the selected container-closed system and remained stable for at least 12 months when stored at -20°C and 2°C–8°C.

[0238] Example 5: Long-term stability study of fusion protein 1. Studies were conducted to investigate the long-term stability of fusion protein 1 formulated with 25 mM histidine, 190 mM trehalose, and 40 mg / mL in 0.03% PS20, pH 6.0, at various temperatures. Fusion protein 1 showed stability for two years after storage at -70°C (Table 26). Fluctuations in EC50 at αvβ3 and α5β1 binding were observed at months 9, 18, and 24 (T9, T18, and T24) and at months 9, 12, and 24 (T9, T12, and T24). These fluctuations were investigated and suggested that they may be due to expired test reagents.

[0239] [Table 26]

[0240] A study is underway to investigate the stability of fusion protein 1, formulated with 25 mM histidine, 190 mM trehalose, and 40 mg / mL in 0.03% PS20, pH 6, after storage at -20°C for 36 months. A summary of the stability results after storage at -20°C for 9 months is shown in Table 27.

[0241] [Table 27]

[0242] Furthermore, studies are underway to investigate the stability of fusion protein 1 formulated with 25 mM histidine, 190 mM trehalose, and 40 mg / mL in 0.03% PS20, pH 6, after storage at 5°C for 36 months. Tables 28 and 29 summarize the stability results after storage at 5°C for 9 months, either upright or upside down.

[0243] [Table 28]

[0244] [Table 29]

[0245] Furthermore, a study was conducted to test the stability of fusion protein 1, formulated with 25 mM histidine, 190 mM trehalose, and 40 mg / mL in 0.03% PS20 at pH 6, after storage at 25°C for 6 months. A summary of the stability results after storage at 25°C for 6 months is shown in Table 30.

[0246] [Table 30]

[0247] Example 6 VEGF-A 165 Vascular leakage score in rabbits treated with a fusion protein 1 formulation after stimulation by [agent]. In vivo studies were conducted to test the efficacy of various doses of fusion protein 1 (40 mg / mL of fusion protein 1 in 25 mM histidine, 190 mM trehalose, and 0.03% PS20, pH 6) formulated into the above-mentioned target formulation 9. Human VEGF-induced retinal vascular leakage was examined in Dutch belt rabbits using a defined leakage score based on standardized fluorescence angiography (FA) scoring images. Leakage scores were classified from 0 to 4 (0, major vessels are very straight and minor vessels are somewhat tortuous; 1, increased tortuousness and / or vasodilation of major vessels; 2, leakage between major vessels; 3, leakage between major and minor vessels, minor vessels are still visible; 4, leakage between major and minor vessels, minor vessels are not visible).

[0248] Each rabbit received a single bilateral intravitreal injection of 50 μL, with each group consisting of a total of 3 rabbits and 6 eyes. On day 0, the rabbits were administered 50 μL of vehicle control, Avastin® (1.25 mg / eye), Eylea® (0.625 mg / eye), and fusion protein 1 at increasing concentrations (i.e., 0.03 mg, 0.1 mg, 0.3 mg, and 1 mg per eye). Subsequently, on day 2, the rabbits were given 1000 ng of human VEGF-A 165 The cells were stimulated, and the leakage score was assessed on day 5 using FA.

[0249] Rabbits treated with a low dose of fusion protein 1 (0.03 mg) showed a reduction in vascular leakage scores (Figure 10). Therefore, a low dose of fusion protein 1 (0.03 mg) effectively reduces vascular leakage.

[0250] The dose of fusion protein 1 was equivalent to that of Avastin® and Eylea® in its ability to inhibit VEGF-induced retinal leakage. These data support the suitability of fusion protein 1 for the treatment of neovascular retinal diseases such as DME, nAMD, and RVO.

Claims

1. a) A fusion protein with a concentration of 0.5 mg / mL to 120 mg / mL, A fusion protein containing SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, b) Trehalose at concentrations of 170 mM to 225 mM, c) A buffer selected from the group consisting of sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and trisodium citrate dihydrate, in a concentration of 10 mM to 50 mM. d) A surfactant at a concentration of 0.01 to 4% w / v, A pharmaceutical preparation containing, The preparation has a pH of 5.5 to 7.5, and optionally further comprises a polysaccharide selected from the group consisting of sodium carboxymethylcellulose, microcrystalline cellulose, or sodium hyaluronate.

2. The pharmaceutical formulation according to claim 1, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188.

3. The pharmaceutical formulation according to claim 1 or claim 2, wherein the surfactant has a concentration of 0.03% w / v.

4. The pharmaceutical formulation according to claim 1 or claim 2, wherein the fusion protein has a concentration of 1 mg / mL to 90 mg / mL.

5. The pharmaceutical preparation according to claim 1 or claim 2, wherein the concentration of the trehalose is 175 mM to 200 mM.

6. The pharmaceutical preparation according to claim 1 or claim 2, wherein the buffering agent is histidine at a concentration of 10 mM to 40 mM.

7. A pharmaceutical preparation according to claim 1 or claim 2, wherein the pH is 5.5 to 7.

0.

8. A pharmaceutical formulation according to claim 1 or claim 2, which is stable for at least 24 months at -70°C, -20°C and / or 5°C.

9. A pharmaceutical formulation according to claim 1 or claim 2, which maintains protein purity and potency for at least six months at -70°C, -20°C, 2 to 8°C and / or 25°C.

10. The pharmaceutical preparation according to claim 1 or claim 2, further comprising a salt in a concentration of 10 mM to 50 mM.

11. The pharmaceutical formulation according to claim 1 or claim 2, further comprising at least one amino acid at a concentration of 10 mM to 50 mM.

12. The pharmaceutical preparation according to claim 10, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

13. The pharmaceutical preparation according to claim 11, wherein the amino acid is selected from the group consisting of arginine, methionine, proline, histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

14. A pharmaceutical formulation for treating an angiogenic disease, fibrous disease, or inflammatory disease related to the target eye, comprising the pharmaceutical formulation described in claim 1.

15. The aforementioned neovascular diseases, fibrous diseases, or inflammatory diseases related to the eye include neovascularization or ischemic uveitis, retinal vasculitis, retinal pigment striata, retinitis pigmentosa, corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy without DME (DR, non-proliferative and proliferative DR), and diabetic retinopathy with DME. A pharmaceutical preparation according to claim 14, selected from (DR, non-proliferative and proliferative DR), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA) and / or vitreomacular traction (VMT).

16. The pharmaceutical preparation according to claim 14 or claim 15, wherein the preparation is administered at a dose of 0.5 to 10 mg / eye.

17. The pharmaceutical formulation according to claim 16, wherein the formulation is administered at a dose of 4 mg / eye.

18. a) A fusion protein with a concentration of 40 mg / mL, Fusion protein containing SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18 b) 25 mM histidine, c) 190 mM trehalose, d) 0.03% polysorbate 20 or polysorbate 80 A pharmaceutical preparation containing, The aforementioned formulation is a pharmaceutical preparation having a pH of 6.

0.

19. The pharmaceutical formulation according to claim 18, which is stable for at least 24 months at -70°C, -20°C and / or 5°C.

20. The pharmaceutical formulation according to claim 18, which maintains protein purity and efficacy for at least six months at -70°C, -20°C and / or 2 to 8°C.

21. The pharmaceutical preparation according to claim 18, further comprising a salt in a concentration of 10 mM to 50 mM.

22. The pharmaceutical formulation according to claim 18, further comprising at least one amino acid in a concentration of 10 mM to 50 mM.

23. The pharmaceutical preparation according to claim 21, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

24. The pharmaceutical formulation according to claim 22, wherein the amino acid is selected from the group consisting of arginine, methionine, proline, histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

25. A pharmaceutical formulation for treating an angiogenic disease, fibrous disease, or inflammatory disease related to the target eye, comprising the pharmaceutical formulation described in claim 18.

26. The aforementioned neovascular diseases, fibrous diseases, or inflammatory diseases related to the eye include neovascularization or ischemic uveitis, retinal vasculitis, retinal pigment striata, retinitis pigmentosa, corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy without DME (DR, non-proliferative and proliferative DR), and diabetic retinopathy with DME. A pharmaceutical formulation according to claim 25, selected from (DR, non-proliferative and proliferative DR), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA) and / or vitreomacular traction (VMT).

27. The pharmaceutical preparation according to claim 25 or claim 26, wherein the preparation is administered at a dose of 0.5 to 10 mg / eye.

28. The pharmaceutical preparation according to claim 27, wherein the preparation is administered at a dose of 4 mg / eye.

29. a) A fusion protein with a concentration of 0.5 mg / mL to 120 mg / mL, A fusion protein containing SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, b) Trehalose at concentrations of 170 mM to 225 mM, c) A buffer selected from the group consisting of sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and trisodium citrate dihydrate, in a concentration of 10 mM to 50 mM. d) A surfactant at a concentration of 0.01 to 4% w / v, A pharmaceutical formulation for treating neovascular disease, fibrous disease, or inflammatory disease related to the target eye, including, A pharmaceutical preparation having a pH of 5.5 to 7.5, and optionally further comprising a polysaccharide selected from the group consisting of sodium carboxymethylcellulose, microcrystalline cellulose, or sodium hyaluronate.

30. The pharmaceutical formulation according to claim 29, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188.

31. The pharmaceutical formulation according to claim 29 or claim 30, wherein the surfactant is present in a concentration of 0.03%.

32. The pharmaceutical formulation according to claim 29 or claim 30, wherein the fusion protein has a concentration of 20 mg / mL to 90 mg / mL.

33. The pharmaceutical formulation according to claim 29 or claim 30, wherein the buffer solution is histidine at a concentration of 20 mM to 40 mM.

34. The pharmaceutical preparation according to claim 29 or claim 30, wherein the pH is 6.0 to 6.

5.

35. The pharmaceutical formulation according to claim 29 or 30, wherein the formulation is stable at -70°C, -20°C and / or 5°C for at least 24 months.

36. The pharmaceutical formulation according to claim 29 or 30, wherein the formulation maintains its protein purity and potency for at least six months at -70°C, -20°C and / or 2 to 8°C.

37. The pharmaceutical formulation according to claim 29 or claim 30, wherein the formulation further comprises a salt in a concentration of 10 mM to 50 mM.

38. The pharmaceutical formulation according to claim 29 or claim 30, wherein the formulation further comprises at least one amino acid at a concentration of 10 mM to 50 mM.

39. The pharmaceutical preparation according to claim 37, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

40. The pharmaceutical preparation according to claim 38, wherein the amino acid is selected from the group consisting of arginine, methionine, proline, histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

41. The aforementioned neovascular diseases, fibrous diseases, or inflammatory diseases related to the eye include neovascularization or ischemic uveitis, retinal vasculitis, retinitis pigmentosa, retinal pigment striata, corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy without DME (DR, non-proliferative and proliferative DR), and diabetic retinopathy with DME. A pharmaceutical formulation according to claim 29 or claim 30, comprising retinopathy (DR, non-proliferative and proliferative DR), diabetic macular edema (DME), exudative and non-exudative age-related macular degeneration (AMD), macular edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatoid proliferation (RAP), polypoid choroidal neovascularization (PCV), vitreomacular adhesion (VMA) and / or vitreomacular traction (VMT).

42. The pharmaceutical preparation according to claim 29 or claim 30, wherein the preparation is administered at a dose of 0.03 to 10 mg / eye.

43. The pharmaceutical formulation according to claim 42, wherein the formulation is administered at a dose of 4 mg / eye.

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