KR20210080733A - A pharmaceutical composition comprising VEGFR fusion protein for preventing and treating angiogenesis related disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition including a fusion protein of immunoglobulin-like domain bound to vascular endothelial growth factor and placental growth factor, as a means for treating cancer or ophthalmic diseases. The present invention also relates to a pharmaceutical formulation including the fusion protein. The fusion protein (KP-VR2) according to the present invention is a polypeptide including: (a) immunoglobulin-like domain 2 of VEGFR1; (b) immunoglobulin-like domain 2 of VEGFR1; and (c) Fc site of human IgG, and has high binding affinity to VEGF and PIGF. In addition, the recombinant fusion protein according to the present invention shows an excellent effect of inhibiting migration and infiltration of vascular endothelial cells and exhibits a significantly high effect of inhibiting proliferation of various tumors and fibroblasts, and thus can be used advantageously as a medicine for treating cancer diseases and ophthalmic diseases caused by angiogenesis.

Description

A pharmaceutical composition comprising a vascular endothelial growth factor receptor fusion protein for preventing and treating angiogenesis-related disease diseases {A pharmaceutical composition comprising VEGFR fusion protein for preventing and treating angiogenesis related disease}

The present invention relates to a vascular endothelial growth factor receptor recombinant fusion protein, which can be used for the preparation of a drug for the prevention, treatment and/or inhibition of angiogenesis-related diseases, and a pharmaceutical composition comprising the same. The fusion protein of the present invention is a multivalent dual-targeting antibody in which the VEGFR extracellular domain is fused to the Fc region of an immunoglobulin-like domain.

Vascular endothelial growth factor (vascular endothelial growth factor, hereinafter VEGF) is a kind of signaling protein, and plays an important role in circulatory system formation (vasculogenesis) and angiogenesis (angiogenesis). VEGF functions as part of a system that stores and supplies oxygen to tissues when blood circulation is inadequate. The general function of VEGF is in embryonic development, muscle after injury or exercise, and the formation of blood vessels that bypass infarcted blood vessels. to create new blood vessels. However, increased amounts of VEGF can cause abnormal angiogenesis. In particular, angiogenesis or angiogenesis is also involved in tumorigenesis and is accompanied by the development of primary or metastatic tumors, and cancer metastasis occurs when cancer cells of the primary cancer are introduced into blood vessels by angiogenesis. Angiogenesis is associated with asthma, dyspnea, endometriosis; acute and chronic inflammation such as atherosclerosis and tissue edema; diseases of infectious agents such as hepatitis and Kaposi's sarcoma; autoimmune diseases such as diabetes, psoriasis, rheumatoid arthritis and thyroiditis; and diseases such as diabetic retinopathy, choroidal neovasularization (CNV), age-related macular degeneration (AMD) and angiofibroma (Carmeliet, P. y Jain, RK. 2000. Nature 407: 249) -257; Kuwano M, et al. 2001. Intern Med 40: 565-572).

VEGF, which plays an important role in angiogenesis, mainly affects the cells constituting the vascular endothelium. According to in vitro results, VEGF stimulates the division and migration of vascular endothelial cells and increases microvascular permeability. VEGF is classified into five types of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PLGF (PlGF, PGF, placenta growth factor) in mammals. VEGF-A promotes angiogenesis, migration of endothelial cells, division, vascular lumen formation, chemotaxis of macrophages and granulocytes, vasodilation, etc., and VEGF-B promotes embryonic angiogenesis, particularly myocardial tissue. promote the formation of VEGF-C promotes lymphangiogenesis, and VEGF-D is required for the development of lymphatic vessels surrounding the lung bronchioles. PlGF plays an important role in vasculogenesis, ischemia, inflammation, wound healing, and angiogenesis in cancer.

VEGF receptor (VEGF receptor, VEGFR) is a receptor using the VEGF as a ligand, and there are three subtypes, such as VEGFR1 (flt-1), VEGFR2 (Kdr/flk-1) and VEGFR3 (flt-4). It exists in the form of a cell membrane receptor (membrane-bound VEGFR, mbVEGFR) or a soluble receptor (soluble VEGFR, sVEGFR) by alternative splicing. The VEGF receptor is normally present in vascular endothelial cells, but is expressed in neurons, Kaposi's sarcoma, and hematopoietic stem cells. When VEGF binds to a VEGF receptor, a signal is transmitted through receptor dimerization along with phosphorylation of receptor tyrosine residues.

VEGF binds with high affinity to its receptors, VEGFR1 and VEGFR2, and mainly transmits a signal through VEGFR2 to induce angiogenesis-related mechanisms such as proliferation and migration of vascular endothelial cells. Thus, VEGF and VEGFR2 have been targets for inhibiting VEGF-induced angiogenesis mechanisms (Ellis and Hicklin, Nature Rev. Cancer, 8:579, 2008; Youssoufian et al., Clin. Cancer Res., 13 :5544s, 2007). A conventionally developed fusion protein-based anti-VEGF, a bi-specific antibody or a multi-specific antibody, is i) a cone composed of VEGFR1 domain 2, VEGFR2 domain 3, domain 4 and IgG1 Fc. Conbercept, ii) VEGF-Trap (Aflibercept) consisting of VEGFR1 domain 2, VEGFR2 domain 3 and IgG1 Fc, and iii) VEGF-Grab consisting of VEGFR1 domain 2, domain 3 and IgG1 Fc.

As a result of the present inventors' diligent efforts to develop multi-target antibodies for anticancer and ophthalmic diseases through inhibition of angiogenesis, surprisingly, they bind to VEGF-A, VEGF-B and PLGF with high sensitivity and specificity, and in vitro and in vivo The present invention was completed by developing a fusion protein of an immunoglobulin-like domain having an excellent inhibitory effect on angiogenesis-related diseases.

Ellis and Hicklin, Nature Rev. Cancer, 8:579, 2008; Youssoufian et al., Clin. Cancer Res., 13:5544s, 2007

As such, an object of the present invention is to provide a pharmaceutical composition for the prevention and/or treatment of angiogenesis-related diseases, including the vascular endothelial growth factor receptor fusion protein.

In addition, an object of the present invention is to provide a pharmaceutical composition comprising a vascular endothelial growth factor receptor fusion protein, and for preventing and/or treating cancer and ophthalmic diseases as diseases related to angiogenesis.

Another object of the present invention is to provide a pharmaceutical formulation for the prevention and/or treatment of angiogenesis-related diseases, including the vascular endothelial growth factor receptor fusion protein.

In addition, an object of the present invention is to provide a pharmaceutical formulation comprising a vascular endothelial growth factor receptor fusion protein, and for preventing and/or treating cancer and eye diseases as diseases related to angiogenesis.

In order to achieve the above object, the present invention provides a vascular endothelial growth factor receptor fusion protein as a tetravalent multi-targeting antibody. The fusion protein of the present invention is a recombinant fusion protein of an immunoglobulin-like domain, composed of (a) an IgG1 Fc domain in which two heavy chains are linked by disulfied bonds and (b) four VEGFR1 immunoglobulin domains 2. and the immunoglobulin domain 2 of VEGFR1 and the immunoglobulin domain 2 of the other VEGFR1 are sequentially fused to each heavy chain of the Fc domain.

In addition, the present invention provides an isolated nucleic acid molecule encoding the recombinant fusion protein.

In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

In another embodiment of the present invention, the present invention provides a host cell transformed with the recombinant expression vector. The recombinant expression vector is inserted into a host cell to form a transformant. Suitable host cells for the recombinant expression vectors include E. coli, Bacillus subtilis (Bacillus subtilis), genus Streptomyces (Streptomyces sp.), Pseudomonas species (Pseudomonas sp . ), Proteus Mirabilis ( Proteus mirabilis ) or genus Staphylococcus ( Staphylococcus sp . ) It may be a prokaryotic cell. In addition, the genus Aspergillus ( Aspergillus sp . ), such as fungi, Pichia pastoris ( Pichia pastoris ), Saccharomyces cerevisiae , Schizosaccharomyces sp . ) and Neurospora crassa crassa ), other lower eukaryotic cells, and eukaryotic cells such as cells of higher eukaryotes such as cells from insects. It may also be derived from plants or mammals. Preferably, monkey kidney cells 7 (COS7: monkey kidney cells) cells, NSO cells, SP2/0, Chinese hamster ovary (CHO: chinese hamster ovary) cells, W138, young hamster kidney (BHK: baby hamster kidney) cells, MDCK, myeloma cell lines, HuT78 cells and HEK293 cells and the like are available.

In the present invention, "transformation" into a host cell includes a method of introducing a nucleic acid into an organism, cell, tissue or organ, and a suitable method can be selected according to the host cell. For example, electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agrobacterium mediated transformation, PEG, dextran sulfate, lipofect Tamine and drying/inhibition mediated transformation methods and the like may be included.

Various recombinant expression vector/host cell combinations can be used to express the fusion protein according to the present invention. Expression vectors suitable for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. . Expression vectors that can be used for bacterial hosts include bacterial plasmids obtained from E. coli such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and derivatives thereof, and plasmids with a wider host range such as RP4. , phage DNA exemplified by a wide variety of phage lambda derivatives such as , λgt10 and λgt11, NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells are plasmids and derivatives thereof. A useful vector for insect cells is pVL941. In the present invention, the recombinant expression vector may be a plasmid, a yeast artificial chromosome (YAC), a yeast episomal plasmid (YEp), a yeast integrative plasmid (YIp), or a recombinant virus.

In one embodiment of the present invention, (a) growing the host cell under conditions capable of producing a fusion polypeptide; And (b) recovering the produced fusion polypeptide, it provides a method for producing a vascular endothelial growth factor receptor fusion protein. The host cell includes a recombinant expression vector in which a nucleotide sequence encoding the fusion polypeptide is operably linked to an expression control sequence. The fusion polypeptide comprises (a) an immunoglobulin-like domain 2 of VEGFR1; (b) immunoglobulin-like domain 2 of VEGFR1; And (c) a recombinant expression vector containing a nucleic acid molecule comprising a nucleotide sequence encoding an Fc region polypeptide of human IgG, can be produced by expressing in a prokaryotic or eukaryotic expression system. The produced fusion polypeptide can be purified by a general method to form a biologically stable protein. For example, purification methods such as dialysis, ion-exchange chromatography, affinity chromatography, high pressure liquid chromatography (HPLC), and polyacrylamide gel electrophesis (PAGE) may be used. , but is not limited thereto.

In addition, the present invention provides a vascular endothelial growth factor receptor fusion protein prepared by the method for producing the vascular endothelial growth factor receptor fusion protein. According to Examples 2 to 4 of the present invention, the fusion protein of the present invention is significantly superior to the prior art aflibercept (Afliberceptⓡ) and bevacizumab (Bevacizumabⓡ) for the target ligands VEGF-A and PlGF. have bonding power. In addition, according to Example 5, when the recombinant fusion protein (KP-VR2) of the present invention is administered to a subject in need of treatment for a VEGF-related disease, it binds with VEGF in the body with very high sensitivity and specificity, thereby anti-VEGF function can be performed. In addition, according to Example 6 of the present invention, the fusion protein (KP-VR2) of the present invention has pharmacokinetic properties similar to that of aflibercept, which is the prior art, and thus replaces the conventionally commercialized anti-VEGF or in combination with it. can do. As such, the present invention provides a pharmaceutical composition for the treatment of tumors comprising a vascular endothelial growth factor receptor fusion protein, and a pharmaceutical composition for the treatment of ocular diseases related to intraocular neovascularization.

In addition, according to Example 7 of the present invention, it was shown that the fusion protein of the present invention inhibited the migration and invasion of vascular endothelial cells (HUVEC) and vascular endothelial-derived cell line (EA-hy923) more effectively than at a lower concentration. . Since cell migration and invasion are important processes for the angiogenesis of vascular endothelial cells, it can be seen that the fusion protein of the present invention can be usefully used as an angiogenesis inhibitor in diseases including cancer and angiogenesis-related eye diseases. In addition, in Examples 8 to 10 of the present invention, the fusion protein (KP-VR2) of the present invention inhibits the growth of various human-derived carcinomas and fibroblasts and their xenografts into nude mice in vitro and in vivo. It shows that the effect is very good.

In one embodiment, examples of tumors that can be treated using the fusion protein of the present invention include epidermoid tumors, squamous tumors such as in the head and neck, colorectal tumors, prostate, breast, lung ( including small cell and non-small cell tumors), pancreas, thyroid, ovarian and liver, Kaposi's sarcoma, central nervous system dysplasia (neuroblastoma, capillary hemangioblastoma, meningioma and brain metastases), melanoma, renal carcinoma, gastrointestinal tumor, rhabdomyolysis sarcoma, glioblastoma, and leiomyosarcoma are presented. In another embodiment of the present invention, the "eye disease related to intraocular neovascularization" is age-related macular degeneration, exudative age-related macular degeneration, choroidal neovascularization, choroidal angiopathy, pathological myopia, diabetic retinopathy, diabetic macular edema. , retinal vessel occlusion, retinopathy of prematurity, or neovascular glaucoma. In addition, the choroidal neovascularization may be myopic choroidal neovascularization, traumatic choroidal neovascularization, choroidal neovascularization caused by uveitis, ocular histoplasmosis, or idiopathic choroidal neovascularization.

In another embodiment of the present invention related thereto, the present invention is a fusion protein of an immunoglobulin-like domain that binds to vascular endothelial growth factor, (a) Fc of IgG1 in which two heavy chains are linked by disulfied bonds domain, and (b) four VEGFR1 immunoglobulin domains 2, wherein the VEGFR1 immunoglobulin domain 2 and the other VEGFR1 immunoglobulin domain 2 are sequentially fused to each heavy chain of the Fc domain, Fc fusion As a pharmaceutical composition comprising a protein as an active ingredient, the Fc fusion protein to the VEGFR1 antigen is 1:2 or more binding, it provides a pharmaceutical composition for the prevention and / or treatment of angiogenesis-related diseases. In another embodiment of the present invention belonging to this category, angiogenesis-related cancer diseases may include colorectal cancer, uterine cancer, stomach cancer, liver cancer, melanoma, lung cancer, kidney cancer, pancreatic cancer, bladder cancer, lymphoma and brain cancer. In addition, angiogenesis-related ophthalmic diseases include uveal melanoma, wet age-related macular degeneration (AMD), diabetic macular edema, retinal vein occlusion, or diabetic retinopathy (diabetic). retinopathy) may be included.

In addition, the present invention (a) immunoglobulin-like domain Fc fusion protein binding to vascular endothelial growth factor (VEGF) as a pharmacologically active ingredient 1 ~ 100 ㎎ / ㎖; (b) 0.01 to 1% (w/v) of polysorbate as a solvent; and (c) sodium phosphate 5-50 mM, sodium citrate 5-50 mM, or sodium phosphate 5-50 mM and sodium citrate 5-50 mM, pH 6.5-7.5. As a pharmaceutical composition for the prevention or treatment of production-related diseases, the Fc fusion protein comprises (i) an Fc domain of IgG1 in which two heavy chains are linked by a disulfied bond, and (ii) four immunoglobulin domains of VEGFR1. Constructed, wherein the immunoglobulin domain 2 of VEGFR1 and the immunoglobulin domain 2 of the other VEGFR1 are sequentially fused to each heavy chain of the Fc domain. Liquid or freeze-dried for the prevention or treatment of angiogenesis-related diseases provided pharmaceutical formulations.

In one related embodiment, the pharmaceutical formulation comprises 1 to 10% (w/v) of one or more stabilizers from the group consisting of sucrose, sorbitol, glycerol, trihalose and mannitol; and 10 to 150 mM of an isotonic agent. In another embodiment belonging to this category, preferably, the Fc fusion protein 25 mg / ml, the polysorbate 0.03% (w / v), the sodium phosphate 10 mM, the sodium citrate 10 mM and the sucrose 5% (w/v) may be included, but is not limited thereto. Also, in another embodiment belonging to this category, preferably, 25 mg/ml of the Fc fusion protein, and 0.03% (w/v) of the polysorbate are included, and the isotonic agent is 40 mM sodium chloride. may be included, but is not limited thereto. In another embodiment belonging to this category, preferably, the Fc fusion protein 20 ~ 100 mg / ml, the polysorbate 0.03% (w / v), the sodium phosphate 5 mM, the sodium citrate 5 mM and The sucrose is 5% (w/v), and the isotonic agent may be 40 mM sodium chloride, but is not limited thereto. In another embodiment belonging to this category, the Fc fusion protein is most preferably at 15 mg/ml, 25 mg/ml, 40 mg/ml, 60 mg/ml, 80 mg/ml and 100 mg/ml. It may be included at a concentration selected from the group consisting of.

In another embodiment of the present invention, a syringe comprising the pharmaceutical composition or pharmaceutical formulation is provided, and the syringe is administered intravenously (intravenous, iv), subcutaneously (sc) or intraperitoneally (intraperitoneal, ip) for administration.

The pharmaceutical composition of the present invention may be administered separately or in parallel with other treatments simultaneously or sequentially depending on the disease to be treated. The pharmaceutical composition of the present invention may include pharmaceutically acceptable excipients, buffers, stabilizers or pharmaceutical carriers regardless of the active ingredient, or other substances well known to those skilled in the art. These substances are non-toxic and do not interfere with the efficacy of the active ingredient, and their precise properties depend on the route of administration: oral, mucosal, or parenteral (eg, intravenous infusion). The fusion protein of the present invention may be administered at a dosage of 0.001 to 5 mg/one eye or 1 to 10 mg/kg.

The vascular endothelial growth factor receptor fusion protein of the present invention is a fusion protein of an immunoglobulin-dead domain that is a tetravalent antibody, and has a structure in which a pair of immunoglobulin domains 2 of VEGFR1 are fused to each heavy chain of a human IgG1 Fc region, VEGF and It has a strong binding affinity to PlGF, an excellent effect of inhibiting the migration and invasion of vascular endothelial cells, and a proliferation inhibitory effect on various carcinomas and fibroblasts. Therefore, the fusion protein of the present invention can be developed as a therapeutic agent for cancer diseases caused by overexpression of vascular endothelial growth factors and ocular diseases related to angiogenesis.

1 schematically shows the fusion protein construct of the present invention.
2a, 2b and 3 show the structure of aflibercept and KP-VR2 (VEGFR fusion protein) of the present invention.
4 is a view showing the size of the molecule by purifying the fusion protein KP-VR2 of the present invention and performing Western blot after SDS-PAGE.
5 is a comparison of the binding affinity of the fusion protein of the present invention, aflibercept and bevacizumab to VEGF-A by an ELISA test.
6 is a comparison of the binding affinity of the fusion protein of the present invention, aflibercept and bevacizumab to PlGF by ELISA test.
7 is a comparison of the maximum binding force to the ligand (VEGFA165, VEGFA121 or PlGF) of the fusion protein KP-VR2 of the present invention.
8 is a SPR test confirming the maximum binding ability of the fusion protein of the present invention and aflibercept to VEGF-A165.
Figures 9a and 9b compare the pharmacokinetic properties of the fusion protein of the present invention according to the route of administration with aflibercept.
Figures 10a and 10b compare the level of the fusion protein of the present invention that inhibits the migration and invasion of umbilical cord-derived vascular endothelial cells (HUVEC) with aflibercept.
11a and 11b are a comparison of the degree of inhibition of migration and invasion of the fusion protein of the present invention in the vascular endothelial cell line (EA-hy926) with aflibercept.
12A and 12B compare the ability of the fusion protein of the present invention to inhibit cell division in 9 types of carcinoma compared to aflibercept.
13 and 14 show the cell division inhibitory ability of the fusion protein of the present invention against anti-VEGF-resistant carcinoma and fibroblasts in comparison with aflibercept.
15 to 17 are comparisons of the growth inhibitory effect of the fusion protein of the present invention and aflibercept on tumors xenografted into nude mice (HT-29, LOVO, and SKUT1B).
18 shows a recombinant expression vector for KP-VR2 of the present invention.
19 shows a recombinant expression vector for KP-VR3 prepared in the present invention.
20 is a comparison result of tumor growth inhibition in pancreatic carcinomas such as ASPC-1 (ATCCⓡ CRL-1682) or MIA PaCa2 cells (ATCCⓡ CRL-1420).
Figure 21 shows the tumor growth inhibitory effect in MKN 45 (KCLB No. 80103), NCI-N87 (ATCCⓡ CRL-25822™), MKN 28 (KCLB No. 80101), or SNU 16 (KCLB No. 00016) gastric cancers. This is the confirmed result.
22 is a result confirming the inhibitory effect of HepG2 (ATCCⓡ HB-8065 ^™ ), SNU423 (ATCCⓡ CRL-2238, A549 (ATCCⓡ CCL-185 ^™ ) or B16F10 (ATCCⓡ CRL-6475 ™) growth.
23 shows Caki-1 (ATCCⓡ HTB-46™), Panc0203 (ATCCⓡ CRL-2553™), PC-3 (ATCCⓡ CRL-1435™) or EL-4 (ATCCⓡ TIB-39) carcinomas. This is the result of confirming the ability to inhibit cell division.
24 is a result of irradiating a laser (laser) to the eye of a rabbit to induce choroidal neovascularization (CNV), and to evaluate the efficacy of the test substance after administration of the test substance.
25 is a result of confirming the impurities generated during storage of the pharmaceutical formulation of the present invention under refrigeration, room temperature and 40° C. conditions by SE-HPLC.
26 is a result of SDS-PAGE analysis of monomers, aggregates, and variants over time while the pharmaceutical formulation of the present invention is refrigerated for 7 days.
27 is a result of SDS-PAGE analysis of monomers, aggregates, and variants over time while the pharmaceutical formulation of the present invention is stored at room temperature for 7 days.
28 is a result of SDS-PAGE analysis of monomers, aggregates, and variants over time while the pharmaceutical formulation of the present invention is stored under harsh conditions for 7 days.
29 is a result of ELISA analysis of monomers, aggregates, and variants over time while refrigerated for 7 days after preparing the pharmaceutical formulation of the present invention to have various pHs.
30 is a result of ELISA analysis of monomers, aggregates, and variants over time, while the pharmaceutical formulation of the present invention was prepared to have various pHs and stored at room temperature for 7 days.
Figure 31 shows the results of ELISA analysis of monomers, aggregates, and variants over time while the pharmaceutical formulation of the present invention was prepared to have various pHs and stored under severe conditions for 7 days.

Hereinafter, the present invention will be described in more detail based on Examples. The following examples are only for easier understanding of the present invention, and the scope of the present invention is not limited by these examples.

Example

Example One: VEGFR fusion protein KP -Production of VR2

human VEGFR2 domain 3 (KP-VR1) fused to the Fc domain of human IgG1; human VEGFR1 domain2 (KP-VR2) (SEQ ID NO: 1) fused to the Fc domain of human IgG1 (SEQ ID NO: 2); human VEGFR2 domain 3 (SEQ ID NO: 3) and VEGFR1 domain 2 (KP-VR3) fused to the Fc domain of human IgG1; Human VEGFR1 domain 2 and VEGFR2 domain 3 (KP-VR4, Aflibercept) fused to the Fc domain of human IgG1 were each cloned into pCHO 1.0 vector (Invitrogen). CHO-S cells (Invitrogen) were transfected with KP-VR1 to KP-VR4 constructs (lipofectamine transformation method, provided by Invitrogen), and the transfected cells were treated with methotrexate and puromycin. was selected as KP-VR2 and KP-VR4 (VEGF-Trap; Aflibercept; trade name: Eylea) were purified by Protein A-Sepharose affinity chromatography. The purified protein was quantified by HPLC analysis, and western blot was performed after SDS-PAGE. Each construct is shown in FIG. 1 . The results of Western blot are shown in FIG. 3 . As shown in Figure 3, it was confirmed that the human VEGFR1 domain 2 (KP-VR2) fused to the Fc domain of human IgG1 of the present invention has a band size of about 125 kDa.

Example 2: of the present invention KP -VR2, Aflibercept ( aflibercept ) and bevacizumab ( bevacizumab )of VEGF - binding affinity for A Measure

The binding affinity of KP-VR2, KP-VR4 (aflibercept) or bevacizumab to VEGF-A ligand was compared. Enzyme immunization assay (ELISA) was used according to the instructions of “BEVACIZUMAB Summary Validation Report” (February 28, 2014). First, a 96-well plate (Nunc) was coated with 50 ng/ml VEGFA165(I) (R&D systems), and KP-VR2, KP-VR4 (aflibercept) or Avastin (bevacizumab) was added in stepwise amounts. (0.0122 ~ 1,000 ng/ml). After washing the plate, it was reacted with peroxidase-conjugated anti-human Fc antibody. Next, 3, 3', 5,5' tetramethylbenzidine (TMB) solution (Roche) was added, and absorbance was measured at 450 nm using an ELISA reader (spectrophotometer, Biorad). The results are shown in FIG. 5 .

In addition, in order to compare the binding amount of KP-VR2 or KP-VR4 (aflibercept) to VEGF-A, a 96-well plate (Nunc) was coated with 2 nM KP-VR2 or KP-VR4 aflibercept and , VEGF165(II) (R&D systems) was added in stepwise increasing amounts (0.03125-64 nM). After washing the plate, it was reacted with anti-human VEGF antibody for 1 hour, and after washing the plate, it was reacted with peroxidase-conjugated anti-goat Ig antibody.

Next, 3, 3', 5,5' tetramethylbenzidine (TMB) solution (Roche) was added, and absorbance was measured at 450 nm using an ELISA reader. The results are shown in FIG. 5 .

Example 3: of the present invention KP -VR2, Aflibercept or of bevacizumab PlGF Comparison of neutralization action

Enzyme immunization was used to determine the binding affinity of KP-VR2, KP-VR4 (aflibercept) or bevacizumab of the present invention to PlGF (placental growth factor). First, a 96 well plate (Nunc) was coated with 50 ng/ml PlGF(I), and KP-VR2 (0.0122 ~ 1,000 ng/ml), aflibercept (0.390625 ~ 16,000 ng/ml) or bevacizumab (0.0122 ~ 1,000 ng/ml) were added in stepwise increments (0.0122 ng/ml ~ 1 uM). After washing the plate, it was reacted with peroxidase-conjugated anti-human Fc antibody. Then, 3,3', 5,5'-tetrabetylbenzidine (TMB) solution (Roche) was added, and absorbance was measured at 450 nm using an ELISA reader. In addition, in order to compare the binding amount of KP-VR2 or KP-VR4 to PlGF, a 96-well plate (Nunc) was coated with 400 ng/ml KP-VR2 or KP-VR4, and the amount of PlGF was increased stepwise ( 0.8 12,500 ng/ml). After incubation with biotinylated-PlGF antibody for 1 hour, the plate was washed and then reacted with peroxidase-conjugated anti-goat Ig antibody (R&D systems). Then, 3,3', 5,5'-tetramethylbenzidine (TMB) solution was added, and absorbance was measured at 450 nm using an ELISA reader. In addition, to compare the binding affinities of KP-VR2 and aflibercept to PlGF, 96-well plates were coated with 50 ng/ml PlGF, and doses of PlGF in stepwise increments with either KP-VR2 or aflibercept were used. (2-31,250 ng/ml) was added. Plates were washed and then reacted with peroxidase-conjugated anti-human Fc antibody. Next, 3,3', 5,5'-tetrabenzidine (TMB) solution was added, and absorbance was measured at 450 nm with an ELISA reader. The results are shown in FIG. 6 .

As shown in FIG. 6 , the titer of KP-VR2 to PlGF increased by about 42-fold compared to that of KP-VR4 (aflibercept), and the maximum binding amount of KP-VR2 to PlGF was KP-VR4 (aflibercept). ) increased by about 200% compared to (FIG. 6). This suggests that KP-VR2 has a remarkably high binding affinity to PlGF. On the other hand, KP-VR2 of the present invention showed a similar level of binding to PlGF as KP-VR4 (aflibercept).

Example 4: of the present invention KP -ligand of VR2 ( VEGFA165 or PlGF ) to compare the maximum combined values for

KP-VR2, KP-VR (aflibercept) or The maximum binding value of bevacizumab to VEGFA165 or PlGF was confirmed through an ELISA test, and the results are shown in Table 1 below (comparison of the binding affinity of KP-VR2, aflibercept and bevacizumab to VEGF and PlGF). It was. As shown in Table 1, it was confirmed that KP-VR2 had about 100% higher binding ability to VEGFA than aflibercept, and about 76% higher binding ability to PlGF.

Ligand binding force ligand Bmax (OD) remark KP-VR2 VEGF-A165 1.30±0.003 106% increase Aflibercept VEGF-A165 0.63±0.008 bevacizumab VEGF-A165 0.55±0.051 KP-VR2 PLGF 1.60±0.03 75.8% increase Aflibercept PLGF 0.91±0.03

In addition, the Octet (Pall ForteBio) test was performed to compare the binding ability of ligands and drugs. First, 200 μl of 2 μg/ml aflibercept and 8 μg/ml KP-VR2 were dispensed on a plate, and VEGF-A (R&D systems) as an analyte was added from 20 nM to 0.315 (20, 10, 5 , 2.5, 1.25, 0.625 and 0.3125) nM, PLGF (R&D systems) from 80 nM to 2.5 nM (80, 40, 20, 10, 5, 2.5, 1.25) diluted by 1/2 and aliquoted in 200 μl did. Aflibercept at a concentration of 2 μg/ml and KP-VR2 at a concentration of 8 μg/ml was attached to the plate, and the binding force was measured using VEGF-A as an analyte. 7A was run by diluting VEGF-A by 1/2 from 20 nM. For the curve fit model, a 1:1 binding model was applied, but in the case of KP-VR2, a 2:1 heterogeneous binding model (KP-VR2) was applied assuming that there are two epitopes. The KD value of aflibercept was measured to be ^{2.69 x 10 - 12.} In the 2:1 heterogeneous binding model, the KD value (<1.00x10-12) beyond the measurement range by the dissociation value in the KD value measured in the binding of KP-VR2 and VEGF-A and the KD value at the level of ^{4.75 x 10 -12} was calculated (Table 2 and Table 3). Table 2 shows the kinetics for VEGF-A, and Table 3 shows the kinetc for PLGA.

Fitting model (Octet) VEGF: receptor KD(M) Kon (1/ms) Kdis(1/s) 1:1 Aflibercept 2.69x10 ^-12 6.01x10 ⁵ 1.64x10 ^-6 2:1 KP-VR2 <1.00x10 ^-12 3.35x10 ⁴ <1.00x10 ^-7 4.75x10 ^-12 6.44x10 ⁴ 3.30x10 ^-7

Fitting model PLGF: Receptor KD(M) Kon (1/ms) Kdis(1/s) 1:1 Aflibercept 2.65x10 ^-10 2.96x10 ⁵ 7.86x10 ^-5 2:1 KP-VR2 7.49x10 ^-10 2.16x10 ⁵ 1.62x10 ^-4 4.85x10 ^-8 1.41x10 ⁵ 6.85x10 ^-3

Example 5: fusion protein KP - with VR2 of aflibercept VEGF -Comparison of maximum bonding force to A165

The Octet (Pall ForteBio) test was performed to compare the maximum binding force between ligands and drugs. 200 μl of aflibercept and KP-VR2 at the same concentration (8 μg/ml) were dispensed into the sensor (ForteBio), and VEGF-A (R&D systems) as an analyte was administered from 40 nM to 1.25 (40, 20, 10, 5). , 2.5, 1.25) diluted by ½ to nM and prepared by aliquoting 200 μl. The maximum binding value of aflibercept to VEGF-A165 was ~ 0.2687 nm, and the maximum binding value of VEGF-A165 in KP-VR2 was ~ 0.5332 nm. The results are shown in Figure 8 and Table 4 (binding affinity of KP-VR2 and KP-VR4 to VEGF). As shown in FIG. 8 , the maximum binding value of KP-VR2 to VEGFA165 was ~0.5332 nm, which was increased by ~98.4% compared to the maximum binding value of aflibercept ~0.2687 nm (Table 4). Therefore, it was confirmed that KP-VR2 of the present invention has a significantly higher level of VEGF-A binding affinity compared to KP-VR4 (aflibercept).

Kinetic binding parameters ligand Rmax(nm) remark KP-VR2 VEGF ~0.5332 98.4% increase Aflibercept VEGF ~0.2687

In addition, the maximum binding value to VEGFA165 or PlGF of KP-VR2 and KP-VR4 (aflibercept) of the present invention was confirmed through an ELISA test, and the results are shown in Table 5 below. Table 5 is a comparison test of binding values to VEGF and PlGF of KP-VR2, aflibercept or bevacizumab. As shown in Table 5, KP-VR2 has a maximum binding amount to VEGFA than aflibercept or bevacizumab. It was 2 times higher, and in the case of PLGF, it was confirmed that binding was 2 times higher than that of aflibercept. Aflibercept or bevacizumab was known to bind a ligand 1:1, and it was confirmed that KP-VR2 could bind a 1:2 ligand.

Molecule Stoichiometry of VEGF-A Stoichiometry of PLGF Bevacizumab 1:1 - Aflibercept 1:1 1:1 KP-VR2 1:2 1:2 Difference in anti-VEGF agents: higher avidity

Example 6. of the present invention KP -VR2's in rats Pharmacokinetics ( pharmacokinetic ) characteristics

To determine the pharmacokinetics (PK) of VR-2 and KP-VR4 (aflibercept), IV (intravenous, intravenous) administration and SC in rats using KP-VR2 and KP-VR4 (aflibercept) (subcutaneous, subcutaneous) administration The ELISA test was performed after dividing into two groups and administering a dose of 1 mg/kg. First, the IV administration group was administered to female SD (Sprague-Dawley) rats (Orient Bio, 6 weeks of age) weighing 180 to 200 g, respectively, immediately after injection of KP-VR2 and KP-VR4 (aflibercept) into the tail vein ( 0H), 5 minutes, 15 minutes, 45 minutes, 3 hours, 5 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 168 hours by collecting blood from the jugular vein of the rat to determine the amount of change in the administered substance (VEGF-TRAP: A VEGF BIOCKER WITH POTENT ANTI TUMOR EFFECTS. PNAS. 2002, 99(17):139311398). Next, the SC administration group was immediately after subcutaneous injection of the test substance (KP-VR2, aflibercept) into the back of the rat's neck to female SD (Orient Bio, 6-week-old, Sprague-Dawley) rats weighing 180 ~ 200 g. (0H), 1 hr, 2 hrs, 4 hrs, 6 hrs, 20 hrs, 22 hrs, 24 hrs, 26 hrs, 28 hrs, 48 hrs, 96 hrs, 168 hrs, 240 hrs, and 336 hrs. The amount of change in the test substance was confirmed by collecting blood from The results are shown in FIG. 9 . As shown in FIGS. 9A and 9B , the KP-VR2 of the present invention has a similar in vivo half-life to that of KP-VR4 (aflibercept) and exhibits similar pharmacokinetic properties.

Example 7: In vascular endothelial cells KP - Analysis of cell migration and invasion inhibition by VR2

Example 7-1. HUVEC (Human umbilical vein endothelial cells) in -VR2 KP, KP-VR4 (Apple River septeu) or bevacizumab-treated cell infiltration deterrent measure derived from a VEGF-A after

In order to measure the degree of inhibition of cell migration and invasion by KP-VR2 of vascular endothelial cells, cell migration was measured using a chemotactic motility assay in a transwell system. First, the transwell system was coated with a growth-reducing factor, Matrigel (Millipore), and then cell invasion was measured by an invasion assay. Quantitative analysis of cell migration and measurement of cell infiltration were performed as follows. Specifically, the lower surface of the transwell was coated with 10 μl of 0.1% gelatin and dried for 24 hours. In order to perform a cell invasion assay (invasion assay), Corning's QCM 24-well cell invasion assay kit (QCM 24-well cell invasion assay kit) was used. After collecting the detached cells with cell invasion assay medium (endothelial cell basal medium, EBM, 0.1% FBS), the number of ^{cells was adjusted to 5 x 10 4} /300 μl cell invasion medium, and each insert (insert) cells were aliquoted. 6 wells were treated with 0-200 nM of KP-VR2, KP-VR4 (aflibercept) (Eylea, trade name), or bevacizumab (Avastin, trade name). 6 wells of VEGF (350 ng/ml) were treated and incubated at 37°C for 48 hours. After completion of the culture, the cells and medium remaining in the insert were removed, and the insert was transferred to a new well. Inserts were placed in 225 μl of cell separation solution and incubated for 30 minutes at 37°C in an incubator. The insert was shaken to completely remove the remaining cells, and 75 μl of lysis buffer/dye solution was added to the cell separation solution and the cell mixture, and then left at room temperature for 15 minutes. 200 μl of the solution was transferred to 96 wells and read with 595 nm fluorescence. The results are shown in FIGS. 10a, 10b, and Table 6 (analysis of cell migration and invasion inhibition by KP-VR2 in vascular endothelial cells).

Inhibition of vascular endothelial cell migration VEGF inhibitor ligand IC ₅₀ (nM) remark KP-VR2 VEGF-A ₁₆₅ 11.00±1.40 Aflibercept VEGF-A ₁₆₅ 20.08±3.95 bevacizumab VEGF-A ₁₆₅ 21.28±2.72

As shown in FIGS. 10a and 10b, VEGF induces cell migration and invasion in HUVEC (Lonza), and KP-VR2, KP-VR4 (aflibercept) or bevacizu) inhibits such cell migration and invasion. It was confirmed that the KP-VR2 of the present invention had better cell migration and invasion inhibitory effects than KP-VR4 (aflibercept) and bevacizumab at the same concentration (13 nM). In addition, as shown in Table 6, the IC _{50 of KP-VR2 of the present invention is about 11 nM, compared with the IC 50} of KP-VR4 (aflibercept) and bevacizumab of 20 nM and 21 nM, respectively. It can be seen that there is a remarkably excellent infiltration inhibitory effect.

Example 7-2. EA - hy926 in cell lines KP -VR2, KP -VR4 ( Aflibercept ) or KP -VR3 ( bevacizumab ) after treatment VEGF -A-derived cell invasion inhibition measurement

First, the EA-hy926 cell line (ATCCⓡ CRL-2922™) was inoculated in a 96-well plate with DMEM medium (Dulbecco's Modified Eagle's Medium Sigma) containing 0.1% FBS at 3.5 x 10 ⁵ cell/ml, and then KP-VR2 (1,500 μg/ml), KP-VR4 (aflibercept) (3,000 μg/ml), and bevacizumab (3,000 μg/ml) were treated and reacted for 24 hours, and the medium not treated with the fusion protein was It was used as a negative control and cultured at _{37° C., 5% CO 2 conditions.} The results are shown in FIGS. 11A and 11B . As shown in FIGS. 11A and 11B , the KP-VR2 of the present invention significantly improved the growth of EA-hy926 cells induced by VEGF compared to KP-VR4 (aflibercept) and bevacizumab even at a lower concentration. effectively suppressed.

Example 8: KP - with VR2 KP -VR4's on carcinoma Comparison of cell division inhibitory ability

In order to confirm the cell division inhibitory ability of KP-VR2 of the present invention for carcinoma, MTS cell proliferation colorimetric assay was performed on 9 types of carcinoma. Table 7 shows the 9 types of carcinoma used in this example.

Kinds biomarker origin Where to buy HT29 VEGF positive Human colorectal adenocarcinoma ATCCⓡ HTB-38™ LoVo VEGF positive Human colorectal adenocarcinoma ATCCⓡ CCL-229™ PLGF positive AGS PLGF positive Human gastric adenocarcinoma ATCCⓡ CRL-1739™ VEGF positive SKUT1b VEGF positive Human uterine sarcoma ATCCⓡ HTB-115™ VEGFR1 positive Caki-1 VEGF positive Renal Clear Cell carcinoma ATCCⓡ HTB-46™ VEGFR1 positive Hy-926 VEGF positive Human Endothelial ATCCⓡ CRL-2922™ VEGFR1 positive HCT 116 VEGF positive Human colorectal carcinoma ATCCⓡ CCL-247™ PANC 02.03 VEGF positive Human Pancreas Adenocarcinoma ATCCⓡ CRL-2553™ SW480 VEGF positive Human colorectal adenocarcinoma ATCCⓡ CCL-228™ PLGF positive PC-3 VEGF positive Human prostate adenocarcinoma ATCCⓡ CRL-1435™ PLGF positive

First, under the conditions of 37°C and 5% CO ₂ , the 9 types of carcinomas HT-29 (ATCCⓡ HTB-38™), LOVO (ATCCⓡ CCL-229™), HCT116 (ATCCⓡ CCL-247™), SKUT1b ( ATCCⓡ HTB-115™), Caki-1 (ATCCⓡ HTB-46™), AGS (ATCCⓡ CRL-1739™), Panc0203 (ATCCⓡ CRL-2553™), SW480 (ATCCⓡ CCL-228™), and PC-3 (ATCCⓡ CRL-1435™) were allowed to reach full confluency. Next, these carcinoma cells were harvested ^{, dispensed at 3 x 10 3} cells/well per well, and cultured at 37°C in RPMI1640 (Sigma) medium containing 10% FBS for 24 hours. Next, the medium was replaced with a medium containing 0.5% FBS, and KP-VR2 or KP-VR4 (aflibercept) was treated at a concentration of 2, 4 or 6 nM and cultured for 72 hours. Finally, MTS reagent (Qiagen) was added, and absorbance was measured at 490 nm, and the results are shown in FIGS. 12A, 12B and Table 8. 12A and 12B, KP-VR2 of the present invention exhibited a significantly superior cell division inhibitory effect than KP-VR4 (aflibercept) on all carcinomas used in the test.

Kinds Approach test method in vitro efficacy KP-VR4 in vitro efficacy KP-VR2 EA-hy926 in vitro efficacy ~ 17% proliferation inhibition ~33% proliferation inhibition HT29 in vitro efficacy - ~ 44% proliferation inhibition LoVo in vitro efficacy ~35% proliferation inhibition ~ 65% proliferation inhibition HCT116 in vitro efficacy - ~ 44% proliferation inhibition SKUT1b in vitro efficacy - ~26% proliferation inhibition CaKi-1 in vitro efficacy - ~ 67% proliferation inhibition PANC0203 in vitro efficacy - ~37% proliferation inhibition SW480 in vitro efficacy - ~38% proliferation inhibition AGS in vitro efficacy - ~34% proliferation inhibition PC-3 in vitro efficacy - ~ 20% inhibition of proliferation

In addition, as shown in Table 8 (cell proliferation inhibitory effect), the KP-VR2 of the present invention had excellent cell proliferation inhibitory activity against 9 types of carcinomas targeting VEGF and PlGF.

Example 9: anti- VEGF tolerance carcinoma and for fibroblasts KP - with VR2 of aflibercept Comparison of cell division inhibition ability

In order to confirm the cell division inhibitory ability of KP-VR2 of the present invention for carcinoma, MTS cell proliferation assay was performed on carcinomas and fibroblasts resistant to anti-VEGFA or anti-VEGFR. Table 9 shows 4 types of carcinoma and 2 types of fibroblasts used in this example.

Kinds biomarker Origin Approach test method
(Stage 1) Approach test method
(Step 2) Where to buy CT26 A model resistant to anti-VEGFR Mouse Colon carcinoma in vitro efficacy Xenograft exam ATCCⓡ CRL-2638™ B16-F10 A model resistant to FOLFIRY(p38) Mouse Skin melanoma in vitro efficacy Xenograft exam ATCCⓡ CRL-6475™ EL4 Anti-VEGF-A refractory mouse lymphoma in vitro efficacy Xenograft exam ATCCⓡ TIB-39 ^™ LCC1 Anti-VEGF-A refractory mouse lung carcinoma in vitro efficacy Xenograft exam ATCCⓡ CRL-1642™ NIH3T3 fibroblast mouse embryo fibroblast in vitro efficacy KCLB NO21658 Hs27 fibroblast human skin fibroblasts in vitro efficacy ATCCⓡ CRL-1634™

First, under the conditions of 37° C. and 5% CO ₂ , the four types of carcinomas (EL-4 (ATCCⓡ TIB-39 ^™ ), LLC1 (ATCC ^ⓡ TIB-39 ^™) , B16F10 (ATCCⓡ CRL-6475™), CT -26 (ATCCⓡ CRL-2638™) and fibroblasts NIH3T3 (Korea Cell Line Bank (KCLB) No 21658) and Hs27 (ATCCⓡ CRL-1634™) were allowed to reach full confluency. And fibroblasts were collected ^{and dispensed at 3 x 10 3} cells/well per well and cultured for 24 hours in RPMI1640 (Sigma) medium containing 10% FBS at 37° C. Then, the medium was replaced with a medium containing 0.5% FBS. and KP-VR2 or KP-VR4 (aflibercept) was treated at a concentration of 2, 4, or 6 nM and incubated for 72 hours. MTS reagent (Qiagen) was added, and absorbance was measured at 490 nm. is shown in Figure 13, Figure 14 and Table 10 (carcinoma and fibroblast).

Kinds Approach test method KP-VR4 KP-VR2 CT26 In vitro efficacy - ~56% proliferation inhibition B16-F10 In vitro efficacy - ~30% proliferation inhibition EL4 In vitro efficacy ~49% proliferation inhibition ~67% proliferation inhibition LLC1 In vitro efficacy ~12% proliferation inhibition ~70% proliferation inhibition NIH3T3 In vitro efficacy ~41% proliferation inhibition ~52% proliferation inhibition Hs27 In vitro efficacy ~15% proliferation inhibition ~25% proliferation inhibition

As shown in FIGS. 13, 14 and 10, KP-VR2 of the present invention has significantly superior ability to inhibit cancer cell proliferation compared to KP-VR4 (aflibercept) (Example 8), and anti-VEGF/ It also showed excellent antiproliferative ability against VEGFR-resistant carcinomas (EL-4, LLC1, B16F10, and CT-26) and fibroblasts (Hs27 and NIH3T3).

Example 10: KP - with VR2 of aflibercept Comparison of tumor growth inhibition in nude mice

For in vivo tests on the tumor growth inhibitory effect of KP-VR2, various tumor xenograft models were used in BALB/c nude mice.

Example 10-1. HT-29 in in vivo xenograft ( xenograft )

First, HT-29 cells (ATCCⓡ HTB-38™) 5 x 10 ⁶ cells/0.2 ㎖ were injected subcutaneously into the back of nude mice (Orient Bio, female 4-week-old), and when the tumor grew to more than 200 ㎣, KP-VR2 (1 mg/kg or 2 mg/kg) and KP-VR4 (aflibercept) (2 mg/kg or 3 mg/kg) were administered intraperitoneally twice a week to mice, respectively, and the same amount was administered intraperitoneally to negative control mice. Phosphate buffered saline (PBS) was administered at the same time and cycle. The volume of the tumor was measured every 3 to 4 days, and the results are shown in FIG. 15 . As shown in FIG. 15 , the inhibitory effect of KP-VR2 of the present invention on the growth of HT-29 colorectal carcinoma increased by about 40% compared to the same amount of aflibercept (KP-VR4) at a dose of 2 mg/kg. (P<0.05). 1 mg/kg KP-VR2 showed similar effects as 3 mg/kg aflibercept (KP-VR4).

Example 10-2. LOVO in in vivo xenograft

Except for using LOVO (ATCCⓡ CCL-229™) instead of HT-29 cells, the same procedure as in Example 10-1 was performed. LOVO 5 x 10 ⁶ cells/0.2 ml was injected subcutaneously into the back of a nude mouse (Samtaco, female 4 weeks old) and when the tumor grew to 200 mm3 or more, KP-VR2 (1 mg/kg) and aflibercept (KP-VR4) ) (1 mg/kg) was administered twice a week to each mouse intraperitoneally. Negative control mice were administered the same amount of PBS (phosphate buffered saline) in the abdominal cavity at the same time and cycle. Changes in tumor volume were measured every 3 to 4 days, and on the final 42 days, tumors were excised and the weights of the tumors were compared. The results are shown in FIGS. 16A and 16B. As shown in FIGS. 16A and 16B , the inhibitory effect of KP-VR2 of the present invention on the growth of LOVO rectal carcinoma was increased by 50-60% compared to the same amount of aflibercept (KP-VR4) at a dose of 1 mg/kg. (P<0.05). The tumor growth inhibitory effect of KP-VR2 of the present invention was observed from the beginning of administration, and the difference showed a tendency to gradually increase. In addition, KP-VR2 of the present invention exhibited a tumor weight reduction effect of about 50% compared to the same amount of aflibercept.

Example 10-3. SKUT1B in in vivo xenograft

In order to further confirm the anticancer activity of KP-VR2 of the present invention, VEGFR1-positive cell line SKUT1B (ATCCⓡ HTB-115™) uterine carcinoma was injected into nude mice instead of HT-29 cells. First, SKUT1B 1 x 10 ⁷ cells/0.2 ml was injected subcutaneously into the back of nude mice, and 1 day later, KP-VR2 (2 mg/kg) and aflibercept (KP-VR4) (2 mg/kg) were administered to the mouse. It was administered intraperitoneally twice a week until day 32, and the same amount of PBS was administered to the negative control mice at the same time and cycle. The tumor volume was measured every 3 to 4 days, and on the last 32 days, the tumor was excised and the weight of the tumor was compared. The results are shown in FIGS. 17A and 17B . As shown in FIGS. 17A and 17B , KP-VR2 of the present invention inhibited the growth of SKUT1B uterine carcinoma by 60 to 70% significantly increased compared to the same amount of aflibercept (KP-VR4) at a dose of 2 mg/kg. effect (P<0.05). In addition, KP-VR2 of the present invention exhibited a tumor weight reduction effect of about 60% compared to the same amount of aflibercept (KP-VR4). Repeated tests were performed under the same conditions, and the results up to the 37th day were reconfirmed. In Fig. 17C, when 1 x 10 ⁷ cells/0.2 ml of SKUT1B was subcutaneously injected into the back of a nude mouse and the tumor size reached 200 mm 3 after 1 week, KP-VR2 (4 mg/kg) and aflibercept (4 mg) /kg) was administered twice a week into the abdominal cavity of each mouse, and the same amount of PBS was administered to the negative control group at the same time and cycle in the abdominal cavity. The KP-VR2 of the present invention showed a significantly increased inhibitory effect on the growth of SKUT1B uterine carcinoma compared with the same amount of aflibercept (P<0.05). The similar test results are shown in FIGS. 17d 17e. 17D and 17E, the KP-VR2 of the present invention significantly increased the growth of SKUT1B uterine carcinoma by 70-80% at a dose of 2 mg/kg compared to the same amount of aflibercept (KP-VR4). It showed an excellent inhibitory effect (P<0.05). In addition, KP-VR2 of the present invention exhibited about 70% tumor weight reduction effect compared to the same amount of aflibercept (KP-VR4).

Example 10-4: ASPC -One ( ATCC ⓡ CRL -1682) or MIA PaCa2 cells (ATCC ⓡ CRL -1420) In pancreatic carcinomas such as Comparative test for tumor growth inhibition

ASPC-1 (ATCCⓡ CRL-1682) and MIA PaCa2 cells (ATCCⓡ CRL-1420) 1 x 10 ⁷ cells/0.2 ml were injected subcutaneously into the back of a nude mouse (Samtaco, 4 weeks old female) and the tumor was over 200 mm3 After confirming that they have grown into , KP-VR2 (5 mg/kg) or bevacizumab injection (5 mg/kg) was administered twice a week to each mouse intraperitoneally, and the same amount of phosphate buffered PBS (phosphate buffered) to the negative control group mice. saline, Gibco) were administered at the same time and cycle. The volume of the tumor was measured every 3 to 4 days, and the results are shown in FIG. 20 . As shown in FIG. 20 , KP-VR2 of the present invention showed a remarkable tumor growth inhibitory effect on the growth of pancreatic carcinoma in comparison with the same amount of bevacizumab at a dose of 5 mg/kg (P<0.001).

Example 10-5: MKN 45 ( KCLB No.80103), NCI-N87 ( ATCC ⓡ CRL -25822™), MKN 28 (KCLB No.80101), or SNU 16 ( KCLB No. 00016) Growth inhibitory effect in gastric cancers Confirm exam

MKN 45, NCI-N87, MKN 28 or SNU16 1 x 10 ⁷ cells/0.2 ㎖ were injected subcutaneously into the back of nude mice, and when the tumor grew to 200 mm3 or more, KP-VR2 (5 mg/kg) and bevacizumab (5 mg/kg) was administered twice a week to each mouse intraperitoneally. Negative control mice were administered the same amount of PBS (phosphate buffered saline) in the abdominal cavity at the same time and cycle. Changes in tumor volume were measured every 3 to 4 days, and on the last day, tumors were excised and the weights of the tumors were compared. The results are shown in FIG. 21 . In FIG. 21B , NCI-N87 carcinoma is a carcinoma resistant to VEGF, showing that it overcomes the limitations of bevacizumab. As shown in FIG. 21 , the KP-VR2 of the present invention exhibited an excellent inhibitory effect of cancer growth on the growth of MKN 45, NCI-N87, MKN28 or SNU16 gastric carcinoma compared to the same amount of bevacizumab at a dose of 5 mg/kg. seemed

Example 10-6: HepG2 ( ATCC ⓡ HB- 8065 ^™ ), SNU423 ( ATCC ⓡ CRL -2238 ™), A549 (ATCC ⓡ CCL-185 ^™ ) or B16F10 ( ATCC ⓡ CRL- 6475 ™) growth inhibition observation

When HepG2 (ATCCⓡ HB-8065), SNU423 (ATCCⓡ CRL-2238) and A549 (ATCCⓡ CCL-185) 5 x 10 ⁶ cells/0.2 ㎖ were injected subcutaneously into the back of nude mice and the tumor grew to over 100 mm3 KP-VR2 (5 mg/kg) and bevacizumab (5 mg/kg) were administered intraperitoneally to mice twice a week, respectively. Negative control mice were administered the same amount of PBS (phosphate buffered saline) in the abdominal cavity at the same time and cycle. Tumor volume change was measured every 3 to 4 days. The results are shown in FIGS. 22A, 22B and 22C. As shown in FIG. 22A , KP-VR2 of the present invention showed a major inhibitory effect on the growth of HepG2 liver cancer compared to the same amount of bevacizumab at a dose of 5 mg/kg. It was also confirmed in FIG. 22B that KP-VR2 of the present invention inhibited the growth of SNU423 liver cancer at a dose of 5 mg/kg compared to the same amount of bevacizumab. In addition, it was confirmed in FIG. 22C that KP-VR2 of the present invention inhibited the growth of A547 lung cancer at a dose of 5 mg/kg compared to the same amount of bevacizumab. In Fig. 22D, 1 x 10 ⁷ cells/0.2 ml of B16F10 (ATCC® CRL-6475) was subcutaneously injected into the back of a nude mouse, and 1 day later, KP-VR2 (2 mg/kg) and aflibercept (2 mg/kg) ) was administered twice a week to each mouse intraperitoneally. Negative control mice were administered the same amount of PBS (phosphate buffered saline) in the abdominal cavity at the same time and cycle. Changes in tumor volume were measured every 3 to 4 days, and on the last day, tumors were excised and the weights of the tumors were compared. The results are shown in FIG. 22D. As shown in FIG. 22 , KP-VR2 of the present invention exhibited an anticancer effect on liver cancer, lung cancer or melanoma.

Example 10-7: Caki -One ( ATCC ⓡ HTB -46™), Panc0203 ( ATCC ⓡ CRL -2553™), PC-3 ( ATCC ⓡ CRL -1435™) or EL-4 ( ATCC ⓡ TIB -39) in cancers Observation of cell division inhibition ability

23 is a result of performing an MTS cell proliferation colorimetric assay in order to confirm the cell division inhibitory ability of KP-VR2 in carcinomas that do not form tumors upon inoculation into nude mice. First, under the conditions of 37° C. and 5% CO ₂ , the four types of carcinomas Caki-1 (ATCCⓡ HTB-46™), Panc0203 (ATCCⓡ CRL-2553™), PC-3 (ATCCⓡ CRL-1435™) or EL-4 (ATCC ^® TIB-39) was allowed to reach full confluency. Next, these carcinoma cells were harvested ^{, dispensed at 3 x 10 3} cells/well per well, and cultured at 37°C in RPMI1640 (Sigma) medium containing 10% FBS for 24 hours. Next, the medium was replaced with a medium containing 0.5% FBS, treated with KP-VR2 or aflibercept at a concentration of 6 μM, and cultured for 72 hours. Finally, MTS reagent (Qiagen) was added, and absorbance was measured at 490 nm, and the results are shown in FIG. 23 . As shown in FIG. 23 , KP-VR2 of the present invention exhibited a significantly superior cell division inhibitory effect compared to aflibercept and bevacizumab lines for all carcinomas used in the test.

Example 10-8: By irradiating a laser into the rabbit's eye choroidal neovascularization (choroidal neovascularization , CNV ) and evaluate the efficacy of the test substance after administration of the test substance

After instillation of mydriatic drug (midriacil 1% eye drop) into the right eye of the animal, anesthesia was performed using Zoletil 50 (VIRBAC, France) and xylazine (Rompunⓡ, Bayer AG, Germany), and the eye was laser ( Elite, Lumenis, USA) was irradiated under the conditions of 532 nm, power 150 mW, duration 0.1 sec, and 6 spots were made at about 6 o'clock with the optic nerve as the center. While the animal was anesthetized, 50 μl of the test substance was intravitreally administered to the right eye of the animal using a syringe equipped with a 31 gagge needle. On Days 0, 3, 7, 10 and 14, after instillation of mydriatic (midriacil 1% eye drop) into the right eye of the animal, Zoletil 50 (VIRBAC, France) and xylazine (Rompunⓡ, Bayer AG, Germany) were administered. After anesthesia was performed, about 1 ml of 2% fluorescein sodium salt solution was injected intravenously, and images were taken within about 2 minutes using a fundus camera (TRC-50IX, TOPCON, Japan). Retinal CNV identification and efficacy evaluation were performed using retinal fluorescence fundus photographs. Image analysis was performed using ImageJ software (NIH, Bethesda, MD), and the fluorescence intensity of the irradiation site was analyzed (FIG. 24A). As a result of retinal fluorescence intensity analysis, the retinal fluorescence intensity levels of the positive control group (G2) and the test substance high dose group (G4) measured on the 7th day (Day 7) after laser irradiation were statistically significant compared to the induced control group (G1). was significantly low (p<0.05), and the retinal fluorescence intensity level of G2 and all test substance administration groups (G3-G4) measured on days 10 and 14 (Day 10 and 14) after laser irradiation was significantly lower than that of G1. (p<0.01 or p<0.05). Figure 24B is a comparison of the residual amount of the drug remaining in the vitreous humor 14 days after the drug administration, the drug remained in proportion to the administered dose.

Example 11: Evaluation of formulation optimal conditions

Example 11-1: Optimum buffer conditions of formulations under refrigeration, room temperature and 40°C storage conditions flat end

In this example, formulations of the following formulations were prepared according to the contents disclosed in International Publication No. WO2017188570, a prior art document: a) 1-100 mg/ml new angiogenesis inhibitor comprising the amino acid sequence of KP-VR2; b) polysorbate 0.01 to 1% (w/v) solvent; c) 10-100 mM sodium chloride isotonic; and d) 5-50 mM sodium phosphate buffer or 5-50 mM citric sodium buffer, optionally further comprising 1-10% (w/v) sucrose as a stabilizer. For the formulation of the vascular endothelial growth factor (VEGF) antagonist, formulation stability according to storage conditions of refrigeration, room temperature and 40° C. was compared. In order to confirm the optimal pH of the liquid formulation containing the vascular endothelial growth factor receptor fusion protein as an active ingredient, a sample of pH 5.0 to 7.5 was prepared. Table 11 shows the formulation and storage conditions.

pH Storage compositions storage conditions analytical method formulation 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 - 20~100 mg/ml KP-VR2
- 5 mM sodiumphosphate
- 5 mM sodium citrate
- 40 mM sodium chloride
- 0.03% polysorbate 20
- 5% sucrose 4℃
25℃
40℃ - SE-HPLC
- SDS-PAGE
- ELISA

Specifically, the final concentration of the vascular endothelial growth factor receptor fusion protein was 60 mg/ml, 5 mM sodium phosphate, 5 mM sodium citrate, 40 mM sodium sodium, 0.03% polysorbate, and pH was 5.0 and 5.5, respectively. , 6.0, 6.5, 7.0 and 7.5, refrigerated, stored at room temperature and 40° C. for 7 days, and impurities generated over time were analyzed by SE-HPLC (size exclusion high performance liquid chromatography). In SE-HPLC analysis, TSK gel G3000swxl column (TOSOH, Japan) was was used to calculate aggregates, which are high molecular weight impurities, and fragments, which are low molecular weight impurities. The results are shown in Tables 12 to 14. In the storage conditions of Table 12 (SE-HPLC, 4 ℃), D/A (%), dimer and total % of agglomerates did not show a significant difference according to pH conditions. Among them, the aggregation content was low in the refrigerated storage conditions of pH 6.5, pH 7.0 and pH 7.5. In the storage conditions of Table 13 (SE-HPLC, RT) at room temperature, the aggregation content was particularly high in the low pH buffer storage condition, and the aggregation content was relatively low in the room temperature storage conditions of pH 6.5, 7.0, and pH 7.5. Under the severe storage conditions shown in Table 14 (SE-HPLC, 40°C), the aggregation content of most samples was high. However, the aggregation content was low in the storage conditions of 6.5, 7.0 and 7.5 buffers.

cold storage pH 5.0 pH5.5 pH6.0 pH6.5 pH7.0 pH7.5 DAY 0 3 7 0 3 7 0 3 7 0 3 7 0 3 7 0 3 7 Monomer
(%) 97.12 96.75 96.2 96.6 97.25 96.84 97.47 97.33 97.63 96.78 97.91 97.44 98.1 97.99 97.61 98.3 98.21 98.02 D/A fragment (%) 2.88 3.25 3.8 3.4 2.75 3.16 2.53 2.67 2.37 3.22 2.09 2.56 1.9 2.01 2.39 1.7 1.79 1.98

room temperature pH 5.0 pH5.5 pH6.0 pH6.5 pH7.0 pH7.5 DAY 0 3 7 0 3 7 0 3 7 0 3 7 0 3 7 0 3 7 Monomer
(%) 97.12 59.79 32.69 96.6 91.73 63.99 97.47 95.43 92.71 96.78 96.81 96.19 98.1 96.9 96.57 98.3 97.3 97.1 D/A fragment (%) 2.88 40.21 67.31 3.4 8.27 36.01 2.53 4.57 7.29 3.22 3.19 3.81 1.9 3.1 3.43 1.7 2.7 2.9

harsh pH 5.0 pH5.5 pH6.0 pH6.5 pH7.0 pH7.5 DAY 0 3 7 0 3 7 0 3 7 0 3 7 0 3 7 0 3 7 Monomer
(%) 97.12 11.41 9.19 96.6 6.78 3.87 97.47 7.17 2.56 96.78 38.45 25.75 98.1 45.36 37.71 98.3 46.51 38.82 D/A fragment (%) 2.88 88.59 90.81 3.4 93.22 96.13 2.53 92.83 97.44 3.22 61.55 74.25 1.9 54.64 62.29 1.7 53.49 61.18

In SE-HPLC analysis, 0.02 M sodium phosphate (pH 7.0) and 0.4 M NaCl buffer were flowed at a rate of 0.5 ml/min using a TSK-gel G3000swxl (7.8 × 300 mm) column (TOSOH, Japan), and absorbance The peak of the vascular endothelial growth factor fusion protein was confirmed at 280 nm. In the storage conditions of Figure 25A, D/A (%), dimers (dimer) and total % of aggregates did not show a significant difference by pH conditions. Among them, the aggregate content was low at pH 6.5, pH 7.0 and pH 7.5 refrigerated storage conditions. In FIG. 25B , the aggregate content was particularly high in the storage condition of a low pH buffer in the room temperature storage condition, and the aggregate content was relatively low in the room temperature storage condition of pH 6.5, 7.0, and pH 7.5. In FIG. 25C , the aggregate content of most samples was high under severe storage conditions (40° C.). However, the aggregate content was low in the storage conditions of 6.5, 7.0 and 7.5 buffers.

Example 11-2: Evaluation of optimal buffer conditions of formulations under refrigeration, room temperature and 40°C storage conditions

The final concentration of the vascular endothelial growth factor receptor fusion protein was 60 mg/ml, 5 mM sodium phosphate, 5 mM sodium citrate, 40 mM sodium sodium, 0.03% polysorbate, and pH was 5.0, 5.5, 6.0, respectively. 6.5, 7.0, and 7.5 were prepared. Several formulations were refrigerated for 7 days, and SDS-PAGE analysis was performed on monomers, aggregates, and variants over time. After adding 5.0 μl of 5 x sample buffer to 20.0 μl of the assay sample diluted to 1 mg/ml, the sample reacted at 100° C. for 5 minutes was used for analysis. The final assay sample was loaded at 10 μl per well. A protein marker (HiMark Pre-stained Protein Standard, Invitrogen) was used as the marker, and was loaded at 5 μl per well. Electrophoresis was performed on Novex Tris-Acetate 3 to 8% gel at 150 V for 1 hour. It was stained with coomassie blue (Merck) and bleached until a clear band appeared. As a result of SDS-PAGE analysis, in refrigerated storage, as compared with FIG. 26 and Tables 12 to 14, the formation of impurities, aggregates and fragments of the formulation was suppressed.

Example 11-3: Evaluation of optimal buffer conditions of formulations under room temperature storage conditions

The final concentration of the vascular endothelial growth factor receptor fusion protein was 60 mg/ml, 5 mM sodium phosphate, 5 mM sodium citrate, 40 mM sodium sodium, and 0.03% polysorbate, and the pH was adjusted to 5.0, 5.5, 6.0, respectively. 6.5, 7.0, and 7.5 were prepared. The prepared formulations were stored at room temperature for 7 days, and the monomers, aggregates, and variants over time were analyzed by SDS-PAGE in the same manner as in the above Examples. As compared in Figures 27A and 27B, the production of impurities, aggregates and fragments of the formulation increased over time. However, for the specific buffered pH 6.5, 7.0 and 7.5 formulations, the production of aggregates and fragments of the formulation did not significantly increase with time even after 1 week of storage at room temperature.

Example 11-4: Evaluation of optimal buffer conditions for formulations under severe storage conditions

The final concentration of the vascular endothelial growth factor receptor fusion protein was 60 mg/ml, 5 mM sodium phosphate, 5 mM sodium citrate, 40 mM sodium sodium, 0.03% polysorbate, and the pH was 5.0, 5.5, 6.0, and 6.5, respectively. , 7.0, and 7.5 were prepared. While these formulations were stored under severe conditions (40° C.) for 7 days, the monomers, aggregates and variants over time were analyzed by SDS-PAGE. As a result, the production of impurities, aggregates and fragments of the formulation increased over time, as shown in control Figures 28A and 28B. However, in the specific buffered pH 6.5, 7.0, and 7.5 formulations, relatively little formation of aggregates and fragments of the formulation was observed even after 1 week of storage at 40°C.

Example 11-5: Evaluation of optimal buffer conditions of formulations under refrigerated storage conditions

The final concentration of the vascular endothelial growth factor receptor fusion protein was 60 mg/ml, 5 mM sodium phosphate, 5 mM sodium citrate, 40 mM sodium sodium, 0.03% polysorbate, and pH was 5.0, 5.5, 6.0, respectively. 6.5, 7.0 and 7.5 were prepared. ELISA analysis of monomers, aggregates, and variants over time was performed while refrigerated storage of these prepared formulations for 7 days. To compare the avidity (avidity) of the buffer pH 5.0, 5.5, 6, 6.5, 7.0 and 7.5 formulations to VEGF-A, 96-well plates (Nunc) were coated with 2 nM KP-VR2, and VEGF165 (R&D systems) ) were added in stepwise increasing amounts (0.0156-64 nM). After washing the plate, it was reacted with anti-human VEGF antibody for 1 hour, and after washing the plate, it was reacted with peroxidase-conjugated anti-goat Ig antibody. Next, 3, 3', 5,5' tetramethylbenzidine (TMB) solution (Roche) was added, and absorbance was measured at 450 nm using an ELISA reader. The results are shown in FIGS. 29A and 29B. As shown in FIGS. 29A and 29B , the generation of impurities, aggregates and fragments of the formulation was inhibited under refrigerated storage conditions.

Example 11-6: Evaluation of optimal buffer conditions of formulations under room temperature storage conditions

The final concentration of the vascular endothelial growth factor receptor fusion protein was 60 mg/ml, 5 mM sodium phosphate, 5 mM sodium citrate, 40 mM sodium sodium, 0.03% polysorbate, and pH was 5.0, 5.5, 6.0, respectively. 6.5, 7.0, and 7.5 were prepared. The prepared formulations were stored at room temperature for 7 days, and the monomers, aggregates, and variants over time were analyzed by ELISA. To compare the affinities of buffer pH 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 formulations to VEGF-A, 96-well plates (Nunc) were coated with 2 nM KP-VR2, and VEGF165 (R&D systems) was applied stepwise. was added in increasing amounts (0.0156 ~ 64 nM). After washing the plate, it was reacted with an anti-human VEGF antibody for 1 hour, and the plate was washed and then reacted with a peroxidase-conjugated anti-goat Ig antibody. Next, 3, 3', 5,5' tetramethylbenzidine (TMB) solution (Roche) was added, and absorbance was measured at 450 nm using an ELISA reader. The results are shown in A and B of FIG. 30 . As a result of storage at room temperature for 7 days, the formation of impurities, aggregates and fragments of the formulation was suppressed in buffer pH 6.5, 7.0 and 7.5 conditions.

Example 11-7: Evaluation of optimal buffer conditions of formulations under severe storage conditions

The final concentration of the vascular endothelial growth factor receptor fusion protein was 60 mg/ml, 5 mM sodium phosphate, 5 mM sodium citrate, 40 mM sodium sodium, 0.03% polysorbate, and pH was 5.0, 5.5, 6.0, respectively. 6.5, 7.0 and 7.5 were prepared. These prepared formulations were stored under severe conditions (40° C.) for 7 days, and the monomers, aggregates, and variants over time were analyzed by ELISA. To compare the affinities of buffer pH 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 formulations to VEGF-A, 96-well plates (Nunc) were coated with 2 nM KP-VR2, and VEGF165 (R&D systems) was applied stepwise. was added in increasing amounts (0.0156 ~ 64 nM). After washing the plate, it was reacted with anti-human VEGF antibody for 1 hour, and the plate was washed and then reacted with peroxidase-conjugated anti-goat Ig antibody. Next, 3, 3', 5,5' tetramethylbenzidine (TMB) solution (Roche) was added, and absorbance was measured at 450 nm using an ELISA reader. The results are shown in A and B of FIG. 31 . As a result of storage under severe conditions for 7 days, the production of impurities, aggregates and fragments of the formulation was inhibited in buffer pH 6.5, 7.0, and 7.5 conditions, and in particular, it was confirmed that the lowest impurity content was found in buffer pH 7.0 conditions.

<110> KOREA PRIME PHARM CO. LTD. <120> A pharmaceutical composition comprising VEGFR fusion protein for preventing and treating angiogenesis related disease <130> KPR19P-0002-KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> VEGFR1 Domain 2 <400> 1 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile 100 <210> 2 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 Fc reagion <400> 2 Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 65 70 75 80 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 115 120 125 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu 130 135 140 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 145 150 155 160 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 165 170 175 Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 180 185 190 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 195 200 205 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 210 215 220 Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 3 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> VEGFR2 Domain 3 <400> 3 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile 100

Claims (9)

(a) 1-100 mg/ml of an immunoglobulin-like domain Fc fusion protein binding to vascular endothelial growth factor (VEGF) as a pharmacologically active ingredient; (b) 0.01 to 1% (w/v) of polysorbate as a solvent; and (c) sodium phosphate 5-50 mM, sodium citrate 5-50 mM, or sodium phosphate 5-50 mM and sodium citrate 5-50 mM, pH 6.5-7.5, angiogenesis-related disease As a pharmaceutical composition for the prevention or treatment of
The Fc fusion protein is composed of (i) an Fc domain of IgG1 in which two heavy chains are linked by a disulfied bond, and (ii) four immunoglobulin domains 2 of VEGFR1, wherein each heavy chain of the Fc domain is The immunoglobulin domain 2 of the VEGFR1 and the immunoglobulin domain 2 of the other VEGFR1 are sequentially fused
A liquid or lyophilized pharmaceutical formulation for the prevention or treatment of angiogenesis-related diseases. The method according to claim 1, comprising 1-10% (w/v) of one or more stabilizers from the group consisting of sucrose, sorbitol, glycerol, trihalose and mannitol; and 10 to 150 mM isotonic agents,
A liquid or lyophilized pharmaceutical formulation for the prevention or treatment of angiogenesis-related diseases. The method according to claim 1, comprising 25 mg/ml of the Fc fusion protein, 0.03% (w/v) of the polysorbate, 10 mM of the sodium phosphate, 10 mM of the sodium citrate, and 5% (w/v) of the sucrose. that is,
A liquid or lyophilized pharmaceutical formulation for the prevention or treatment of angiogenesis-related diseases. According to claim 2, wherein the Fc fusion protein 25 mg / ml, the polysorbate 0.03% (w / v), and the isotonic agent comprising 40 mM, and the isotonic agent is sodium chloride,
A liquid or lyophilized pharmaceutical formulation for the prevention or treatment of angiogenesis-related diseases. The method according to claim 2, wherein the Fc fusion protein 20-100 mg/ml, the polysorbate 0.03% (w/v), the sodium phosphate 5 mM, the sodium citrate 5 mM, the sucrose 5% (w/v) , and 40 mM of the isotonic agent, wherein the isotonic agent is sodium chloride,
A liquid or lyophilized pharmaceutical formulation for the prevention or treatment of angiogenesis-related diseases. The method according to any one of claims 1 to 5, wherein the Fc fusion protein is composed of 15 mg/ml, 25 mg/ml, 40 mg/ml, 60 mg/ml, 80 mg/ml and 100 mg/ml. A liquid or lyophilized pharmaceutical formulation for the prevention or treatment of angiogenesis-related diseases, which is included at a concentration selected from the group. A syringe comprising the pharmaceutical formulation of claim 1 . A syringe comprising the pharmaceutical formulation of claim 6. The syringe of claim 7, for intravenous (intravenous, iv), subcutaneous (sc) or intraperitoneal (intraperitoneal, ip) administration.

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