Fusion protein that binds VEGF and TIE2 and its use

Abstract

The present disclosure refers to a fusion protein having an antibody or antigen-binding fragment thereof against Tie-2 and a vascular endothelial growth factor (VEGF) binding domain, a method for making the fusion protein, and pharmaceutical compositions and methods for preventing or treating angiogenic diseases or modulating angiogenesis, endothelial signaling, inflammation and / or vascular leakage comprising the fusion protein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 299,177, filed on January 13, 2022, U.S. Provisional Patent Application No. 63 / 310,359, filed on February 15, 2022, and U.S. Provisional Patent Application No. 63 / 335,805, filed on April 28, 2022. The entire disclosure of these prior applications is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in XML format. The XML copy created on January 11, 2023, is named "2023 - 01 - 12_01262 - 0004 - 00PCT_ST26.xml" and has a size of 48,489 bytes. The information in the electronic form of the sequence listing is hereby incorporated by reference in its entirety.

[0003] Field The present disclosure relates to a fusion protein comprising an anti - Tie - 2 antibody or an antigen - binding fragment thereof and a vascular endothelial growth factor (VEGF) binding domain, which simultaneously binds to Tie2 and VEGF, a nucleic acid encoding the fusion protein, a vector containing the nucleic acid, a cell transformed with the vector, a method for preparing the fusion protein, methods and compositions for regulating angiogenesis, inflammation, and vascular leakage. In some embodiments, the present disclosure relates to methods and pharmaceutical compositions for preventing or treating angiogenesis diseases and vascular diseases.

Background Art

[0004] Introduction and Summary Angiogenesis is a dynamic process regulated by various factors during the development, growth, maintenance, and homeostasis of an organism. Newly formed blood vessels in this process function as transport channels for various biomaterials such as nutrients, oxygen, and hormones in surrounding cells. Functionally and structurally abnormal blood vessels are the direct or indirect cause of the onset and progression of various diseases. Tumor vessels, due to their functional and structural defects, exacerbate hypoxia, leading to tumor progression and metastasis to other tissues, and impair the delivery of anticancer drugs to the center of the tumor mass. Defective blood vessels are also found in various other diseases and conditions besides cancer. Examples include various eye diseases (e.g., diabetic macular edema, exudative age-related macular degeneration, diabetic retinopathy), viral infections, and acute inflammatory responses such as sepsis. Therefore, if therapeutic agents capable of normalizing pathological blood vessels are available, they can be applied to the treatment of various patients / animals with vascular abnormalities.

[0005] The angiopoietin family plays a crucial role in the formation and maintenance of blood vessels and consists of four angiopoietins (Ang1, Ang2, Ang3, and Ang4). Angiopoietin-1 (Ang1) binds to the Tie2 receptor on the surface of vascular endothelial cells, phosphorylating and activating it, leading to vascular stabilization and suppression of vascular leakage. On the other hand, angiopoietin-2 (Ang2) also binds to the Tie2 receptor but acts as an antagonist, inducing inactivation of the Tie2 receptor, inhibiting binding by Ang1, leading to vascular destabilization and leakage, which in turn tends to promote the growth of new blood vessels. Ang2 expression levels have been reported to be significantly increased in the blood of cancer patients, those with eye diseases, viral and bacterial infections, and inflammatory diseases (Saharinen P et al., 2017, Nature Review Drug Discovery 16:635-661). However, Ang2 is also known to act as an agonist that induces activation of the Tie2 receptor in several processes, including lymphangiogenesis and maintenance, suggesting that Ang2 plays various functions depending on the situation.

[0006] To date, many biopharmaceutical companies have shown interest in the development and clinical trials of various anti-Ang2 antibodies (e.g., U.S. Patent Nos. 7,658,924 and 8,987,420). These Ang2 antibodies have been reported to inhibit the binding of Ang2 to Tie2, and their Ang2 neutralizing effect has been reported to prevent the formation of new blood vessels. The anti-angiogenic and anticancer activities of these anti-Ang2 antibodies have been demonstrated in many preclinical models, and a variety of anti-Ang2 antibodies have been clinically tested in patients with various cancers. However, their anticancer effects have been demonstrated to be insufficient. For example, a phase 3 clinical trial conducted by Amgen showed that the anticancer effect of Ang2 antibodies in ovarian cancer patients was minimal (Marth C et al, 2017, Eur.J.Cancer, 70:111-121). In addition to cancer models, the Ang2 neutralizing antibody nesbukumaab was tested in ocular patients, but it failed to improve the efficacy of Eylea® (anti-VEGF) in a phase 2 clinical trial. For example, see the press release "Regeneron provides update on EYLEA(R)(aflibercept) injection and nesvacumab(ang2 antibody) combination program," available at investor.regeneron.com / news-releases / news-release-details / regeneron-provides-update-eylear-aflibercept-injection-and.

[0007] In contrast to the Ang2 neutralization approach described above, direct Tie2 activation has also been considered an alternative approach to inhibit angiogenesis and suppress vascular permeability. Recombinant proteins that directly bind to the Tie2 receptor and induce Tie2 phosphorylation and activation have also been developed and tested in many preclinical cancer and ocular models. Examples include cartilage oligomeric matrix protein (COMP)-Angl (Cho et al., 2004, PNAS 101(15):5547-5552) and vasculotide (David S et al., 2011, Am J Physiol Lung Cell Mol Physiol 300(6):L851-L862). While these drugs exhibit anti-angiogenic and anti-permeability activity, they tend to have very short half-lives and unstable physicochemical properties. Furthermore, a small molecule compound (AKB-9778) has been developed as an inhibitor of VE-PTP, a phosphatase that inactivates Tie2 by removing phosphate groups from phosphorylated Tie2 (Goel S et al., 2013, J Natl Cancer Inst 105(16):1188-1201). This compound indirectly increases Tie2 activity by inhibiting VE-PTP, but it has the drawback of also activating other receptors. See, for example, Frye M. et al., 2015, J Exp. Med., 212(13):2267-2287; Hayashi M, et al., 2013, Nature Communication, 4:1672; and Mellberg S. et al., 2009, FASEB J., 23(5):1490-1502). In addition, Tie2 antibodies with agonist activity have been developed. See, for example, U.S. Patent No. 6,365,154 and U.S. Patent Application Publication No. 20170174789. These antibodies increased endothelial cell survival and inhibited vascular leakage. Interestingly, herbal extracts have been shown to activate Tie2 activity and have been claimed for use in skincare cosmetics. See, for example, Japanese Patent Publication Nos. 2011102273, 2018043949, and 2015168656.

[0008] Tie2 is a receptor protein that promotes vascular differentiation and stabilization and is highly expressed in blood vessels. When activated, the Tie2 receptor stabilizes blood vessels and allows surrounding supporting cells to be attracted. For example, activated Tie2 in cancer blood vessels normalizes cancer blood vessels, reduces vascular leakage, eliminates increased hypoxia within the tumor, and supplies sufficient oxygen by increasing blood flow into the tumor, thereby increasing the delivery of other anticancer drugs and the penetration of immune cells.

[0009] Vascular endothelial growth factor (VEGF) inhibitors are the first-line standard treatment for diabetic macular edema (DME) and exudative age-related macular degeneration (wAMD), but their effectiveness is limited by a significant number of intraocular administrations and a population with considerably inadequate responses. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 7,658,924 [Patent Document 2] U.S. Patent No. 8,987,420 [Patent Document 3] U.S. Patent No. 6,365,154 [Patent Document 4] U.S. Patent Application Publication No. 2017 / 0174789 [Patent Document 5] Japanese Patent Publication No. 2011102273 [Patent Document 6] Japanese Patent Publication No. 2018043949 [Patent Document 7] Japanese Patent Publication No. 2015168656 [Non-patent literature]

[0011] [Non-Patent Document 1] Saharinen P et al.,2017,Nature Review Drug Discovery 16:635-661 [Non-Patent Document 2] Marth C et al,2017,Eur.J.Cancer,70:111-121 [Non-Patent Document 3] Cho et al., 2004, PNAS 101(15):5547-5552 [Non-Patent Document 4] David S et al.,2011,Am J Physiol Lung Cell Mol Physiol 300(6):L851-L862 [Non-Patent Document 5] Goel S et al.,2013,J Natl Cancer Inst 105(16):1188-1201 [Non-Patent Document 6] Frye M. et al.,2015,J Exp.Med.,212(13):2267-2287 [Non-Patent Document 7] Hayashi M,et al.,2013,Nature Communication,4:1672 [Non-Patent Document 8] Mellberg S. et al.,2009,FASEB J.,23(5):1490-1502 [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] Therefore, there is a need for improved methods to modulate angiogenesis, endothelial signaling, inflammation and / or vascular leakage, and / or to treat angiogenic diseases. For example, there is a need for treatments for angiogenic diseases such as cancer that involve improved efficacy, affinity, half-life, stability, pharmacodynamics, duration, and / or reduced frequency of treatment to alleviate patient burden and medication adherence issues. This disclosure aims to satisfy one or more of these needs, provide other benefits, or at least provide the public with a useful option. [Means for solving the problem]

[0013] This disclosure provides a fusion protein comprising an anti-Tie2 antibody or its antigen-binding fragment and a vascular endothelial growth factor (VEGF)-binding domain. In some embodiments, the fusion protein binds to a Tie2 Ig3-FNIII(1-3) domain, including SEQ ID NOs: 2, 3, or 4, and VEGF.

[0014] In one embodiment, the VEGF-binding domain includes the VEGF receptor extracellular domain.

[0015] In one embodiment, the VEGF-binding domain is an antagonist VEGF-binding domain. Therefore, in one embodiment, the VEGF-binding domain antagonizes (i.e., inhibits) the activity of VEGF in the cell. The VEGF-binding domain achieves its antagonist effect by binding to VEGF and, as a result, preventing VEGF from exerting its activity.

[0016] In one embodiment, the VEGF-binding domain includes the extracellular domains of two VEGF receptor isoforms, for example, the extracellular domains of both VEGF receptor 1 and VEGF receptor 2 (e.g., the Ig2 domain of VEGF receptor 1 and the Ig3 domain of VEGF receptor 2).

[0017] In one embodiment, the VEGF-binding domain comprises an anti-VEGF antibody or an antibody-binding fragment thereof. In a further embodiment, the anti-VEGF antibody or fragment thereof comprises a variable-binding domain of bevacizumab, ranibizumab, or HuMab G6-31 (U.S. Patent Application Publication No. 2007 / 0141065).

[0018] In some embodiments, the present disclosure includes a vascular endothelial growth factor (VEGF) binding domain comprising the vascular endothelial growth factor (VEGF)-A binding domain of VEGF receptor 1 (VEGFR1) of SEQ ID NO: 13 and the vascular endothelial growth factor (VEGF)-A binding domain of VEGF receptor 2 (VEGFR2) of SEQ ID NO: 14, An anti-Tie2 antibody or its antibody-conjugated fragment, A fusion protein containing, Here, the fusion protein provides a fusion protein that binds to a Tie2 Ig3-FNIII(1-3) domain containing SEQ ID NOs. 2, 3, or 4 and to VEGF.

[0019] In one embodiment, the anti-Tie2 antibody or its antigen-binding fragment is an agonist anti-Tie2 antibody or its antigen-binding fragment. Therefore, in one embodiment, the anti-Tie2 antibody or its antigen-binding fragment binds to Tie2 and provides, preserves, or enhances Tie2 or Tie2 activity on the cell surface membrane.

[0020] In one embodiment, the VEGF-binding domain is ligated to the C-terminus of the heavy chain (HC) of an anti-Tie2 antibody or its antigen-binding fragment.

[0021] In one embodiment, the fusion protein is conjugated to amino acids 633-644 (SEQ ID NO: 19) and / or amino acids 713-726 (SEQ ID NO: 20) of Tie2 in the amino acid sequence of SEQ ID NO: 1. In another embodiment, the anti-Tie2 antibody or its antibody-conjugated fragment is conjugated to amino acids 633-644 (SEQ ID NO: 19) and / or amino acids 713-726 (SEQ ID NO: 20) of Tie2 in the amino acid sequence of SEQ ID NO: 1.

[0022] In one embodiment, the anti-Tie2 antibody has an IgG1 isotype.

[0023] In one embodiment, the anti-Tie2 antibody or its antigen-binding fragment includes a heavy chain variable region containing a heavy chain CDR containing the amino acid sequences of SEQ ID NOs. 5 to 7, and a light chain variable region containing a light chain CDR containing the amino acid sequences of SEQ ID NOs. 8 to 10.

[0024] In one embodiment, the VEGF-binding domain includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 15.

[0025] In one embodiment, the VEGF-binding domain includes the amino acid sequence of SEQ ID NO: 15.

[0026] In one embodiment, the fusion protein includes a linker between a VEGF-binding domain (e.g., a VEGF receptor) and an anti-Tie2 antibody or a fragment thereof. In a further embodiment, the linker includes 5 to 50 amino acid residues, e.g., 10 to 40 residues, e.g., 15 to 30 residues, e.g., 20 residues.

[0027] In one embodiment, the fusion protein includes a linker between a VEGF-binding domain (e.g., a VEGF receptor) and an anti-Tie2 antibody or a fragment thereof, wherein the linker includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 16.

[0028] In one embodiment, the fusion protein includes a linker between a VEGF-binding domain (e.g., a VEGF receptor) and an anti-Tie2 antibody or a fragment thereof, the linker containing the amino acid sequence of SEQ ID NO: 16.

[0029] In one embodiment, the fusion protein includes a linker between a VEGF-binding domain (e.g., a VEGF receptor) and an anti-Tie2 antibody or a fragment thereof, wherein the linker includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 25.

[0030] In one embodiment, the fusion protein includes a linker between a VEGF-binding domain (e.g., a VEGF receptor) and an anti-Tie2 antibody or a fragment thereof, the linker containing the amino acid sequence of SEQ ID NO: 25.

[0031] In one embodiment, the fusion protein includes a CH domain containing a sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 17.

[0032] In one embodiment, the fusion protein includes a CH domain containing the amino acid sequence of SEQ ID NO: 17.

[0033] In one embodiment, the fusion protein contains one or more mutations in the constant region domain of the heavy chain to reduce or eliminate efficient interaction with Fc receptors on other immune cells in the body. In one embodiment, one or more mutations include altering L234, L235, G236, and G237 (EU numbering) to LAGA mutations, FEGG mutations, AAGG mutations, AAGA mutations, LALA mutations, or combinations thereof. In one embodiment, one or more mutations include an LALA mutation and mutations at K322 (e.g., K322A) and P331 (e.g., P331S) (EU numbering).

[0034] In one embodiment, the fusion protein contains one or more mutations in L234, L235, H310, M252, I253, S254, T256, H433, N434 and / or H435 (EU numbering). In one embodiment, the fusion protein contains one or more mutations in L234A, L235A and / or H310A. In one embodiment, the fusion protein contains one or more mutations in M252 (e.g., M252Y), I253 (e.g., I253A, I253M or I253V), S254 (e.g., S254T), T256 (e.g., T256D), H433 (e.g., H433K), N434 (e.g., N434F) and / or H435 (e.g., H435A, H435Q or H435R).

[0035] In one embodiment, the fusion protein includes a linker between the VEGF-binding domain and the anti-Tie2 antibody or antibody fragment, located on the C-terminal side of one or more heavy chain constant domains of the anti-Tie2 antibody or its antigen-binding fragment, and in an N-terminus to C-terminus order, for example, the linker includes a sequence having 5 to 50 residues, e.g., 10 to 40 residues, e.g., 15 to 30 residues, e.g., 20 residues, and the VEGF-binding domain includes the amino acid sequence of SEQ ID NO: 15.

[0036] In one embodiment, the fusion protein includes a linker between the VEGF-binding domain and the anti-Tie2 antibody or antibody fragment, located on the C-terminal side of one or more heavy chain constant domains of the anti-Tie2 antibody or its antigen-binding fragment, and in an N-terminus to C-terminus order, wherein the linker includes the amino acid sequence of SEQ ID NO: 16 or 25, and the VEGF-binding domain includes the amino acid sequence of SEQ ID NO: 15.

[0037] In one embodiment, the fusion protein includes a CH domain containing the amino acid sequence of SEQ ID NO: 17, and a linker between the VEGF-binding domain and an anti-Tie2 antibody or an antibody fragment thereof containing the amino acid sequence of SEQ ID NO: 16 or 25, wherein the VEGF-binding domain contains the amino acid sequence of SEQ ID NO: 15.

[0038] In one embodiment, the fusion protein includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19.

[0039] In one embodiment, the fusion protein includes a heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 11.

[0040] In one embodiment, the fusion protein includes a heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 26.

[0041] In one embodiment, the fusion protein includes a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

[0042] In one embodiment, the fusion protein includes a heavy chain containing the amino acid sequence of SEQ ID NO: 26.

[0043] In one embodiment, the fusion protein includes a light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with respect to the amino acid sequence of SEQ ID NO: 12.

[0044] In one embodiment, the fusion protein includes a light chain containing the amino acid sequence of SEQ ID NO: 12.

[0045] In one embodiment, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 11 and a light chain containing the amino acid sequence of SEQ ID NO: 12.

[0046] In one embodiment, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 26 and a light chain containing the amino acid sequence of SEQ ID NO: 12.

[0047] In one embodiment, the fusion protein is 3E -9 Affinity K less than M D (M) binds to the Tie2 Ig3-FNIII(1-3) domain containing sequence numbers 2, 3, or 4.

[0048] In one embodiment, the fusion protein is PEGylated. PEGylation (or PEGylation) refers to the covalent attachment or fusion of a chain containing multiple polyethylene glycol (PEG, also called macrogol) units (e.g., a PEG polymer). In one embodiment, the PEG has a molecular weight of about 40 kDa or about 20 kDa.

[0049] In one embodiment, the fusion protein is site-specifically pegylated. In one embodiment, the fusion protein is site-specifically pegylated on a cysteine ​​residue. In one embodiment, the fusion protein further comprises the sequence of SEQ ID NO: 22 and is site-specifically pegylated on a cysteine ​​residue of the sequence of SEQ ID NO: 22.

[0050] In one embodiment, the sequence of SEQ ID NO: 22 is located at the C-terminus of the heavy chain. In one embodiment, the heavy chain includes the sequence of SEQ ID NO: 23. In one embodiment, the heavy chain includes the sequence of SEQ ID NO: 24.

[0051] In one embodiment, the fusion protein further comprises one or more half-life extension modulators. In one embodiment, the one or more half-life extension modulators comprise a chemical, biopolymer, or peptide that increases the half-life of the fusion protein.

[0052] In one embodiment, a polypeptide comprising a chain monomer of the fusion protein of the present invention is provided.

[0053] In one embodiment, the polypeptide comprises the heavy chain monomer of the fusion protein of the present invention. In a further embodiment, the polypeptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO: 11 or 26, for example, at least 75% identity, for example, at least 80% identity, for example, at least 85% identity, for example, at least 90% identity, for example, at least 91% identity, for example, at least 92% identity, for example, at least 93% identity, for example, at least 94% identity, for example, at least 95% identity, for example, at least 96% identity, for example, at least 97% identity, for example, at least 98% identity, for example, at least 99% identity, for example, 100% identity. In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 11. In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 26.

[0054] In one embodiment, the polypeptide comprises the light chain monomer of the fusion protein of the present invention. In a further embodiment, the polypeptide comprises a sequence having at least 70% identity with respect to SEQ ID NO: 12, for example, at least 75% identity, for example, at least 80% identity, for example, at least 85% identity, for example, at least 90% identity, for example, at least 91% identity, for example, at least 92% identity, for example, at least 93% identity, for example, at least 94% identity, for example, at least 95% identity, for example, at least 96% identity, for example, at least 97% identity, for example, at least 98% identity, for example, at least 99% identity, for example, 100% identity. In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 12.

[0055] As used herein, “biopolymer” refers to a polymer produced from natural sources, either chemically synthesized from biological materials or entirely biosynthesized by organisms or microorganisms. In some embodiments, biopolymers include, but are not limited to, polypeptides or proteins (i.e., polymers of amino acids, e.g., collagen, actin, and fibrin), polynucleotides (i.e., polymers of nucleic acids, e.g., DNA or RNA), polysaccharides (i.e., carbohydrates and glycosylated molecules, e.g., cellulose, starch, or alginates), natural rubber (polymers of isoprene), subarin, lignin (complex polyphenol polymers), kutin and kutan (complex polymers of long-chain fatty acids), melanin, metabolites, and other structural molecules.

[0056] In one embodiment, one or more half-life extension modulators include a biopolymer containing PEG (polyethylene glycol), hyaluronic acid (HA), or phosphorylcholine; albumin; albumin-binding peptides and / or HA-binding protein fragments.

[0057] In another embodiment, the present disclosure provides nucleic acids encoding a fusion protein.

[0058] In another embodiment, the disclosure provides a nucleic acid encoding a polypeptide comprising a chain monomer of the fusion protein of the present invention. In one embodiment, the disclosure provides a nucleic acid encoding the heavy chain of the fusion protein. In another embodiment, the disclosure provides a nucleic acid encoding the light chain of the fusion protein.

[0059] In one embodiment, the nucleic acid molecule includes a sequence having at least 70% identity with respect to sequence number 27 or 29, for example, at least 75% identity, for example, at least 80% identity, for example, at least 85% identity, for example, at least 90% identity, for example, at least 91% identity, for example, at least 92% identity, for example, at least 93% identity, for example, at least 94% identity, for example, at least 95% identity, for example, at least 96% identity, for example, at least 97% identity, for example, at least 98% identity, for example, at least 99% identity, for example, 100% identity. In one embodiment, the nucleic acid molecule consists of sequence number 27 or 29.

[0060] In one embodiment, the nucleic acid molecule includes a sequence having at least 70% identity with respect to sequence number 28, for example, at least 75% identity, for example, at least 80% identity, for example, at least 85% identity, for example, at least 90% identity, for example, at least 91% identity, for example, at least 92% identity, for example, at least 93% identity, for example, at least 94% identity, for example, at least 95% identity, for example, at least 96% identity, for example, at least 97% identity, for example, at least 98% identity, for example, at least 99% identity, for example, 100% identity. In one embodiment, the nucleic acid molecule consists of sequence number 28.

[0061] In one embodiment, the Disclosure provides a set of one or more polynucleotides, each of which encodes at least one monomer chain of the fusion protein of the present invention, thereby encoding both such chains (i.e., the light chain and the heavy chain) of the fusion protein.

[0062] In another embodiment, the disclosure provides an expression vector comprising nucleic acids. In another embodiment, a vector is provided comprising nucleic acids encoding one heavy chain sequence and one light chain sequence of a fusion protein.

[0063] In one embodiment, a set of one or more vectors is provided that collectively comprises one or more sets of polynucleotides of the present invention, such that both chains of the fusion protein (i.e., the light chain and the heavy chain) are encoded in the set of vectors.

[0064] In one embodiment, the vector is a virus selected from animal viruses, such as reverse transcriptase viruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses, varicella viruses, baculoviruses, papillomaviruses, aneroviruses, and papovaviruses.

[0065] In one embodiment, the expression vector further comprises a human cytomegalovirus IE1 (CMV-IE1) promoter / enhancer.

[0066] In another embodiment, the present disclosure provides cells transformed with an expression vector.

[0067] In another embodiment, the disclosure provides a method for producing a fusion protein that binds Tie2 and VEGF, comprising the steps of culturing cells transformed with an expression vector and recovering the fusion protein from the cultured cells. In yet another embodiment, the disclosure provides a method for producing the fusion protein of the present invention by expression from a vector or a set of vectors.

[0068] In another embodiment, the present disclosure provides a method for preventing or treating angiogenic disease, comprising administering an effective amount of a fusion protein to a subject in need of preventing or treating angiogenic disease.

[0069] In another embodiment, the present disclosure provides the use of a fusion protein for the manufacture of a pharmaceutical for treating angiogenic diseases in subjects requiring treatment of angiogenic diseases.

[0070] In another embodiment, the disclosure provides a fusion protein for use in the treatment of angiogenic or vascular disease in subjects requiring treatment of angiogenic or vascular disease.

[0071] In some embodiments, neovascular disease or vascular disease is cancer, metastasis, diabetic retinopathy, retinopathy of prematurity, diabetic macular edema, corneal graft rejection, macular degeneration, glaucoma such as neovascular glaucoma, systemic erythroderma, proliferative retinopathy, psoriasis, hemophilic arthropathy, nephrosclerosis, atherosclerotic plaque capillary formation, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases, or neurodegenerative diseases.

[0072] In some embodiments, cancer is esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin lymphoma, lymphoma, or multiple myeloid hematological malignancy.

[0073] In some embodiments, the present disclosure provides methods for modulating angiogenesis, endothelial signaling, inflammation and / or vascular leakage, comprising administering an effective amount of a fusion protein to a subject requiring modulation of angiogenesis, endothelial signaling, inflammation and / or vascular leakage.

[0074] In some embodiments, the inflammation originates from sepsis, acute respiratory distress syndrome, and / or a viral infectious disease.

[0075] In some embodiments, the subject is a human. In some embodiments, the subject is a pet animal such as a mammalian companion animal. In some embodiments, the mammalian companion animal is a dog, cat, rabbit, ferret, horse, mule, donkey or hamster or other domesticated pet.

[0076] In some embodiments, the disclosure provides a pharmaceutical composition comprising a fusion protein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0077] Additional objectives and benefits are partially described in the following description and can be partially understood from the description or acquired through practice. These objectives and benefits are realized and achieved by the elements and combinations specifically indicated in the attached claims.

[0078] Please understand that the general descriptions above and the detailed descriptions below are illustrative and explanatory only and do not limit the scope of the claims. Section headings are provided solely for the convenience of the reader and do not limit this disclosure.

[0079] The accompanying drawings incorporated herein and constituting part of this specification illustrate specific embodiments and, together with the descriptions, are useful in illustrating the principles described herein. [Brief explanation of the drawing]

[0080] [Figure 1A] Figures 1A–1C provide sensorgrams of IGT-427 binding to human (Figure 1A), rabbit (Figure 1B), and mouse (Figure 1C) Tie2-Fc fusion proteins. [Figure 1B] Figures 1A–1C provide sensorgrams of IGT-427 binding to human (Figure 1A), rabbit (Figure 1B), and mouse (Figure 1C) Tie2-Fc fusion proteins. [Figure 1C] Figures 1A–1C provide sensorgrams of IGT-427 binding to human (Figure 1A), rabbit (Figure 1B), and mouse (Figure 1C) Tie2-Fc fusion proteins. [Figure 2A]Figures 2A-2C show the inhibition of VEGFR2 phosphorylation and AKT activation by IGT-427. Human umbilical vein endothelial cells (HUVECs) were serum-starved for 4 hours and incubated with the indicated concentrations of IGT-427 (Figure 2A) or aflibercept (Figure 2B) for 30 minutes, followed by treatment with recombinant human VEGF for 2 minutes. Cell lysates were subjected to SDS-PAGE / Western blotting, and blots were probed with anti-phospho-VEGFR2 (Tyr1175), anti-VEGFR2, anti-phospho-Akt (S473), or total Akt antibody. The VEGF reporter assay showed equivalent inhibitory effects of IGT-427, falisimab, and aflibercept (Figure 2C). [Figure 2B] Figures 2A-2C show the inhibition of VEGFR2 phosphorylation and AKT activation by IGT-427. Human umbilical vein endothelial cells (HUVECs) were serum-starved for 4 hours and incubated with the indicated concentrations of IGT-427 (Figure 2A) or aflibercept (Figure 2B) for 30 minutes, followed by treatment with recombinant human VEGF for 2 minutes. Cell lysates were subjected to SDS-PAGE / Western blotting, and blots were probed with anti-phospho-VEGFR2 (Tyr1175), anti-VEGFR2, anti-phospho-Akt (S473), or total Akt antibody. The VEGF reporter assay showed equivalent inhibitory effects of IGT-427, falisimab, and aflibercept (Figure 2C). [Figure 2C] Figures 2A-2C show the inhibition of VEGFR2 phosphorylation and AKT activation by IGT-427. Human umbilical vein endothelial cells (HUVECs) were serum-starved for 4 hours and incubated with the indicated concentrations of IGT-427 (Figure 2A) or aflibercept (Figure 2B) for 30 minutes, followed by treatment with recombinant human VEGF for 2 minutes. Cell lysates were subjected to SDS-PAGE / Western blotting, and blots were probed with anti-phospho-VEGFR2 (Tyr1175), anti-VEGFR2, anti-phospho-Akt (S473), or total Akt antibody. The VEGF reporter assay showed equivalent inhibitory effects of IGT-427, falisimab, and aflibercept (Figure 2C). [Figure 3]Figure 3 shows the more potent and sustained levels of phospho-Tie2 signaling by IGT-427 compared to angiopoietin-1. Chinese hamster ovary (CHO) cells overexpressing human Tie2 were serum-starved for 4 hours and then treated with 10 nM IGT-427 or angiopoietin-1 for the indicated durations. Solubilized cells were subjected to phospho-Tie2 and total Tie2 ELISA assays. [Figure 4] Figure 4 shows dose-dependent activation of Akt signaling by IGT-427 in HUVEC. [Figure 5] Figure 5 shows that IGT-427 overcomes and evades the effect of angiopoietin-2 on Tie2 signaling in HUVEC, leading to further activation of Akt signaling in the presence of pre-treated Ang2. [Figure 6] Figure 6 shows that IGT-427 simultaneously binds to Tie2 and VEGF, which have formed a complex with Ang2. Ang2 was captured on a CM5 chip, and subsequently, human Tie2, IGT-427, and human VEGF were injected using surface plasmon resonance (SPR) (Biocore®) analysis. [Figure 7] Figure 7 shows the inhibition of TNF-α-induced apoptosis by IGT-427. HUVEC cells were pretreated with IGT-427 or Eylea® for 1 hour, followed by treatment with TNF (50 ng / ml) for 24 hours. Apoptotic cells were stained with APO-BrdU® TUNEL Assay Kit (Thermo Fisher, A23210) and evaluated using Attune (Thermo Fisher). Values ​​are mean ± standard error. One-way ANOVA showed ***p<0.001 and ****p<0.0001. [Figure 8] Figure 8 shows the cell surface levels of Tie2 on Chinese hamster ovary cells (CHO-Tie2) overexpressing human Tie2, treated with various drugs at different time points. [Figure 9]Figure 9 shows that IGT-427 inhibits Tie2 cleavage by MMP14. Recombinant human MMP14, human Tie2-ECD-human IgG Fc fusion protein, or IGT-427 were mixed and incubated as shown. [Figure 10] Figure 10 shows that IGT-427 inhibits sTie2 production in basal HUVEC or TNF-α-treated HUVEC. sTie2 levels were measured by a Tie2 ELISA assay. [Figure 11] Figure 11 shows that IGT-427 restores endothelial barrier integrity impaired by VEGF treatment. TEER (transendothelial electrical resistance) was evaluated in HUVEC. [Figure 12] Figure 12 shows the suppression of choroidal neovascularization (CNV) by intravitreous injection of IGT-427 or Eylea(R) in a laser-induced CNV model. Intravitreous administration of antibodies (50 μl injection volume / eye, Eylea(R) (800 μg), IGT-427 (885 μg), control IgG (716 μg)) was performed on day 0 after laser photocoagulation. The molar ratio of Eylea(R), IGT-427, and control IgG was 1:0.65:0.68. Fluorescence intensity in the leakage region around CNV was measured in FA images taken on day 14 after laser photocoagulation. For each group, 4 to 7 rabbits were tested, and 6 laser-induced CNV lesions were tested in each rabbit. CNV lesions (n=24 to 42) were imaged for each group. Values ​​are mean ± SEM. One-way ANOVA yields *p < 0.05 and **p < 0.001. [Figure 13] Figure 13 shows a schematic diagram of the IGT-427 variant for PEGylation. All constructs have a common light chain with five different heavy chains that produce two unstable cysteines per antibody. [Figure 14]Figure 14 shows SDS-PAGE data for optimized PEGylation conditions for five IGT-427 variants. PRO593, PRO594, and PRO595 undergo complete conversion without any unmodified antibodies in the reaction mixture. The reaction mixtures for PRO592 and PRO596 contain some unmodified antibodies at the end of the reaction. [Figure 15] Figure 15 shows the SPR binding of IGT-427 and PEGylated variants to the Tie2 and VEGF surfaces. Although there are slight differences in the binding signals to either antigen, the PEGylated variants bind to both antigens similarly compared to unmodified IGT-427. [Figure 16] Figure 16 shows the SEC-HPLC chromatograms of the PK study test substances for five eyes. All constructs were pure above 90%, with the exception of a 20 kDa PEGylated species containing 15% high molecular weight species. [Figure 17] Figure 17 shows unreduced SDS-PAGE data for ocular PK study test substances. Lane 1: Eylea(R); Lane 2: Falicimab; Lane 3: IGT-427; Lane 4: (2×20kDa linear PEG)-IGT-427; Lane 5: (2×40kDa branched PEG)-IGT-427. [Figure 18A] Figures 18A–18C show SPR binding data of ocular PK test substances to rabbit VEGF (Figure 18A), Tie2 (Figure 18B), and Ang2 (Figure 18C). A summary of the measured binding constants is provided in a table. [Figure 18B] Figures 18A–18C show SPR binding data of ocular PK test substances to rabbit VEGF (Figure 18A), Tie2 (Figure 18B), and Ang2 (Figure 18C). A summary of the measured binding constants is provided in a table. [Figure 18C] Figures 18A–18C show SPR binding data of ocular PK test substances to rabbit VEGF (Figure 18A), Tie2 (Figure 18B), and Ang2 (Figure 18C). A summary of the measured binding constants is provided in a table. [Figure 19-1]Figure 19 shows a summary of total drug measurements from the vitreous humor of New Zealand white rabbits. Measured drug levels from ELISA quantification are plotted over time. Each data point represents a pair of measurements from a single rabbit eye. Exponential fits to each dataset are shown in red along with the fitted parameters, and the extracted eye half-lives are shown below each curve. For the PEGylated IGT-427 variant, 14-day measurements are included in the plot but have been excluded from the fitting and extracted half-life analysis. A summary of the observed half-lives for each test substance is shown in the lower right. [Figure 19-2] Figure 19 shows a summary of total drug measurements from the vitreous humor of New Zealand white rabbits. Measured drug levels from ELISA quantification are plotted over time. Each data point represents a pair of measurements from a single rabbit eye. Exponential fits to each dataset are shown in red along with the fitted parameters, and the extracted eye half-lives are shown below each curve. For the PEGylated IGT-427 variant, 14-day measurements are included in the plot but have been excluded from the fitting and extracted half-life analysis. A summary of the observed half-lives for each test substance is shown in the lower right. [Figure 19-3] Figure 19 shows a summary of total drug measurements from the vitreous humor of New Zealand white rabbits. Measured drug levels from ELISA quantification are plotted over time. Each data point represents a pair of measurements from a single rabbit eye. Exponential fits to each dataset are shown in red along with the fitted parameters, and the extracted eye half-lives are shown below each curve. For the PEGylated IGT-427 variant, 14-day measurements are included in the plot but have been excluded from the fitting and extracted half-life analysis. A summary of the observed half-lives for each test substance is shown in the lower right. [Modes for carrying out the invention]

[0081] Array description Table 1 provides a list of specific sequences referenced herein. [Table 1]

[0082] TIFF0007884072000002.tif232151

[0083] TIFF0007884072000003.tif233151

[0084] TIFF0007884072000004.tif232149

[0085] TIFF0007884072000005.tif232151

[0086] TIFF0007884072000006.tif233148

[0087] Description of the Embodiment definition Unless otherwise specified, the following terms and phrases used herein are intended to have the meanings set forth below.

[0088] As used herein, the term “anti-Tie2 antibody” means an antibody that specifically binds to Tie2, and includes an antigen-binding fragment of the antibody molecule in addition to a complete antibody that specifically binds to Tie2. In some embodiments, the complete antibody has a structure of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The constant region of the heavy chain has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) forms, with subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) forms.

[0089] An "antigen-binding fragment" or "antibody fragment of an antibody" refers to a fragment capable of binding to an antigen, and includes Fab, F(ab'), F(ab')2, and Fv. Of the antibody fragments, Fab has variable regions of the light chain and heavy chain, a constant region of the light chain, and one antigen-binding site having the structure of the first CH1 of the heavy chain.

[0090] Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain. F(ab')2 antibodies are produced by the formation of disulfide bonds between cysteine ​​residues within the hinge region of Fab'. Fv is the smallest antibody fragment, containing only the variable regions of the heavy chain and the variable regions of the light chain. Double-chain Fv (double-stranded Fv) is formed by non-covalent bonding between the heavy chain and light chain variable regions, while single-chain Fv (scFv) is generally formed via a covalently bonded peptide linker between the heavy chain and light chain variable regions, or by direct linking at the C-terminus by forming a dimer-like structure similar to double-chain Fv. This fragment can be obtained by proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the whole antibody using papain, and the F(ab')2 fragment can be obtained by cleavage with pepsin), and can also be produced by genetic engineering techniques.

[0091] The antibody may be in the form of, for example, Fv (e.g., scFv) or complete antibody. Furthermore, the constant region of the heavy chain may be selected from any isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). For example, the constant region may be gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The constant region of the light chain may be kappa or lambda.

[0092] Antibodies include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv(scFV), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(sdFV), and anti-idiotype (anti-Id) antibodies, or epitope-linked fragments of the above antibodies.

[0093] The term "heavy chain" in "HC" means a full-length heavy chain or fragment thereof comprising a variable domain VH and three constant domains CH1, CH2, and CH3, having an amino acid sequence with a sufficiently variable region to provide antigen specificity. Furthermore, as used herein, "light chain" or "LC" means a full-length light chain or fragment thereof comprising a variable domain VL and a constant domain CL, having an amino acid sequence with a sufficiently variable region to provide antigen specificity. The heavy chain constant domain (for example, human IgG1) may contain one or more modifications to reduce or eliminate efficient interaction with the Fc receptor on immune cells in the body. One or more such mutations can suppress its binding to the Fc receptor and reduce or eliminate antibody-induced cytotoxicity. Such modifications include L 234 L 235 G 236 G 237 Residues (EU numbering) may be changed to LAGA mutations, FEGG mutations, AAGG mutations, AAGA mutations, LALA mutations, or combinations thereof. In some embodiments, the IgG constant region includes mutations in K322 and P331 (EU numbering) (e.g., in combination with any of the aforementioned mutations) to reduce or eliminate immune cell-mediated cytotoxicity. In some embodiments, the IgG constant region includes LALA mutations and mutations in K322 and P331 (e.g., K322A and P331S) (EU numbering) to reduce or eliminate immune cell-mediated cytotoxicity.

[0094] A monoclonal antibody is an antibody produced by a single clone of a cell or cell line and consists of an identical antibody molecule. Monoclonal antibodies can have a monovalent affinity and bind only to the same epitope. In contrast, polyclonal antibodies bind to multiple epitopes and are usually produced by several different antibody-secreting plasma cell lines. Bispecific monoclonal antibodies can also be designed by increasing the target of a single monoclonal antibody to two epitopes.

[0095] An "epitope" refers to a protein determinant (e.g., a part of an antigen recognized by an antibody) to which an antibody can specifically bind. Epitopes can be a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally possess specific charge properties and specific three-dimensional structural properties.

[0096] A “humanized” form of a non-human (e.g., mouse) antibody is a chimeric antibody comprising one or more amino acid sequences (e.g., one or more CDR sequences, such as a sequence of six CDRs) derived from a non-human antibody (donor or source antibody) and other minimal sequences derived from a non-human immunoglobulin. In some embodiments, the humanized antibody is a human immunoglobulin (receptor antibody) in which its hypervariable region is replaced by residues derived from the hypervariable region of a non-human primate, mouse, rat, rabbit, or non-human primate (receptor antibody) having the desired specificity, affinity, and capabilities of the residues derived from the recipient's hypervariable region. For humanization, one or more residues in the framework domain (FR) may be replaced by corresponding residues from a non-human donor antibody. This may help maintain the proper three-dimensional configuration of the (one or more) transplanted CDRs, thereby improving affinity and antibody stability. The humanized antibody may, for example, substitute or additionally, contain novel residues not present in the original recipient or donor antibody to further improve the antibody’s performance.

[0097] This includes any “chimeric” antibody (immunoglobulin) exhibiting desired biological activity, and fragments of such antibody, wherein a portion of the heavy chain and / or light chain originates from a particular species or is identical or homologous to a corresponding sequence in an antibody belonging to a subclass, while the remaining chains (one or more) originate from another species or belong to another antibody class or are identical to a corresponding sequence in an antibody belonging to a subclass.

[0098] As used herein, “antibody variable domain” refers to a domain containing complementarity-determining regions (CDRs; e.g., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. The “framework region” (FR) is the variable domain segment outside the CDR. Each variable domain typically has four FRs, which are identified as FR1, FR2, FR3, and FR4.

[0099] The "complementarity-determining regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refer to the amino acid residues in the antibody's variable domain that are necessary for antigen binding. Each variable domain typically contains three CDR regions, identified as CDR1, CDR2, and CDR3.

[0100] As used herein, “half-life extension modulator” refers to a chemical substance, biopolymer, peptide, polypeptide, or protein fragment that can be added to an antibody or antibody fragment to increase the half-life of the antibody or antibody fragment. Half-life extension modulators may include biopolymers containing PEG (polyethylene glycol), hyaluronic acid (HA), or phosphorylcholine; albumin; albumin-binding peptides; or HA-binding protein fragments.

[0101] Half-life can also be reduced, for example, by eliminating the interaction between the antibody or antibody fragment and neonatal Fc receptor (FcRn)-mediated reuse. For example, combinatorial mutations at multiple sites are known to affect the half-life of an antibody. In some embodiments, mutations include M252 (e.g., M252Y), I253 (e.g., I253A, I253M, or I253V), S254 (e.g., S254T), T256 (e.g., T256D), H310 (e.g., H310A), H433 (e.g., H433K), N434 (e.g., N434F), and / or H435 (e.g., H435A, H435Q, or H435R) (EU numbering). In some embodiments, mutations include L235A, L236A, and H310A (EU numbering). A reduced systemic half-life may be advantageous in the treatment of non-systemic diseases, such as eye diseases, where minimal systemic exposure and a reduced systemic half-life are desirable due to the established on-target toxicity issues of VEGF inhibition (hemorrhage, hypertension, etc.).

[0102] As used herein, "or" has an inclusive meaning (i.e., equivalent to and / or) unless the context clearly indicates otherwise.

[0103] As used herein, “fusion protein” encompasses any polypeptide comprising sequences from multiple sources. A fusion protein may be produced, for example, from a gene fusion (e.g., a polynucleotide encoding the sequence of the fusion protein) or by chemically linking polypeptides that have been produced or synthesized separately.

[0104] "Angiogenic diseases," also known as "angiogenesis-related diseases," refer to diseases associated with the onset or progression of angiogenesis.

[0105] As used herein, “Subject” can be a human or an animal. For example, the subject may be a human. In some embodiments, the subject may be a mammal. In some embodiments, the subject may include a companion animal (also called a “pet”). The term “companion animal” means a domesticated animal that can be kept as a pet or for other amusement purposes, and includes, but is not limited to, dogs, cats, rabbits, ferrets, horses, donkeys, mules, and hamsters. In some embodiments, the companion animal is a mammalian companion animal.

[0106] As used herein, the term “prevention” refers to any action by which the administration of a fusion protein or composition inhibits, delays the onset of, or reduces the likelihood of the development of a disease of interest. The terms “treatment” or “therapy” refer to any action that reduces, delays, or alleviates the symptoms of a disease of interest, or that cures, reduces the severity of, or slows the progression of a disease of interest.

[0107] This disclosure describes nucleic acid sequences and amino acid sequences that have a certain degree of identity with respect to a given nucleic acid sequence or amino acid sequence (reference sequence).

[0108] Sequence identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between those sequences. Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between those sequences.

[0109] The terms “% identical,” “% identity,” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences to be compared. The percentage is purely statistical, and the difference between the two sequences may be randomly distributed across the entire length of the sequences to be compared, but not necessarily randomly distributed across the entire length of the sequences to be compared. Comparison of two sequences is typically performed by comparing them with respect to a segment or “window of comparison” after optimal alignment to identify the local regions of the corresponding sequences. Optimal alignment for comparison can be performed manually, or with the help of a local phasic algorithm by Smith and Waterman, 1981, Ads App.Math.2, 482, a local phasic algorithm by Needleman and Wunsch, 1970, J.Mol.Biol.48, 443, a similarity search algorithm by Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 88, 2444, or with the help of computer programs using said algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, GAP, BESTFIT, FASTA, and TFASTA in Wisconsin).

[0110] Percentage identity is obtained by determining the number of identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0111] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the total length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given in some embodiments for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides in the sequence. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0112] Each nucleic acid sequence or amino acid sequence having a certain degree of identity with a given nucleic acid sequence or amino acid sequence may have at least one functional property of the given sequence, and in some cases, it is functionally equivalent to the given sequence. One important property is the ability to act as a cytokine, particularly when administered to a target. In some embodiments, each nucleic acid sequence or amino acid sequence having a certain degree of identity with a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0113] Where used herein, the term “about” indicates a degree of variation, e.g., within 10%, 5%, 2%, or 1%, that does not substantially affect the characteristics of the subject matter described. Therefore, unless otherwise indicated, the numerical parameters described in the following specification and the appended claims are approximations that may vary depending on the desired characteristics to be obtained. Each numerical parameter should be interpreted at least in light of the number of significant figures reported and by applying common rounding techniques, and not as an attempt to limit the application of the doctrine of equivalents to the claims.

[0114] overview Fusion proteins that specifically bind to both Tie2 and VEGF are provided herein. These fusion proteins are designed to address both the limitations of current therapies for Tie2 and VEGF, as well as the limitations of other currently available therapies for angiogenic or vascular diseases. As a result, fusion proteins comprising an anti-Tie2 antibody or its antigen-binding fragment and a vascular endothelial growth factor (VEGF)-binding domain are provided. Due to their dual functions of VEGF inhibition and Tie2 activation, these fusion proteins can play a beneficial role as therapeutic agents for angiogenic or vascular diseases.

[0115] Fusion protein This disclosure relates to a fusion protein comprising a Tie2 antibody or its antigen-binding fragment and a VEGF-binding domain. In some embodiments, the Tie-2 antibody or its antigen-binding fragment binds to a Tie2 Ig3-FNIII(1-3) domain containing the sequence of SEQ ID NO: 2.

[0116] In some embodiments, the Tie2 antibody or its antigen-binding fragment includes a heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs. 5 to 7, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs. 8 to 10.

[0117] In some embodiments, the fusion protein binds to the Tie2 Ig3-FNIII(1-3) domain, which includes SEQ ID NOs. 2, 3, or 4, and to VEGF.

[0118] In some embodiments, the fusion protein is a vascular endothelial growth factor (VEGF) binding domain, the VEGF binding domain comprising a vascular endothelial growth factor (VEGF) binding domain including the vascular endothelial growth factor (VEGF)-A binding domain of VEGF receptor 1 (VEGFR1) of SEQ ID NO: 13 and the vascular endothelial growth factor (VEGF)-A binding domain of VEGF receptor 2 (VEGFR2) of SEQ ID NO: 14, and an anti-Tie2 antibody or an antibody-binding fragment thereof, wherein the fusion protein binds to a Tie2 Ig3-FNIII(1-3) domain including SEQ ID NOs: 2, 3, or 4 and VEGF.

[0119] In some embodiments, the VEGF-binding domain comprises an anti-VEGF antibody or an antibody-binding fragment thereof. In further embodiments, the anti-VEGF antibody or fragment thereof comprises a variable-binding domain of bevacizumab, ranibizumab, or HuMab G6-31 (U.S. Patent Application Publication No. 2007 / 0141065).

[0120] In one embodiment, the VEGF-binding domain is ligated to the C-terminus of the heavy chain (HC) of an anti-Tie2 antibody or its antigen-binding fragment.

[0121] In one embodiment, the fusion protein is conjugated to amino acids 633-644 (SEQ ID NO: 20) and / or amino acids 713-726 (SEQ ID NO: 21) of Tie2 having the amino acid sequence of SEQ ID NO: 1. In another embodiment, the anti-Tie2 antibody or its antibody-conjugated fragment is conjugated to amino acids 633-644 (SEQ ID NO: 19) and / or amino acids 713-726 (SEQ ID NO: 20) of Tie2 having the amino acid sequence of SEQ ID NO: 1.

[0122] In one embodiment, the anti-Tie2 antibody has an IgG1 isotype.

[0123] In one embodiment, the VEGF-binding domain includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 15. In one embodiment, the VEGF-binding domain includes the amino acid sequence of SEQ ID NO: 15.

[0124] In one embodiment, the fusion protein comprises four polypeptide chains, two of which are a pair of heavy chains and two of which are a pair of light chains. Preferably, the heavy chains contain a Tie2-binding domain at the N-terminus that binds to a Tie2 Ig3-FNIII(1-3) domain, including SEQ ID NOs. 2, 3, or 4, and a VEGF-binding domain at the C-terminus. Preferably, the Tie2-binding domain is an anti-Tie2 antibody, the pair of heavy chains in the fusion protein contains the heavy chain of the antibody, and the pair of light chains in the fusion protein contains the light chain of the antibody.

[0125] In one embodiment, the fusion protein is a. A dimer of the first and second heavy chain monomers, wherein each heavy chain monomer is arranged from the N-terminus to the C-terminus. i.ii. A heavy chain variable domain (VH) domain that binds Tie2 together with the light chain variable domain, linked to the constant heavy chain domain (CH), A constant heavy chain domain (CH) containing a CH1 domain and an Fc region or fragment thereof, linked to the VEGF-binding domain of ii.iii. iii. VEGF-binding domains including the extracellular domain of the VEGF receptor (such as the VEGF-A binding domains of VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2)) Dimers of first and second heavy chain monomers, comprising a single-chain polypeptide containing; b. First and second light chain monomers, each light chain monomer comprising a variable light chain domain (VL) that binds Tie2 together with VH, linked from the N-terminus to the C-terminus to a constant light chain domain (CL1); Includes, The first and second monomers dimerize via their Fc regions or fragments thereof; The Tie2 binding domain is formed by pairing each heavy chain monomer with one of the light chain monomers, such that the VH and CH1 domains of each heavy chain monomer pair with the VL and CL1 domains of the light chain monomer.

[0126] In one embodiment, the fusion protein includes a linker between the VEGF receptor and an anti-Tie2 antibody or an antibody fragment thereof. In a further embodiment, the linker includes 5 to 50 residues, for example 10 to 40 residues, for example 15 to 30 residues, for example 20 residues.

[0127] In some embodiments, the fusion protein includes a linker between the VEGF-binding domain and an anti-Tie2 antibody or a fragment thereof, the linker containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the linker contains the sequence of SEQ ID NO: 16.

[0128] In some embodiments, the fusion protein includes a linker between the VEGF receptor and an anti-Tie2 antibody or an antibody fragment thereof, the linker containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, the linker contains the sequence of SEQ ID NO: 25.

[0129] In one embodiment, the fusion protein includes a linker between the VEGF-binding domain and the anti-Tie2 antibody or antibody fragment, located on the C-terminal side of one or more heavy chain constant domains of the anti-Tie2 antibody or its antigen-binding fragment, and in an N-terminus to C-terminus order, for example, the linker includes a sequence having 5 to 50 amino acid residues, for example 10 to 40 residues, for example 15 to 30 residues, for example 20 residues.

[0130] In one embodiment, the fusion protein includes a linker between the VEGF-binding domain and the anti-Tie2 antibody or antibody fragment, located on the C-terminal side of one or more heavy chain constant domains of the anti-Tie2 antibody or its antigen-binding fragment, and in an N-terminus to C-terminus order, wherein the linker includes the amino acid sequence of SEQ ID NO: 16 or 25, and the VEGF-binding domain includes the amino acid sequence of SEQ ID NO: 15.

[0131] In one embodiment, the fusion protein includes a CH domain containing the amino acid sequence of SEQ ID NO: 17, and a linker between the VEGF-binding domain and an anti-Tie2 antibody or an antibody fragment thereof containing the amino acid sequence of SEQ ID NO: 16 or 25, wherein the VEGF-binding domain contains the amino acid sequence of SEQ ID NO: 15.

[0132] In one embodiment, the fusion protein includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19.

[0133] In one embodiment, the fusion protein includes a heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 11. In one embodiment, the fusion protein includes a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

[0134] In one embodiment, the fusion protein includes a heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 26. In one embodiment, the fusion protein includes a heavy chain containing the amino acid sequence of SEQ ID NO: 26.

[0135] In one embodiment, the fusion protein includes a light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 12. In one embodiment, the fusion protein includes a light chain containing the amino acid sequence of SEQ ID NO: 12. In one embodiment, the fusion protein includes a heavy chain containing the amino acid sequence of SEQ ID NO: 11 and a light chain containing the amino acid sequence of SEQ ID NO: 12.

[0136] In one embodiment, the fusion protein comprises a CH domain comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the fusion protein comprises a CH domain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the fusion protein comprises one or more modifications or mutations in the constant region domain of the heavy chain to reduce or eliminate efficient interaction with Fc receptors on other immune cells in the body constant region domain. In some embodiments, the one or more modifications or mutations are L 234 L 235 G 236 G 237 (EU numbering) changed to a LAGA mutation, a FEGG mutation, an AAGG mutation, an AAGA mutation, a LALA mutation or a combination thereof. In some embodiments, the IgG constant region comprises any of the aforementioned mutations (e.g., the LALA mutation) and mutations at K322 and P331 (EU numbering) to reduce and eliminate cell damage mediated by immune cells.

[0137] The Tie2 antibody is monovalent or bivalent and contains a single chain or a double chain. Functionally, the binding affinity or dissociation constant (K D ) is the molar (M) concentration of the ligand at which half of the ligand binding sites on the protein are occupied in the system equilibrium state. The dissociation constant (K D ) is calculated by dividing the dissociation rate (K off ) value by the association rate (K on ) value. In the context of the dissociation constant, a lower value is consistent with a stronger binding. The K D of the Tie2 antibody is in the range of 10 -5 M to 10 -12 M. For example, the binding affinity (K D ) of the Tie2 antibody is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10-5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 It is M.

[0138] The exemplary fusion protein IGT-427 described herein simultaneously represses VEGF signaling and activates the Tie2 signaling pathway. IGT-427 exhibits a divalent interaction of less than 1 nM, as well as cell surface interactions. D Human Tie-2 is bound, and K is less than 10 pM. D It binds to human VEGF. IGT-427 binds to surface VEGF with an apparent affinity of less than 500 pM. IGT-427 binds to surface VEGF with K DThe concentration is less than 10 pM. IGT-427 shows comparable high affinity to rabbit orthologs.

[0139] In one embodiment, the fusion protein is 3E -9 M(Here, E -X The negative exponent and the number of zeros are represented, for example, 3E -9 This is the same as .0000000003. ) Less than 3.5E -9 Less than M, 4E -10 Affinity K less than M or less than M D (M) binds to the Tie2 Ig3-FNIII(1-3) domain containing sequence numbers 2, 3, or 4. K is less than a given amount. D value, that K D This means that the quantity is numerically smaller than the given quantity, indicating a stronger bond.

[0140] IGT-427 may offer superior efficacy compared to drugs that inhibit VEGF only, and / or may have more potent Tie-2 agonist activity compared to Ang2 inhibitors.

[0141] The Tie2 antibody or antibody fragment in the fusion protein may contain its biological equivalent to the extent that it can specifically recognize Tie2. For example, the Tie2 antibody or antibody fragment in the fusion protein may include changes to the amino acid sequence to further improve the antibody's binding affinity and / or other biological properties. Such modifications may include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. These amino acid variations may be made based on the relative similarity of amino acid substituents, such as the hydrophobicity, hydrophilicity, charge, and size of the amino acid side chains, in order to provide conservative substitutions. Analysis of the size, shape, and type of amino acid side chain substituents has shown that arginine, lysine, and histidine are all positively charged residues, alanine, glycine, and serine have similar sizes, and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, it can be said that arginine, lysine, and histidine, alanine, glycine, and serine; as well as phenylalanine, tryptophan, and tyrosine, are conservative substitutions.

[0142] Taking into account the above variations having biologically equivalent activity, in some embodiments the amino acid sequence of the fusion protein includes the sequences of the six CDRs in SEQ ID NOs. 5-10, and / or the heavy and light chains of SEQ ID NOs. 11 and 12, or any sequence exhibiting substantial identity. Substantial identity means at least 80% identity, e.g., at least 85% identity, 90% identity, 95% identity, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity.

[0143] Based on this, the fusion protein or its components may have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity when compared to the specified or all sequences described herein.

[0144] In one embodiment, the fusion protein is pegylated. In one embodiment, the fusion protein is modified with a half-life extension modulator, such as PEG, hyaluronic acid, or phosphorylcholine. In one embodiment, the fusion protein is site-specifically pegylated. In one embodiment, the fusion protein is site-specifically pegylated on a cysteine ​​residue. In one embodiment, the fusion protein is site-specifically modified (e.g., on a cysteine ​​residue) with a half-life extension modulator that may include PEG, hyaluronic acid, or phosphorylcholine. Alternatively, the half-life extension modulator may include albumin, albumin-binding peptides, and / or HA-binding protein fragments.

[0145] In one embodiment, the fusion protein further comprises the sequence of SEQ ID NO: 22, and the cysteine ​​residues of the sequence of SEQ ID NO: 22 are site-specifically modified (e.g., pegylated) with one of the half-life extension modulators described herein. In one embodiment, the sequence of SEQ ID NO: 22 is located at the C-terminus of the heavy chain. In one embodiment, the heavy chain comprises the sequence of SEQ ID NO: 23. In one embodiment, the heavy chain comprises the sequence of SEQ ID NO: 24.

[0146] In one embodiment, PEG has a molecular weight of approximately 40 kDa.

[0147] In one embodiment, a polypeptide comprising a chain monomer of the fusion protein of the present invention is provided.

[0148] In one embodiment, the polypeptide comprises the heavy chain monomer of the fusion protein of the present invention. In a further embodiment, the polypeptide comprises a sequence having at least 70% identity to the sequence of SEQ ID NO: 11 or 26, for example, at least 75% identity, for example, at least 80% identity, for example, at least 85% identity, for example, at least 90% identity, for example, at least 91% identity, for example, at least 92% identity, for example, at least 93% identity, for example, at least 94% identity, for example, at least 95% identity, for example, at least 96% identity, for example, at least 97% identity, for example, at least 98% identity, for example, at least 99% identity, for example, 100% identity. In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 11. In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 26.

[0149] In one embodiment, the polypeptide comprises the light chain monomer of the fusion protein of the present invention. In a further embodiment, the polypeptide comprises a sequence having at least 70% identity with respect to SEQ ID NO: 12, for example, at least 75% identity, for example, at least 80% identity, for example, at least 85% identity, for example, at least 90% identity, for example, at least 91% identity, for example, at least 92% identity, for example, at least 93% identity, for example, at least 94% identity, for example, at least 95% identity, for example, at least 96% identity, for example, at least 97% identity, for example, at least 98% identity, for example, at least 99% identity, for example, 100% identity. In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 12.

[0150] Polynucleotides; manufacturing methods; vectors and host cells In another aspect, the disclosure relates to nucleic acids encoding fusion proteins.

[0151] In another embodiment, the disclosure provides a nucleic acid encoding a polypeptide comprising a chain monomer of the fusion protein of the present invention. In one embodiment, the disclosure provides a nucleic acid encoding the heavy chain of the fusion protein. In another embodiment, the disclosure provides a nucleic acid encoding the light chain of the fusion protein.

[0152] In one embodiment, the nucleic acid molecule includes a sequence having at least 70% identity with respect to sequence number 27 or 29, for example, at least 75% identity, for example, at least 80% identity, for example, at least 85% identity, for example, at least 90% identity, for example, at least 91% identity, for example, at least 92% identity, for example, at least 93% identity, for example, at least 94% identity, for example, at least 95% identity, for example, at least 96% identity, for example, at least 97% identity, for example, at least 98% identity, for example, at least 99% identity, for example, 100% identity. In one embodiment, the nucleic acid molecule consists of sequence number 27 or 29.

[0153] In one embodiment, the nucleic acid molecule includes a sequence having at least 70% identity with respect to sequence number 28, for example, at least 75% identity, for example, at least 80% identity, for example, at least 85% identity, for example, at least 90% identity, for example, at least 91% identity, for example, at least 92% identity, for example, at least 93% identity, for example, at least 94% identity, for example, at least 95% identity, for example, at least 96% identity, for example, at least 97% identity, for example, at least 98% identity, for example, at least 99% identity, for example, 100% identity. In one embodiment, the nucleic acid molecule consists of sequence number 28.

[0154] In one embodiment, the Disclosure provides a set of one or more polynucleotides, each of which encodes at least one monomer chain of the fusion protein of the present invention, thereby encoding both such chains (i.e., the light chain and the heavy chain) of the fusion protein.

[0155] Fusion proteins can be recombinantly produced by constructing or isolating the nucleic acid encoding the fusion protein. Further cloning (DNA amplification) may be performed by isolating the nucleic acid and inserting it into a replicable vector, or further expression may be performed. Based on this, the disclosure relates in another embodiment to a nucleic acid-containing vector.

[0156] In some embodiments, this disclosure relates to nucleic acids, including fusion proteins. "Nucleic acids" comprehensively encompass DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acids, include native nucleotides and analogues having modified sugar or base moieties. The sequences of nucleic acids encoding heavy chain variable regions and light chain variable regions may be modified. Modifications include the addition, deletion, or non-conservative or conserved substitution of nucleotides.

[0157] The DNA encoding the fusion protein can be readily isolated or synthesized using conventional processes (for example, by using oligonucleotide probes that can specifically bind to the DNA encoding the heavy and light chains). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0158] In some embodiments, the disclosure provides an expression vector comprising nucleic acids encoding a fusion protein. In other embodiments, a vector is provided comprising nucleic acids encoding one heavy chain sequence and one light chain sequence of a fusion protein.

[0159] In one embodiment, a set of one or more vectors is provided that collectively comprises one or more sets of polynucleotides of the present invention, such that both chains of the fusion protein (i.e., the light chain and the heavy chain) are encoded in the set of vectors.

[0160] In one embodiment, the vector is a virus selected from animal viruses, such as reverse transcriptase viruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses, varicella-zoster viruses, baculoviruses, papillomaviruses, and papovaviruses.

[0161] In some embodiments, the expression vector further comprises a promoter / enhancer such as a human cytomegalovirus IE1 (CMV-IE1) promoter / enhancer.

[0162] As used herein, the term “vector” as a means for expressing a target gene in a host cell includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. The nucleic acid encoding the antibody in the vector is operablely ligated to a promoter.

[0163] As used herein, the terms “operably linked” or “linked” mean a functional linkage between a nucleic acid expression regulatory sequence (e.g., an array of promoters, signal sequences, or transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to control the transcription and / or translation of the other nucleic acid sequence.

[0164] In some embodiments, host cells comprising polynucleotides encoding the fusion proteins described herein are provided herein. In some embodiments, host cells comprising vectors comprising polynucleotides encoding the fusion proteins described herein are provided herein. The host cells may be prokaryotic or eukaryotic cells. The host cells may be isolated, cultured, for example, or not part of a multicellular organism. In some embodiments, the host cells are members of a cell line. In some embodiments, the host cells are mammalian cells. In some embodiments, the host cells are immortalized mammalian cells.

[0165] In the case of prokaryotic cells as hosts, strong promoters capable of processing transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter) are generally included, along with ribosome binding sites for translation initiation and transcription / translation termination sequences. Furthermore, in the case of eukaryotic cells as hosts, for example, promoters derived from mammalian cell genomes (e.g., metallothione promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Roussarcoma virus (RSV) promoter) can be used, and generally, polyadenylated sequences may be included as transcription termination sequences.

[0166] In some cases, the vector may be fused with other sequences to facilitate the purification of the expressed antibody. Examples of sequences that can be fused include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6×His (hexahistidine; Qiagen, USA).

[0167] The vector contains antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0168] In another aspect, this disclosure relates to cells transformed with the expression vector described above. The cells used to produce antibodies may be, but are not limited to, prokaryotes, yeasts, and higher eukaryotes.

[0169] Prokaryotic host cells such as Escherichia coli, Bacillus strains including Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0170] Exemplary useful animal host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0171] In one embodiment, the present disclosure provides a method for producing a fusion protein that binds Tie2 and VEGF, comprising the steps of: culturing cells containing a nucleic acid such as an expression vector that encodes the fusion protein (for example, cells transformed with an expression vector containing a nucleic acid that encodes the fusion protein); and recovering the fusion protein from the cultured cells.

[0172] Cells can be cultured in a variety of media. Any commercially available culture medium can be used without restriction. All other essential supplements known to those skilled in the art may be included in appropriate concentrations. Culture conditions such as temperature and pH, and the host cells to be selected, are known to those skilled in the art.

[0173] The antibody or its antigen-binding fragment can be recovered, for example, by centrifugation or ultrafiltration, and by removing impurities, for example, by affinity chromatography. Further additional purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography may be used.

[0174] Use of fusion proteins; treatment In another embodiment, this disclosure relates to methods and compositions for preventing or treating angiogenic or vascular disease by administering an effective amount of fusion protein. In yet another embodiment, this embodiment relates to methods and compositions for regulating angiogenesis, endothelial signaling, inflammation and / or vascular leakage by administering fusion protein to subjects who require regulation of angiogenesis, endothelial signaling, inflammation and / or vascular leakage. In some embodiments, the fusion proteins described herein are for therapeutic use.

[0175] Angiogenesis refers to the formation or growth of new blood vessels from existing blood vessels. Examples of neovascular diseases or angiogenesis-related diseases include, but are not limited to, cancer, metastasis, diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, corneal graft rejection, macular degeneration, glaucoma including neovascular glaucoma, systemic erythroderma, proliferative retinopathy, psoriasis, hemophilic arthropathy, capillary formation in atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases, and neurodegenerative diseases. Furthermore, similar cancers include, but are not limited to, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin lymphoma, lymphoma, and multiple myeloid hematological malignancies.

[0176] Fusion proteins may be administered to modulate angiogenesis, endothelial signaling, inflammation, and / or vascular leakage. For example, fusion proteins may be administered to increase the integrity of vascular endothelial cell membranes and / or reduce vascular leakage. Vascular leakage is known to contribute to visual impairment in several common eye disorders, including but not limited to diabetic macular edema (DME), diabetic retinopathy (DR), and age-related macular degeneration (AMD). In some embodiments, inflammation may originate from sepsis, respiratory distress syndrome, and / or viral infectious diseases.

[0177] Fusion proteins and their compositions may be administered to mammals, including rats, mice, livestock, pets, and humans. In some embodiments, the subject is human. In some embodiments, the subject is pets, such as mammalian pets. In some embodiments, the pets are dogs, cats, rabbits, ferrets, horses, mules, donkeys, or hamsters. Fusion proteins or their compositions may be administered daily. Fusion proteins or their compositions may be administered once every 1, 2, 3, 4, 6, or 12 months. In one embodiment, the fusion protein is administered once every 6 months (i.e., twice a year). Administration is by typically accepted routes, e.g., oral, rectal, intravitreous, intravenous, subcutaneous, intrauterine, or intracerebrovascular. In some embodiments, administration is by intravitreous injection. In one embodiment, 1 mg to 10 mg of the fusion protein is administered daily, monthly, or every 6 months by intravitreous injection. In one embodiment, less than 10 mg of the fusion protein is administered by intravitreous injection approximately once every six months (for example, twice a year).

[0178] Pharmaceutical composition; administration In some embodiments, the composition comprising the fusion protein is a pharmaceutical composition. In some embodiments, the composition comprises a suitable vehicle, excipient, or diluent commonly used in the art.

[0179] Suitable vehicles, excipients, and diluents are described in Remington: The Science and Practice of Pharmacy (19th ed.), Argennaro, Mack Publishing Company, Easton, Pa. 1995. Suitable pharmaceutically acceptable vehicles, excipients, or diluents include, for example, one or more of water, physiological saline, phosphate-buffered saline, dextrose, histidine, glycerol, sucrose, polysorbate, ethanol, and combinations thereof. The pH of the solution is preferably about 5 to about 8, more preferably about 6 to about 7. It will be apparent to those skilled in the art that certain carriers may be more preferable, for example, depending on the route of administration and concentration.

[0180] Pharmaceutical compositions having a pharmaceutically acceptable vehicle may be in various oral or parenteral dosage forms, such as tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, lyophilized products, and suppositories. Pharmaceutical compositions may contain diluents or excipients that can be compounded in combination, such as fillers, thickeners, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may be in the form of tablets, pills, powders, granules, capsules, etc. In connection with binding, compounds may be formulated by combining one or more excipients such as starch, calcium carbonate, sucrose, lactose, or gelatin. Simple excipients and lubricants such as magnesium stearate and talc may be used further.

[0181] Liquid preparations for oral administration may be suspensions, oral solutions, emulsions, syrups, etc. Excipients, such as simple diluents like water or wet paraffin, various wetting agents, sweeteners, fragrances, and preservatives, may be included in the liquid formulations. Furthermore, pharmaceutical compositions may be in parenteral dosage forms such as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized products, and suppositories. Injectable propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and esters such as ethyl oleate may be suitable for insoluble solvents and suspensions. Basic substances for suppositories include vitepsol, macrogol, Tween 61, cocoa butter, lauric butter, and glycerol gelatin.

[0182] The composition is administered in a pharmaceutically effective dose. Terms used herein, such as "pharmaceutically effective dose," refer to a sufficient amount of the pharmaceutical composition for disease treatment that has an appropriate benefit / risk ratio, which may be applicable to all medical treatments. The effective dose may vary depending on various factors, including parameters such as disease severity, patient / animal age and sex, disease type, drug activity, drug sensitivity, administration time, route of administration, secretion rate, duration of treatment, and other factors. The composition may also be administered in a single dose or divided into multiple doses. Considering these factors, it is important to administer the minimum amount sufficient to achieve the maximum effect without side effects. The dosage of the pharmaceutical composition is not particularly limited but will vary depending on various factors, including the patient / animal's health condition and weight, disease severity, drug type, route of administration, and administration time.

[0183] The composition may be administered to mammals, including rats, mice, livestock, pets, and humans, once or more times daily via typically accepted routes, such as orally, rectally, intravenously, subcutaneously, intrauterine, intravitreous, or intravascularly. In some embodiments, the composition is administered by intravitreous injection. In some embodiments, the subject is human. In some embodiments, the subject is pets, such as mammalian pets. In some embodiments, the pets are dogs, cats, rabbits, ferrets, horses, mules, donkeys, hamsters, or other domesticated pets.

[0184] In other respects, this disclosure refers to methods for the prevention or treatment of angiogenic or vascular diseases, and anti-angiogenic methods, which include a step of administering an antibody or the above composition to an individual requiring it.

[0185] The fusion protein may be provided in a pharmaceutical composition.

[0186] The methods of this disclosure include procedures for administering a pharmaceutically effective dose of a pharmaceutical composition to an individual requiring inhibition of angiogenesis. The individual may be, but is not limited to, a mammal such as a dog, cat, ferret, cattle, horse, rabbit, mouse, rat, or human. For example, in one embodiment, the individual may be a chicken, turkey, or other non-mammalian. The pharmaceutical composition may be administered by appropriate means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary or intranasal, and, if necessary, intralesional for topical treatment. In some embodiments, the dose of the pharmaceutical composition may vary depending on a variety of factors, including, but not limited to, the individual's health condition and weight, the severity of the disease, the type of drug, the route of administration, and the time of administration, and may be readily determined by those skilled in the art.

[0187] In other embodiments, the disclosure refers to methods for the prevention or treatment of cancer, including procedures for administering compositions or antibodies to individuals requiring antibodies and compositions or pharmaceutical compositions for the prevention or treatment of cancer.

[0188] Cancers are not limited to those that can be treated with the antibodies of this disclosure. Specifically, the antibodies can prevent the development or progression of cancer by inhibiting angiogenesis. Examples of cancers include, but are not limited to, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin lymphoma, lymphoma, and multiple myeloid cancers, as well as hematological cancers.

[0189] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those understood by those skilled in the art.

[0190] [Examples] The following examples are intended merely to illustrate the present invention and are not intended to limit it.

[0191] [Example 1] Preparation of fusion proteins A. Design of structures We designed a fusion protein, IGT-427, in which the VEGF-A binding domains of VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) were fused to the C-terminus of the heavy chain (HC) of an anti-Tie2 antibody. The construct was then used to create a protein expression vector containing a human cytomegalovirus IE1 (CMV-IE1) promoter / enhancer. The amino acid sequences of the heavy chain (HC) and light chain (LC) are shown in Table 1 above.

[0192] Expression and purification of B.IGT-427 Based on the manufacturer's instructions (Gibco), plasmid DNA encoding IGT-427 was transiently and co-transfected into ExpiCHO-S cells. Equal volumes of heavy and light chain DNA were used. The transfected cells were then incubated at 32°C in a humidified atmosphere of 5% CO2 in air with shaking for the Max titer protocol. After 12 days of incubation, the culture medium containing the secreted fusion protein was collected, the cells were removed by centrifugation, and the culture supernatant was collected and filtered. The fusion protein was purified using an AKTAPure purification system (Cytiva) with a HiTrap MabSelect Sure Protein A (Cytiva) affinity column in binding / wash buffer (20 mM sodium phosphate, pH 7.2 and 150 mM NaCl) and elution buffer (25 mM sodium acetate, pH 3.3), and thus immediately neutralized by adding 100 μl of 1 M Tris-HCl, pH 8.8 per 1 ml fraction. Next, the pooled fractions were buffered with PBS, pH 7.4 using a centrifugal filter unit (Amicon). The resulting final sample was stored until further use. Simultaneously, the fusion protein was subjected to quality control studies including SDS-PAGE (Invitrogen®, Novex®, WedgeWell), SEC-HPLC (Agilent Infinity 1260 and 300Å pore size column), endotoxin measurement (Charles River Cartridge <0.05 EU / ml sensitivity), and surface plasmon resonance (SPR Biacore).

[0193] [Example 2] Preparation of orthologous Tie2 protein A. Design of structures The Ig3 domain and FNIII(1-3) domain of three orthologue proteins of Tie2 (human, rabbit, and mouse) were selected as binding proteins to IGT-427. The domains of each Tie2 orthologue were cloned into a pFuse-mouse IgG1 Fc2 vector, which consists of an elongation factor 1-alpha (EF-1α) promoter and an IL2 signal peptide, along with the consecutive C-terminal hexa-histidine and thrombin protease cleavage sites (LVPRGS) between Tie2 and the IgG1 Fc sequence. The sequences of the human (SEQ ID NO: 2), rabbit (SEQ ID NO: 3), and mouse (SEQ ID NO: 4) Tie2 orthologues are shown in Table 1 above.

[0194] Expression and purification of B.Tie2 orthologue To produce Tie2 orthologues, Expi293F (Gibco) cells, which can efficiently produce recombinant proteins, were used. Orthologue Tie2 protein was transiently expressed in the Expi293F cell line by referring to the manufacturer's instructions (Gibco). After 4 days of transfection and incubation at 37°C in a humid atmosphere of 8% CO2 air with shaking, the resulting culture medium was collected and cells were removed by centrifugation. The culture supernatant containing the secreted antibodies was isolated and stored at 4°C, or immediately purified using an AKTAPure purifier (Cytiva) equipped with an affinity column (HiTrap MabSelect Sure Protein A, Cytiva) in two different buffer systems: binding and washing (20 mM sodium phosphate, pH 7.2 and 150 mM NaCl) and elution buffer (25 mM sodium acetate, pH 3.3). The Tie2 protein was immediately neutralized by adding 100 μl of 1 M Tris-HCl, pH 8.8 per 1 ml fraction. The fractions were pooled and then buffered with PBS, pH 7.4 through a protein centrifuge filter (Amicon) and stored at -80°C. In the next process, the protein was analyzed for quality control studies using SDS-PAGE (Invitrogen® Novex® WedgeWell), SEC-HPLC (Agilent Infinity 1260 and 300 Å pore size column) and surface plasmon resonance (SPR).

[0195] [Example 3] Affinity measurement of the fusion protein (IGT-427) against the Tie2 ortholog. The affinity of the fusion protein to the orthologous Tie2 protein was measured using an SPR system (Biacore 3000). CM5 SPR chips (Cytiva) were modified with either monomeric or dimeric forms of human, rabbit, or mouse Tie2 using EDC / NHS chemistry (here, the dimeric form was an Fc fusion). After activating the sensor chips with EDC (0.2M) and NHS (0.05M) for 7 minutes, 2.5 μg mL of 10 mM sodium acetate at pH 5 was added to achieve an antigen surface density of approximately 200 RU. -1 The Tie2 construct was injected. The surface was then deactivated by injecting 1M ethanolamine·HCl pH 8.5 for 7 minutes. Activation and deactivation reagents were purchased from Cytiva. After immobilization of the Tie2 construct, the surface was prepared with two 5-second pulses of 300 mM phosphate. Fusion proteins in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 2 mg / mL BSA, and 0.05% polysorbate-20) were injected at 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, and 1.2 nM concentrations at 30 μL / min for 5 minutes, and dissociation was monitored for 20 minutes. Surface regeneration was performed with a 10-second injection of 300 mM phosphate. The sensorgrams were fitted to a 1:1 Langmuir junction model using Scrubber software (BioLogic). D The values ​​and SPR sensorgrams are shown in Table 2 and Figures 1A-1C below, respectively. [Table 2]

[0196] [Example 4] Inhibition of VEGFR2 phosphorylation by IGT-427 In a 60mm culture dish, in EGM-2 medium (Lonza), HUVEC (1 × 10) was incubated at 37°C. 5Cells were cultured (cells / ml). To induce starvation, 90% confluent cells were incubated with supplement-free EBM-2 medium for 4 hours. Starved HUVECs were pretreated with IGT-427 or Eylea(R) at the indicated concentrations for 30 minutes, followed by treatment with recombinant human VEGF for 2 minutes. Cells were washed with cold PBS and treated with lysis buffer (10mM Tris-Cl pH 7.4, 150mM NaCl, 5mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor) and lysed at 4°C for 20 minutes. Cell solubilates were then prepared by centrifugation at 13000 rpm for 15 minutes. Protein concentrations in the supernatant were quantified by BCA assay. Cell solubilates were prepared by adding 4× SDS sample buffer, and the cell solubilates were subjected to SDS PAGE to transfer proteins to a nitrocellulose membrane (GE).

[0197] To investigate the phosphorylation of VEGFR2 and Akt, blots were blocked with 5% skim milk-containing TBS-T at room temperature (RT) for 1 hour and incubated with anti-phospho-VEGFR2 antibody (Tyr1175) or anti-phospho-Akt (S473) antibody at 4°C for approximately 16 hours. The signals for phospho-VEGFR2 (Tyr1175) or pAkt (S473) were visualized by chemiluminescence sensitization (ECL). The membranes were then incubated in stripping buffer (Thermo) for 15 minutes, and subsequently probed again with anti-VEGFR2 antibody or anti-Akt antibody to determine the amounts of total VEGFR2 and total Akt. VEGF-inducible VEGFR2 phosphorylation at Tyr1175 was inhibited in a concentration-dependent manner by IGT-427, and this inhibition was comparable to that by Eylea(R) (Figures 2A and 2B). Furthermore, IGT-427 induced Akt phosphorylation in a dose-dependent manner.

[0198] A VEGF reporter assay was performed using the VEGF Reporter Bioassay Kit (PROMEGA®). Briefly, 0.4 ml of KDR / NFAT-RE HEK293 cells were added to 4.6 ml of DMEM medium containing 10% FBS (assay buffer), and 25 μl of the cell suspension was seeded onto a white flat-bottom 96-well assay plate (Corning). IGT-427, falisimab, or aflibercept were added at a 3-fold high concentration of 50 nM and in 3-fold serial dilutions in 25 μl of assay buffer. Recombinant human VEGF at a 3-fold high concentration of 40 ng / ml in 25 μl of assay buffer was added to the plate. After incubation at 37°C for 6 hours, 75 μl of the BIO-GLO® reagent included in the kit was added, and luminescence was measured using the GloMax® Discovery System (PROMEGA®). The IC50 values ​​for IGT-427, falisimab, and aflibercept were approximately 0.48 nM, 0.32 nM, and 0.75 nM, respectively (Figure 2C), suggesting that VEGF inhibition by IGT-427 is equivalent to inhibition by either falisimab or aflibercept.

[0199] [Example 5] IGT-427 enables stronger and more sustained activation of Tie2. CHO cells overexpressing full-length human Tie2 (CHO-hTie2, 1 × 10⁵ cells / ml) were cultured in DMDM ​​medium (Thermo Fisher) in 60 mm culture dishes at 37°C. To induce starvation, 90% confluent cells were incubated with supplement-free DMEM medium for 4 hours. Starved CHO cells were treated with 10 nM IGT-427 or recombinant human angiopoietin-1 (R&D Systems®) for various durations (30 minutes, 1 hour, 2 hours, 6 hours, and 24 hours). Cells were washed with cold PBS and treated with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor), and lysed at 4°C for 20 minutes. Next, cell lysates were prepared by centrifugation at 13,000 rpm for 15 minutes. The lysates were then used to detect the levels of phospho-Tie2 and total Tie2 signals using the pTie2 and total Tie2 ELISA assay kit (R&D Systems(R)). The phospho-Tie2 signal was normalized by the total Tie2 signal. IGT-427 showed a stronger and more sustained pTie2 signal compared to angiopoietin-1 (Figure 3).

[0200] [Example 6] Dose-dependent activation of Akt signaling by IGT-427 HUVEC cells (1 × 10⁵ cells / ml) were cultured in EBM-2 medium (Lonza) at 37°C in 60 mm culture dishes. To induce starvation, 90% confluent cells were incubated with supplement-free EBM-2 medium for 4 hours. Starved HUVEC cells were treated with various concentrations of IGT-427 (0.3, 1, 3, 10, 30, 100 nM) for 30 minutes. Cells were washed with cold PBS and treated with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor), and lysed at 4°C for 20 minutes. Cell lysates were then prepared by centrifugation at 13000 rpm for 15 minutes. Protein concentrations were quantified by BCA assay. Cell lysates were prepared by adding 4×SDS sample buffer, and the lysates were subjected to SDS PAGE to transfer the proteins to a nitrocellulose membrane (GE). To investigate Akt phosphorylation, the blot was blocked with 5% skim milk-containing TBS-T at room temperature (RT) for 1 hour and incubated with anti-phosphoAkt(S473) antibody at 4°C for approximately 16 hours. The pAkt(S473) signal was visualized by sensitized chemiluminescence (ECL). The membrane was then incubated in stripping buffer (Thermo) for 15 minutes, and subsequently probed again with anti-Akt antibody to determine the total amount of Akt. As shown in Figure 4, a dose-dependent escalation of phospho-Akt signaling by IGT-427 was observed.

[0201] [Example 7] IGT-427 bypasses Ang2 signaling. HUVEC cells (1 × 10⁵ cells / ml) were cultured in EBM-2 medium (Lonza) at 37°C in 60 mm culture dishes. To induce starvation, 90% confluent cells were incubated with supplement-free EBM-2 medium for 4 hours. Starved HUVEC cells were pretreated with recombinant human angiopoietin-2 (R&D Systems®) at concentrations of 0.01, 0.1, and 1 nM for 30 minutes, followed by treatment with 2 × 20 kDa pegylated IGT-427 at a concentration of 30 nM for 60 minutes. Cells were washed with cold PBS and treated with lysis buffer (10 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, protease inhibitor, phosphatase inhibitor) and lysed at 4°C for 20 minutes. Next, cell lysates were prepared by centrifugation at 13,000 rpm for 15 minutes. Protein concentration was quantified by BCA assay. Cell lysates were prepared by adding 4× SDS sample buffer, and the cell lysates were subjected to SDS PAGE to transfer the proteins to a nitrocellulose membrane (GE). To investigate Akt phosphorylation, the blot was blocked with 5% skim milk-containing TBS-T at room temperature (RT) for 1 hour and incubated with anti-phosphoAkt(S473) antibody at 4°C for approximately 16 hours. The pAkt(S473) signal was visualized by sensitized chemiluminescence (ECL). The membrane was then incubated in stripping buffer (Thermo) for 15 minutes, and subsequently probed again with anti-Akt antibody to determine the total amount of Akt. Recombinant angiopoietin-2 (Ang2) induced weak Akt phosphorylation in a concentration-dependent manner. However, IGT-427 further increased Akt phosphorylation in the presence of recombinant angiopoietin-2, demonstrating that IGT-427 overcomes Ang2 signaling (Figure 5).

[0202] [Example 8] IGT-427 double bond capacity The double-binding properties of IGT-427 to its antigens, Tie2 and VEGF, were investigated using a surface plasmon resonance (SPR) system (BIACORE® 3000). Recombinant human full-length Ang2 (R&D Systems®) was captured on a CM5 SPR chip (Cytiva), and human dimerized Tie2 (100 nM) was injected first, followed by sequential injection of IGT-427 (150 nM) and human VEGF (20 nM) (R&D Systems®). Sensorgrams showed a sequential increase in signal, demonstrating that IGT-427 can simultaneously bind to the Ang2-Tie2 complex and VEGF (Figure 6).

[0203] [Example 9] Inhibition of TNF-α-induced apoptosis by IGT-427 HUVEC cells (1 × 10⁵ cells / ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60 mm culture dishes. Cells were pre-treated to 90% confluent with IGT-427 or Eylea® for 60 minutes, followed by treatment with recombinant human TNF-α (50 ng / ml) for 24 hours. Apoptotic cells were stained with APO-BrdU® TUNEL Assay Kit (Thermo Fisher, A23210) and identified by Attune (Thermo Fisher). Briefly, cells were suspended in 0.5 mL of PBS, 5 mL of 1% (w / v) paraformaldehyde in PBS was added to the cell suspension, and the cell suspension was placed on ice for 15 minutes. Cells were centrifuged at 300 × g for 5 minutes and washed twice with 5 mL of PBS. The cells were resuspended in 0.5 mL of PBS, 5 mL of ice-cold 70% (v / v) ethanol was added to the cell suspension, and the cell suspension was placed in a -20°C freezer for at least 30 minutes. To remove the 70% (v / v) ethanol, the cell suspension was centrifuged at 300 × g for 5 minutes, and then the cell pellet was resuspended in 1 mL of wash buffer provided in the kit. The cell pellet was incubated in 50 μL of DNA labeling solution (10 μL of reaction buffer, 0.75 μL of TdT enzyme, 8.0 μL of BrdUTP, and 31.25 μL of dH2O) at 37°C for 60 minutes. At the end of the incubation time, 1.0 mL of rinse buffer was added to the cells, and the cells were centrifuged at 300 × g for 5 minutes. After further washing with rinse buffer, cells were incubated in 100 μL of antibody staining solution (5.0 μL of Alexa Fluor® 488 dye-labeled anti-BrdU antibody and 95 μL of rinse buffer) at room temperature for 30 minutes. Apoptotic cells were analyzed by Attune flow cytometry (Thermo Fisher). TNF-α induced apoptosis in HUVEC cells, and IGT-427 significantly inhibited TNF-α-induced apoptosis, while Eylea® did not (Figure 7).

[0204] [Example 10] Surface Tie2 level measurement As preparation for the assay, CHO-hTie2 cells were grown on a 10 cm tissue culture dish until confluent. The CHO-hTie2 cells were washed with Dulbecco's phosphate-buffered saline (DPBS), detached by treatment with trypsin, counted, centrifuged at 200 g for 5 minutes, and resuspended at a density of 0.05 × 10⁶ cells / mL. To seed the assay plates, 1 mL of the cell suspension was dispensed into each well of a 24-well tissue culture plate, and the plates were incubated overnight at 370°C. The following day, the culture medium was removed from the tissue culture plates and replaced with either DMEM alone or 0.5 mL of DMEM containing either 10 nM angiopoietin 1 (R&D Systems(R) #923-AN), 10 nM IGT-301 (ITP-006), or 10 nM IGT-427 (ITP-016) for the unstimulated group. The assay plates were returned to a 370°C tissue culture incubator for the specified time (0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, or 24 hours).

[0205] After incubation, the tissue culture plates were placed on ice, washed with cold DPBS, and detached by adding 0.5 mL of non-enzymatic cell dissociation buffer (PeproTech(R)#CPD-125). The cells were incubated on ice for 5 minutes, and then collected by adding 1 mL of FACS buffer (0.5% BSA in DPBS) and gently pipetting to detach any cells still adhering to the culture surface. The resulting cell suspension was collected in a 5 mL Falcon FACS tube, pelletized by centrifugation at 300 g for 5 minutes, and the supernatant was aspirated. After washing the cells once with 1 mL of FACS buffer and aspirating the supernatant, approximately 100 μL of residual FACS buffer remained in the tube. The cells were then stained by adding a 100 μL staining cocktail consisting of 99.5 μL of FACS buffer and 0.5 μL of PE-Tie2 (BioLegend(R)#334205). The sample was covered with Parafilm and incubated in the dark at 40°C for 30 minutes.

[0206] After incubation, cells were washed twice with 1 mL of FACS buffer as described above, then resuspended in 400 μL of final volume of FACS buffer and fixed for immediate analysis or analysis the following day. For fixation, cells were washed twice as described above, resuspended in 200 μL of 4% paraformaldehyde at 40°C in the dark for 30 minutes, washed once with 1 mL of FACS buffer, resuspended in 400 μL of final volume of FACS buffer, covered and stored overnight at 40°C. Samples were acquired using an Attune NXT Acoustic flow cytometer (Thermo Fisher), and files were analyzed using FlowJo® (Tree Star, Inc.). IGT-427 treatment resulted in slower disappearance and greater presence of surface Tie2 compared to angiopoietin-1 (Ang1), an endogenous Tie2 agonist (Figure 8).

[0207] [Example 11] Block Tie2 disconnection. Tie2 is known to be cleaved and downregulated in human, mouse, and endothelial cells in various inflammatory conditions, leading to increased levels of soluble Tie2 (sTie2) (Thamm et al., Critical Care Medicine, 2018. 46:e928-e936). Blocking a wide range of matrix metalloproteinases (MMPs) is known to be sufficient to prevent Tie2 cleavage and vascular leakage (Thamm et al., Critical Care Medicine, 2018. 46:e928-e936; Sung et al., 2011. The Journal of Clinical Endocrinology & Metabolism 96:E1148-E1152; Findley et al., 2007, Arteriosclerosis, Thrombosis, and Vascular Biology 27:2619-2626). MMP-14 is considered a major Tie2 cleavage, and Tie2 is cleaved at multiple sites within the fibronectin type 3 domain by matrix metalloproteinase-14 (Idowu, TO et al., eLife, August 24, 2020, 9:e59520).

[0208] Recombinant human MMP-14 (R&D Systems(R), catalog # 918-MP-010, 0.5 ug) was mixed with dimeric human Tie2 extracellular domain (ECD)-human IgG fusion protein (hTie2-ECD, 5 ug) in the absence or presence of IGT-427 (same molar concentration as hTie2-ECD). The mixture was incubated at 30°C for 16 hours and subjected to SDS-PAGE (reducing conditions). Consistent with previous reports, co-incubation of MMP-14 and human Tie2 resulted in a significant reduction of intact hTie2-ECD and the appearance of shorter, cleaved fragments. However, IGT-427 significantly blocked the cleavage and reduction of intact human Tie2-ECD protein by MMP-14 (Figure 9).

[0209] While we do not wish to be bound by theory, based on this evidence, it is thought that the bispecificity of an anti-Tie2 antibody or antigen-binding fragment that binds to the Tie2 Ig3-FNIII(1-3) domain, including the amino acid sequence of SEQ ID NO: 2, 3, or 4, or more specifically, SEQ ID NO: 20 and / or SEQ ID NO: 21, and a VEGF-binding domain, reduces Tie2 shedding by combining the effect of blocking the binding of MMP-14 to human Tie2 with the effect of inhibiting the cleavage of the human Tie2-ECD protein caused by VEGF. This provides a particularly beneficial result of retaining Tie2 on the cell surface membrane.

[0210] [Example 12] Soluble Tie2 (sTie2) measurement HUVEC cells (1 × 10⁵ cells / ml) were cultured in EGM-2 medium (Lonza) at 37°C in 60 mm culture dishes. 90% confluent cells were pretreated with 10 nM IGT-427, falisimab, or recombinant human angiopoietin-1 (R&D Systems®) for 60 minutes, followed by treatment with recombinant human TNF-α (50 ng / ml) for 24 hours. The cell supernatant was collected and centrifuged at 13000 rpm for 10 minutes. Soluble Tie2 in the cell supernatant was measured using the Total Tie2 ELISA Assay Kit (R&D Systems®). IGT-427 suppressed soluble Tie2 levels under both basal and TNF-α-induced conditions, while the other agents did not (Figure 10).

[0211] [Example 13] TEER (Transendothelial Electrical Resistance) Assay HUVEC (2 × 10⁵ cells) were cultured at 37°C in a cell culture insert in a 24-well plate (Corning) in EGM-2 medium (Lonza). After 2 days of incubation, the medium was changed to EGM-2 medium containing 0.5% FBS. After another 2 days of incubation, the cell culture insert was placed in a CellZscope(R) (NanoAnalytics), and TEER (transendothelial electrical resistance) was continuously measured in a CO₂ incubator at 37°C. After confirming cell barrier formation, recombinant human VEGF (R&D) at a concentration of 10 ng / ml was added to the lower compartment. Various drugs, such as IGT-427, falisimab, aflibercept, or recombinant human angiopoietin-1 (R&D) at a concentration of 10 nM, were added to the lower compartment, and TEER was monitored for 48 hours after VEGF treatment. VEGF disrupts the integrity of the endothelial barrier, and IGT-427 was superior to falisimab, aflibercept, and Ang1 in restoring endothelial barrier integrity from VEGF-induced damage (Figure 11).

[0212] [Example 14] Inhibition of CNV (choroidal neovascularization) by intravitreous injection of IGT-427. Chinchilla rabbits (male, 2.0-2.5 kg) were anesthetized by applying eye drops (Mydriacyl eye drops, 1%) to the right eyeball. A laser (Elite, Lumenis(R), USA) was then shone onto the right eyeball at 532 nm, 150 mW output, and a duration of 0.1 seconds to generate six spots around the optic nerve. IGT-427, Eylea(R), or control IgG (50 μl injection volume / eye, Eylea(R) (800 μg), IGT-427 (885 μg), control IgG (716 μg)) was administered intravitreously to the right eye of the anesthetized animals using a syringe equipped with a 3-gauge needle on the day of choroidal neovascularization (CNV) induction. The molar ratio of Eylea(R), IGT-427, and control IgG was 1:0.65:0.68. Different molar values ​​were used due to several limitations on concentration and volume. On days 0, 7, and 14, animals were anesthetized by applying eye drops (Mydriacyl eye drops, 1%) to the right eyeball, and 1 mL of fluorescein sodium salt solution (2%) was intravenously injected. Fundus images were taken within 2 minutes using a fundus camera (TRC-50IX, TOPCON, Japan). Retinal CNV area and efficacy were evaluated using retinal fluorescein fundus photographs, and image analysis was performed using ImageJ software (NIH, Bethesda, Maryland) to verify the fluorescence intensity of CNV lesion sites. IGT-427 suppressed retinal leakage by 30% compared to control IgG. On the other hand, Eylea(R) was able to inhibit retinal leakage by 20% (Figure 12).

[0213] [Example 15] IGT-427 variant for PEGylation To extend the half-life of IGT-427, the heavy and light chain plasmids of IGT-427 were modified to generate five different antibody constructs for PEGylation (outlined in Figure 13). All five variants have a light chain with the C214S mutation (EU numbering) that removes the inter-chain disulfide bond between the heavy and light chains, in combination with the C218S mutation (EU numbering) on each heavy chain. Additionally, one or both of the heavy chain cysteines within the hinge region of IGT-427 were replaced with serine, leaving either one or no inter-chain disulfide bonds between the heavy chains. In the case of PRO592 and PRO596, a single disulfide bond remains in the reduced and PEGylated state. In the case of PRO593, PRO594, and PRO595, all inter-chain disulfide bonds were removed and new cysteine residues were introduced for PEGylation. PRO593 has a cysteine introduced at the C-terminus of the heavy chain, while PRO594 and PRO595 have cysteines introduced in the linker domain between the Fc domain and the VEGF trap domain, respectively. Together, these variants form a set of constructs with diverse PEGylation sites that were tested for PEGylation efficiency, antigen binding, and biological activity. Each IGT-427 variant was expressed using the Expi293 transient expression system. The clarified cell culture supernatant was purified using a HiTrap mAbselect PrismA column (Cytiva). The eluted antibody was dialyzed into 1×PBS and concentrated to 10 mg / mL for PEGylation studies.

[0214] [Example 16] PEGylation Reaction Screening Each of the 10 mg / mL IGT-427 variants was reduced with 10 or 20 mM cysteamine for 1 hour at room temperature and then buffer-exchanged into PEGylation buffer (50 mM sodium phosphate, 150 mM NaCl, 2.5 mM EDTA, pH 7.2). 10 molar equivalents of 20 kDa linear PEG-maleimide were added to the buffer-exchanged antibody and the PEGylation reaction was allowed to proceed for 1.5 hours at room temperature. Analysis of the reaction mixture by non-reducing SDS-PAGE revealed that these variants showed up to approximately 50% conversion to doubly PEGylated species.

[0215] To further optimize the PEGylation efficiency, reduction conditions were screened using PRO593, a variant that showed the best conversion under mild reduction conditions. PRO593 at 10 mg / mL was reduced with 10 or 100 mM cysteamine for 1 hour, 1 or 10 mM DTT for 15 minutes, or 2 or 20-fold molar excess of TCEP (tris(2-carboxyethyl)phosphine) for 15 minutes. After each reduction, PRO593 was buffer-exchanged into the PEGylation buffer and PEGylated with a 10-fold molar excess of 20 kDa linear PEG-maleimide for 1.5 hours. From this set of reduction conditions, it was observed that a 20-fold molar excess of TCEP resulted in nearly complete conversion of PRO593 to the species with two PEG additions. Next, all five IGT-427 variants were reduced with a 20-fold molar excess of TCEP for 15 minutes at room temperature, followed by buffer-exchange into the PEGylation buffer and PEGylation with a 10-fold molar excess of 20 kDa linear PEG-maleimide. The results of the optimized reaction conditions are shown in Figure 14. Antibodies with two PEG additions were purified from the reaction mixture using a HiTrap SP-HP cation exchange chromatography column (Cytiva) and gradient salt elution. The PEGylated antibodies were dialyzed into 1×PBS, concentrated to 10 mg / mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

[0216] [Example 17] Characterization of PEGylated IGT-427 Variants Binding to Tie2 or VEGF was characterized by SPR (Figure 15). A 100 nM solution of each antibody was injected at 30 μL / min for 300 seconds onto a surface containing immobilized Tie2 or VEGF. The difference in binding was determined using the relative binding signal after 300 seconds.

[0217] [Example 18] Ocular PK Studies To evaluate the effects of PEGylation and PEG molecular weight on the ocular pharmacokinetics of IGT-427, 500 μg intravitreal injections of Eylea®, falisimab, IGT-427, (2 × 20 kDa PEG)-IGT-427, and (2 × 40 kDa PEG)-IGT-427 were administered to male New Zealand white rabbits. Three or four animals were administered each test substance group on day 0. On days 1, 3, 7, and 14 after administration, blood was collected from one of the three or four animals, and after euthanasia by intravenous barbiturate overdose, both eyes were collected, rapidly frozen in liquid nitrogen, and dissected for collection of aqueous humor, vitreous fluid, retina, and choroid. Vitreous fluid samples were diluted 1:5 with 1 × PBST without homogenization and stored at -70°C until further analysis.

[0218] Zaltrap(R) was purchased from a commercial source, dialyzed against Eylea(R) preparation buffer (10 mM sodium phosphate, 40 mM NaCl, 0.03% polysorbate-20, 5% sucrose), and diluted to 10 mg / mL with the same buffer.

[0219] Falicimab was expressed using an Expi293 transient expression system (ThermoFisher). The clarified cell culture supernatant was purified using a HiTrap MabSelect PrismA column (Cytiva) with 2% ethanol washing to remove endotoxins, followed by pH gradient elution to remove incompletely assembled antibodies. The eluted antibodies were dialyzed in 1×PBS, concentrated to 10 mg / mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

[0220] IGT-427 was expressed using the Expi293 transient expression system. The clarified cell culture supernatant was purified using a HiTrap MabSelect PrismA column (Cytiva) with 2% ethanol washing to remove endotoxins. The eluted antibody was dialyzed in 1×PBS, concentrated to 10 mg / mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

[0221] PEGylated IGT-427. PRO593 was expressed using an Expi293 transient expression system (ThermoFisher). The clarified cell culture supernatant was purified using a HiTrap MabSelect PrismA column (Cytiva) with 2% ethanol washing to remove endotoxins. The eluted antibody was dialyzed in 1×PBS and concentrated to 10 mg / mL for PEGylation. The concentrated antibody was reduced with 20-fold molar excess TCEP, then desalted in PEGylation buffer and incubated with either 10-fold molar excess 20 kDa linear or 40 kDa branched PEG maleimide at room temperature for 1.5 hours. The PEGylation reaction mixture was purified using a HiTrap SP-HP column (Cytiva) at pH 4.6 using a NaCl gradient. The eluted PEGylated antibody was dialyzed in PBS, concentrated to 10 mg / mL, formulated with 0.01% polysorbate-20, and filtered through a 0.2 μm filter.

[0222] [Example 19] Characterization of samples for PK research Non-reducing SDS-PAGE analysis of each test substance was performed by mixing 5 μg of protein with 4× LDS (lithium dodecyl sulfate), electrophoresis on a 4-12% Bis-Tris gel at 150 V for 60 minutes without heating, and then staining with SafeStain (ThermoFisher). SEC-HPLC analysis was performed by loading 5 μg of each test substance onto a Zenix(R) size exclusion column (SEC)-300 at 0.5 mL / min in 100 mM arginine, 1× PBS, pH 6.7. Binding to rabbit Tie2 and VEGF was characterized by SPR.

[0223] SPR was used to evaluate the binding of PEGylated IGT-427 variants to either Tie2 or VEGF. Figure 3 shows sensorgrams obtained from 100 nM injection of the variants onto the surface of human Tie2 or VEGF. Compared to unmodified IGT-427, there is a dramatic decrease in binding signal due to the presence of PEG. However, the relative binding signal of each variant to the remaining variants can be used as a measure of relative affinity to the antigen. Although there are slight differences in the binding signal of each variant, overall, binding to either Tie2 or VEGF is largely independent of the PEGylation site.

[0224] All test substances were formulated at 10 mg / mL and, with the exception of PEGylated IGT-427, a 20 kDa compound containing 15% high molecular weight species, were found to be over 90% pure by SEC-HPLC (Figure 16) and unreduced SDS-PAGE (Figure 17). Endotoxin levels were all less than 0.1 EU / mg, and all test substances bound their respective antigens with the expected affinity (Figures 18A-18C).

[0225] [Example 20] Total drug ELISA for vitreous body fluid measurement Depending on the administered drug, total drug levels in the vitreous fluid were quantified using three different ELISA methods. IGT-427 and its PEGylated version were captured on mouse anti-human IgG coated plates and detected with Tie2-HRP conjugates. Eylea(R) was captured on mouse anti-human IgG coated plates and detected with polyclonal goat anti-human IgG-HRP conjugates. Finally, falisimab was captured on VEGF coated plates and detected with polyclonal goat anti-human IgG-HRP conjugates. For all ELISA analyses, samples were assayed in pairs at dilutions of 1:1250 to 1:62500, and the luminescence of the assay plates was read using a Molecular Devices SpectraMax(R) M5 plate reader. For all ELISA analyses, the sample luminescence values ​​were within the linear range of the standard curve. The entire standard curve was fitted using a four-parameter equation, and the sample luminescence values ​​were converted to concentrations by four-parameter fitting.

[0226] Anti-human IgG capture / Tie2 detection ELISA. High-binding plates were coated with 1 μg / mL anti-human IgG in carbonate buffer (pH 9.5) and left overnight at 4°C. The coated plates were washed with washing buffer (1×PBST (Phosphate Buffered Saline with TWEEN(R)20) containing 150 mM NaCl) and blocked at 37°C for 1 hour with 5% BSA in PBST (shaking at 420 RPM). The blocked plates were washed, and samples, standard curves, and blanks were incubated on the plates at room temperature for 1.5 hours (shaking at 420 RPM). After sample incubation, the plates were washed and incubated with 1 μg / mL biotinylated human Tie2 at room temperature for 1 hour (covered and shaken at 420 RPM). The plates were washed again and incubated with streptavidin-HRP for 1 hour in the dark. The plates were washed one last time and then colored with chemiluminescent HRP substrate. After 30 seconds, all emission wavelengths were read using a plate reader.

[0227] Anti-human IgG capture / anti-huFc detection ELISA. High-binding plates were coated with 1 μg / mL anti-human IgG in carbonate buffer (pH 9.5) and left overnight at 4°C. The coated plates were washed with washing buffer and blocked with 1% BSA in PBST at 4°C for 4 hours. The blocked plates were washed, and after the final wash, the plates were dried at 4°C. Samples, standard curves, and blanks were incubated on the plates overnight at 4°C. After sample incubation, the plates were washed and incubated with polyclonal goat anti-human IgG-HRP conjugate at room temperature for 1 hour (covered and shaken at 420 RPM). The plates were given a final wash and then colored with chemiluminescent HRP substrate. All emission wavelengths were read with a plate reader after 30 seconds.

[0228] VEGF capture / anti-huFc detection ELISA. High-binding plates were coated with 1 μg / mL of VEGF in carbonate buffer (pH 9.5) and left overnight at 4°C. The coated plates were washed with washing buffer and blocked with 1% BSA in PBST at 4°C for 4 hours. The blocked plates were washed, and after the final wash, the plates were dried at 4°C. Samples, standard curves, and blanks were incubated on the plates overnight at 4°C. After sample incubation, the plates were washed and incubated with polyclonal goat anti-human IgG-HRP conjugate at room temperature for 1 hour (covered and shaken at 420 RPM). The plates were given a final wash and then colored with chemiluminescent HRP substrate. All emission wavelengths were read with a plate reader after 30 seconds.

[0229] [Example 21] Measurement of pharmacokinetics in the eye The total drug levels of each test substance were measured in vitreous fluid on days 1, 3, 7, and 14 after administration. At each time point, eyes were collected from 3 or 4 animals, yielding either 6 or 8 vitreous fluid samples per test substance per time point. Total drug levels from animals administered with IGT-427 or PEGylated IGT-427 were quantified by anti-human IgG capture and Tie2 detection ELISA. Total drug levels from animals administered with Eylea(R) were quantified by anti-human IgG capture and anti-human Fc detection ELISA. Finally, total drug levels from animals administered with falisimab were quantified by VEGF capture and anti-human Fc detection ELISA. The results of each of these ELISAs are shown in Figure 19. Eylea(R) was measured in the vitreous humor and had a Cmax value of 433.8 μg / mL and a half-life of 4.2 days. Falicimab was measured in the vitreous humor and showed a Cmax of 154.2 μg / mL and a half-life of 4.4 days. IGT-427 was measured in the vitreous humor and showed a Cmax of 217.3 μg / mL and a half-life of 3.8 days. IGT-427 modified with two 20 kDa linear PEG molecules was measured in the vitreous humor and showed a Cmax of 406.6 μg / mL and a half-life of 8.3 days. IGT-427 modified with two 40 kDa branched PEG molecules was measured in the vitreous humor and showed a Cmax of 268.8 μg / mL and a half-life of 8.0 days. [Industrial applicability]

[0230] Fusion proteins that bind to Tie2 and VEGF can bind to Tie2 and VEGF with high affinity, maintain cross-reactivity to humans, mice, and rabbits, and exhibit desired antigen reactivity. Furthermore, by inducing Tie2 phosphorylation, Tie2 receptor activation, and VEGF inhibition, fusion proteins that bind to Tie2 and VEGF can be used to prevent or treat target angiogenic or vascular diseases.

[0231] Equal parts The foregoing specification is considered to be sufficient to enable a person skilled in the art to carry out the embodiments. The foregoing description and examples detail particular embodiments and describe the best mode contemplated by the inventors. However, it will be understood that, however detailed the foregoing may be in words, the embodiments may be carried out in many ways and should be construed in accordance with the appended claims and their equivalents.

[0232] When terms such as at least and about are present prior to a list of numerical values or ranges, those terms modify all of the values or ranges provided in the list. In some cases, the term about may include a numerical value rounded to the nearest significant digit. Any recited range includes its endpoints unless the contrary is expressly stated, for example, "5 to 50" includes the values 5 and 50.

Claims

1. (a) A vascular endothelial growth factor (VEGF) binding domain comprising a VEGF receptor extracellular domain, wherein the VEGF receptor extracellular domain comprises a VEGF binding domain comprising the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14 in order from the N-terminus to the C-terminus, and (b) an anti-Tie2 antibody, (i) Heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 18; (ii) Light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 19; and (iii) An anti-Tie2 antibody containing an IgG1 isotype constant region including a constant heavy chain domain (CH), A fusion protein containing; Here, the VEGF-binding domain is linked to the C-terminus of the CH of the anti-Tie2 antibody by a linker. Fusion protein.

2. The aforementioned fusion protein comprises a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 26, The fusion protein according to claim 1, comprising a light chain (LC) containing the amino acid sequence of SEQ ID NO:

12.

3. The fusion protein according to claim 1, wherein the CH comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

17.

4. The fusion protein according to claim 3, wherein the CH comprises the amino acid sequence of SEQ ID NO:

17.

5. The IgG1 isotype constant region includes one or more mutations that reduce or eliminate interaction with the Fc receptor, where, The fusion protein according to claim 1, wherein the one or more mutations are selected from the group consisting of K322, P331, L234, L235, H310, M252, I253, S254, T256, H433, N434, and H435, and the amino acid position numbering follows EU numbering.

6. The fusion protein according to claim 1, wherein the linker includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

25.

7. The fusion protein according to claim 6, wherein the linker comprises the amino acid sequence of SEQ ID NO:

25.

8. (a) HC containing the following in the order from the N-terminus to the C-terminus: (i) VH containing the amino acid sequence of SEQ ID NO: 18, (ii) CH containing the amino acid sequence of SEQ ID NO: 17, (iii) A linker containing the amino acid sequence of Sequence ID No. 25, and (iv) The extracellular domain of the VEGF receptor, comprising the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14 in order from the N-terminus to the C-terminus, and (b) LC containing the following in order from the N-terminus to the C-terminus: (i) VL containing the amino acid sequence of SEQ ID NO: 19, and (ii) A constant light chain domain (CL) containing the amino acid sequence of SEQ ID NO: 30; A fusion protein containing [the specified ingredient].

9. One or more nucleic acids encoding the fusion protein according to any one of claims 1 to 8.

10. One or more expression vectors comprising one or more nucleic acids as described in claim 9.

11. A cell comprising one or more expression vectors as described in claim 10.

12. (a) A first nucleic acid encoding the HC of a fusion protein, wherein the HC comprises, in order from the N-terminus to the C-terminus: (i) VH containing the amino acid sequence of SEQ ID NO: 18, (ii) CH containing the amino acid sequence of SEQ ID NO: 17, (iii) A linker containing the amino acid sequence of Sequence ID No. 25, and (iv) The extracellular domain of the VEGF receptor, comprising the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14 in order from the N-terminus to the C-terminus, (b) A second nucleic acid encoding the LC of the fusion protein, wherein the LC comprises, in order from the N-terminus to the C-terminus: (i) VL containing the amino acid sequence of SEQ ID NO: 19, and (ii) CL containing the amino acid sequence of SEQ ID NO: 30; A nucleic acid composition containing the following:

13. (a) A first nucleic acid encoding HC containing the nucleic acid sequence of Sequence ID No. 29, and (b) A second nucleic acid that encodes an LC containing the nucleic acid sequence of sequence number 28, The nucleic acid composition according to claim 12.

14. The nucleic acid composition according to claim 12, wherein HC comprises the amino acid sequence of SEQ ID NO: 26, and LC comprises the amino acid sequence of SEQ ID NO:

12.

15. One or more expression vectors comprising the nucleic acid composition according to any one of claims 12 to 14.

16. A cell comprising one or more expression vectors according to claim 15, wherein one or more expression vectors encode both the HC and LC of a fusion protein.

17. (a) HC of a fusion protein, wherein the HC comprises, in order from the N-terminus to the C-terminus: (i) VH containing the amino acid sequence of SEQ ID NO: 18, (ii) CH containing the amino acid sequence of SEQ ID NO: 17, (iii) A linker containing the amino acid sequence of Sequence ID No. 25, and (iv) The extracellular domain of the VEGF receptor, comprising the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14 in order from the N-terminus to the C-terminus, and (b) LC of a fusion protein, wherein the LC comprises, in order from the N-terminus to the C-terminus: (i) VL containing the amino acid sequence of SEQ ID NO: 19, and (ii) CL containing the amino acid sequence of SEQ ID NO: 30; An isolated nucleic acid that codes for [something].

18. The isolated nucleic acid according to claim 17, comprising the nucleic acid sequence of SEQ ID NO: 29 and the nucleic acid sequence of SEQ ID NO:

28.

19. The isolated nucleic acid according to claim 17, wherein HC comprises the amino acid sequence of SEQ ID NO: 26, and LC comprises the amino acid sequence of SEQ ID NO:

12.

20. One or more expression vectors comprising an isolated nucleic acid according to any one of claims 17 to 19.

21. A cell comprising one or more expression vectors according to claim 20, wherein one or more expression vectors encode both HC and LC.

22. A method for producing a fusion protein that binds Tie2 and VEGF, A step of culturing the cells described in claim 11 to produce a fusion protein, A step of recovering the fusion protein from cultured cells and Methods that include...

23. A method for producing a fusion protein that binds Tie2 and VEGF, A step of culturing the cells described in claim 16 or 21 to produce a fusion protein, A step of recovering the fusion protein from cultured cells and Methods that include...

24. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier, diluent, or excipient.

25. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 8, for use in the treatment of diabetic macular edema in patients in need.

26. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 8, for use in the treatment of macular degeneration in patients in need.

27. The pharmaceutical composition according to claim 26, wherein the macular degeneration is neovascular age-related macular degeneration.

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