Inhibitors of angiogenic factors

Abstract

This invention provides a recombinant human Fc fusion protein useful for treating or controlling ocular diseases, conditions, or disorders that have an etiology of abnormal angiogenesis. [Solution] The recombinant human Fc fusion protein comprises an Ig-like domain of a first human VEGF receptor, a different Ig-like domain of a second human VEGF receptor, at least yet another Ig-like domain of a third human VEGF receptor, and the Fc portion of human IgG1.

Description

[Technical Field]

[0001]

[0001] The present invention relates to inhibitors of angiogenic factors. More specifically, the present invention relates to inhibitors of members of the family of vascular endothelial growth factors ("VEGF family members"). More specifically, the present invention relates to inhibitors of the activity of VEGF family members, the use of such inhibitors and methods for producing them. [Background technology]

[0002]

[0002] The main cellular components of the mammalian vascular system are endothelial cells, smooth muscle cells, and pericytes. Endothelial cells form the inner lining of all blood vessels in mammals and constitute a non-thrombus-forming interface between blood and tissue. Therefore, the proliferation of endothelial cells is an important component for the development of new capillaries and blood vessels, and such development is an essential process for the growth and / or regeneration of mammalian tissues.

[0003]

[0003] The family of secreted polypeptides has been shown to play a crucial role in promoting endothelial cell proliferation and angiogenesis. The pathological characteristic of unregulated angiogenesis caused by VEGF overexpression is increased vascular permeability, which leads to fluid leakage into surrounding tissues and swelling of those tissues. In mammals, this family consists of five related growth factors with highly conserved receptor-binding structures: vascular endothelial growth factors A-D ("VEGF-A", "VEGF-B", "VEGF-C", and "VEGF-D") and placental growth factor ("PGF"). In this disclosure, this family of growth factors is also referred to as the VEGF family. These growth factors act through a family of cognitive receptor tyrosine kinases that are present only on the surface of vascular endothelial cells: VEGF receptor-1 (also known as "VEGFR-1" or "flt-1"), VEGF receptor-2 ("VEGFR-2," known as "KDR" in humans and "flk-1" in mice), and VEGF receptor-3 (also known as "VEGFR-3" or "flt-4"), thereby stimulating angiogenesis.

[0004]

[0004] VEGF-A (sometimes simply called VEGF) has emerged as the most important member of this family of growth factors. Human VEGF-A is expressed in various tissues as multiple homodimeric forms (121, 145, 165, 183, 189, and 206 amino acids per monomer), in which case each form arises as a result of alternative splicing of a single RNA transcript.

[0005]

[0005] Since VEGF promotes the proliferation and angiogenesis of vascular endothelial cells, it may be effective for the treatment of a number of conditions if its proliferation-promoting activity to vascular endothelial cells is beneficially important in the treatment of, for example, ulcers, vascular injuries, and myocardial infarction.

[0006]

[0006] However, in contrast, while vascular endothelial proliferation is desirable under certain circumstances, vascular endothelial proliferation and angiogenesis are also undesirable components of a variety of diseases and disorders, including tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, posterior lens fibrosis, neovascular glaucoma, neovascular age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues, and chronic inflammation. Individuals suffering from any of these disorders would likely want to inhibit, or at least substantially reduce, the endothelial proliferation activity of VEGF.

[0007]

[0007] Each of the flt-1, KDR, and flt-4 tyrosine kinase receptors has seven extracellular immunoglobulin-like ("Ig-like") domains available for ligand binding, It has a transmembrane domain that functions to fix the receptor to the surface of cells expressing the receptor, and an intracellular catalytic tyrosine kinase domain. Flt-1 binds to VEGF-A, VEGF-B, and PlGF. KDR binds to VEGF-A, VEGF-C, and VEGF-D. Flt-4 binds to VEGF-C and VEGF-D. [Overview of the Initiative]

[0008]

[0008] In light of the role of VEGF family growth factors in vascular endothelial proliferation and angiogenesis, and the roles these processes play in a wide variety of diseases and disorders, it is desirable to have pharmacological means to reduce or inhibit one or more of the biological activity of these growth factors in patients whose pathology is rooted in abnormal angiogenesis. It is also desirable to have pharmacological means for improved treatment or control of pathologies rooted in abnormal angiogenesis.

[0009]

[0009] As used herein, the term “control” also includes reduction, mitigation, improvement, or prevention.

[0010]

[0010] Overall, the present invention provides fused or chimeric polypeptides or proteins, methods for producing them and compositions comprising them, and methods for treating or controlling at least one condition of interest in which the pathogenesis is abnormal angiogenesis.

[0011]

[0011] In one embodiment, the present invention provides a fusion polypeptide or chimeric polypeptide that substantially binds to one or more VEGF family members; thereby reducing or inhibiting their binding to VEGF receptors.

[0012]

[0012] In another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises an Ig-like domain of a first VEGF receptor, an Ig-like domain of a second VEGF receptor, and at least one Ig-like domain of a third VEGF receptor.

[0013]

[0013] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1 (or flt-1), or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2 (or KDR), or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3 (or flt-4), or substantially Ig-like domains 1 and 2.

[0014]

[0014] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1, 2, and 3 of human VEGFR-3, or substantially Ig-like domains 1, 2, and 3.

[0015]

[0015] In yet another embodiment, human VEGFR-3 is wild-type human VEGFR-3 having the amino acid sequence listed in SEQ ID NO: 13; the Ig-like domain 1 of human VEGFR-3 contained in any of the fusion proteins of the present invention has the amino acid sequence listed in SEQ ID NO: 14. The fusion protein of the present invention comprises the Ig-like domain 1 of human VEGFR-3, or substantially the Ig-like domain 1; where the amino acid sequence from positions 80 to 82 of SEQ ID NO: 14 is NDT, NDS, NXT, or NXS; X is any amino acid. In one embodiment, X is a naturally occurring amino acid.

[0016]

[0016] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) the Ig-like domain 2 of human VEGFR-1, or substantially the Ig-like domain 2; (b (c) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; where the amino acid sequence of human VEGFR-3 is listed in SEQ ID NO: 13; where the amino acid sequence from positions 104 to 106 is NDT, NDS, NXT, or NXS; where X is any amino acid. In one embodiment, X is a naturally occurring amino acid.

[0017]

[0017] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1 (or flt-1), or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2 (or KDR), or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3 (or flt-4), or substantially Ig-like domains 1 and 2; and is linked to a polymerizing component.

[0018]

[0018] In one embodiment, the polymer-forming component includes a portion of the Fc domain of human IgG1.

[0019]

[0019] In another embodiment, the polymerizing component comprises the last two heavy chain domains at the amino terminus of IgG1; in other words, substantially the Fc CH2 domain and CH3 domain of human IgG1.

[0020]

[0020] In yet another aspect, the present invention provides isolated nucleic acid molecules encoding the fusion polypeptide or chimeric polypeptide.

[0021]

[0021] In yet another aspect, the present invention provides a vector comprising the nucleic acid molecule, including an expression vector comprising the nucleic acid molecule operably linked to an expression control sequence.

[0022]

[0022] In yet another aspect, the present invention provides a host-vector system for generating the fusion polypeptide or chimeric polypeptide, comprising an expression vector in a suitable host cell.

[0023]

[0023] In another aspect, the present invention provides a method for producing a fusion polypeptide or a chimeric polypeptide, comprising the steps of (a) growing cells of a host-vector system under conditions that enable the production of a fusion polypeptide or a chimeric polypeptide; and (b) recovering the fusion polypeptide or chimeric polypeptide thus produced.

[0024]

[0024] In yet another aspect, the present invention provides a method for treating or controlling in a subject at least one disease, condition, or disorder having an etiology of abnormal angiogenesis.

[0025]

[0025] In yet another aspect, the present invention provides a method for treating or controlling in a subject at least one ocular disease, condition, or disorder having an etiology of abnormal angiogenesis.

[0026]

[0026] Other features and advantages of the present invention will become apparent from the following embodiments and claims. [Brief explanation of the drawing]

[0027] [Figure 1]

[0027] This figure schematically shows the fusion polypeptide of the present invention, which includes Ig-like domain 2 of VEGFR-1, Ig-like domain 3 of VEGFR-2, Ig-like domains 1 and 2 of VEGFR-3, and an Fc domain. [Figure 2]

[0028] This figure shows the results of SDS-PAGE and SEC-HPLC of transient expression of EB-101 in HEK293 cells. [Figure 3]

[0029] This figure shows the results of SDS-PAGE and SEC-HPLC on transient expression of EB-101 in CHO cells. [Figure 4]

[0030] This figure shows a comparison of the binding affinity of EB-101 and aflibercept to human recombinant human VEGF-A165 using SPR Biacore. [Figure 5]

[0031] This figure shows a comparison of the binding affinity of EB-101 and aflibercept to human VEGF-B167 / 189 using SPR Biacore. [Figure 6]

[0032] This figure shows a comparison of the binding affinity of EB-101 and aflibercept to human VEGF-C using SPR Biacore. [Figure 7]

[0033] This figure shows a comparison of the binding affinity of EB-101 and aflibercept to human VEGF-D using SPR Biacore. [Figure 8]

[0034] This figure shows a comparison of the binding affinity of EB-101 and aflibercept to human PlGF using SPR Biacore. [Figure 9]

[0035] This figure compares the effects of EB-101, aflibercept, and bevacizumab on inhibiting VEGF-A165-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells. [Figure 10]

[0036] This figure compares the effects of EB-101 and aflibercept on inhibiting VEGF-C-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells. [Figure 11]

[0037] This figure shows a comparison of the inhibitory effects of EB-101 on VEGF-C-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells, with recombinant human VEGFR-3 (rhVEGFR-3-7ECD) containing aflibercept and seven extracellular domains. [Figure 12]

[0038] This figure shows the quantitative analysis of EB-101, along with aflibercept and recombinant human VEGFR-3 (rhVEGFR-3) containing seven extracellular domains, on the effect of inhibiting VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells stimulated by VEGF-A165 and VEGF-C. [Figure 13]

[0039] This figure compares the effects of EB-101, aflibercept, and bevacizumab on VEGF-A165-mediated proliferation of human lymphatic endothelial cells (HLECs). [Figure 14]

[0040] This figure compares the effects of EB-101 on VEGF-C-mediated HLEC proliferation with recombinant human VEGFR3 containing aflibercept and seven extracellular domains (rhVEGFR3 ECD). [Figure 15]

[0041] This figure shows the quantitative analysis of EB-101, aflibercept, and recombinant human VEGFR3 (rhVEGFR3 ECD) containing seven extracellular domains regarding VEGF-A165 plus VEGF-C mediated HLEC proliferation. [Figure 16]

[0042] This figure schematically shows the fusion polypeptide of the present invention, which includes Ig-like domain 2 of VEGFR-1, Ig-like domain 3 of VEGFR-2, Ig-like domains 1, 2, and 3 of VEGFR-3, and an Fc domain. [Figure 17]

[0043] This figure compares the effects of EB-101BIb on VEGF-A-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells with aflibercept, EB-101, and EB-101BIa. [Figure 18]

[0044] This figure compares the effects of recombinant human VEGF-C and -D on VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells with EB-101BIb, aflibercept, EB-101, and EB-101BIa. [Figure 19]

[0045] This figure shows a comparison of the inhibitory effects of EB-101BIb on VEGF-A, -C, and -D-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells, with aflibercept, EB-101, and a combination of recombinant human VEGFR-3 and aflibercept. [Figure 20]

[0046] This figure compares the effects of EB-101BIb, aflibercept, and EB-101BIa on VEGF-A165-mediated proliferation of human lymphatic endothelial cells (HLECs). [Figure 21]

[0047] This figure compares the effects of EB-101BIb, aflibercept, EB-101BIa, and recombinant human VEGFR-3 containing seven extracellular domains on VEGF-C and VEGF-D-mediated HLEC proliferation. [Figure 22]

[0048] This figure shows a comparison of the effects of EB-101BIb on the proliferation of VEGF-A, -C, and -D-mediated human lymphoid endothelial cells (HLECs) with aflibercept, recombinant human VEGFR-3 containing seven extracellular domains (seven ECDs), and a combination of VEGFR-3 and aflibercept. [Modes for carrying out the invention]

[0028]

[0049] In this disclosure, the terms “fusion polypeptide,” “fusion protein,” “chimeric polypeptide,” and “chimeric protein” are used interchangeably.

[0029]

[0050] The present invention provides a binding construct that binds to at least one member of the VEGF family ("VEGF family member"), thereby substantially rendering the VEGF family member unavailable for binding to VEGF receptors on endothelial cells. As a result, the binding construct of the present invention substantially inhibits the biological activity of the VEGF family member in promoting angiogenesis, thereby controlling pathological conditions having a pathogenesis related to abnormal angiogenesis.

[0030]

[0051] The conjugation constructs of the present invention include or consist of fused or chimeric polypeptides or proteins, comprising one or more conjugation units that associate with each other by covalent or other forms of attachment. The conjugation constructs of the present invention can bind to VEGF family members or parts thereof, and bind thereto with high affinity. The conjugation units preferably comprise at least one peptide or polypeptide. The conjugation units preferably comprise a single polypeptide, however, if a single polypeptide is not sufficient to bind to a particular growth factor, the conjugation units may comprise multiple polypeptides. If two or more conjugation units or polypeptide segments are present in a given conjugation construct, the conjugation units may be bound to each other directly or through a linker. The conjugation constructs may further comprise heterologous peptides or other chemical moieties. Such additions may modify the properties of the conjugation construct, such as stability, solubility, toxicity, serum half-life, immunogenicity, detectability, or other properties.

[0031]

[0052] The term "high affinity" is used in a physiological context relating to the relative affinity of a binding construct to a VEGF family member in vivo in mammals, e.g., clinical laboratory animals, domesticated livestock or pets, or humans. The VEGF members targeted in this invention typically have a sub-nanomole dissociation constant (K). d ) has characteristic affinity for those receptors in vivo, as measured from the perspective of ). For the purposes of the present invention, the binding construct of the present invention is K of the native growth factor receptor pair. d K that is approximately 1x or less, or approximately 5x or less, or approximately 10x or less, or approximately 50x or less, or approximately 100x or less, or approximately 500x or less, or approximately 1000x or less d And it can then bind to that target VEGF family member.

[0032]

[0053] In one embodiment, the fused or chimeric polypeptide or protein of the present invention comprises a first polypeptide binding unit, a second polypeptide binding unit, and a third polypeptide binding unit, which are operably linked to one another. The polypeptide binding units may be referred to herein by abbreviation as “binding units.” Overall, the first, second, and third binding units can be linked to one another directly or through intervening linkers in any order.

[0033]

[0054] In another embodiment, a targeted VEGF family member can bind to one or more binding units of a fusion polypeptide or chimeric polypeptide.

[0034]

[0055] In yet another embodiment, the binding unit substantially comprises the Ig-like domain of the VEGF receptor.

[0035]

[0056] In another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises a first binding unit comprising an Ig-like domain of a first VEGF receptor; a second binding unit comprising an Ig-like domain of a second VEGF receptor; and a third binding unit comprising at least one Ig-like domain of a third VEGF receptor.

[0036]

[0057] In another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises an Ig-like domain of a first VEGF receptor, an Ig-like domain of a second VEGF receptor, and at least one Ig-like domain of a third VEGF receptor; wherein the third VEGF receptor comprises human VEGFR-3 (or flt-4) having the amino acid sequence listed in SEQ ID NO: 13, provided that the amino acids at positions 105 and 106 of SEQ ID NO: 13 are substitutable, so that the amino acid sequence from positions 104 to 106 of SEQ ID NO: 13 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0037]

[0058] In yet another embodiment, the fusion polypeptide of the present invention comprises (a) an Ig-like domain of a first VEGF receptor having an amino acid sequence that is at least 90% identical to Ig-like domain 2 of human VEGFR-1; (b) an Ig-like domain of a second VEGF receptor having an amino acid sequence that is at least 90% identical to Ig-like domain 3 of human VEGFR-2; and (c) a third VEGF receptor having an amino acid sequence that is at least 90% identical to Ig-like domains 1 and 2 of human VEGFR-3. It contains at least one Ig-like domain, where human VEGFR-3 has the amino acid sequence listed in SEQ ID NO: 13, provided that the amino acids at positions 105 and 106 of SEQ ID NO: 13 are substitutable, so that the amino acid sequence from positions 104 to 106 of SEQ ID NO: 13 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0038]

[0059] In yet another embodiment, the fusion polypeptide of the present invention comprises (a) an Ig-like domain of a first VEGF receptor having an amino acid sequence that is at least 95% identical to Ig-like domain 2 of human VEGFR-1; (b) an Ig-like domain of a second VEGF receptor having an amino acid sequence that is at least 95% identical to Ig-like domain 3 of human VEGFR-2; and (c) at least one Ig-like domain of a third VEGF receptor having an amino acid sequence that is at least 95% identical to Ig-like domains 1 and 2 of human VEGFR-3, wherein human VEGFR-3 has the amino acid sequence listed in SEQ ID NO: 13, provided that the amino acids at positions 105 and 106 of SEQ ID NO: 13 are substitutable, thereby the amino acid sequence at positions 104-106 of SEQ ID NO: 13 is NDT, NDS, NXT, or NXS. Herein, X is a condition that it is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0039]

[0060] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1 (or flt-1), or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2 (or KDR), or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3 (or flt-4), or substantially Ig-like domains 1 and 2; or Ig-like domains 1, 2, and 3 of human VEGFR-3, or substantially Ig-like domains 1, 2, and 3.

[0040]

[0061] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; or Ig-like domains 1, 2, and 3 of human VEGFR-3, or substantially Ig-like domain 1 This includes 2, and 3; where human VEGFR-3 has the amino acid sequence listed in SEQ ID NO: 13, provided that the amino acids at positions 105 and 106 of SEQ ID NO: 13 are substitutable, so that the amino acid sequence from positions 104 to 106 of SEQ ID NO: 13 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0041]

[0062] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; or Ig-like domains 1, 2, and 3 of human VEGFR-3, or substantially Ig-like domains 1, 2, and 3; wherein human VEGFR-3 has the amino acid sequence listed in SEQ ID NO: 13, provided that the amino acid sequence from positions 104 to 106 of SEQ ID NO: 13 is unsubstituted and remains an NDT.

[0042]

[0063] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; or Ig-like domains 1, 2, and 3 of human VEGFR-3, or substantially Ig-like domain This includes 1, 2, and 3; where the Ig-like domain 1 of human VEGFR-3 has the amino acid sequence listed in SEQ ID NO: 14; where the amino acids at positions 81 and 82 of SEQ ID NO: 14 are substitutable, so that the amino acid sequence from positions 80 to 82 of SEQ ID NO: 14 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0043]

[0064] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; or Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; where Ig-like domain of human VEGFR-3 1 has the amino acid sequence listed in SEQ ID NO: 14; where the amino acids at positions 81 and 82 of SEQ ID NO: 14 are substitutable, so that the amino acid sequence from positions 80 to 82 of SEQ ID NO: 14 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0044]

[0065] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; or Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; wherein Ig-like domain 1 of human VEGFR-3 has the amino acid sequence listed in SEQ ID NO: 14; where the amino acid sequence from positions 80 to 82 of SEQ ID NO: 14 is unsubstituted and remains NDT.

[0045]

[0066] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; or Ig-like domains 1, 2, and 3 of human VEGFR-3, or substantially Ig-like domains 1, 2, and 3; wherein the Ig-like domain 1 of human VEGFR-3 included in the fusion polypeptide or chimeric polypeptide has the amino acid sequence listed in Sequence ID No. 14.

[0046]

[0067] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; or Ig-like domains 1, 2, and 3 of human VEGFR-3, or substantially Ig-like domains 1, 2, and 3; wherein the Ig-like domain 1 of human VEGFR-3 included in the fusion polypeptide or chimeric polypeptide has the amino acid sequence listed in SEQ ID NO: 14, and the amino acid sequence at position 1 is unsubstituted and remains NDT.

[0047]

[0068] In yet another embodiment, the fusion polypeptide or chimeric polypeptide of the present invention comprises (a) Ig-like domain 2 of human VEGFR-1, or substantially Ig-like domain 2; (b) Ig-like domain 3 of human VEGFR-2, or substantially Ig-like domain 3; and (c) Ig-like domains 1 and 2 of human VEGFR-3, or substantially Ig-like domains 1 and 2; linked to a polymerizing component; wherein human VEGFR-3 has the amino acid sequence listed in SEQ ID NO: 13, provided that the amino acids at positions 105 and 106 of SEQ ID NO: 13 are substitutable, so that the amino acid sequence from positions 104 to 106 of SEQ ID NO: 13 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0048]

[0069] In one embodiment, the polymer-forming component substantially comprises a portion of the Fc domain of human IgG1.

[0049]

[0070] In another embodiment, the polymerizing component substantially comprises a portion of the Fc domain of the heavy chain of human IgG1. In one embodiment, such portion of the Fc domain of the heavy chain of human IgG1 The fraction contains the last two domains at the carboxyl terminus of the IgG monochain.

[0050]

[0071] In some embodiments, two or more binding units act together to bind to a single ligand molecule (in this case, the ligand may include monomers or dimers). In some other embodiments, the binding units act independently, i.e., each binding unit binds to a separate ligand molecule.

[0051]

[0072] In one embodiment, the fusion protein or chimeric protein comprises (a) Ig-like domain 2 or substantially Ig-like domain 2 of human VEGFR-1, wherein (b) is operably ligated to Ig-like domain 3 or substantially Ig-like domain 3 of human VEGFR-2, and (b) is operably ligated to (c)(1) Ig-like domains 1 and 2 or substantially Ig-like domains 1 and 2 of human VEGFR-3, or (c)(2) Ig-like domains 1, 2 and 3 or substantially Ig-like domains 1, 2 and 3 of human VEGFR-3, and (c)(1) or (c)(2) is operably ligated to a portion of the Fc domain of IgG1.

[0052]

[0073] In one embodiment, a polypeptide linker is inserted between the last binding unit and the portion of the Fc domain of IgG1.

[0053]

[0074] In one embodiment, the fusion protein or chimeric protein comprises (b) Ig-like domain 3 or substantially Ig-like domain 3 of human VEGFR-2, (a) Ig-like domain 2 or substantially Ig-like domain 2 of human VEGFR-1, wherein (b) is operably ligated to (c)(1) Ig-like domains 1 and 2 or substantially Ig-like domains 1 and 2 of human VEGFR-3, or (c)(2) Ig-like domains 1, 2 and 3 or substantially Ig-like domains 1, 2 and 3 of human VEGFR-3, wherein (c)(1) or (c)(2) is operably ligated to a portion of the Fc domain of IgG1; Thus, the Ig-like domain 2 of human VEGFR-1 ("VEGFR-1-D2") has the amino acid sequence listed in SEQ ID NO: 2; the Ig-like domain 3 of human VEGFR-2 ("VEGFR-2-D3") has the amino acid sequence listed in SEQ ID NO: 4; the Ig-like domains 1 and 2 of human VEGFR-3 ("VEGFR-3-D1D2") have the amino acid sequences listed in SEQ ID NO: 6; the Ig-like domains 1, 2, and 3 of human VEGFR-3 ("VEGFR-3-D1D2D3") have the amino acid sequences listed in SEQ ID NO: 15; and the Fc domain portion of IgG1 ("IgG1Fc") has the amino acid sequence listed in SEQ ID NO: 8.

[0054]

[0075] In another embodiment, the fusion or chimeric polypeptide or protein of the present invention further comprises a polypeptide linker inserted between the carboxyl terminus of VEGFR-3-D1D2 and the amino terminus of IgG1Fc; wherein the linker has the amino acid sequence listed in SEQ ID NO: 10.

[0055]

[0076] In yet another embodiment, the fusion or chimeric polypeptide or protein of the present invention may further comprise a polypeptide leader sequence preceding the amino terminus of VEGFR-1-D2.

[0056]

[0077] In yet another embodiment, the fusion or chimeric polypeptide or protein of the present invention comprises an amino acid sequence that is at least 90% identical to each of SEQ ID NOs: 2, 4, 6, 15, 8, and 10; where the amino acids at positions 81 and 82 of SEQ ID NOs: 6 and 15 are substitutable, thereby the amino acid sequence from positions 80 to 82 of SEQ ID NOs: 6 and 15 is NDT, NDS, NXT, or NXS, where X is A, R, N, C, E, Q, G, H, I, L, K, These are amino acids selected from the group consisting of M, F, P, S, T, W, Y, and V.

[0057]

[0078] In yet another embodiment, the fusion or chimeric polypeptide or protein of the present invention comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 8, and SEQ ID NO: 10; where the amino acids at positions 81 and 82 of SEQ ID NO: 6 and SEQ ID NO: 15 are substitutable, thereby the amino acid sequence from positions 80 to 82 of SEQ ID NO: 6 and SEQ ID NO: 15 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0058]

[0079] In yet another embodiment, the fusion or chimeric polypeptide or protein of the present invention comprises the amino acid sequence enumerated in SEQ ID NO: 12 or SEQ ID NO: 16. Fusion proteins having SEQ ID NO: 12 and SEQ ID NO: 16 were prepared for testing and are referred herein to as "EB-101" and "EB-101BIb," respectively.

[0059]

[0080] In yet another embodiment, the fusion or chimeric polypeptide or protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 or SEQ ID NO: 16; where the amino acids at positions 286 and 287 of SEQ ID NO: 12 or SEQ ID NO: 16 are substitutable, thereby the amino acid sequence from positions 285 to 287 of SEQ ID NO: 12 or SEQ ID NO: 16 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0060]

[0081] In yet another embodiment, the fusion or chimeric polypeptide or protein of the present invention comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 or SEQ ID NO: 16, wherein the amino acids at positions 285-287 of SEQ ID NO: 12 or SEQ ID NO: 16 are unsubstituted.

[0061]

[0082] In yet another embodiment, the fusion or chimeric polypeptide or protein of the present invention comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 12 or SEQ ID NO: 16; where the amino acids at positions 286 and 287 of SEQ ID NO: 12 or SEQ ID NO: 16 are substitutable, thereby the amino acid sequence from positions 285 to 287 of SEQ ID NO: 12 or SEQ ID NO: 16 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0062]

[0083] In yet another embodiment, the fusion or chimeric polypeptide or protein of the present invention comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 12 or SEQ ID NO: 16, wherein the amino acids at positions 285-287 of SEQ ID NO: 12 or SEQ ID NO: 16 are unsubstituted.

[0063]

[0084] In yet another aspect, one or more amino acid substitutions can be made in any of the above amino acid sequences. Preferably, such substitutions are conservative substitutions, in which case one amino acid from one of the following groups is substituted with another amino acid from the same group: (1) A, S, T; (2) D, E; (3) N, Q; (4) R, K; (5) I, L, M, V; and (6) F, Y, W; such substitutions are selected to substantially preserve the binding activity of the fusion polypeptide. In one embodiment, the fusion polypeptide of the invention having conservative substitutions has a K d value less than about 120% of the K d value before such substitution. Preferably, the K d value is less than about 110% of the K d value before such substitution. More preferably, the K d value is less than about 105% of the K d value before such substitution.

[0064]

[0085] Most conserved substitutions are not expected to create radical changes in the characteristics of one or more Ig-like domains of the fusion polypeptide. However, when it is difficult to predict the exact effect of a substitution in advance, one of ordinary skill in the art will understand that the effect will be evaluated by routine screening assays. For example, Ig-like domain variants are typically produced by site-directed mutagenesis of the nucleic acid encoding the intact fusion polypeptide, expression of the variant nucleic acid in recombinant cell culture, purification of the variant fusion polypeptide from the cell culture, and detection of the ability of the variant fusion polypeptide to specifically bind to the VEGF ligand. An exemplary binding assay that can be used to determine whether one or more specific substitutions in one or more Ig-like domains affect the ability of the fusion polypeptide to bind to and inhibit the activity of a VEGF family member is described in the paper by Park et al., J. Biol. Chem. 269:25646-25654 (1994).

[0065]

[0086] The VEGFR-1-D2 binding unit of the fusion protein can bind with high affinity to free VEGF-A, VEGF-B, and PGF (Davis-Smyth et al., EMBO J., 15(18):4919 (1996)). The VEGFR-2-D3 binding unit of the fusion protein can bind with high affinity to free VEGF-A, VEGF-C, and VEGF-D (Stuttfeld et al., 61(9):915 (2009)). The VEGFR-3-D1D2 or VEGFR-3-D1D2D3 (VEGFR-3 Ig-like domains 1, 2, and 3) binding units of the fusion protein can bind with high affinity to free VEGF-C and VEGF-D. Therefore, the fusion protein of the present invention can substantially inhibit the angiogenic activity of these VEGF family members on endothelial cells at disease sites.

[0066]

[0087] In yet another aspect, the present invention provides isolated nucleic acid molecules encoding the fusion polypeptide or chimeric polypeptide.

[0067]

[0088] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding the fusion polypeptide or chimeric polypeptide; wherein the isolated nucleic acid molecule comprises (a) a nucleic acid sequence encoding the Ig-like domain 2 of human VEGFR-1 (or flt-1), or substantially the Ig-like domain 2; (b) a nucleic acid sequence encoding the Ig-like domain 3 of human VEGFR-2 (or KDR), or substantially the Ig-like domain 3; and (c) a nucleic acid sequence encoding the Ig-like domains 1 and 2 of human VEGFR-3 (or flt-4), or substantially the Ig-like domains 1 and 2.

[0068]

[0089] In yet another aspect, the present invention provides isolated nucleic acid molecules encoding the fusion polypeptide or chimeric polypeptide, wherein the isolated nucleic acid molecule comprises (a) a nucleic acid sequence encoding Ig-like domain 2 of human VEGFR-1 (or flt-1), or substantially Ig-like domain 2; (b) a nucleic acid sequence encoding Ig-like domain 3 of human VEGFR-2 (or KDR), or substantially Ig-like domain 3; (c)(1) a nucleic acid sequence encoding Ig-like domains 1 and 2 of human VEGFR-3 (or flt-4), or substantially Ig-like domains 1 and 2; or (c)(2) a nucleic acid sequence encoding Ig-like domains 1, 2 and 3 of human VEGFR-3 (or flt-4), or substantially Ig-like domains 1, 2 and 3; and (d) a nucleic acid sequence encoding a portion of the Fc domain of IgG1.

[0069]

[0090] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding the fusion polypeptide or chimeric polypeptide; wherein the isolated nucleic acid molecule (a) the Ig-like domain 2 of human VEGFR-1 (or flt-1), or substantially Ig-like (b) a nucleic acid sequence having the sequence that codes for domain 2 and is listed in Sequence ID No. 1; (c) a nucleic acid sequence having the sequence that codes for the Ig-like domain 3 of human VEGFR-2 (or KDR), or substantially the Ig-like domain 3 and is listed in Sequence ID No. 3; (d) a nucleic acid sequence having the sequence that codes for the Ig-like domains 1 and 2 of human VEGFR-3 (or flt-4), or substantially the Ig-like domains 1 and 2 and is listed in Sequence ID No. 5; and (d) a nucleic acid sequence having the sequence that codes for a portion of the Fc domain of IgG1 and is listed in Sequence ID No. 7.

[0070]

[0091] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding the fusion polypeptide or chimeric polypeptide, wherein the isolated nucleic acid molecule comprises (a) a nucleic acid sequence encoding the amino acid sequence listed in SEQ ID NO: 2; (b) a nucleic acid sequence encoding the amino acid sequence listed in SEQ ID NO: 4; (c) a nucleic acid sequence encoding the amino acid sequence listed in SEQ ID NO: 6 or SEQ ID NO: 15; and (d) a nucleic acid sequence encoding a portion of the amino acid sequence of the Fc domain of IgG1 listed in SEQ ID NO: 8.

[0071]

[0092] In yet another embodiment, the isolated nucleic acid molecule further comprises a linked nucleic acid sequence encoding a polypeptide linker; where the linked nucleic acid has the sequence listed in SEQ ID NO: 9 and is inserted between SEQ ID NO: 5 and SEQ ID NO: 7.

[0072]

[0093] In yet another embodiment, the isolated nucleic acid may further include a leading nucleic acid sequence that encodes a polypeptide leader sequence. If present in the isolated nucleic acid, the leading nucleic acid sequence precedes SEQ ID NO: 1.

[0073]

[0094] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding the fusion polypeptide or chimeric polypeptide; wherein the isolated nucleic acid molecule comprises the nucleic acid sequence listed in Sequence ID No. 11.

[0074]

[0095] In yet another embodiment, the present invention provides isolated nucleic acid molecules encoding fused or chimeric polypeptides or proteins; wherein the fused or chimeric polypeptide or protein has SEQ ID NO: 12 or SEQ ID NO: 16.

[0075]

[0096] In another embodiment, the present invention provides isolated nucleic acid molecules encoding the fusion polypeptide or chimeric polypeptide; wherein the isolated nucleic acid molecule comprises a nucleic acid sequence that differs in one or more codons from a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11 as a result of degeneracy of the genetic code. Such a variety of nucleic acid sequences are within the scope of the present invention.

[0076]

[0097] In yet another embodiment, the present invention provides a vector comprising any of the nucleic acid molecules, including an expression vector comprising any of the nucleic acid molecules operably linked to an expression control sequence. Embodiments of the vector include a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11. In particular, the vector of the present invention includes a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 11.

[0077]

[0098] In yet another aspect, the present invention provides a host-vector system for generating the fusion polypeptide or chimeric polypeptide, comprising an expression vector in a suitable host cell.

[0078]

[0099] In one embodiment, the present invention provides the construction of a nucleic acid molecule encoding a fusion polypeptide disclosed herein, the nucleic acid being inserted into a vector capable of expressing the fusion polypeptide when introduced into a suitable host cell. Suitable host cells include, but are not limited to, those present in the vector. These include bacterial cells, yeast cells, insect cells, and mammalian cells. Expression vectors encoding chimeric polypeptide molecules under the control of transcription / translational regulatory signals can be constructed using any method known to those skilled in the art for the insertion of DNA fragments into vectors. These methods may include in vitro recombinant DNA techniques and synthetic techniques, as well as in vivo recombination (genetic recombination) (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Ausubel et al., Greene). (See Publ.Assoc.,Wiley-Interscience, NY)

[0079]

[0100] The expression of nucleic acid molecules encoding the fusion polypeptide of the present invention is the fusion polypeptide. The expression of the fusion polypeptide described herein can be regulated by a second nucleic acid sequence (promoter) so that it is expressed in a host transformed with the nucleic acid molecule. For example, the expression of the fusion polypeptide described herein can be controlled by any promoter / enhancer element known in the art.

[0080]

[0101] Generally, they contain replicons and regulatory sequences derived from species compatible with the host cell. Plasmid vectors are used in relation to such hosts. Vectors typically have replication sites as well as marking sequences that can provide phenotypic selection in transformed cells. For example, Escherichia coli (E. coli) is usually transformed using pBR322 plasmids derived from the E. coli species (see, e.g., Bolivar et al., Gene, 2:95 (1977)). The pBR322 plasmid contains ampicillin and tetracycline resistance genes, thus providing an easy means of identifying transformed cells. The pBR322 plasmid or other microbial plasmids or phages also need to contain, or be modifiable to contain, a promoter that can be used by the microbial body for protein expression.

[0081]

[0102] Among the promoters most commonly used in recombinant DNA construction are β-LA These include kutamase (penicillinase) and lactose promoter systems, as well as tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res., 8:4057 (1980)). While these are the most commonly used, other microbial promoters have been discovered and utilized. For example, the tac promoter is a synthetically produced DNA promoter generated from a combination of promoters derived from the trp operon and the lac operon (de Boer et al., PNAS, (1983-01-80(1):21-25 (1983)). This is commonly used for protein synthesis in E. coli (Amann et al., Gene, 25:167 (1983)). Any of these promoters can be used in relation to the method for generating fusion polypeptides of the present invention.

[0082]

[0103] In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, but several other strains are also commonly available. For expression in Saccharomyces, plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)) is commonly used. Other plasmids are disclosed in U.S. Patent No. 4,615,974; Struhl et al., PNAS, 76(3):1035 (1979). Plasmid YRp7 contains the trp1 gene, which provides a selection marker for yeast mutant strains lacking the ability to grow without tryptophan, such as ATCC No. 44,076 or RH218 (Jones, Genetics, 85:23 (1977)). The characteristics of the yeast host cell genome and The presence of trp1 damage then provides an effective environment for detecting transformation by proliferation in the absence of tryptophan.

[0083]

[0104] In yeast vectors, the appropriate promoter sequence is 3-phosphoglycerate quina This includes promoters for enzymes such as alcohol dehydrogenase (Hitzeman et al., J. Biol. Chem., 255:2073 (1980)), or other glycoseptic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, and glucokinase (Romanos et al., Yeast, 8:423 (1992); Weinhandl et al., Microb. Cell factories, 13:5 (2014)). When constructing an appropriate expression plasmid, the stop sequences associated with these genes are also ligated into the 3' of the expression vector of the sequence to be expressed, resulting in polyadenylation and termination of mRNA. Other promoters with further advantages in terms of transcription controlled by growth conditions include the promoter regions for alcohol dehydrogenase 2, as well as enzymes responsible for maltose and galactose utilization (Romanos et al., Weinhandl et al., cited above). Any plasmid vector containing yeast-compatible promoter, origin, and termination sequences is suitable.

[0084]

[0105] In addition to microorganisms, cultures of cells derived from multicellular organisms are also used as hosts. It is possible. In principle, any such cell culture is feasible, whether derived from vertebrate or invertebrate cultures. However, there is great interest in vertebrate cells, and the proliferation of vertebrate cells in culture (tissue culture) has become a common procedure in recent years (Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973)). Examples of such useful host cell lines include VERO cells and HeLa cells, Chinese hamster ovary (CHO) cell lines, and the W138, BHK, COS-7,293, and MDCK cell lines. Expression vectors for such cells typically include (if necessary) an origin of replication and a promoter located before the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences.

[0085]

[0106] When used in mammalian cells, the regulatory function on the expression vector depends on the viral material. These are often provided. For example, commonly used promoters are derived from polyomas and adenovirus 2, most frequently from Simianvirus 40 (SV40). Early and late promoters of the SV40 virus are particularly useful because both promoters can be easily obtained from the above virus as fragments that also contain the SV40 virus origin of replication (Fiers et al., Nature, 273: p. 113 (1978)). Smaller or larger SV40 fragments can also be used if they contain a roughly 250 bp sequence extending from the HindIII site to the BglI site located at the origin of replication of the above virus. Furthermore, if such regulatory sequences are compatible with the host cell system, it is possible, and often desirable, to use promoters or regulatory sequences that are normally associated with the desired gene sequence.

[0086]

[0107] Therefore, according to the present invention, encoding the fusion polypeptide described herein Transfection into a host is performed using an expression vector containing nucleic acids that can be replicated in bacterial, yeast, insect, or mammalian cells, thereby directing the expression of such nucleic acids to produce a fusion polypeptide, which can then be recovered in a biologically active form. As used herein, the biologically active form includes a form that can bind to at least one VEGF family member.

[0087]

[0108] In some embodiments, the host cell is E. coli, COS cells, or Chinese Hamster ovary ("CHO") cells can be used. In some other embodiments, the host cells can be HEK293 cells.

[0088] Vector construction

[0109] The construction of a suitable vector containing the desired coding sequence and control sequence is Standard ligation techniques are used. Isolated plasmids or DNA fragments are cleaved, prepared, and ligated into the desired form for forming the required plasmid. The methods used are independent of the DNA source or intended host. Cleavage is performed by treatment with restriction enzymes (or multiple restriction enzymes) in a suitable buffer.

[0089]

[0110] VEGFR-1, VEGFR-2, or VEGR-3 (1 or more) Nucleic acid sequences substantially encoding the g-like domain can be generated according to the method disclosed in U.S. Patent No. 6,897,294.

[0090]

[0111] Ig-like domain 2 of VEGFR-1, Ig-like domain 3 of VEGFR-2, Furthermore, nucleic acid sequences substantially encoding Ig-like domains 1 and 2 of VEGR-3 are tandem ligated in a desired order. This construct is then ligated to the N-terminus of a nucleic acid sequence encoding a desired Fc portion of IgG1. The entire nucleic acid sequence is then placed into a vector containing a promoter in the gene's reading frame and compatible with the proposed host cell. Several plasmids, such as those described in U.S. Patents 4,456,748; 5,460,811; 5,888,808; and 6,333,147, may be used to generate the fusion polypeptide of the present invention.

[0091]

[0112] In one embodiment, the fusion polypeptide of the present invention is used in the U.S. 7,070,95 It is produced according to the method described in No. 9. The entire nucleic acid sequence of the fusion polypeptide ("VEGFR-1-D2-VEGFR-2-D3-VEGFR-3-D1D2-Fc" or "VEGFR-1-D2-VEGFR-2-D3-VEGFR-3-D1D2D3-Fc") is inserted into the expression vector pEE14.1 (Lonza Biologics) having a CMV promoter. CHO K1 cells are transfected with the pEE14.1 / VEGFR-1-D2-VEGFR-2-D3-VEGFR-3-D1D2-Fc DNA construct or the pEE14.1 / VEGFR-1-D2-VEGFR-2-D3-VEGFR-3-D1D2D3-Fc DNA construct and then grown. As described in U.S. Patent No. 7,070,959, fusion polypeptides obtained from CHO cells or HEK293 cells are purified and characterized by a binding assay.

[0092]

[0113] In one embodiment, the fusion polypeptide of the present invention is K d ≤10 -9 M with VEGF It can bind to family members. In another embodiment, the fusion polypeptide of the present invention is K d ≤ 5 × 10 -10 It can bind to VEGF family members at M. In yet another embodiment, the fusion polypeptide of the present invention is Kd ≤10 -10 It can be joined to VEGF family members using M.

[0093]

[0114] In one embodiment, the present invention relates to diseases, conditions, or disorders having an etiology of abnormal angiogenesis. The present invention provides compounds, compositions, and methods for treating or controlling harm.

[0094]

[0115] In another aspect, the present invention relates to at least one pathogenesis having abnormal angiogenesis The present invention provides a method for treating or controlling an ocular disease, condition, or disorder in a subject. The method comprises the step of administering a composition comprising a fusion polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 15, 12, and 16 to a subject requiring such treatment or control.

[0095]

[0116] In yet another embodiment, the method is as enumerated in Sequence ID No. 12 or Sequence ID No. 16 The procedure includes administering to a subject a composition comprising a fusion polypeptide comprising an amino acid sequence that is at least 90% identical to an amino acid sequence, provided that the amino acids at positions 286 and 287 of SEQ ID NO: 12 or SEQ ID NO: 16 are substitutable, thereby the amino acid sequence from positions 285 to 287 of SEQ ID NO: 12 or SEQ ID NO: 16 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0096]

[0117] In yet another embodiment, the method is as enumerated in Sequence ID No. 12 or Sequence ID No. 16 The step of administering to a subject a composition comprising a fusion polypeptide containing an amino acid sequence that is at least 90% identical to an amino acid sequence, wherein the amino acid sequence at positions 285-287 of SEQ ID NO: 12 or SEQ ID NO: 16 is unsubstituted and remains NDT.

[0097]

[0118] In yet another embodiment, the method is as enumerated in Sequence ID No. 12 or Sequence ID No. 16 The method includes administering to a subject a composition comprising a fusion polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid sequence, provided that the amino acids at positions 286 and 287 of SEQ ID NO: 12 or SEQ ID NO: 16 are substitutable, thereby the amino acid sequence from positions 285 to 287 of SEQ ID NO: 12 or SEQ ID NO: 16 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0098]

[0119] In yet another embodiment, the method is as enumerated in Sequence ID No. 12 or Sequence ID No. 16 The method includes administering to a subject a composition comprising a fusion polypeptide containing an amino acid sequence that is at least 95% identical to an amino acid sequence, provided that the amino acid sequence at positions 285-287 of SEQ ID NO: 12 or SEQ ID NO: 16 is unsubstituted and remains NDT.

[0099]

[0120] In yet another embodiment, the ocular disease, condition, or disorder is choroidal neovascularization. Macular edema caused by a condition selected from the group consisting of polypoid choroidal vasculopathy, retinal neovascularization, vascular leakage, retinal edema, diabetic macular edema, retinal vein occlusion and non-infectious posterior uveitis (including myopic choroidal neovascularization), diabetic retinopathy, corneal neovascularization, corneal inflammation, myopic neovascularization, neovascular glaucoma, and neovascular age-related macular degeneration (exudative age-related macular degeneration caused by retinal neovascularization).

[0121] In another aspect, the present invention relates to at least one pathogenesis having abnormal angiogenesis The present invention provides for the use of fusion polypeptides for the preparation or manufacture of pharmaceuticals for treating or controlling ocular diseases, conditions, or disorders in a subject; wherein the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, SEQ ID NOs: 4, SEQ ID NOs: 6, SEQ ID NOs: 15, SEQ ID NOs: 12, and SEQ ID NOs: 16.

[0100]

[0122] In yet another aspect, the present invention relates to a condition having a pathogenesis of abnormal angiogenesis, at least The present invention provides for the use of a fusion polypeptide for the preparation or manufacture of a pharmacopoeia to treat or control a single ocular disease, condition, or disorder in a subject; wherein the fusion polypeptide is the amino acid sequence listed in SEQ ID NO: 12 or SEQ ID NO: 16, wherein the amino acids at positions 286 and 287 of SEQ ID NO: 12 or SEQ ID NO: 16 are substitutable, so that the amino acid sequence from positions 285 to 287 of SEQ ID NO: 12 or SEQ ID NO: 16 is NDT, NDS, NXT, or NXS, where X is selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. It contains an amino acid sequence that is at least 90% identical to an amino acid sequence, provided that it is an amino acid.

[0101]

[0123] In yet another embodiment, the fusion polypeptide for use is SEQ ID NO: 12 or an amino acid sequence listed in SEQ ID NO: 16, wherein the amino acid sequence at positions 285-287 of SEQ ID NO: 12 or SEQ ID NO: 16 is unsubstituted and remains an NDT, and includes an amino acid sequence that is at least 90% identical to the amino acid sequence.

[0102]

[0124] In yet another aspect, the present invention relates to a condition having a pathogenesis of abnormal angiogenesis, at least The present invention provides for the use of a fusion polypeptide for the preparation or manufacture of a pharmacopoeia to treat or control a disease, condition, or disorder of one eye in a subject; wherein the fusion polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence enumerated in SEQ ID NO: 12 or SEQ ID NO: 16, provided that the amino acids at positions 286 and 287 of SEQ ID NO: 12 or SEQ ID NO: 16 are substitutable, thereby the amino acid sequence at positions 285-287 of SEQ ID NO: 12 or SEQ ID NO: 16 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0103]

[0125] In yet another embodiment, the fusion polypeptide for use is SEQ ID NO: 12 or an amino acid sequence listed in SEQ ID NO: 16, wherein the amino acid sequence at positions 285-287 of SEQ ID NO: 12 or SEQ ID NO: 16 is unsubstituted and remains an NDT, and includes an amino acid sequence that is at least 95% identical to the amino acid sequence.

[0104]

[0126] In yet another embodiment, the ocular disease, condition, or disorder is disclosed herein. It is selected from a group consisting of diseases, conditions, or disorders.

[0105]

[0127] In one embodiment, the subject is a fused polypropylene in doses of approximately 25 to 4000 micrograms. The peptide is administered. In another embodiment, the subject is administered a fusion polypeptide in doses of approximately 50-4000, approximately 100-4000, approximately 500-4000, approximately 1000-4000, approximately 2000-4000, approximately 50-3000, approximately 50-2000, approximately 50-1000, or approximately 50-500 micrograms.

[0106]

[0128] In yet another embodiment, a composition comprising a fusion polypeptide is used as eye drops or an ophthalmic injection. These are in the form of injections (e.g., intravitreous, intraacular, periorbital, subtenon's capsule, subretinal, or choroidal). Such compositions include ophthalmic compositions. The fusion polypeptides of the present invention may also be incorporated into medical devices that can be implanted in or near diseased tissue.

[0107]

[0129] In one embodiment, anterior disease, condition, or disorder; for example, corneal neovascularization, cornea When treating or controlling inflammation or neovascular glaucoma, compositions comprising fusion polypeptides may be in the form of eye drops or intracavitary or subconjunctival injections. In another embodiment, when treating or controlling posterior segment diseases, conditions, or disorders; for example, choroidal neovascularization (including myopic choroidal neovascularization), neovascular age-related macular degeneration, vascular leakage, retinal edema, or diabetic retinopathy, compositions comprising fusion polypeptides may be in the form of intravitreal injections.

[0108]

[0130] In yet another aspect, eye drops are used to substantially treat a disease, condition, or disorder. The drug is administered to the subject at least once a day, at least once a week, or at least once a month until control is achieved.

[0109]

[0131] In yet another embodiment, the composition is used for at least one month, at least two months, and less The drug is administered to the subject for a period of at least three months, or at least six months.

[0110]

[0132] In yet another embodiment, intravitreal injection or into or near the affected tissue The injection is administered to the target patient according to a regimen recommended by the physician for that particular patient. For example, the injection may be administered at least once per month, at least once every two months, at least once every three months, or at least once every six months until the disease, condition, or disorder is substantially treated or controlled. In one embodiment, the treatment may be administered more frequently at first, and then less frequently after a certain period, as may be determined by the physician.

[0111]

[0133] The concentration of the fusion polypeptide of the present invention in such ophthalmic composition is about 0.1 to about The range can be up to 200 mg / ml (or alternatively, from approximately 0.25 to approximately 200 mg / ml, or from approximately 0.25 to approximately 160 mg / ml, or from approximately 0.5 to approximately 100 mg / ml, or from approximately 0.25 to approximately 50 mg / ml, or from approximately 0.5 to approximately 200 mg / ml, or from approximately 0.5 to approximately 160 mg / ml, or from approximately 0.5 to approximately 100 mg / ml, or from approximately 0.5 to approximately 50 mg / ml, or from approximately 1 to approximately 200 mg / ml, or from 1 to approximately 160 mg / ml, or from approximately 0.5 to approximately 100 mg / ml, or from approximately 1 to approximately 50 mg / ml).

[0112]

[0134] In yet another embodiment, a method for preparing the composition of the present invention is (a) the present invention The process includes the step of combining (b) a certain amount of the fusion polypeptide with a physiologically acceptable carrier.

[0113]

[0135] In one embodiment, such physiologically acceptable carrier is sterile saline or It can be a physiologically acceptable buffer. In another embodiment, such a carrier comprises a hydrophobic medium such as a pharmaceutically acceptable oil. In yet another embodiment, the carrier comprises an emulsion of a hydrophobic material and water. In yet another embodiment, the fusion polypeptide of the present invention may be associated with or linked to a high molecular weight substance to provide a long circulation time.

[0114]

[0136] Physiologically acceptable buffers include, but are not limited to, phosphate buffers. Alternatively, a Tris-HCl buffer (containing tris(hydroxymethyl)aminomethane and HCl) may be included. For example, a Tris-HCl buffer having a pH of 7.4 contains 3 g / l of tris(hydroxymethyl)aminomethane and 0.76 g / l of HCl. In yet another embodiment, the buffer is 10 × phosphate-buffered saline ("PBS") or a 5 × PBS solution.

[0115]

[0137] Other buffering agents may also be used in some situations, for example, with a pK of 7.5 at 25°C. a and about HEPES (N-{2-hydroxyethyl}piperazine-N'-{2-ethanesulfonic acid}) has a pH in the range of 6.8 to 8.2; pK of 7.1 at 25°C. a and BES (N,N-bis{2-hydroxyethyl}2-aminoethanesulfonic acid) with a pH in the range of approximately 6.4 to 7.8; pK of 7.2 at 25°C. a and MOPS (3-{N-morpholino}propanesulfonic acid) with a pH in the range of approximately 6.5 to 7.9; pK of 7.4 at 25°C. a and TES (N-tris{hydroxymethyl}-methyl-2-aminoethanesulfonic acid) with a pH in the range of approximately 6.8 to 8.2; pK of 7.6 at 25°C. a and MOBS (4-{N-morpholino}butanesulfonic acid) with a pH in the range of approximately 6.9 to 8.3; pK of 7.52 at 25°C. a and DIPSO(3-(N,N-bis{2-hydroxyethyl}amino)-2-hydroxypropane)) with a pH in the range of approximately 7-8.2; 7 at 25°C. 61 pK a TAPSO(2-hydroxy-3{tris(hydroxymethyl)methylamino}-1-propanesulfonic acid)) has a pH in the range of approximately 7-8.2 and a pK of 8.4 at 25°C. a and TAPS ({(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino}-1-propanesulfonic acid) with a pH in the range of approximately 7.7 to 9.1; pK of 8.9 at 25°C. aand TABS (N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid) with a pH in the range of approximately 8.2 to 9.6; pK of 9.0 at 25°C. a and AMPSO(N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid) with a pH in the range of approximately 8.3 to 9.7; pK of 9.5 at 25°C a and CHES(2-cyclohexylamino)ethanesulfonic acid with a pH in the range of approximately 8.6 to 10.0; pK of 9.6 at 25°C a and CAPSO(3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid) with a pH in the range of approximately 8.9 to 10.3; or 10.4 pK at 25°C. a Furthermore, buffers based on CAPS (3-(cyclohexylamino)-1-propanesulfonic acid) with a pH in the range of approximately 9.7 to 11.1 may also be found to be appropriate or desirable.

[0116]

[0138] In certain embodiments, the composition of the present invention ranges from about 4 to about 6.8, Alternatively, the composition may be formulated in a buffer having an acidic pH, such as between approximately 5 and 6.8. In such embodiments, the buffering capacity of the composition is preferably such that it allows the composition to rapidly reach a physiological pH after administration to the patient.

[0117]

[0139] In addition to buffering agents, the composition of the present invention contains surfactants, stabilizers, preservatives, cosolvents, The material may include a material selected from the group consisting of humectants, emollients, chelating agents, tonicity modifiers, and antioxidants.

[0118]

[0140] In one embodiment, any of these materials that may be used in the composition of the present invention are ophthalmic It is a generally acceptable material.

[0119]

[0141] Water-soluble preservatives that can be used include sodium bisulfite, sodium bisulfate, and thi These include sodium sulfite, benzalkonium chloride, chlorobutanol, thimerosal, ethyl alcohol, methylparaben, polyvinyl alcohol, benzyl alcohol, and phenylethyl alcohol. These agents may be present in individual amounts ranging from about 0.001% to about 5% by weight (preferably about 0.01% to about 2% by weight). Suitable water-soluble buffers that may be used are sodium carbonate, sodium borate, sodium phosphate, sodium acetate, sodium bicarbonate, etc., as approved by the U.S. Food and Drug Administration ("USFDA") for the desired route of administration.

[0120]

[0142] Non-exclusive examples of surfactants include, but are not limited to, nonionic surfactants. Examples of surfactants include polysorbates (e.g., polysorbate 20, polysorbate 80), 4-(1,1,3,3-tetramethylbutyl)phenol / poly(oxyethylene) polymers (e.g., polymers sold under the trademark Tyloxapol), poly(oxyethylene)-poly(oxypropylene) block copolymers, glycolic acid esters of fatty acids, and mixtures thereof.

[0121]

[0143] In one embodiment, the pH of the composition is in the range of about 4 to about 11. Alternatively, The pH of the composition is in the range of approximately 6 to approximately 8, or approximately 6.5 to approximately 8.

[0122]

[0144] In another embodiment, the composition has a pH of about 7. Alternatively, the composition has a pH of about 7 to about It has a pH range up to 7.5.

[0123]

[0145] In yet another embodiment, the composition has a pH of approximately 7.4.

[0124]

[0146] In yet another embodiment, the composition also facilitates the administration of the composition to a subject. The composition may include viscosity-modifying compounds designed to enhance bioavailability in or to the subject. In yet another embodiment, the viscosity-modifying compound may be selected so that the composition does not easily disperse after being administered to the eye environment. Such compounds can enhance the viscosity of the composition and may include, but are not limited to: monomer polyols such as glycerol, propylene glycol, and ethylene glycol; polymer polyols such as polyethylene glycol; various polymers of the cellulose family such as hydroxypropyl methylcellulose ("HPMC"), carboxymethylcellulose ("CMC") sodium, and hydroxypropylcellulose ("HPC"); polysaccharides such as hyaluronic acid and its salts, chondroitin sulfate and its salts, and dextran, e.g., dextran 70; water-soluble proteins such as gelatin; vinyl polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and povidone; carbomers such as carbomer 934P, carbomer 941, carbomer 940, or carbomer 974P; and acrylic acid polymers. Generally, the desired viscosity can range from about 1 to about 400 centipoise ("cps") or mPa.s.

[0125]

[0147] Non-exclusive examples of chelating agents include ethylenediaminetetraacetic acid ("EDTA"), Examples include diethylenetriaminepentakis(methylphosphonic acid), etidronic acid, and the tetrasodium salt of etidronic acid (also known as "HAP").

[0126]

[0148] The buffer itself is a "tonic" buffer that roughly maintains the eye drop solution at a specific ion concentration and pH. These are "adjusting agents" and "pH adjusters," but further "tonicity adjusters" may be added to adjust the final tonicity of the solution. Such tonicity adjusters are well known to those skilled in the art and include, but are not limited to, mannitol, sorbitol, dextrose, sucrose, urea, propylene glycol, and glycerin. Various salts may also be used, including monovalent cation halide salts (e.g., NaCl or KCl). Typically, the tonicity of the formulations of the present invention ranges from about 200 to 400 mOsm / kg. Alternatively, the tonicity of the formulations of the present invention ranges from about 220 to 400 mOsm / kg, or about 220 to 350 mOsm / kg, or about 220 to 300 mOsm / kg, or about 250 to 350 mOsm / kg.

[0127]

[0149] Non-specific examples of antioxidants include ascorbic acid (vitamin C) and others. Examples include salts and esters of; tocopherols (e.g., α-tocopherol) and tocotrienols (vitamin E) and their salts and esters (e.g., vitamin E TGPS (D-α-tocopheryl polyethylene glycol 1000 succinate)); glutathione; lipoic acid; uric acid; butylated hydroxyanisole ("BHA"); butylated hydroxytoluene ("BHT"); tertiary butylhydroquinone ("TBHQ"); and polyphenol antioxidants (e.g., gallic acid, cinnamic acid, flavonoids, and their salts, esters, and derivatives).

[0128]

[0150] Non-specific examples of tranquilizers include sucrose, mannitol, sorbitol, And trehalose is another example.

[0129]

[0151] The proportions of various components or mixtures in the following examples were adjusted as appropriate. Please understand that this is possible.

[0130]

[0152] In another embodiment, the fusion polypeptide of the present invention and an appropriate amount of one or more The desired excipient is incorporated into a formulation for topical administration or injection into a portion of the eye, such as the anterior or posterior segment, or into the vitreous humor. The injectable formulation is intended for use for a period longer than approximately one week (or approximately one week). Preferably, it may include a carrier that provides sustained release of the active ingredient for a period of time longer than 2, 3, 4, 5, or 6 months.

[0131]

[0153] In yet another embodiment, a combination comprising the fusion polypeptide of the present invention and a desired excipient The product is freeze-dried and reconstituted in a physiologically acceptable liquid carrier substantially immediately before administration to the subject.

[0132]

[0154] In one embodiment, the compound or composition of the present invention is an electrode such as 25-35 gauge. It can be injected with a fine-gauge needle. Typically, a composition in an amount ranging from about 25 μl to about 100 μl containing about 25 to 4000 μg of the fusion polypeptide of the present invention is administered to the patient. In one embodiment, the fusion polypeptide has an amino acid sequence substantially identical to the amino acid sequence listed in SEQ ID NO: 12 or SEQ ID NO: 16, provided that the amino acids at positions 286 and 287 of SEQ ID NO: 12 or SEQ ID NO: 16 are substitutable, thereby the amino acid sequence from positions 285 to 287 of SEQ ID NO: 12 or SEQ ID NO: 16 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In another embodiment, the fusion polypeptide has an amino acid sequence as listed in SEQ ID NO: 12 or SEQ ID NO: 16. The concentration of such fusion polypeptide is selected from the range disclosed above.

[0133]

[0155] In yet another embodiment, the fusion polypeptide of the present invention comprises an ophthalmic biodegradable material. The active ingredient is incorporated into a device, which is implanted in the posterior ocular tissue of the subject to provide long-term treatment or control (e.g., longer than about one week, or longer than about one, two, three, four, five, or six months) of a neovascular disease, condition, or disorder. Such a device may be implanted in the ocular or periorbital tissue of the subject by a skilled physician. Non-limiting examples of ophthalmic implant systems or devices for sustained release of active ingredients are disclosed in U.S. Patents 5,378,475; 5,773,019; 5,902,598; 6,001,386; 6,051,576; and 6,726,918.

[0134]

[0156] In yet another aspect, treating neovascular disease, condition, or disorder of the eye A method for control includes the step of administering a composition comprising the fusion polypeptide of the present invention to a subject requiring it.

[0135]

[0157] In yet another aspect, treating neovascular disease, condition, or disorder of the eye A method for control includes administering a composition comprising a fusion polypeptide having the amino acid sequence as enumerated in SEQ ID NO: 12 or SEQ ID NO: 16 to a subject requiring it.

[0136]

[0158] In yet another aspect, the eye has a pathogenesis involving abnormal neovascularization in the posterior segment of the eye. A method for treating or controlling angiogenic disease, condition, or disorder comprises the step of intravitreal injection of a composition comprising a fusion polypeptide having the amino acid sequence as enumerated in SEQ ID NO: 12 or SEQ ID NO: 16 into a subject requiring it.

[0137]

[0159] In another embodiment, such disease, condition, or disorder is choroidal angiogenesis, polyangiogenesis, polyangiogenesis The group is selected from the following: pipoid choroidal vasculopathy, myopic neovascularization, retinal neovascularization including retinopathy of prematurity, vascular leakage, retinal edema including macular edema caused by diabetic macular edema and other retinal conditions such as retinal vein occlusion and non-infectious posterior uveitis, diabetic retinopathy including proliferative diabetic retinopathy, corneal neovascularization, and neovascular glaucoma.

[0138]

[0160] In yet another embodiment, the composition of the present invention is used once a week, once a month, once a year, and annually. It is administered twice a day, four times a year, or at an appropriate frequency determined to be appropriate for treating or controlling anterior segment inflammatory disease, condition, or disorder.

[0139]

[0161] In yet another embodiment, the fusion polypeptide or fusion protein of the present invention also It may also be used for the treatment or control of other non-ocular diseases or conditions that are promoted by or have pathogenesis related to abnormal angiogenesis, such as cancer, psoriasis, rheumatoid arthritis, and atherosclerosis. Such treatment or control may be carried out, for example, by systemic administration.

[0140]

[0162] In yet another aspect, the present invention relates to cancer, psoriasis, rheumatoid arthritis, and atherosclerosis. The present invention provides the use of fusion polypeptides or fusion proteins for the preparation or manufacture of pharmaceuticals for treating or controlling non-ocular diseases or conditions that are promoted by or have a pathogenesis related to abnormal angiogenesis, such as ocular arteriosclerosis.

[0141]

[0163] Cancers suitable for treatment according to the present invention include, but are not limited to, cancers, lymphomas, and other cancers. This includes tumors, blastomas, sarcomas, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancers, as well as B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL)), small lymphoma Examples include pacific (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade non-cleavage cell NHL, giant lesion NHL, mantle cell lymphoma, AIDS-associated lymphoma, and Valdenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorders (PTLD); as well as abnormal angiogenesis, edema (e.g., edema associated with brain tumors), and Meggs syndrome. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, colon cancer, non-small cell lung cancer, non-Hodgkin lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid cancer, head and neck cancer, malignant melanoma, ovarian cancer, mesothelioma, and multiple myeloma. In another embodiment, the cancer is colorectal cancer. Cancerous conditions suitable for treatment according to the present invention include metastatic cancer. The method of the present invention is particularly suitable for the treatment of vascularized tumors. [Examples]

[0142] Example 1 Composition 1

[0164] The composition of the present invention contains 10 to 100 mg / mL of the fusion protein EB-101. The composition is prepared by combining a 10 mM histidine HCl buffer, 10% (by weight) α,α-trehalose, and 0.01% (by weight) polysorbate 20. In some embodiments, the composition may also contain materials selected from the group consisting of antioxidants, chelating agents, and preservatives, as well as combinations thereof. The composition has a pH in the range of about 5–6 and is suitable for intraocular administration, including intravitreous administration.

[0143] Example 2 Composition 2

[0165] The composition of the present invention contains 10 to 100 mg / mL of the fusion protein EB-101. It is prepared by combining 10 mM phosphate buffer, 40 mM NaCl, 5% sucrose, and 0.03% polysorbate 20. The composition has a pH in the range of approximately 5.5 to 6.5 and is suitable for intraocular administration, including intravitreous administration.

[0144] Example 3 Transient expression of EB-101 in HEK293 cells

[0166] This encodes the amino acid sequence of EB-101 as indicated by Sequence ID No. 12. We synthesized the gene and constructed a vector for protein expression. Using the expression vector, we transiently transfected HEK293 cells with synthetic culture medium. The resulting protein was purified by Protein A affinity column and ultrafiltration, and then subjected to 0.2 μm sterile filtration to obtain high-purity bulk. When EB-101 expression in HEK293 cells was analyzed by size exclusion chromatography (SEC-HPLC), it produced a protein with a MW of approximately 200 kDa (unreduced form by SDS-PAGE) and a purity of 96%. See Figure 2.

[0145] Example 4 Transient expression of EB-101 in CHO cells.

[0167] This encodes the amino acid sequence of EB-101 as indicated by Sequence ID No. 12. The gene was synthesized, and a vector for protein expression was constructed. Using the expression vector, transient transfection was performed into CHO cells using synthetic culture medium. The generated protein was purified by Protein A affinity column and ultrafiltration, and then subjected to 0.2 μm sterile filtration to obtain high-purity bulk. When EB-101 expression in CHO cells was analyzed by SEC-HPLC, it produced a protein of approximately 200 kDa MW (unreduced form by SDS-PAGE) and 100% purity. See Figure 3.

[0146]

[0168] Example 5 Human recombinant human VEGF-A using SPR Biacore 165 VEGF-B 167 / 189 A comparison of the binding affinity of EB-101 to VEGF-C, VEGF-D, and PlGF between EB-101 and aflibercept. See Figures 4-8.

[0147]

[0169] Immobilization of anti-human IgG(Fc) antibodies on the surface of the CM5 sensor chip allows for S PR Biacore assays were performed, and the level of ligand immobilization was determined by SPR Biacore assays. The amount of anti-Fc antibody coupled onto the CM5 sensor chip was approximately 7,000–14,000 response units ("RU"). EB-101 or aflibercept was then injected into the sample channel, resulting in a capture level of approximately 400 RU. Finally, various concentrations of the test substance (VEGF-A in this assay), diluted with the flowing buffer, were injected for affinity and kinetics measurements. Binding affinity and / or kinetics were measured using a 1:1 binding model. VEGF-A to EB-101 165 The reaction rate constant for the bond was as follows: Equilibrium dissociation constant K D = 7.51 × 10 -12 M(7.51pM), binding rate constant K a = 5.06 × 10 7 M -1 s -1 , dissociation rate constant Kd = 3.64 × 10 -4 s -1 VEGF-A to aflibercept 165 The reaction rate constant for the bond was as follows: Equilibrium dissociation constant K D = 3.36 × 10 -11 M(33.6pM), binding rate constant K a = 1.36 × 10 7 M -1 s -1 , dissociation rate constant K d = 4.58 × 10 -4 s -1 .

[0148]

[0170] VEGF-B for EB-101 167 / 189 The reaction rate constants for the bond are as follows: The equilibrium dissociation constant K was: D = 1.38 × 10 -10 M(138pM), binding rate determination number K a = 2.20 × 10 6 M -1 s -1 , dissociation rate constant K d = 3.03 × 10 -4 s -1 VEGF-B to aflibercept 167 / 189 The reaction rate constant for the bond was as follows: Equilibrium dissociation constant K D = 1.73 × 10 -11 M(17.3pM), binding rate constant K a = 3.27 × 10 6 M -1 s -1 , dissociation rate constant K d = 5.67 × 10 -5 s -1 .

[0149]

[0171] The reaction rate constants for VEGF-C binding to EB-101 were as follows: equilibrium dissociation constant K D = 4.64 × 10 -10 M(0.464nM), binding rate constant K a = 3.11 × 106 M -1 s -1 , dissociation rate constant K d = 1.44×10 -3 s -1 . Binding of VEGF-C to aflibercept could not be detected.

[0150]

[0172] Binding of VEGF-D to EB-101 or aflibercept could not be detected .

[0151]

[0173] The reaction rate constants for PlGF binding to EB-101 were as follows: equilibrium dissociation constant K D = 2.09×10 -10 M (209 pM), association rate constant K a = 1.68×10 7 M[[ID=3-4]] -1 s -1 , dissociation rate constant K d = 3.15×10 -3 s -1 . The reaction rate constants for PlGF binding to aflibercept were as follows: equilibrium dissociation constant K D = 4.84×10 -10 M (484 pM), association rate constant K a = 1.26×10 6 M -1 s -1 , dissociation rate constant K d = 6.10×10 -4 s -1 .

[0152]

Table 1

[0153]

[0174] Therefore, the binding of EB-101 to these VEGF family members is [[ID=7G]]better than that of aflibercept.

[0154] Example 6 VEGF-A in VEGFR-2-NFAT-RE luciferase reporter cells 165A comparison of EB-101, aflibercept, and bevacizumab regarding their effects on inhibiting VEGFR-2 signaling pathways.

[0175] Recombinant human VEGF-A 165 Mix with a series of dilutions of the test compound and leave at room temperature for 3 minutes. Incubate for 0 minutes, then 4 x 10 4 VEGFR-2(KDR)-NFAT-RE luciferase reporter cells were added to wells containing individual cells, followed by incubation at 37°C for 6 hours. After adding 50 μl of Bright-Lite, the luciferase signal was detected using a plate reader. VEGF-A 165 The effect of EB-101 on inhibiting VEGFR-2 signaling was comparable to that achieved by aflibercept or bevacizumab. See Figure 9.

[0155] Example 7 A comparison of EB-101 and aflibercept regarding their effects on inhibiting VEGF-C-mediated VEGFR2 signaling in VEGFR2-NFAT-RE luciferase reporter cells.

[0176] Recombinant human VEGF-C was mixed with a serial dilution of the test compound and left at room temperature for 30 minutes. Incubate, 4 x 10 4 VEGFR-2(KDR)-NFAT-RE luciferase reporter cells were added to wells containing one cell each, followed by incubation at 37°C for 6 hours. After the addition of 50 μl of Bright-Lite, the luciferase signal was detected using a plate reader. EB-101, rather than aflibercept, exhibited concentration-dependent inhibition of VEGF-C-mediated VEGFR-2 signaling. See Figure 10.

[0156] Example 8 A comparison of the inhibitory effects of EB-101 on VEGF-C-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells, and recombinant human VEGFR-3 (rhVEGFR-3-7ECD) containing aflibercept and seven extracellular domains.

[0177] Recombinant human VEGF-C was mixed with a serial dilution of the test compound and left at room temperature for 30 minutes. Incubate, 4 x 10 4 VEGFR-2(KDR)-NFAT-RE luciferase reporter cells were added to wells containing one cell / well, followed by incubation at 37°C for 6 hours. After the addition of 50 μl of Bright-Lite, the luciferase signal was detected using a plate reader. EB-101 and rhVEGFR-3, rather than aflibercept, exhibited dose-dependent inhibition of VEGF-C-mediated VEGFR-2 signaling. See Figure 11.

[0157] Example 9 VEGF-A 165 Quantitative analysis of EB-101, along with aflibercept and recombinant human VEGFR-3 (rhVEGFR-3) containing seven extracellular domains, to investigate its effect on inhibiting VEGFR-2 signaling in VEGF-C-stimulated VEGFR-2-NFAT-RE luciferase reporter cells.

[0178] The test compound was subjected to VEGF-A at 60 ng / ml. 165 and 25 ng / ml Mix with a cocktail containing VEGF-C, incubate at room temperature for 30 minutes, and then divide the resulting solution into 4 × 10⁻⁶ units. 4 Each well containing one VEGFR-2-NFAT-RE luciferase reporter cell was added, followed by incubation at 37°C for 6 hours. After adding 50 μl of Bright-Lite, the luciferase signal was detected using a plate reader. EB-101, and the combination of rhVEGFR-3 and aflibercept, showed VEGF-A 165Plus effectively inhibited VEGF-C-mediated VEGFR-2 signaling, whereas aflibercept or rhVEGFR3 alone did not inhibit VEGF-A 165 Furthermore, it showed only limited effects on inhibiting VEGF-C-mediated VEGFR-2 signaling. See Figure 12.

[0158] Example 10 VEGF-A in human lymphoid endothelial cells (HLECs) 165 A comparison of the effects of EB-101 on vector-mediated proliferation with aflibercept and bevacizumab.

[0179] The original HLEC was seeded into a 96-well plate at a density of 2500 cells / well. Then, it was cultured overnight at 37°C. The next day, recombinant human VEGF-A 165 The test compound was mixed with a serial dilution and incubated at room temperature for 30 minutes. The resulting solution was then added to each well and incubated at 37°C for 72 hours. 10% Alamar Blue dye was added to each well and incubated for 5 hours. Cell proliferation was then evaluated by fluorescence reading using a plate reader. VEGF-A 165 The effect of EB-101 on inhibiting HLEC proliferation was comparable to that achieved by aflibercept and bevacizumab. See Figure 13.

[0159]

[0180] Example 11 This study compares the effects of EB-101 on VEGF-C-mediated HLEC proliferation with recombinant human VEGFR-3 (rhVEGFR-3ECD) containing aflibercept and seven extracellular domains.

[0181] The original HLEC was seeded into a 96-well plate at a density of 2500 cells / well. The cells were then cultured overnight in an incubator at 37°C. The following day, recombinant human VEGF-C was mixed with serial dilutions of the test compound and incubated at room temperature for 30 minutes. The resulting solution was then added to each well and incubated at 37°C for 72 hours. 10% Alamar Blue dye was added to each well and incubated for 5 hours, after which cell proliferation was evaluated by fluorescence reading using a plate reader. EB-101 and rhVEGFR-3, rather than aflibercept, showed concentration-dependent inhibition of VEGF-C-mediated HLEC proliferation. See Figure 14.

[0160] Example 12 VEGF-A 165 Quantitative analysis of EB-101, aflibercept, and recombinant human VEGFR-3 (rhVEGFR-3 ECD) containing seven extracellular domains regarding VEGF-C-mediated human lymphatic endothelial cell (HLEC) proliferation.

[0182] The original HLEC was seeded into a 96-well plate at a density of 2500 cells / well. Then, it was incubated overnight at 37°C in an incubator. The next day, 60 ng / ml of VEGF-A was added. 165 A cocktail containing 25 ng / ml of VEGF-C was mixed with the test compound and incubated at room temperature for 30 minutes. The resulting solution was then added to each well and incubated at 37°C for 72 hours. 10% Alamar Blue dye was added and incubated for 5 hours, after which cell proliferation was evaluated by fluorescence reading using a plate reader. EB-101, and the combination of rhVEGFR-3 and aflibercept, showed VEGF-A 165 And it effectively inhibited VEGF-C-mediated VEGFR2 signaling, whereas aflibercept or rhVEGFR3 alone did not inhibit VEGF-A 165 Furthermore, it exhibited only limited inhibition of VEGF-C-mediated HLEC proliferation. See Figure 15.

[0161] Example 13 Molecular design and expression of EB-101BIb.

[0183] EB-101BIb binds to VEGFR-1's binding domain 2 and VEGFR-2's binding domain. This is a recombinant human Fc fusion protein containing domain 3 and domains 1-3 of VEGFR-3. A gene encoding the amino acid sequence of EB-101BIb, as indicated by Sequence ID No. 16, was synthesized, and a vector for protein expression was constructed. Transient transfection of CHO cells was performed using the expression vector in synthetic culture medium. The resulting protein was purified by Protein A affinity column and ultrafiltration, followed by 0.2 μm sterile filtration to obtain high-purity bulk protein. EB-101BIb expression in CHO cells, as analyzed by SEC-HPLC, produced a protein with approximately 200 kDa MW (unreduced form by SDS-PAGE) and 88% purity. See Figure 16.

[0162] Example 14 A comparison of EB-101BIb, aflibercept, EB-101, and EB-101BIa on the effects of EB-101BIb on VEGF-A-mediated VEGFR2 signaling in VEGFR2-NFAT-RE luciferase reporter cells.

[0184] Recombinant human VEGF-A 165 Mix with a series of dilutions of the test compound and leave at room temperature for 3 minutes. Incubate for 0 minutes, then 4 x 10 4 Each well containing one VEGFR2(KDR)-NFAT-RE luciferase reporter cell was then added, followed by incubation at 37°C for 6 hours. After adding 50 μl of Bright-Lite, the luciferase signal was detected using a plate reader. VEGF-A 165The effect of EB-101BIb on inhibiting VEGFR-2 signaling was comparable to that of aflibercept, EB-101, and EB-101BIa. The latter two molecules contained VEGFR-1 binding domain 2, VEGFR-2 domain 3, and VEGFR-3 domains 1-2, but lacked VEGFR-3 domain 3, and were expressed in HEK293 (EB-101) cells and CHO (EB-101BIa) cells, respectively. See Figure 17.

[0163] Example 15 The effects of EB-101BIb on VEGF-C and -D-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells were compared with aflibercept, EB-101, and EB-101BIa.

[0185] Recombinant human VEGF-C and -D were mixed with a series of dilutions of the test compound at room temperature. Incubate for 30 minutes, then 4 x 10 4 VEGFR-2(KDR)-NFAT-RE luciferase reporter cells were added to wells containing one cell / well, followed by incubation at 37°C for 6 hours. After the addition of 50 μl of Bright-Lite, the luciferase signal was detected using a plate reader. EB-101BIb showed concentration-dependent inhibition of VEGF-C and -D-mediated VEGFR-2 signaling, while EB-101 and EB-101BIa showed limited inhibition of VEGF-C and -D-mediated VEGFR-2 signaling. Aflibercept showed no effect on VEGF-C and -D-mediated VEGFR-2 signaling. EB-101 and EB-101BIa contained VEGFR-1 binding domain 2, VEGFR-2 domain 3, and VEGFR-3 domains 1-2, but lacked VEGFR-3 domain 3, and were expressed in HEK293 (EB-101) cells and CHO (EB-101BIa) cells, respectively. See Figure 18.

[0164] Example 16 The effects of EB-101BIb on VEGF-A, -C, and -D-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells were compared with aflibercept, EB-101, and a combination of recombinant human VEGFR-3 and aflibercept.

[0186] The test compound was subjected to VEGF-A at 60 ng / ml. 165 and 25 ng / ml Mix with a cocktail containing VEGF-C and 150 ng / ml of VEGF-D, incubate at room temperature for 30 minutes, then 4 × 10⁻⁶ of the resulting solution. 4 Each well containing one VEGFR-2-NFAT-RE luciferase reporter cell / well was added, followed by incubation at 37°C for 6 hours. After the addition of 50 μl of Bright-Lite, the luciferase signal was detected using a plate reader. Aflibercept showed little inhibition of VEGFR-2 signaling, while EB-101BIb showed inhibition of VEGF-A 165 VEGFR-2-NFAT-RE luciferase reporter cells stimulated with a cocktail consisting of -C and -D showed concentration-dependent inhibition of VEGFR-2 signaling (Figure 19A). EB-101BIb inhibits VEGF-A 165 EB-101 completely inhibited VEGF-C and VEGF-D-mediated VEGFR-2 signaling. EB-101 showed limited inhibitory effects, while aflibercept had minimal inhibition of VEGF-A, -C, and -D-mediated VEGFR-2 signaling compared to EB-101BIb and EB-101. The complete inhibition induced by EB-101BIb was achieved by the combination of recombinant human VEGFR-3 and EB-101. This was reproduced (Figure 19B). EB-101 contains the binding domain 2 of VEGFR-1, domain 3 of VEGFR-2, and domains 1-2 of VEGFR-3, but does not contain domain 3 of VEGFR-3. EB-101BIb contains the binding domain 2 of VEGFR-1, domain 3 of VEGFR-2, and domains 1-3 of VEGFR-3. See Figure 19.

[0165] Example 17 VEGF-A in human lymphatic endothelial cells (HLECs) 165 A comparison of the effects of EB-101BIb on vector propagation with aflibercept and EB-101BIa.

[0187] The original HLEC was seeded into a 96-well plate at a density of 2500 cells / well. Then, it was cultured overnight at 37°C. The next day, recombinant human VEGF-A 165 The test compound was mixed with a serial dilution and incubated at room temperature for 30 minutes. The resulting solution was then added to each well and incubated at 37°C for 72 hours. 10% Alamar Blue dye was added to each well and incubated for 5 hours. Cell proliferation was then evaluated by fluorescence reading using a plate reader. VEGF-A 165 The effect of EB-101BIb on inhibiting HLEC proliferation was comparable to that achieved by aflibercept and EB-101BIa. See Figure 20.

[0166] Example 18 A comparison of the effects of EB-101BIb on VEGF-C and VEGF-D-mediated HLEC proliferation with aflibercept, EB-101BIa, and recombinant human VEGFR-3 containing seven extracellular domains.

[0188] The original HLEC was seeded into a 96-well plate at a density of 2500 cells / well. The cells were then cultured overnight in an incubator at 37°C. The following day, recombinant human VEGF-C (25 ng / ml) and VEGF-D (150 ng / ml) were mixed with serial dilutions of the test compounds and incubated at room temperature for 30 minutes. The resulting solution was then added to each well and incubated at 37°C for 72 hours. 10% Alamar Blue dye was added to each well and incubated for 5 hours, after which cell proliferation was evaluated by fluorescence reading using a plate reader. EB-101BIb and rhVEGFR-3 showed a more potent effect on inhibiting VEGF-C and VEGF-D-mediated HLEC proliferation compared to the EB-101BIa treatment group. See Figure 21.

[0167] Example 19 The effects of EB-101BIb on the proliferation of VEGF-A, -C, and -D-mediated human lymphoid endothelial cells (HLECs) were compared with aflibercept, recombinant human VEGFR-3 containing seven extracellular domains (seven ECDs), and a combination of VEGFR-3 and aflibercept.

[0189] The original HLEC was seeded into a 96-well plate at a density of 2500 cells / well. Then, it was incubated overnight at 37°C in an incubator. The next day, recombinant human VEGF-A 165 A cocktail containing VEGF-C and VEGF-D was mixed with an equal volume of a sequential dilution of the test compound and incubated at room temperature for 30 minutes (VEGF-A 165 Final concentrations of -C and -D: 60, 25, and 150 ng / ml, respectively). The resulting solutions were added to nine wells in each group and incubated at 37°C for 72 hours. 10% Alamar Blue dye was added to each well and incubated for 5 hours, after which cell proliferation was assessed by fluorescence reading using a plate reader. Aflibercept or VEGFR-3 alone showed limited inhibition, while EB-101BIb, or a combination of VEGFR-3 and aflibercept, showed VEGF-A 165 These completely inhibited HLEC proliferation stimulated by -C and -D. See Figure 22.

[0168] Sequence Listing of Nucleic Acids and Amino Acids

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[0205] [Chemical]

[0184] List of Formulas [Chemical] [Chemical]

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[0185]

[0206] Although specific embodiments of the present invention have been described above, the scope of the attached claims is as follows: Those skilled in the art will understand that many equivalents, modifications, substitutions, and alterations can be made to them without departing from the spirit and scope of the invention as defined within the scope.

Claims

1. A recombinant human Fc fusion protein comprising a) an Ig-like domain of a first VEGF receptor, b) an Ig-like domain of a second VEGF receptor, and c) at least one Ig-like domain of a third VEGF receptor, The third VEGF receptor comprises human VEGF-3 (flt-4) having the amino acid sequence listed in SEQ ID NO: 13, wherein the amino acids at positions 105 and 106 of SEQ ID NO: 13 are substitutable, so that the amino acid sequence from positions 104 to 106 of SEQ ID NO: 13 is NDT, NDS, NXT, or NXS, where X is an amino acid selected from the group consisting of A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. The Ig-like domain of the first VEGF receptor comprises an amino acid sequence that is at least 95% identical to the Ig-like domain 2 of human VEGF-1 (flt-1); The Ig-like domain of the second VEGF receptor contains an amino acid sequence that is at least 95% identical to the Ig-like domain 3 of human VEGF-2 (KDR); At least one Ig-like domain of the third VEGF receptor comprises an amino acid sequence that is at least 95% identical to Ig-like domains 1, 2, and 3 of human VEGF-3. The recombinant human Fc fusion protein.

2. d) The fusion protein according to claim 1, further comprising a polymer-forming component linked to the Ig-like domain.

3. The fusion protein according to claim 2, wherein the polymer-forming component comprises a portion of the Fc domain of human IgG1.

4. The Ig-like domain 2 of human VEGFR-1 comprises the amino acid sequence listed in Sequence ID No. 2; The Ig-like domain 3 of the human VEGFR-2 comprises the amino acid sequence listed in Sequence ID No. 4; The Ig-like domains 1, 2, and 3 of the aforementioned human VEGFR-3 comprise the amino acid sequences listed in Sequence ID No. 15; A portion of the Fc domain of human IgG1 contains the amino acid sequence listed in Sequence ID No.

8. The fusion protein according to claim 3.

5. A recombinant human Fc fusion protein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence listed in Sequence ID No.

16.

6. The fusion protein according to claim 5, comprising an amino acid sequence that is at least 95% identical to the amino acid sequence listed in Sequence ID No.

16.

7. The fusion protein according to claim 5, comprising the amino acid sequence listed in Sequence ID No.

16.

8. An isolated nucleic acid molecule encoding the fusion polypeptide described in any one of claims 1 to 7.

9. A vector comprising the nucleic acid molecule described in Claim 8.

10. The vector according to claim 9, comprising the nucleic acid molecule operably linked to an expression control sequence.

11. A host-vector system comprising the expression vector described in claim 10 in a host cell.

12. A method for producing a substantially purified fusion polypeptide, (a) Proliferating cells of the host-vector system according to claim 11 under conditions that enable the production of fusion polypeptides; (b) a step of recovering the fusion polypeptide or chimeric polypeptide to produce the recovered fusion polypeptide; (c) The step of purifying the recovered fusion polypeptide to produce a substantially purified fusion polypeptide. The method, including the method described above.

13. A pharmaceutical composition for use in a subject requiring treatment or control of at least one disease, condition, or disorder having an etiology of abnormal angiogenesis, wherein The pharmaceutical composition comprising a fusion protein containing an amino acid sequence that is at least 90% identical to the amino acid sequence listed in Sequence ID No.

16.

14. The pharmaceutical composition according to claim 13, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence listed in SEQ ID NO:

16.

15. The pharmaceutical composition according to claim 13, wherein the fusion protein has the amino acid sequence listed in SEQ ID NO:

16.

16. The pharmaceutical composition according to claim 15, wherein the disease, condition, or disorder is selected from the group consisting of choroidal neovascularization, polypoid choroidal vasculopathy, myopic neovascularization, retinal neovascularization, retinopathy of prematurity, vascular leakage, retinal edema, diabetic macular edema, retinal vein occlusion or macular edema caused by non-infectious posterior uveitis, diabetic retinopathy, proliferative diabetic retinopathy, corneal neovascularization, and neovascular glaucoma.

17. The pharmaceutical composition according to claim 16, comprising a fusion polypeptide in a dose of about 25 to 4000 micrograms.

18. The pharmaceutical composition according to claim 17, wherein the composition comprises eye drops, intravitreal injection, subretinal injection, or choroidal injection.

19. The pharmaceutical composition according to claim 17, wherein the composition is administered intraocularly to a target for a period of at least one month.

20. The pharmaceutical composition according to claim 17, wherein the composition is administered intraocularly at a frequency of at least once per month.

21. The pharmaceutical composition according to claim 15, wherein the disease, condition, or disorder is a non-ocular disease, condition, or disorder having an etiology of abnormal angiogenesis.

22. The pharmaceutical composition according to claim 21, wherein the disease, condition, or disorder is selected from the group consisting of cancer, psoriasis, rheumatoid arthritis, and atherosclerosis.

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