WO2024092717A1 - 纤维黏蛋白第iii型结构域衍生的蛋白质及其应用 - Google Patents

纤维黏蛋白第iii型结构域衍生的蛋白质及其应用 Download PDF

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WO2024092717A1
WO2024092717A1 PCT/CN2022/129857 CN2022129857W WO2024092717A1 WO 2024092717 A1 WO2024092717 A1 WO 2024092717A1 CN 2022129857 W CN2022129857 W CN 2022129857W WO 2024092717 A1 WO2024092717 A1 WO 2024092717A1
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cysteine
mutation
substituted
substitution
protein
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PCT/CN2022/129857
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French (fr)
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庄伟哲
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庄伟哲
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the present invention relates to protein engineering, and in particular to a protein derived from the type III domain of fibronectin and its application, including: a method for treating or preventing diseases or disorders caused by or associated with the activity or signal transduction of type II vascular endothelial growth factor receptor 2 (VEGFR2), or a method for treating or preventing diseases or disorders caused by angiogenesis.
  • VAGFR2 vascular endothelial growth factor receptor 2
  • the 10th human fibronectin type III domain (10Fn3) is one of the non-antibody scaffolds currently widely used to prepare novel binding proteins.
  • 10Fn3 The 10th human fibronectin type III domain is one of the non-antibody scaffolds currently widely used to prepare novel binding proteins.
  • Several molecules based on 10Fn3 are gradually being developed and tried for the treatment of diseases, but most are still in the clinical trial stage.
  • 10Fn3 has many features that are superior to immunoglobulin-derived systems. Based on the overall ⁇ -sandwich fold, 10Fn3 belongs to the immunoglobulin superfamily. The three surface loop regions near the N-terminus are structurally similar to the three antigen recognition loop regions or complementarity-determining regions (CDRs) of the immunoglobulin variable region. However, unlike the typical regions of immunoglobulins, 10Fn3 does not contain disulfide bonds. Moreover, the thermal transfer temperature of 10Fn3 is greater than 80°C, and the configuration is stable. In addition, 10Fn3 has reversible and rapid unfolding and refolding characteristics.
  • 10Fn3 has about 94 amino acid residues, which is smaller than the antigen binding unit (VHH) of heavy chain antibodies.
  • VHH antigen binding unit
  • U.S. Patent Application No. 11/448,171 entitled “INHIBITORS OF TYPE 2 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS”, proposes a type 2 vascular endothelial growth factor receptor binding protein C7.
  • the protein C7 uses 10Fn3 as the main structure, and the difference from the wild type is that the three surface loop region sequences 23 DAPAVTVRY, 51 PGSKST, and 75 VTGRGDSPASSKP of the wild type are replaced by 23 RHPHFPTRY, 51 PLQPPT, and 75 VTDGRNGRLLSIP respectively.
  • the protein C7 can be used as an antagonist of the type 2 vascular endothelial growth factor receptor and has the potential to become an anti-cancer drug.
  • due to insufficient thermal stability and low solubility, its development as a drug is hindered.
  • protein C7 was modified to obtain a variant protein C7-NM.
  • the present invention is accomplished based on the following matters: using the software Disulfide by Design 2.0 (DbD2) and referring to parameters such as B-factor, ⁇ 3 torsion angle, and energy to design the introduction of disulfide bonds in the structure of protein C7-NM to obtain a variant with disulfide bonds, which can improve thermal stability and solubility without affecting the activity of binding to specific proteins, thereby having the potential to be used as a biopharmaceutical.
  • DbD2 Disulfide by Design 2.0
  • parameters such as B-factor, ⁇ 3 torsion angle, and energy to design the introduction of disulfide bonds in the structure of protein C7-NM to obtain a variant with disulfide bonds, which can improve thermal stability and solubility without affecting the activity of binding to specific proteins, thereby having the potential to be used as a biopharmaceutical.
  • the present invention provides a protein comprising a 10th human fibronectin type III domain, and comprising a first mutation that replaces an amino acid site with cysteine and a second mutation that replaces another amino acid site with another cysteine, wherein the first mutation and the second mutation occur in other regions other than the FG loop of the 10th human fibronectin type III domain, whereby the cysteine replaced by the first mutation and the cysteine replaced by the second mutation form a disulfide bond.
  • the 10th human fibronectin type III domain comprises an amino acid sequence as shown in SEQ ID NO: 1.
  • the protein further comprises a mutation that replaces the amino acid sequence of loop BC with RHPHFPTRY, a mutation that replaces the amino acid sequence of loop DE with PLQPPT, and a mutation that replaces the amino acid sequence of loop FG with VTDGRNGRLLSIP.
  • the first mutation occurs in any one of ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop BC, loop CD, loop DE, and loop EF of the tenth human fibronectin type III domain
  • the second mutation occurs in any one of ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop BC, loop CD, loop DE, and loop EF of the tenth human fibronectin type III domain.
  • the first mutation occurs in any one of ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop CD, and loop EF of the tenth human fibronectin type III domain
  • the second mutation occurs in any one of ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop CD, and loop EF of the tenth human fibronectin type III domain.
  • the first mutation and the second mutation occur in the same region or in different regions.
  • the first mutation comprises: substitution of leucine at position 8 with cysteine, substitution of serine at position 17 with cysteine, substitution of leucine at position 19 with cysteine, substitution of isoleucine at position 34 with cysteine, substitution of threonine at position 35 with cysteine, substitution of tyrosine at position 36 with cysteine, substitution of glycine at position 37 with cysteine, substitution of threonine at position 39 with cysteine, substitution of lysine at position 63 with cysteine, or substitution of aspartic acid at position 67 with cysteine
  • the second mutation comprises: substitution of tryptophan at position 22 for cysteine, substitution of valine at position 45 for cysteine, substitution of phenylalanine at position 48 for cysteine, substitution of threonine at position 58 for cysteine, substitution of serine at position 60 for cysteine, substitution of valine at position 66 for cysteine, substitution of aspartic acid at position 67 for cysteine, substitution of
  • the first mutation comprises: leucine at position 8 is substituted with cysteine, and the second mutation comprises: tryptophan at position 22 is substituted with cysteine; the first mutation comprises: leucine at position 8 is substituted with cysteine, and the second mutation comprises: serine at position 89 is substituted with cysteine; the first mutation comprises: serine at position 17 is substituted with cysteine, and the second mutation comprises: serine at position 60 is substituted with cysteine; the first mutation comprises: leucine at position 19 is substituted with cysteine, and the second mutation comprises: threonine at position 58 is substituted with cysteine; the first mutation comprises: isoleucine at position 34 is substituted with cysteine, and the second mutation comprises: phenylalanine at position 48 is substituted with cysteine; the first mutation comprises: threonine at position 35 is substituted with cysteine, and the second mutation comprises: valine at position 45 is substituted with cysteine; the first mutation comprises: position 36 The first mutation
  • the protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 14.
  • the protein comprises an amino acid sequence as shown in SEQ ID NO: 11.
  • the protein includes a mutation that substitutes alanine at position 12 with glutamate, a mutation that substitutes threonine at position 14 with serine, a mutation that substitutes leucine at position 18 with isoleucine, and a mutation that substitutes leucine at position 19 with glutamate.
  • the first mutation comprises: leucine at position 8 is substituted with cysteine, serine at position 17 is substituted with cysteine, isoleucine at position 34 is substituted with cysteine, threonine at position 35 is substituted with cysteine, tyrosine at position 36 is substituted with cysteine, glycine at position 37 is substituted with cysteine, threonine at position 39 is substituted with cysteine, lysine at position 63 is substituted with cysteine, or aspartic acid at position 67 is substituted with cysteine, and the second mutation comprises: tryptophan at position 22 is substituted with cysteine, valine at position 45 is substituted with cysteine, phenylalanine at position 48 is substituted with cysteine, under the conditions that the protein comprises a mutation in which alanine at position 12 is substituted with glutamic acid, a mutation in which threonine at position 14 is substituted with serine, a mutation in which leucine at
  • the first mutation comprises: leucine at position 8 is substituted with cysteine, and the second mutation comprises: tryptophan at position 22 is substituted with cysteine; the first mutation comprises: leucine at position 8 is substituted with cysteine, and the second mutation comprises: serine at position 89 is substituted with cysteine; the first mutation comprises: serine at position 17 is substituted with cysteine, and the second mutation comprises: serine at position 60 is substituted with cysteine; the first mutation comprises: isoleucine at position 34 is substituted with cysteine, and the second mutation comprises: phenylalanine at position 48 is substituted with cysteine; the first mutation comprises: threonine at position 35 is substituted with cysteine, and the second mutation comprises: valine at position 45 is substituted with cysteine; the first mutation comprises: tyrosine at position 36 is substituted with cysteine, and the second mutation comprises: isoleucine at position 70 is substituted with cysteine ; the first mutation comprises substitution of g
  • the protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 17 to 26.
  • the protein further comprises a third mutation that replaces another amino acid site with another cysteine and a fourth mutation that replaces another amino acid site with another cysteine, the third mutation and the fourth mutation occur in other regions outside the loop FG of the 10th human fibronectin type III domain, whereby the cysteine substituted by the third mutation and the cysteine substituted by the fourth mutation form a disulfide bond.
  • the third mutation occurs in any one of ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop BC, loop CD, loop DE, and loop EF of the tenth human fibronectin type III domain
  • the fourth mutation occurs in any one of ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop BC, loop CD, loop DE, and loop EF of the tenth human fibronectin type III domain.
  • the third mutation occurs in any one of ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop CD, and loop EF of the tenth human fibronectin type III domain
  • the fourth mutation occurs in any one of ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop CD, and loop EF of the tenth human fibronectin type III domain.
  • the first mutation, the second mutation, the third mutation, and the fourth mutation occur in different regions.
  • the third mutation comprises: substitution of leucine at position 8 for cysteine, substitution of serine at position 17 for cysteine, substitution of leucine at position 19 for cysteine, substitution of isoleucine at position 34 for cysteine, substitution of threonine at position 35 for cysteine, substitution of tyrosine at position 36 for cysteine, substitution of glycine at position 37 for cysteine, substitution of threonine at position 39 for cysteine, substitution of lysine at position 63 for cysteine, or substitution of aspartic acid at position 67 for cysteine
  • the fourth mutation comprises: substitution of tryptophan at position 22 for cysteine, substitution of valine at position 45 for cysteine, substitution of phenylalanine at position 48 for cysteine, substitution of threonine at position 58 for cysteine, substitution of serine at position 60 for cysteine, substitution of valine at position 66 for cysteine, substitution of aspartic acid at position 67 for cysteine,
  • the first mutation comprises: substitution of leucine at position 8 with cysteine
  • the second mutation comprises: substitution of serine at position 89 with cysteine
  • the third mutation comprises: substitution of serine at position 17 with cysteine
  • the fourth mutation comprises: substitution of serine at position 60 with cysteine; or the first mutation comprises: substitution of leucine at position 8 with cysteine
  • the second mutation comprises: substitution of serine at position 89 with cysteine
  • the third mutation comprises: substitution of threonine at position 39 with cysteine
  • the fourth mutation comprises: substitution of aspartic acid at position 67 with cysteine.
  • the protein comprises an amino acid sequence as shown in SEQ ID NO: 15 or 16.
  • the protein includes a mutation that replaces alanine at position 12 with glutamate, a mutation that replaces threonine at position 14 with serine, a mutation that replaces leucine at position 18 with isoleucine, and a mutation that replaces leucine at position 19 with glutamate.
  • the first mutation comprises: leucine at position 8 is substituted with cysteine, serine at position 17 is substituted with cysteine, isoleucine at position 34 is substituted with cysteine, threonine at position 35 is substituted with cysteine, tyrosine at position 36 is substituted with cysteine, glycine at position 37 is substituted with cysteine, threonine at position 39 is substituted with cysteine, lysine at position 63 is substituted with cysteine, or aspartic acid at position 67 is substituted with cysteine, and the second mutation comprises: tryptophan at position 22 is substituted with cysteine, valine at position 45 is substituted with cysteine, phenylalanine at position 48 is substituted with cysteine, threonine at position 58 is substituted with cysteine, serine at position 60 is substituted with cysteine, valine at position 66 is substituted with cysteine, aspartic acid at position 67 is substituted with cysteine
  • the first mutation comprises: leucine at position 8 is substituted with cysteine, serine at position 17 is substituted with cysteine, isoleucine at position 34 is substituted with cysteine, threonine at position 35 is substituted with cysteine, tyrosine at position 36 is substituted with cysteine, glycine at position 37 is substituted with cysteine, threonine at position 39 is substituted with cysteine, lysine at position 63 is substituted with cysteine, or aspartic acid at position 67 is substituted with cysteine;
  • the second mutation comprises: tryptophan at position 22 is substituted with cysteine, valine at position 45 is substituted with cysteine, phenylalanine at position 48 is substituted with cysteine, threonine at position 58 is substituted with cysteine, and tyrosine at position 36 is substituted with cysteine.
  • the protein comprises a mutation in which the alanine at position 12 is substituted for glutamate, a mutation in which the threonine at position 14 is substituted for serine, a mutation in which the leucine at position 18 is substituted for isoleucine, and a mutation in which the leucine at position 19 is substituted for glutamate.
  • the first mutation comprises: leucine at position 8 is substituted by cysteine
  • the second mutation comprises: serine at position 89 is substituted by cysteine
  • the third mutation comprises: serine at position 17 is substituted by cysteine
  • the fourth mutation comprises: serine at position 60 is substituted by cysteine
  • the first mutation comprises: leucine at position 8 is substituted by cysteine
  • the second mutation comprises: serine at position 89 is substituted by cysteine
  • the third mutation comprises: threonine at position 39 is substituted by cysteine
  • the fourth mutation comprises: aspartic acid at position 67 is substituted by cysteine
  • the protein comprises a mutation that replaces alanine at position 12 by glutamate, a mutation that replaces threonine at position 14 by serine, a mutation that replaces leucine at position 18 by isoleucine, and a mutation that replaces leucine at position 19 by glutamate.
  • the protein comprises an amino acid sequence as shown in SEQ ID NO: 27 or 28.
  • the protein is used to bind to DLL4, EGFR, VEGFR2, or IGF-1R.
  • the protein of the present invention is based on the 10th human fibronectin type III domain, and introduces a disulfide bond at a specific position of the main body to improve thermal stability and solubility.
  • the protein of the present invention can further utilize amino acid sequence substitution to construct a protein binding interface to bind to specific proteins, such as DLL4, EGFR, VEGFR2, or IGF-1R, to act as a receptor antagonist, and has the potential to be used as a biopharmaceutical.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising: the protein as described above; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is an oral administration formulation, an injectable administration formulation, an inhalation administration formulation, or a topical or transdermal administration formulation.
  • the present invention further provides a use of the pharmaceutical composition, which is used for preparing a medicine for treating or preventing a disease or disorder caused by or associated with type II vascular endothelial growth factor receptor activity or signal transduction.
  • diseases or disorders caused by or associated with type II VEGF receptor activity or signaling include: autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, dysfibrosis, or cancer.
  • the cancer comprises renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.
  • the present invention further provides a method for treating or preventing diseases or disorders caused by or associated with type II vascular endothelial growth factor receptor activity or signal transduction, comprising: administering the pharmaceutical composition as described above to an individual in need thereof, thereby binding to the type II vascular endothelial growth factor receptor of the individual to inhibit its activity.
  • diseases or disorders caused by or associated with VEGF receptor type II activity or signaling include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs syndrome, hydropericardium, pleural effusion, diabetes, endometriosis, dysfibrosis, or cancer.
  • the cancer comprises renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.
  • the present invention further provides a use of the above pharmaceutical composition, which is used for preparing a medicine for treating or preventing diseases or disorders caused by angiogenesis.
  • diseases or disorders caused by angiogenesis include: autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, pleural effusion, diabetes, endometriosis, dysfibrosis, or cancer.
  • the cancer comprises renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.
  • the present invention further provides a method for treating or preventing diseases or disorders caused by angiogenesis, comprising: administering the pharmaceutical composition as described above to an individual in need thereof, thereby binding to the individual's type II vascular endothelial growth factor receptor to inhibit angiogenesis.
  • diseases or disorders caused by angiogenesis include: autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, pleural effusion, diabetes, endometriosis, dysplastic fibrosis, or cancer.
  • the cancer comprises renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.
  • the present invention further provides a nucleic acid comprising a nucleotide sequence encoding the protein as described above.
  • the present invention further provides a host cell comprising the nucleic acid described above.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell is Escherichia coli
  • the eukaryotic cell is CHO cell, COS cell, or HEK293 cell.
  • the present invention also provides a method for preparing the protein as described above, which comprises culturing the host cell as described above to make it express the protein.
  • Figure 1 is a protein structure diagram showing the three-dimensional structure of protein C7-NM;
  • FIG2 is a protein structure diagram showing the position of the disulfide bond that can be designed in the three-dimensional structure of protein C7-NM;
  • FIG3 is a graph showing the results of differential scanning calorimetry, comparing the melting point temperatures of protein C7 and its variant proteins
  • FIG4 is a graph showing differential scanning calorimetry results, comparing the melting temperatures of proteins C7 and C7-SL1-LL2;
  • FIG5 is a graph showing solubility results, comparing the solubility of proteins C7 and C7-SL1-LL2;
  • FIG. 6 is a diagram showing the results of enzyme-bound immunosorbent assay, comparing the affinity of proteins C7 and C7-SL1-LL2 for binding to VEGF receptor type II.
  • protein herein includes wild-type proteins expressed by natural cells, recombinant proteins expressed by genetic engineering techniques, or synthetic proteins obtained by chemical methods. At least one amino acid may be substituted, deleted, and/or inserted into the protein sequence without affecting the original activity.
  • amino acids include D-amino acids or L-amino acids.
  • D- and L- represent the absolute configuration of amino acids, rather than the specific rotation direction of plane polarization.
  • this article uses the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission to represent amino acids. Protein sequences are represented by a string of multiple single-letter symbols, and the order of the single-letter symbols corresponds to the order of amino acids from the N-terminus to the C-terminus of the protein.
  • substitutions between amino acids with similar properties for example: acidic amino acids can replace each other, i.e. aspartate and glutamate; basic amino acids can replace each other, i.e. lysine, arginine, and histidine; non-polar amino acids can replace each other, i.e.
  • uncharged polar amino acids can replace each other, i.e. glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine.
  • amino acids of related side chains for example: aliphatic-hydroxy amino acids can replace each other, i.e., serine and threonine; amide-containing amino acids can replace each other, i.e., asparagine and glutamine; aliphatic amino acids can replace each other, i.e., alanine, valine, leucine, and isoleucine; aromatic amino acids can replace each other, i.e., phenylalanine, tryptophan, and tyrosine.
  • the "10th human fibronectin type III domain" referred to herein comprises, from N-terminus to C-terminus, the N-terminal region, ⁇ -strand A, loop AB, ⁇ -strand B, loop BC, ⁇ -strand C, loop CD, ⁇ -strand D, loop DE, ⁇ -strand E, loop EF, ⁇ -strand F, loop FG, ⁇ -strand G, and the C-terminal region, and has at least 94 amino acids without any disulfide bonds, such as: SEQ ID NO: 1.
  • N-terminal region is defined as the amino acid segment from position 1 to 7
  • ⁇ -strand A is defined as the amino acid segment from position 8 to 13
  • loop AB is defined as the amino acid segment from position 14 to 17
  • ⁇ -strand B is defined as the amino acid segment from position 18 to 22.
  • the amino acid fragment of ⁇ -strand C is defined as the amino acid fragment of positions 32 to 36
  • the loop CD is defined as the amino acid fragment of positions 37 to 47
  • the ⁇ -strand D is defined as the amino acid fragment of positions 48 to 50
  • the loop DE is defined as the amino acid fragment of positions 51 to 56
  • the ⁇ -strand E is defined as the amino acid fragment of positions 57 to 62
  • the loop EF is defined as the amino acid fragment of positions 63 to 67
  • the ⁇ -strand F is defined as the amino acid fragment of positions 68 to 74
  • the loop FG is defined as the amino acid fragment of positions 75 to 87
  • the ⁇ -strand G is defined as the amino acid fragment of positions 88 to 92
  • the N-terminal region contains 1 VSDVPRD, ⁇ strand A contains 8 LEVVAA, loop AB contains 14 TPTS, ⁇ strand B contains 18 LLISW, loop BC contains 23 DAPAVTVRY, ⁇ strand C contains 32 YRITY, loop CD contains 37 GETGGNSPVQE, ⁇ strand D contains 48 FTV, loop DE contains 51 PGSKST, ⁇ strand E contains 57 ATISGL, loop EF contains 63 KPGVD, ⁇ strand F contains 68 YTITVYA, loop FG contains 75 VTGRGDSPASSKP, ⁇ strand G contains 88 ISINY, and the C-terminal region contains 93 RT.
  • the "protein C7" referred to herein refers to a variant of the type III domain of human fibronectin 10, which can bind to the type II vascular endothelial growth factor receptor to inhibit its activity, such as: SEQ ID NO: 2.
  • the amino acid sequence corresponding to the wild-type loop BC is 23 RHPHFPTRY
  • the amino acid sequence corresponding to the wild-type loop DE is 51 PLQPPT
  • the amino acid sequence corresponding to the wild-type loop FG is 75 VTDGRNGRLLSIP.
  • the three mutant regions above constitute the binding interface of the type II vascular endothelial growth factor receptor.
  • protein C7-NM refers to a variant of protein C7, such as SEQ ID NO: 3.
  • amino acid corresponding to position 12 of the reference sequence is glutamic acid
  • amino acid corresponding to position 14 of the reference sequence is serine
  • amino acid corresponding to position 18 of the reference sequence is isoleucine
  • amino acid corresponding to position 19 of the reference sequence is glutamic acid.
  • VEGF receptor type II refers to a transmembrane receptor tyrosine kinase that regulates angiogenesis induced by VEGF-A and VEGF-B.
  • VEGF receptor type II is synonymous with kinase insert domain receptor (KDR) and fetal liver kinase 1 (FLK-1) and can be used interchangeably.
  • KDR kinase insert domain receptor
  • FLK-1 fetal liver kinase 1
  • treatment refers to therapeutic intervention to cure or improve a disease, including complete or partial cure or improvement.
  • prevention refers to completely or nearly completely preventing a disease. For example, when a disease does not exist or is suspected to exist but has not yet occurred, preventive intervention can be performed to prevent the disease from occurring.
  • the "pharmaceutically acceptable carrier” referred to herein refers to an additive that is suitable for contact with an individual within the scope of sound medical judgment, has no excessive toxicity, irritation, allergic reaction, or other problems or complications, and has a reasonable benefit-risk ratio, such as a filler, diluent, agglutinating agent, binder, lubricant, glidant, stabilizer, colorant, wetting agent, or disintegrant.
  • Protein consisting mainly of the type III domain of human fibronectin
  • the first embodiment of the present invention discloses a protein, which is based on the 10th human fibronectin type III domain, and introduces disulfide bonds by amino acid substitution at specific positions of the main body, thereby improving the thermal stability and solubility of the protein.
  • amino acid substitution can be further used to construct a protein binding interface to bind to specific proteins, such as: DLL4, EGFR, VEGFR2, or IGF-1R.
  • specific proteins such as: DLL4, EGFR, VEGFR2, or IGF-1R.
  • the protein of this embodiment comprises the 10th human fibronectin type III domain, and comprises a first mutation that replaces one amino acid site with cysteine and a second mutation that replaces another amino acid site with another cysteine, the first mutation and the second mutation occur in other regions outside the loop FG of the 10th human fibronectin type III domain, whereby the cysteine replaced by the first mutation and the cysteine replaced by the second mutation form a disulfide bond.
  • the 10th human fibronectin type III domain comprises an amino acid sequence as shown in SEQ ID NO: 1.
  • the first mutation may occur in ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop BC, loop CD, loop DE, or loop EF
  • the second mutation may occur in ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop BC, loop CD, loop DE, or loop EF.
  • the first mutation occurs in ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop CD, or loop EF
  • the second mutation occurs in ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop CD, or loop EF.
  • the first mutation and the second mutation may occur in one of the same or two different regions in the above group of regions.
  • the first mutation and the second mutation may both occur in loop CD or loop EF.
  • the first mutation may occur in ⁇ strand A and the second mutation may occur in ⁇ strand B; the first mutation may occur in ⁇ strand A and the second mutation may occur in ⁇ strand G; the first mutation may occur in loop AB and the second mutation may occur in ⁇ strand E; the first mutation may occur in ⁇ strand B and the second mutation may occur in ⁇ strand E; the first mutation may occur in ⁇ strand C and the second mutation may occur in ⁇ strand D; the first mutation may occur in ⁇ strand C and the second mutation may occur in loop CD; the first mutation may occur in ⁇ strand C and the second mutation may occur in ⁇ strand F; the first mutation may occur in loop CD and the second mutation may occur in loop EF; or the first mutation may occur in loop EF and the second mutation may occur in ⁇ strand G.
  • the first mutation may include: substitution of leucine at position 8 with cysteine, substitution of serine at position 17 with cysteine, substitution of leucine at position 19 with cysteine, substitution of isoleucine at position 34 with cysteine, substitution of threonine at position 35 with cysteine, substitution of tyrosine at position 36 with cysteine, substitution of glycine at position 37 with cysteine, substitution of threonine at position 39 with cysteine, substitution of lysine at position 63 with cysteine, or substitution of threonine at position 67 with cysteine.
  • Aspartic acid is substituted with cysteine
  • the second mutation may include: tryptophan at position 22 is substituted with cysteine, valine at position 45 is substituted with cysteine, phenylalanine at position 48 is substituted with cysteine, threonine at position 58 is substituted with cysteine, serine at position 60 is substituted with cysteine, valine at position 66 is substituted with cysteine, aspartic acid at position 67 is substituted with cysteine, isoleucine at position 70 is substituted with cysteine, serine at position 89 is substituted with cysteine, or asparagine at position 91 is substituted with cysteine.
  • the first mutation may include: leucine at position 8 is substituted with cysteine, and the second mutation may include: tryptophan at position 22 is substituted with cysteine; the first mutation may include: leucine at position 8 is substituted with cysteine, and the second mutation may include: serine at position 89 is substituted with cysteine; the first mutation may include: position 17
  • the first mutation may include: the serine at position is substituted with cysteine, and the second mutation may include: the serine at position 60 is substituted with cysteine; the first mutation may include: the leucine at position 19 is substituted with cysteine, and the second mutation may include: the threonine at position 58 is substituted with cysteine; the first mutation may include: the isoleucine at position 34 is substituted with cysteine, and the second mutation may include: the phenylalanine at position 48 is substituted with cysteine; the first mutation may include: the threonine at position 35 is substituted with cysteine, and the second mutation
  • the protein of this embodiment may further include a third mutation that replaces another amino acid site with another cysteine and a fourth mutation that replaces another amino acid site with another cysteine, the third mutation and the fourth mutation occur in other regions other than loop FG of the 10th human fibronectin type III domain, whereby the cysteine replaced by the third mutation and the cysteine replaced by the fourth mutation form a disulfide bond. Under the condition of containing at least two disulfide bonds, the thermal stability and solubility of the protein can be further improved.
  • the third mutation may occur in ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop BC, loop CD, loop DE, or loop EF
  • the fourth mutation may occur in ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop BC, loop CD, loop DE, or loop EF.
  • the third mutation occurs in ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop CD, or loop EF
  • the fourth mutation occurs in ⁇ strand A, ⁇ strand B, ⁇ strand C, ⁇ strand D, ⁇ strand E, ⁇ strand F, ⁇ strand G, loop AB, loop CD, or loop EF.
  • the first mutation, the second mutation, the third mutation, and the fourth mutation may occur in four different regions in the above group of regions.
  • the first mutation may occur in ⁇ strand A
  • the second mutation may occur in ⁇ strand G
  • the third mutation may occur in loop AB
  • the fourth mutation may occur in ⁇ strand E
  • the first mutation may occur in ⁇ strand A
  • the second mutation may occur in ⁇ strand G
  • the third mutation may occur in loop CD
  • the fourth mutation may occur in loop EF.
  • the third mutation may include: substitution of leucine at position 8 with cysteine, substitution of serine at position 17 with cysteine, substitution of leucine at position 19 with cysteine, substitution of isoleucine at position 34 with cysteine, substitution of threonine at position 35 with cysteine, substitution of tyrosine at position 36 with cysteine, substitution of glycine at position 37 with cysteine, substitution of threonine at position 39 with cysteine, substitution of lysine at position 63 with cysteine, or substitution of threonine at position 67 with cysteine.
  • the fourth mutation may include: substitution of tryptophan at position 22 for cysteine, substitution of valine at position 45 for cysteine, substitution of phenylalanine at position 48 for cysteine, substitution of threonine at position 58 for cysteine, substitution of serine at position 60 for cysteine, substitution of valine at position 66 for cysteine, substitution of aspartic acid at position 67 for cysteine, substitution of isoleucine at position 70 for cysteine, substitution of serine at position 89 for cysteine, or substitution of asparagine at position 91 for cysteine.
  • the first mutation may include: leucine at position 8 is substituted by cysteine
  • the second mutation may include: serine at position 89 is substituted by cysteine
  • the third mutation may include: serine at position 17 is substituted by cysteine
  • the fourth mutation may include: serine at position 60 is substituted by cysteine; or the first mutation may include: leucine at position 8 is substituted by cysteine
  • the second mutation may include: serine at position 89 is substituted by cysteine
  • the third mutation may include: threonine at position 39 is substituted by cysteine
  • the fourth mutation may include: aspartic acid at position 67 is substituted by cysteine.
  • the protein of the present embodiment can bind to DLL4, EGFR, VEGFR2, or IGF-1R.
  • the protein of the present embodiment can further include a mutation that replaces the amino acid sequence of loop BC with RHPHFPTRY, a mutation that replaces the amino acid sequence of loop DE with PLQPPT, and a mutation that replaces the amino acid sequence of loop FG with VTDGRNGRLLSIP.
  • the protein of this embodiment comprises an amino acid sequence as shown in SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. More preferably, the protein of this embodiment comprises an amino acid sequence as shown in SEQ ID NO: 11, 15, or 16.
  • protein C7-NM can improve thermal stability and solubility.
  • the protein of this embodiment can be modified with reference to the amino acid sequence of protein C7-NM.
  • the protein of this embodiment may further include a mutation replacing alanine at position 12 with glutamate, a mutation replacing threonine at position 14 with serine, a mutation replacing leucine at position 18 with isoleucine, and a mutation replacing leucine at position 19 with glutamate.
  • the first mutation comprises: substitution of leucine at position 8 with cysteine, substitution of serine at position 17 with cysteine, substitution of isoleucine at position 34 with cysteine, substitution of threonine at position 35 with cysteine, substitution of tyrosine at position 36 with cysteine, substitution of glycine at position 37 with cysteine, substitution of threonine at position 39 with cysteine, substitution of lysine at position 63 with cysteine, or substitution of aspartic acid at position 67 with cysteine;
  • the second mutation comprises: substitution of tryptophan at position 22 with cysteine, substitution of valine at position 45 with cysteine, substitution of phenylalanine at position 48 with cysteine, substitution of tyrosine at position 36 with cysteine, substitution of glycine at position 37 with cysteine, substitution of threonine at position 39 with cysteine, substitution of lysine at position 63 with cysteine, or substitution of aspartic acid at position 67
  • the protein of the present embodiment may further include a mutation replacing the alanine at position 12 by glutamate, a mutation replacing the threonine at position 14 by serine, a mutation replacing the leucine at position 18 by isoleucine, and a mutation replacing the leucine at position 19 by glutamate.
  • the first mutation comprises: leucine at position 8 is substituted with cysteine, and the second mutation comprises: tryptophan at position 22 is substituted with cysteine; the first mutation comprises: leucine at position 8 is substituted with cysteine, and the second mutation comprises: serine at position 89 is substituted with cysteine; the first mutation comprises: serine at position 17 is substituted with cysteine, and the second mutation comprises: serine at position 60 is substituted with cysteine; the first mutation comprises: isoleucine at position 34 is substituted with cysteine, and the second mutation comprises: phenylalanine at position 48 is substituted with cysteine; the first mutation comprises: threonine at position 35 is substituted with cysteine, and the second mutation comprises: valine at position 45 is substituted with cysteine; the first mutation comprises: tyrosine at position 36 is substituted with cysteine, and the second mutation comprises: isoleucine at position 70 is substituted with cysteine; the first mutation comprises: glycine
  • the protein of this embodiment comprises an amino acid sequence as shown in SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.
  • the protein of this embodiment may further include a mutation replacing alanine at position 12 with glutamate, a mutation replacing threonine at position 14 with serine, a mutation replacing leucine at position 18 with isoleucine, and a mutation replacing leucine at position 19 with glutamate.
  • the first mutation comprises: substitution of leucine at position 8 with cysteine, substitution of serine at position 17 with cysteine, substitution of isoleucine at position 34 with cysteine, substitution of threonine at position 35 with cysteine, substitution of tyrosine at position 36 with cysteine, substitution of glycine at position 37 with cysteine, substitution of threonine at position 39 with cysteine, substitution of lysine at position 63 with cysteine, or substitution of aspartic acid at position 67 with cysteine
  • the second mutation comprises: substitution of tryptophan at position 22 with cysteine, substitution of valine at position 45 with cysteine, substitution of phenylalanine at position 48 with cysteine.
  • the first mutation comprises: substituted leucine at position 8 for cysteine, substituted threonine at position 17 for cysteine, substituted isoleucine at position 34 for cysteine, substituted threonine at position 35 for cysteine, substituted tyrosine at position 36 for cysteine, substituted valine at position 66 for cysteine, substituted aspartic acid at position 67 for cysteine, substituted isoleucine at position 70 for cysteine, substituted serine at position 89 for cysteine, or substituted asparagine at position 91 for cysteine
  • the third mutation comprises: substituted leucine at position 8 for cysteine, substituted serine at position 17 for cysteine, substituted isoleucine at position 34 for cysteine, substituted threonine at position 35 for cysteine, substituted tyrosine at position 36 for cysteine, and substituted threonine at position 37 for cysteine.
  • the first mutation comprises: substitution of tryptophan at position 22 for cysteine, substitution of valine at position 45 for cysteine, substitution of phenylalanine at position 48 for cysteine, substitution of threonine at position 58 for cysteine, substitution of serine at position 60 for cysteine, substitution of valine at position 66 for cysteine, and substitution of threonine at position 58 for cysteine.
  • the protein of the present embodiment may further include a mutation in which alanine at position 12 is substituted by glutamate, a mutation in which threonine at position 14 is substituted by serine, a mutation in which leucine at position 18 is substituted by isoleucine, and a mutation in which leucine at position 19 is substituted by glutamate.
  • the protein of this embodiment may further comprise a mutation of substitution of alanine at position 12 with glutamate, a mutation of substitution of threonine at position 14 with serine, a mutation of substitution of leucine at position 18 with isoleucine, and a mutation of substitution of leucine at position 19 with glutamate.
  • the protein of this embodiment comprises an amino acid sequence as shown in SEQ ID NO: 27 or 28.
  • the protein of this embodiment can be prepared by genetic engineering technology or chemical methods, such as solid phase synthesis or solution synthesis.
  • the protein of this embodiment can be subsequently isolated or purified by ammonium sulfate or ethanol precipitation, acid extraction, ion exchange chromatography, affinity chromatography, or lectin chromatography, preferably by high performance liquid chromatography.
  • the protein of this embodiment may further have a hydrophilic group to improve water solubility or circulation half-life.
  • the hydrophilic group may be connected to the N-terminus of the protein.
  • the hydrophilic group is polyethylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyvinyl alcohol, or polyglucose. More preferably, the hydrophilic group is polyethylene glycol composed of 2 to 40 ethylene glycol repeating units.
  • the protein of this embodiment may further contain a purification tag to assist in purification.
  • the purification tag may be connected to the N-terminus or C-terminus of the protein.
  • the purification tag is a histidine tag (His-tag), a glutathione S-transferase tag (GST-tag), a maltose binding protein tag (MBP-tag), a transcription termination/anti-termination protein (NusA-tag), or a small ubiquitin-related modifier tag (SUMO-tag).
  • the second embodiment of the present invention discloses a pharmaceutical composition, which contains the protein of the first embodiment, and can be administered to an individual to bind to its specific protein, so as to act as an antagonist of the specific protein, thereby inhibiting the activity of the specific protein or blocking the related signal transmission.
  • the pharmaceutical composition of this embodiment comprises: the protein of the first embodiment; and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can make the pharmaceutical composition present different forms as a whole or be suitable for different routes of administration.
  • the pharmaceutical composition is an oral administration formulation, an injection administration formulation, an inhalation administration formulation, or a topical or transdermal administration formulation, so as to be used for different routes of administration.
  • the pharmaceutical composition is a tablet, a capsule, a granule, a powder, a solution, a syrup, a suspension, or an emulsion.
  • the pharmaceutically acceptable carrier may be an excipient, a filler, a diluent, an agglutinating agent, a binder, a lubricant, a glidant, a stabilizer, a colorant, a wetting agent, or a disintegrant.
  • excipients may be sodium citrate, calcium carbonate, or calcium phosphate
  • examples of fillers may be lactose or high molecular weight polyethylene glycol
  • examples of diluents may be water, ethanol, propylene glycol, or glycerol
  • examples of binders may be sucrose, gelatin, or gum arabic
  • examples of lubricants may be magnesium stearate, calcium stearate, zinc stearate, sodium stearate, stearic acid, aluminum stearate, leucine, glyceryl behenate, or hydrogenated vegetable oil
  • examples of glidants may be sodium aluminosilicate, calcium silicate, microcrystalline cellulose, corn starch, or polysaccharide.
  • Powder sodium benzoate, calcium carbonate, magnesium carbonate, talc, calcium stearate, magnesium stearate, zinc stearate, magnesium lauryl sulfate, or magnesium oxide
  • stabilizers may be citric acid or ascorbic acid
  • colorants may be titanium dioxide or iron oxide
  • wetting agents may be Pluronic F68, Tween 20, or Tween 80
  • disintegrants may be potato starch, cassava starch, or silicates.
  • the third embodiment of the present invention discloses a use of the pharmaceutical composition of the second embodiment, which is used to prepare a medicine for treating or preventing a disease or disorder caused by or associated with the activity or signaling of the type II vascular endothelial growth factor receptor.
  • the prepared medicine can be administered to an individual to inhibit the activity of the type II vascular endothelial growth factor receptor or block its signaling. That is, the prepared medicine can be administered to an individual who needs to treat or prevent a disease or disorder caused by or associated with the activity or signaling of the type II vascular endothelial growth factor receptor, so as to achieve the therapeutic or preventive effect by inhibiting the activity of the type II vascular endothelial growth factor receptor of the individual or blocking its signaling.
  • the drug can be administered in different ways, such as oral administration, injection, inhalation, or topical or transdermal administration.
  • Diseases or disorders caused by or associated with VEGF receptor type II activity or signaling may include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs syndrome, hydropericardium, hydropleural, diabetes, endometriosis, dysfibrosis, or cancer.
  • cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.
  • the fourth embodiment of the present invention discloses a method for treating or preventing a disease or disorder caused by or associated with type II vascular endothelial growth factor receptor activity or signal transduction, comprising: administering the pharmaceutical composition of the second embodiment to an individual in need of such treatment or prevention, thereby binding to the type II vascular endothelial growth factor receptor of the individual to inhibit its activity or block its signal transduction.
  • the drug can be administered in different ways, such as oral administration, injection, inhalation, or topical or transdermal administration.
  • Diseases or disorders caused by or associated with type II VEGF receptor activity or signaling may include: autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs syndrome, hydropericardium, hydropleural, diabetes, endometriosis, dysfibrosis, or cancer.
  • cancer includes: renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.
  • the fifth embodiment of the present invention discloses a use of the pharmaceutical composition of the second embodiment, which is used to prepare a medicine for treating or preventing a disease or disorder caused by angiogenesis.
  • the prepared medicine can be administered to an individual to inhibit angiogenesis. That is, the prepared medicine can be administered to an individual who needs to treat or prevent a disease or disorder caused by angiogenesis, so as to achieve a therapeutic or preventive effect by inhibiting angiogenesis in the individual.
  • the drug can be administered in different ways, such as oral administration, injection, inhalation, or topical or transdermal administration.
  • Diseases or disorders caused by angiogenesis may include: autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, dysfibrosis, or cancer.
  • cancer includes: renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.
  • the sixth embodiment of the present invention discloses a method for treating or preventing a disease or disorder caused by angiogenesis, comprising: administering the pharmaceutical composition of the second embodiment to a subject in need of such treatment or prevention, thereby binding to the subject's type II vascular endothelial growth factor receptor to inhibit angiogenesis.
  • the drug can be administered in different ways, such as oral administration, injection, inhalation, or topical or transdermal administration.
  • Diseases or disorders caused by angiogenesis may include: autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, dysfibrosis, or cancer.
  • cancer includes: renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.
  • the seventh embodiment of the present invention discloses a nucleic acid comprising a nucleotide sequence for encoding the protein of the first embodiment.
  • the nucleic acid may further comprise a promoter, which is operably linked to the nucleotide sequence encoding the protein.
  • operably linked means that two or more nucleic acid sequences are in a functional relationship with each other.
  • the eighth embodiment of the present invention discloses a host cell comprising the nucleic acid of the seventh embodiment. Since the host cell of this embodiment contains a nucleotide sequence encoding a protein, the protein can be produced by culturing the host cell.
  • the host cell can be a prokaryotic cell or a eukaryotic cell.
  • An example of a prokaryotic cell can be Escherichia coli, and an example of a eukaryotic cell can be a CHO cell, a COS cell, or a HEK293 cell.
  • the ninth embodiment of the present invention discloses a method for preparing the protein of the first embodiment, which comprises culturing the host cell of the eighth embodiment to make it express the protein.
  • An appropriate inducer can be selected according to the promoter to induce the cell to express the protein.
  • protein C7-NM has 8 ⁇ strands, and a ring structure is formed between two adjacent ⁇ strands.
  • Protein C7-NM is used as the parent protein, and the software Disulfide by Design 2.0 (DbD2) is used to introduce disulfide bonds into the structural design of the parent protein with reference to parameters such as B factor, ⁇ 3 torsion angle, and energy.
  • DbD2 Disulfide by Design 2.0
  • the positions of disulfide bonds can be designed to be between two ⁇ strands, between two rings, and between a ⁇ strand and a ring of the parent protein.
  • Table 2 the amino acid sequence of the variant protein with disulfide bonds is presented, which is obtained by using the amino acid sequence of protein C7 as the reference sequence and performing amino acid substitution according to the designed disulfide bond position.
  • Protein C7 and its variant proteins were expressed in E. coli.
  • protein C7 and its variant proteins were prepared using pET21a expression vector and BL21(DE3)pLysS strain or SHuffle strain, wherein the culture medium of BL21(DE3)pLysS strain was LB medium, and the culture medium of SHuffle strain was LLB medium.
  • the BL21(DE3)pLysS strain was cultured in 5 mL of culture medium at 37°C for about 16 to 18 hours (the SHuffle strain was cultured at 30°C).
  • the BL21(DE3)pLysS strain was transferred to 500 mL of culture medium and cultured at 37°C for about 4 hours (the SHuffle strain was cultured at 30°C for about 6 hours).
  • 500 ⁇ L of IPTG (concentration 1 M) was added and the BL21(DE3)pLysS strain was induced to express protein at 25°C for about 16 to 18 hours (the SHuffle strain was induced at 16°C for about 48 hours), and then the cell pellet was collected by centrifugation.
  • the bacteria were dissolved in binding Buffer A (50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0) and the cells were disrupted with a French filter press (pressure maintained at 1500 psi). The supernatant was obtained by high-speed centrifugation and loaded onto a nickel ion chelating column pre-equilibrated with Buffer A.
  • the target protein was extracted using a gradient of extraction buffer (300 mM imidazole in Buffer A, pH 7.0). After extraction, the target protein was confirmed by glycine SDS-PAGE. Finally, the recombinant protein was dialyzed with PBS and stored at -80°C until use.
  • Differential scanning calorimetry was used to measure the melting point of proteins.
  • the melting point difference between protein C7-SL1 and protein C7 is 37.0°C, which is higher than the melting point difference between other proteins with a single disulfide bond and protein C7, indicating that protein C7-SL1 has better thermal stability.
  • proteins C7-SL1-LL2 and C7-SL1-LL3 which are based on protein C7-SL1 and introduce other disulfide bonds, can further improve thermal stability.
  • the protein solubility was determined by ammonium sulfate precipitation.
  • the PBS solubility of protein C7-SL1 is about 6 times that of protein C7, which is higher than other proteins with a single disulfide bond, indicating that protein C7-SL1 has good solubility.
  • proteins C7-SL1-LL2 and C7-SL1-LL3 which are based on protein C7-SL1 and introduce other disulfide bonds, can further increase the PBS solubility to about 7 times that of protein C7.
  • the cell proliferation assay was used to analyze the inhibitory activity of proteins on the proliferation of human umbilical vein endothelial cells (HUVEC). As shown in Table 3, the inhibitory activity of protein C7-SL1 on HUVEC proliferation was not significantly different from that of protein C7, indicating that protein C7-SL1 has the ability to inhibit angiogenesis. As shown in Table 3, the inhibitory activity of proteins C7-SL1-LL2 and C7-SL1-LL3, which are obtained by using protein C7-SL1 as the main body and introducing other disulfide bonds, on HUVEC proliferation was also not significantly different from that of protein C7, indicating that proteins C7-SL1-LL2 and C7-SL1-LL3 also have the ability to inhibit angiogenesis.
  • HUVEC human umbilical vein endothelial cells
  • the affinity of the protein binding to the type II vascular endothelial growth factor receptor was analyzed by enzyme-linked immunosorbent assay (ELISA). As shown in Table 3, the affinity of protein C7-SL1 binding to the type II vascular endothelial growth factor receptor was not significantly different from that of protein C7, indicating that protein C7-SL1 has the ability to bind to the type II vascular endothelial growth factor receptor.
  • the affinity of proteins C7-SL1-LL2 and C7-SL1-LL3 obtained by using protein C7-SL1 as the main body and introducing other disulfide bonds to bind to the type II vascular endothelial growth factor receptor was also not significantly different from that of protein C7, and the affinity of protein C7-SL1-LL2 binding to the type II vascular endothelial growth factor receptor was slightly better than that of protein C7, indicating that proteins C7-SL1-LL2 and C7-SL1-LL3 also have the ability to bind to the type II vascular endothelial growth factor receptor.
  • ND means not measurable, because it is formed in inclusion bodies or not tested

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Abstract

提供了一种纤维黏蛋白第Ⅲ型结构域衍生的蛋白质及其应用，蛋白质包含第10人类纤维黏蛋白第IIⅢ型结构域，且包含将一氨基酸位点取代为一半胱胺酸的第一突变及将另一氨基酸位点取代为另一半胱胺酸的第二突变，第一突变与第二突变发生于第10人类纤维黏蛋白第Ⅲ型结构域的环FG以外的其他区域，借此第一突变取代成的半胱胺酸与第二突变取代成的半胱胺酸形成双硫键。

Description

纤维黏蛋白第III型结构域衍生的蛋白质及其应用 技术领域
本发明涉及蛋白质工程，特别关于一种纤维黏蛋白第III型结构域衍生的蛋白质及其应用，包括：治疗或预防第二型血管内皮生长因子受体(vascular endothelial growth factor receptor 2，VEGFR2)活性或讯息传递导致或与其相关联的疾病或失调的方法、或治疗或预防血管新生导致的疾病或失调的方法。
背景技术
近年致力开发蛋白质支架为基础的分子辨识模块来取代利用免疫球蛋白建构的模块，以期克服利用免疫球蛋白作为分子辨识模块主体所产生的既定限制，如：过大尺寸、复杂的异二聚体结构、及需要正确形成双硫键等。此种蛋白质工程的基本理论在于：透过于非抗体的适当蛋白质框架或分子支架建构出结合界面，以开发具有与抗体相当的亲和性与特异性的分子。
第10人类纤维黏蛋白第III型结构域(10th human fibronectin type III domain，10Fn3)为目前广泛使用于制备新颖结合蛋白质的非抗体支架之一。数种10Fn3为主体的分子正逐渐开发并尝试于疾病的治疗，惟多数仍处于临床试验阶段。
10Fn3具备许多优于免疫球蛋白衍生系统的特征。基于整体的β-三明治(β-sandwich)折迭，10Fn3属于免疫球蛋白的超家族成员之一，近于N端的三个表面环状区域于结构上与免疫球蛋白变异区域的三个抗原辩识环区域或互补决定区(complementarity-determining region，CDR)类似。然而，与免疫球蛋白的典型区域不同，10Fn3不含双硫键。而且，10Fn3的热转移温度大于80℃，构型稳定。另外，10Fn3具备可逆且快速的展开与再折迭特性。
10Fn3具有约94个氨基酸残基，小于重链抗体的抗原结合单元(VHH)。上述特性使10Fn3可与不同的分子表现系统与简单有效的生产方法兼容。
美国专利申请号11/448,171，专利名称为“INHIBITORS OF TYPE 2 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS”，提出一种第二型血管内皮生长因子受体的结合蛋白质C7。蛋白质C7以10Fn3为结构主体，与野生型的差异在于：将野生型的三个表面环状区域序列 23DAPAVTVRY、 51PGSKST、与 75VTGRGDSPASSKP各以 23RHPHFPTRY、 51PLQPPT、与 75VTDGRNGRLLSIP取代。透过上述三个突变区域作为第二型血管内皮生长因子受体的结合位点，使蛋白质C7可作为第二型血管内皮生长因子受体的拮抗剂，并有成为抗癌药物的潜力。然而，碍于热稳定性不足且溶解度低，使其于药物的发展受阻。
因此，开发高热稳定性以及高溶解度的以10Fn3为结构主体的蛋白质支架确实为本发明所属技术领域人士积极解决的课题之一。
发明内容
先前改良蛋白质C7来取得变异蛋白质C7-NM。本发明乃根据以下事项完成：利用软件Disulfide by Design 2.0(DbD2)并参考B因子(B-factor)、χ 3扭转角(χ 3angle)、与能量等参数来于蛋白质C7-NM的结构设计双硫键的引入，以取得具双硫键的变异体，其可于不影响与特定蛋白质结合的活性下来提升热稳定性与溶解度，从而具有作为生物药的潜力。
本发明提出一种蛋白质，其包含第10人类纤维黏蛋白第III型结构域，且包含将一氨基酸位点取代为一半胱胺酸的第一突变及将另一氨基酸位点取代为另一半胱胺酸的第二突变，第一突变与第二突变发生于第10人类纤维黏蛋白第III型结构域的环FG以外的其他区域，借此第一突变取代成的半胱胺酸与第二突变取代成的半胱胺酸形成双硫键。
示例地，第10人类纤维黏蛋白第III型结构域包含一如SEQ ID NO：1所示的氨基酸序列。
示例地，蛋白质更包含将环BC的氨基酸序列取代为RHPHFPTRY的突变、将环DE的氨基酸序列取代为PLQPPT的突变、以及将环FG的氨基酸序列取代为VTDGRNGRLLSIP的突变。
示例地，第一突变发生于第10人类纤维黏蛋白第III型结构域的β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环BC、环CD、环DE、与环EF中的任一者，第二突变发生于第10人类纤维黏蛋白第III型结构域的β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环BC、环CD、环DE、与环EF中的任一者。
示例地，第一突变发生于第10人类纤维黏蛋白第III型结构域的β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环CD、与环EF中的任一者，第二突变发生于第10人类纤维黏蛋白第III型结构域的β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环CD、与环EF中的任一者。
示例地，第一突变与第二突变发生于相同区域或不同区域。
示例地，第一突变包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置19的白胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第二突变包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸。
示例地，第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置22的色胺酸取代为半胱胺酸；第一突变包含：位置8的白胺酸取代 为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸；第一突变包含：位置17的丝胺酸取代为半胱胺酸，第二突变包含：位置60的丝胺酸取代为半胱胺酸；第一突变包含：位置19的白胺酸取代为半胱胺酸，第二突变包含：位置58的苏胺酸取代为半胱胺酸；第一突变包含：位置34的异白胺酸取代为半胱胺酸，第二突变包含：位置48的苯丙胺酸取代为半胱胺酸；第一突变包含：位置35的苏胺酸取代为半胱胺酸，第二突变包含：位置45的缬胺酸取代为半胱胺酸；第一突变包含：位置36的酪胺酸取代为半胱胺酸，第二突变包含：位置70的异白胺酸取代为半胱胺酸；第一突变包含：位置37的甘胺酸取代为半胱胺酸，第二突变包含：位置45的缬胺酸取代为半胱胺酸；第一突变包含：位置39的苏胺酸取代为半胱胺酸，第二突变包含：位置67的天门冬胺酸取代为半胱胺酸；第一突变包含：位置63的离胺酸取代为半胱胺酸，第二突变包含：位置66的缬胺酸取代为半胱胺酸；或第一突变包含：位置67的天门冬胺酸取代为半胱胺酸，第二突变包含：位置91的天门冬酰胺取代为半胱胺酸。
示例地，蛋白质包含一如SEQ ID NOs：4至14中任一所示的氨基酸序列。
示例地，蛋白质包含一如SEQ ID NO：11所示的氨基酸序列。
示例地，于第一突变及第二突变未包含位置19的白胺酸取代为半胱胺酸的条件下，蛋白质包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。
示例地，于第一突变包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第二突变包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸 取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸的条件下，蛋白质包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。
示例地，于第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置22的色胺酸取代为半胱胺酸；第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸；第一突变包含：位置17的丝胺酸取代为半胱胺酸，第二突变包含：位置60的丝胺酸取代为半胱胺酸；第一突变包含：位置34的异白胺酸取代为半胱胺酸，第二突变包含：位置48的苯丙胺酸取代为半胱胺酸；第一突变包含：位置35的苏胺酸取代为半胱胺酸，第二突变包含：位置45的缬胺酸取代为半胱胺酸；第一突变包含：位置36的酪胺酸取代为半胱胺酸，第二突变包含：位置70的异白胺酸取代为半胱胺酸；第一突变包含：位置37的甘胺酸取代为半胱胺酸，第二突变包含：位置45的缬胺酸取代为半胱胺酸；第一突变包含：位置39的苏胺酸取代为半胱胺酸，第二突变包含：位置67的天门冬胺酸取代为半胱胺酸；第一突变包含：位置63的离胺酸取代为半胱胺酸，第二突变包含：位置66的缬胺酸取代为半胱胺酸；或第一突变包含：位置67的天门冬胺酸取代为半胱胺酸，第二突变包含：位置91的天门冬酰胺取代为半胱胺酸的条件下，蛋白质包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。
示例地，蛋白质包含一如SEQ ID NOs：17至26中任一所示的氨基酸序列。
示例地，蛋白质更包含将又一氨基酸位点取代为又一半胱胺酸的第三突变及将再一氨基酸位点取代为再一半胱胺酸的第四突变，第三突变与第四突变发生于第10人类纤维黏蛋白第III型结构域的环FG以外的其他区域，借此第三突变取代成的半胱胺酸与第四突变取代成的半胱胺酸形成双硫键。
示例地，第三突变发生于第10人类纤维黏蛋白第III型结构域的β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环BC、环CD、环DE、与环EF中的任一者，第四突变发生于第10人类纤维黏蛋白第III型结构域的β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环BC、环CD、环DE、与环EF中的任一者。
示例地，第三突变发生于第10人类纤维黏蛋白第III型结构域的β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环CD、与环EF中的任一者，第四突变发生于第10人类纤维黏蛋白第III型结构域的β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环CD、与环EF中的任一者。
示例地，第一突变、第二突变、第三突变、与第四突变发生于不同区域。
示例地，第三突变包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置19的白胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第四突变包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸。
示例地，第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸，第三突变包含：位置17的丝胺酸取代为半胱胺酸，第四突变包含：位置60的丝胺酸取代为半胱胺酸；或第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸，第三突变包含：位置39的苏胺酸取代为半胱胺酸，第四突变包含：位置67的天门冬胺酸取代为半胱胺酸。
示例地，蛋白质包含一如SEQ ID NO：15或16所示的氨基酸序列。
示例地，于第一突变、第二突变、第三突变、及第四突变未包含位置19的白胺酸取代为半胱胺酸的条件下，蛋白质包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。
示例地，于第一突变包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第二突变包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸，第三突变包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第四突变包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半 胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸的条件下，蛋白质包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。
示例地，于第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸，第三突变包含：位置17的丝胺酸取代为半胱胺酸，第四突变包含：位置60的丝胺酸取代为半胱胺酸；或第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸，第三突变包含：位置39的苏胺酸取代为半胱胺酸，第四突变包含：位置67的天门冬胺酸取代为半胱胺酸的条件下，蛋白质包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。
示例地，蛋白质包含一如SEQ ID NO：27或28所示的氨基酸序列。
示例地，蛋白质用于结合至DLL4、EGFR、VEGFR2、或IGF-1R。
本发明的蛋白质以第10人类纤维黏蛋白第III型结构域为主体，并于主体特定位置引入双硫键来提升热稳定性与溶解度。本发明的蛋白质可进一步地利用氨基酸序列的取代来建构蛋白质结合界面，以与特定蛋白质结合，如：DLL4、EGFR、VEGFR2、或IGF-1R，以作为受体拮抗剂，具有作为生物药的潜力。
本发明另提出一种医药组合物，其包含：如上所述的蛋白质；以及一医药上可接受的载体。
示例地，医药组合物为口服投予配制物、注射投予配制物、吸入投予配制物、或局部或经皮投予配制物。
本发明又提出一种上述医药组合物的用途，其为用于制备治疗或预防第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调的医药。
示例地，第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调包含：自体免疫失调症、心脏失调症、视网膜病变(retinopathy)、肾病、成血管细胞瘤(hemangioblastoma)、血管瘤(hemangioma)、甲状腺增生(thyroid hyperplasia)、慢性炎症、梅格斯氏征候群(Meigs syndrome)、心包膜积水(pericardial effusion)、肋膜积水(pleural effusion)、糖尿病、子宫内膜异位、不良纤维化、或癌症。
示例地，癌症包含：肾癌、胰腺癌、乳癌、头颈部癌、前列腺癌、恶性胶质瘤、骨肉瘤、大肠直肠癌、胃癌、恶性间皮瘤、多发性骨髓瘤、卵巢癌、小细胞肺癌、非小细胞肺癌、滑膜肉瘤、甲状腺癌、或黑色素瘤。
本发明更提出一种治疗或预防第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调的方法，其包含：投予如上所述的医药组合物至一有此需求的个体，借此结合至个体的第二型血管内皮生长因子受体以抑制其活性。
示例地，第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调包含：自体免疫失调症、心脏失调症、视网膜病变、肾病、成血管细胞瘤、血管瘤、甲状腺增生、慢性炎症、梅格斯氏征候群、心包膜积水、肋膜积水、糖尿病、子宫内膜异位、不良纤维化、或癌症。
示例地，癌症包含：肾癌、胰腺癌、乳癌、头颈部癌、前列腺癌、恶性胶质瘤、骨肉瘤、大肠直肠癌、胃癌、恶性间皮瘤、多发性骨髓瘤、卵巢癌、小细胞肺癌、非小细胞肺癌、滑膜肉瘤、甲状腺癌、或黑色素瘤。
本发明更提出一种上述医药组合物的用途，其为用于制备治疗或预防血管新生导致的疾病或失调的医药。
示例地，血管新生导致的疾病或失调包含：自体免疫失调症、心脏失调症、视网膜病变、肾病、成血管细胞瘤、血管瘤、甲状腺增生、慢性炎症、梅 格斯氏征候群、心包膜积水、肋膜积水、糖尿病、子宫内膜异位、不良纤维化、或癌症。
示例地，癌症包含：肾癌、胰腺癌、乳癌、头颈部癌、前列腺癌、恶性胶质瘤、骨肉瘤、大肠直肠癌、胃癌、恶性间皮瘤、多发性骨髓瘤、卵巢癌、小细胞肺癌、非小细胞肺癌、滑膜肉瘤、甲状腺癌、或黑色素瘤。
本发明复提出一种治疗或预防血管新生导致的疾病或失调的方法，其包含：投予如上所述的医药组合物至一有此需求的个体，借此结合至个体的第二型血管内皮生长因子受体以抑制血管新生。
示例地，血管新生导致的疾病或失调包含：自体免疫失调症、心脏失调症、视网膜病变、肾病、成血管细胞瘤、血管瘤、甲状腺增生、慢性炎症、梅格斯氏征候群、心包膜积水、肋膜积水、糖尿病、子宫内膜异位、不良纤维化、或癌症。
示例地，癌症包含：肾癌、胰腺癌、乳癌、头颈部癌、前列腺癌、恶性胶质瘤、骨肉瘤、大肠直肠癌、胃癌、恶性间皮瘤、多发性骨髓瘤、卵巢癌、小细胞肺癌、非小细胞肺癌、滑膜肉瘤、甲状腺癌、或黑色素瘤。
本发明又提出一种核酸，其包含用于编码如上所述的蛋白质的核苷酸序列。
本发明再提出一种宿主细胞，其包含如上所述的核酸。
示例地，宿主细胞为原核细胞或真核细胞。
示例地，原核细胞为大肠杆菌，真核细胞为CHO细胞、COS细胞、或HEK293细胞。
本发明尚提出一种制备如上所述的蛋白质的方法，其包含培养如上所述的宿主细胞使其表现蛋白质。
附图说明
图1为蛋白质结构图，呈现蛋白质C7-NM的三维结构；
图2为蛋白质结构图，呈现蛋白质C7-NM于三维结构可设计双硫键的位置；
图3为差示扫描量热法结果图，比较蛋白质C7及其变异蛋白质的熔点温度；
图4为差示扫描量热法结果图，比较蛋白质C7及C7-SL1-LL2的熔点温度；
图5为溶解度结果图，比较蛋白质C7及C7-SL1-LL2的溶解度；
图6为酵素结合免疫吸附分析法结果图，比较蛋白质C7及C7-SL1-LL2结合至第二型血管内皮生长因子受体的亲和力。
具体实施方式
为让本发明上述及/或其他目的、功效、特征更明显易懂，下文特举较佳实施方式，作详细说明于下：
一、术语的定义
本文所指的“蛋白质”若无特殊定义下，包含天然细胞表现的野生蛋白质、利用基因工程技术表达的重组蛋白质、或利用化学方式取得的合成蛋白质。于不影响原有的活性下，可于蛋白质序列中取代、删除、且/或插入至少一个氨基酸。
本文所指的“氨基酸”若无特殊定义下，包含D-氨基酸或L-氨基酸。D-与L-表示氨基酸的绝对构型，而非平面偏光的特定旋转方向。若无特殊情况下，本文采用IUPAC-IUB Biochemical Nomenclature Commission推荐的单字母符号来表示氨基酸。蛋白质序列由多个单字母符号组成的字符串表示，单字母符号顺序对应氨基酸自蛋白质N端至C端的方向的顺序。若单字母符号之前有上标数字，其表示对应氨基酸位于蛋白质自N端起算的位置顺序；举例而言， 23DAPAVTVRY表示天门冬胺酸位于蛋白质的位置23；其余以此类推，不再赘述。
蛋白质序列的取代、删除、及/或插入可发生于蛋白质的非骨架区域，此通常不影响原有的活性。另外，蛋白质序列的取代可包含保留性氨基酸的取代，保留性取代意指具有相似特性或相关侧链的氨基酸间的取代。相似特性的氨基酸间的取代，例如：酸性氨基酸可互相替代，即天门冬胺酸(aspartate)、麸胺酸(glutamate)；碱性氨基酸可互相替代，即离胺酸(lysine)、精胺酸(arginine)、与组胺酸(histidine)；非极性氨基酸可互相替代，即丙胺酸(alanine)、缬胺酸(valine)、白胺酸(leucine)、异白胺酸(isoleucine)、脯胺酸(proline)、苯丙胺酸(phenylalanine)、甲硫胺酸(methionine)、与色胺酸(tryptophan)；非带电极性氨基酸可互相替代，即甘胺酸(glycine)、天门冬酰胺(asparagine)、麸酰胺酸(glutamine)、半胱胺酸(cysteine)、丝胺酸(serine)、苏胺酸(threonine)、与酪胺酸(tyrosine)。相关侧链的氨基酸间的取代，例如：脂肪羟基类(aliphatic-hydroxy)氨基酸可互相替代，即丝胺酸与苏胺酸；含酰胺类氨基酸(amide-containing)氨基酸可互相替代，即天门冬酰胺与麸酰胺酸；脂肪类氨基酸可互相替代，即丙胺酸、缬胺酸、白胺酸、与异白胺酸；芳香类(aromatic)氨基酸可互相替代，即苯丙胺酸、色胺酸、与酪胺酸。
本文所指的“第10人类纤维黏蛋白第III型结构域”若无特殊定义下，自N端至C端依序含有N端区域、β股A(β-strand A)、环AB(loop AB)、β股B(β-strand B)、环BC(loop BC)、β股C(β-strand C)、环CD(loop CD)、β股D(β-strand D)、环DE(loop DE)、β股E(β-strand E)、环EF(loop EF)、β股F(β-strand F)、环FG(loop FG)、β股G(β-strand G)、与C端区域，至少有94个氨基酸，不具任何双硫键，如：SEQ ID NO：1。参照美国专利申请号13/757,664，专利名称为「FIBRONECTIN BINDING DOMAINS WITH REDUCED IMMUNOGENICITY」，N端区域定义为位置1至7的氨基酸片段，β股A定义为位置8至13的氨基酸片段，环AB定义为位置14至17的氨基酸片段，β股B定义为位置18至22 的氨基酸片段，环BC定义为位置23至31的氨基酸片段，β股C定义为位置32至36的氨基酸片段，环CD定义为位置37至47的氨基酸片段，β股D定义为位置48至50的氨基酸片段，环DE定义为位置51至56的氨基酸片段，β股E定义为位置57至62的氨基酸片段，环EF定义为位置63至67的氨基酸片段，β股F定义为位置68至74的氨基酸片段，环FG定义为位置75至87的氨基酸片段，β股G定义为位置88至92的氨基酸片段，C端区域定义为位置93及94的氨基酸片段；环BC、环DE、与环FG位于分子的一侧，环AB、环CD、与环EF位于分子的另一侧。举例而言，N端区域包含 1VSDVPRD，β股A包含 8LEVVAA，环AB包含 14TPTS，β股B包含 18LLISW，环BC包含 23DAPAVTVRY，β股C包含 32YRITY，环CD包含 37GETGGNSPVQE，β股D包含 48FTV，环DE包含 51PGSKST，β股E包含 57ATISGL，环EF包含 63KPGVD，β股F包含 68YTITVYA，环FG包含 75VTGRGDSPASSKP，β股G包含 88ISINY，C端区域包含 93RT。
本文所指的「蛋白质C7」若无特殊定义下，是指第10人类纤维黏蛋白第III型结构域的变异体，可结合至第二型血管内皮生长因子受体以抑制其活性，如：SEQ ID NO：2。申言之，其对应于野生型的环BC的氨基酸序列为 23RHPHFPTRY，对应于野生型的环DE的氨基酸序列为 51PLQPPT，对应于野生型的环FG的氨基酸序列为 75VTDGRNGRLLSIP，透过上述三个突变区域构成第二型血管内皮生长因子受体的结合界面。
本文所指的「蛋白质C7-NM」若无特殊定义下，是指蛋白质C7的变异体，如：SEQ ID NO：3。申言之，其对应于参考序列位置12的氨基酸为麸胺酸，对应于参考序列位置14的氨基酸为丝胺酸，对应于参考序列位置18的氨基酸为异白胺酸，对应于参考序列位置19的氨基酸为麸酰胺酸。
本文所指的“第二型血管内皮生长因子受体”若无特殊定义下，是指一跨膜受体酪胺酸激酶(transmembrane receptor tyrosine kinase)，其可调控VEGF-A与VEGF-B导致的血管新生。第二型血管内皮生长因子受体与激酶插入 结构域受体(kinase insert domain receptor，KDR)及胎肝激酶(fetal liver kinase 1，FLK-1)同义，可交替使用。
本文所指的“治疗”若无特殊定义下，是指治疗性介入以治愈或改善病症，即包含完全或局部治愈或改善。
本文所指的“预防”若无特殊定义下，是指完全地或近乎完全地防止病症。举例而言，于未有病症或疑似有病症但尚未造成病症时，可预防性介入以避免病症发生。
本文所指的“医药上可接受的载体”若无特殊定义下，是指于健全的医疗判断范围内，适合与个体接触且无过量毒性、刺激性、过敏反应、或其他问题或并发症，并具有合理利弊比例的添加物，如：填充剂、稀释剂、凝集剂、接合剂、润滑剂、助流剂、稳定剂、着色剂、润湿剂、或崩解剂。
二、第10人类纤维黏蛋白第III型结构域为主体的蛋白质
本发明第一实施方式揭示一种蛋白质，其以第10人类纤维黏蛋白第III型结构域为主体，并将主体特定位置的氨基酸取代引入双硫键，借此提升蛋白质的热稳定性与溶解度。基于上述的高热稳定性与高溶解度及第10人类纤维黏蛋白第III型结构域的原有特性，可进一步地利用氨基酸取代来建构蛋白质结合界面，以与特定蛋白质结合，如：DLL4、EGFR、VEGFR2、或IGF-1R。借此，本实施方式的蛋白质可作为受体拮抗剂，作为生物药使用。
本实施方式的蛋白质包含第10人类纤维黏蛋白第III型结构域，且包含将一氨基酸位点取代为一半胱胺酸的第一突变及将另一氨基酸位点取代为另一半胱胺酸的第二突变，第一突变与第二突变发生于第10人类纤维黏蛋白第III型结构域的环FG以外的其他区域，借此第一突变取代成的半胱胺酸与第二突变取代成的半胱胺酸形成双硫键。较佳地，第10人类纤维黏蛋白第III型结构域包含一如SEQ ID NO：1所示的氨基酸序列。
依第10人类纤维黏蛋白第III型结构域的区域而言，第一突变可发生于β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环BC、环CD、环DE、或环EF，第二突变可发生于β股A、β股B、β股C、β 股D、β股E、β股F、β股G、环AB、环BC、环CD、环DE、或环EF。较佳地，第一突变发生于β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环CD、或环EF，第二突变发生于β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环CD、或环EF。
第一突变与第二突变可发生于以上区域群组中的一相同者或二不同者。举例而言，第一突变与第二突变均可发生环CD或环EF。再举例而言，第一突变可发生于β股A，第二突变可发生于β股B；第一突变可发生于β股A，第二突变可发生于β股G；第一突变可发生于环AB，第二突变可发生于β股E；第一突变可发生于β股B，第二突变可发生于β股E；第一突变可发生于β股C，第二突变可发生于β股D；第一突变可发生于β股C，第二突变可发生于环CD；第一突变可发生于β股C，第二突变可发生于β股F；第一突变可发生于环CD，第二突变可发生于环EF；或第一突变可发生于环EF，第二突变可发生于β股G。
依第10人类纤维黏蛋白第III型结构域的氨基酸序列而言，第一突变可包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置19的白胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第二突变可包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸。具体而言，第一突变可包含：位置8的白胺酸取代为半胱胺酸，第二突变可包含：位置22的色胺酸取代为半胱胺酸；第一突变可包含：位置8的白胺酸取代为半胱胺酸，第二突变可包含：位置89的丝胺酸取代为半胱胺酸；第一突变可包含：位置17 的丝胺酸取代为半胱胺酸，第二突变可包含：位置60的丝胺酸取代为半胱胺酸；第一突变可包含：位置19的白胺酸取代为半胱胺酸，第二突变可包含：位置58的苏胺酸取代为半胱胺酸；第一突变可包含：位置34的异白胺酸取代为半胱胺酸，第二突变可包含：位置48的苯丙胺酸取代为半胱胺酸；第一突变可包含：位置35的苏胺酸取代为半胱胺酸，第二突变可包含：位置45的缬胺酸取代为半胱胺酸；第一突变可包含：位置36的酪胺酸取代为半胱胺酸，第二突变可包含：位置70的异白胺酸取代为半胱胺酸；第一突变可包含：位置37的甘胺酸取代为半胱胺酸，第二突变可包含：位置45的缬胺酸取代为半胱胺酸；第一突变可包含：位置39的苏胺酸取代为半胱胺酸，第二突变可包含：位置67的天门冬胺酸取代为半胱胺酸；第一突变可包含：位置63的离胺酸取代为半胱胺酸，第二突变可包含：位置66的缬胺酸取代为半胱胺酸；或第一突变可包含：位置67的天门冬胺酸取代为半胱胺酸，第二突变可包含：位置91的天门冬酰胺取代为半胱胺酸。
本实施方式的蛋白质更可包含将又一氨基酸位点取代为又一半胱胺酸的第三突变及将再一氨基酸位点取代为再一半胱胺酸的第四突变，第三突变与第四突变发生于第10人类纤维黏蛋白第III型结构域的环FG以外的其他区域，借此第三突变取代成的半胱胺酸与第四突变取代成的半胱胺酸形成双硫键。于含有至少二个双硫键的条件下，蛋白质可进一步提升热稳定性与溶解度。
依第10人类纤维黏蛋白第III型结构域的区域而言，第三突变可发生于β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环BC、环CD、环DE、或环EF，第四突变可发生β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环BC、环CD、环DE、或环EF。较佳地，第三突变发生于β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环CD、或环EF，第四突变发生于β股A、β股B、β股C、β股D、β股E、β股F、β股G、环AB、环CD、或环EF。
第一突变、第二突变、第三突变、与第四突变可发生于以上区域群组中的四不同者。举例而言，第一突变可发生于β股A，第二突变可发生于β股 G，第三突变可发生于环AB，第四突变可发生于β股E；或第一突变可发生于β股A，第二突变可发生于β股G，第三突变可发生于环CD，第四突变可发生于环EF。
依第10人类纤维黏蛋白第III型结构域的氨基酸序列而言，第三突变可包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置19的白胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第四突变可包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸。具体而言，第一突变可包含：位置8的白胺酸取代为半胱胺酸，第二突变可包含：位置89的丝胺酸取代为半胱胺酸，第三突变可包含：位置17的丝胺酸取代为半胱胺酸，第四突变可包含：位置60的丝胺酸取代为半胱胺酸；或第一突变可包含：位置8的白胺酸取代为半胱胺酸，第二突变可包含：位置89的丝胺酸取代为半胱胺酸，第三突变可包含：位置39的苏胺酸取代为半胱胺酸，第四突变可包含：位置67的天门冬胺酸取代为半胱胺酸。
如前所述，本实施方式的蛋白质可结合至DLL4、EGFR、VEGFR2、或IGF-1R。为与VEGFR2结合，本实施方式的蛋白质另可包含将环BC的氨基酸序列取代为RHPHFPTRY的突变、将环DE的氨基酸序列取代为PLQPPT的突变、以及将环FG的氨基酸序列取代为VTDGRNGRLLSIP的突变。
较佳地，本实施方式的蛋白质包含一如SEQ ID NO：4、5、6、7、8、9、10、11、12、13、14、15、或16所示的氨基酸序列。更佳地，本实施方式的蛋白质包含一如SEQ ID NO：11、15、或16所示的氨基酸序列。
先前发现蛋白质C7-NM可提高热稳定性与溶解度。本实施方式的蛋白质可参照蛋白质C7-NM的氨基酸序列进行修饰。
于第一突变及第二突变未包含位置19的白胺酸取代为半胱胺酸的条件下，本实施方式的蛋白质可进一步地包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。具体而言，于第一突变包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第二突变包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸的条件下，本实施方式的蛋白质可进一步地包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。更具体而言，于第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置22的色胺酸取代为半胱胺酸；第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸；第一突变包含：位置17的丝胺酸取代为半胱胺酸，第二突变包含：位置60的丝胺酸取代为半胱胺酸；第一突变包含：位置34的异白胺酸取代为半胱胺酸，第二突变包含：位置48的苯丙胺酸取代为半胱胺酸；第一突变包含：位置35的苏胺酸取代为半胱胺酸，第二突变包含：位置45的缬胺酸取代为半胱胺酸；第一突变包含：位置36的酪胺酸取代为半胱胺酸，第二突变包含：位置70的异白胺酸取代为半胱胺酸；第一突变包含：位置37的甘胺 酸取代为半胱胺酸，第二突变包含：位置45的缬胺酸取代为半胱胺酸；第一突变包含：位置39的苏胺酸取代为半胱胺酸，第二突变包含：位置67的天门冬胺酸取代为半胱胺酸；第一突变包含：位置63的离胺酸取代为半胱胺酸，第二突变包含：位置66的缬胺酸取代为半胱胺酸；或第一突变包含：位置67的天门冬胺酸取代为半胱胺酸，第二突变包含：位置91的天门冬酰胺取代为半胱胺酸的条件下，本实施方式的蛋白质可进一步地包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。
较佳地，本实施方式的蛋白质包含一如SEQ ID NO：17、18、19、20、21、22、23、24、25、或26所示的氨基酸序列。
于第一突变、第二突变、第三突变、及第四突变未包含位置19的白胺酸取代为半胱胺酸的条件下，本实施方式的蛋白质可另包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。具体而言，于第一突变包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第二突变包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸，第三突变包含：位置8的白胺酸取代为半胱胺酸、位置17的丝胺酸取代为半胱胺酸、位置34的异白胺酸取代为半胱胺酸、位置35的苏胺酸取代为半胱胺酸、位置36的酪胺酸取代为 半胱胺酸、位置37的甘胺酸取代为半胱胺酸、位置39的苏胺酸取代为半胱胺酸、位置63的离胺酸取代为半胱胺酸、或位置67的天门冬胺酸取代为半胱胺酸，第四突变包含：位置22的色胺酸取代为半胱胺酸、位置45的缬胺酸取代为半胱胺酸、位置48的苯丙胺酸取代为半胱胺酸、位置58的苏胺酸取代为半胱胺酸、位置60的丝胺酸取代为半胱胺酸、位置66的缬胺酸取代为半胱胺酸、位置67的天门冬胺酸取代为半胱胺酸、位置70的异白胺酸取代为半胱胺酸、位置89的丝胺酸取代为半胱胺酸、或位置91的天门冬酰胺取代为半胱胺酸的条件下，本实施方式的蛋白质可进一步包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。更具体而言，于第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸，第三突变包含：位置17的丝胺酸取代为半胱胺酸，第四突变包含：位置60的丝胺酸取代为半胱胺酸；或第一突变包含：位置8的白胺酸取代为半胱胺酸，第二突变包含：位置89的丝胺酸取代为半胱胺酸，第三突变包含：位置39的苏胺酸取代为半胱胺酸，第四突变包含：位置67的天门冬胺酸取代为半胱胺酸的条件下，本实施方式的蛋白质可再包含将位置12的丙胺酸取代为麸胺酸的突变、将位置14的苏胺酸取代为丝胺酸的突变、将位置18的白胺酸取代为异白胺酸的突变、以及将位置19的白胺酸取代为麸酰胺酸的突变。
较佳地，本实施方式的蛋白质包含一如SEQ ID NO：27或28所示的氨基酸序列。
本实施方式的蛋白质可透过基因工程技术或化学方式制备，化学方式如：固相合成或溶液合成。可于后续采用硫酸铵或乙醇沉淀、酸萃取、离子交换层析、亲和层析、或凝集素层析等方法分离取得或纯化本实施方式的蛋白质，较佳地采用高效液相层析。
本实施方式的蛋白质更可具有一亲水性基团，藉以提升水溶性或循环半衰期。亲水性基团可连接蛋白质的N端。较佳地，亲水性基团为聚乙二醇、聚丙 二醇、聚乳酸、聚乙醇酸、聚乙烯醇、或聚葡萄醣。更佳地，亲水性基团为2至40个乙二醇重复单元组成的聚乙二醇。
本实施方式的蛋白质更可含有一纯化标签，藉以协助纯化。纯化标签可连接蛋白质的N端或C端。较佳地，纯化卷标为组胺酸卷标(His-tag)、谷胱甘肽S-转移酶标签(GST-tag)、麦芽糖结合蛋白标签(MBP-tag)、转录终止/抗终止蛋白(NusA-tag)、或小泛素相关修饰物标签(SUMO-tag)。
三、医药组合物
本发明第二实施方式揭示一种医药组合物，其含有第一实施方式的蛋白质，故可投予至个体以与其特定蛋白质结合，以作为特定蛋白质的拮抗剂，从而抑制特定蛋白质的活性或阻断相关的讯息传递。本实施方式的医药组合物包含：第一实施方式的蛋白质；以及一医药上可接受的载体。
医药上可接受的载体可使医药组合物整体呈现不同型态或适用于不同投药途径。较佳地，医药组合物为口服投予配制物、注射投予配制物、吸入投予配制物、或局部或经皮投予配制物，以使用于不同投药途径。较佳地，医药组合物为锭剂、胶囊、颗粒剂、散剂、溶液剂、糖浆剂、悬液剂、或乳剂。
医药上可接受的载体可为赋形剂、填充剂、稀释剂、凝集剂、接合剂、润滑剂、助流剂、稳定剂、着色剂、润湿剂、或崩解剂。赋形剂的实例可为柠檬酸钠、碳酸钙、或磷酸钙，填充剂的实例可为乳糖或高分子量聚乙二醇，稀释剂的实例可为水、乙醇、丙二醇、或甘油，接合剂的实例可为蔗糖、明胶、或阿拉伯胶，润滑剂的实例可为硬脂酸镁、硬脂酸钙、硬脂酸锌、硬脂酸钠、硬脂酸、硬脂酸铝、白胺酸、山嵛酸甘油酯、或氢化植物油，助流剂的实例可为铝硅酸钠、硅酸钙、微晶纤维素、玉米淀粉、苯甲酸钠、碳酸钙、碳酸镁、滑石、硬脂酸钙、硬脂酸镁、硬脂酸锌、月桂基硫酸镁、或氧化镁，稳定剂的实例可为柠檬酸或抗坏血酸，着色剂的实例可为二氧化钛或氧化铁，润湿剂的实例可为普朗尼克F68(Pluronic F68)、吐温20(Tween 20)、或吐温80(Tween 80)，崩解剂的实例可为马铃薯淀粉、树薯淀粉、或硅酸盐。
四、医药用途
本发明第三实施方式揭示一种第二实施方式的医药组合物的用途，其为用于制备治疗或预防第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调的医药。可投予制备的医药至个体，以抑制第二型血管内皮生长因子受体活性或阻断其讯息传递。亦即，可投予制备的医药至有治疗或预防第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调需求的个体，以透过抑制个体的第二型血管内皮生长因子受体活性或阻断其讯息传递，来达到治疗或预防功效。
可采用不同的方式投药，如：口服给药、注射给药、吸入给药、或局部或经皮给药。
第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调可包含：自体免疫失调症、心脏失调症、视网膜病变、肾病、成血管细胞瘤、血管瘤、甲状腺增生、慢性炎症、梅格斯氏征候群、心包膜积水、肋膜积水、糖尿病、子宫内膜异位、不良纤维化、或癌症。较佳地，癌症包含：肾癌、胰腺癌、乳癌、头颈部癌、前列腺癌、恶性胶质瘤、骨肉瘤、大肠直肠癌、胃癌、恶性间皮瘤、多发性骨髓瘤、卵巢癌、小细胞肺癌、非小细胞肺癌、滑膜肉瘤、甲状腺癌、或黑色素瘤。
本发明第四实施方式揭示一种治疗或预防第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调的方法，其包含：投予第二实施方式的医药组合物至一有此治疗或预防需求的个体，借此结合至个体的第二型血管内皮生长因子受体以抑制其活性或阻断其讯息传递。
可采用不同的方式投药，如：口服给药、注射给药、吸入给药、或局部或经皮给药。
第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调可包含：自体免疫失调症、心脏失调症、视网膜病变、肾病、成血管细胞瘤、血管瘤、甲状腺增生、慢性炎症、梅格斯氏征候群、心包膜积水、肋膜积水、糖尿病、子宫内膜异位、不良纤维化、或癌症。较佳地，癌症包含：肾癌、胰腺癌、乳癌、头颈部癌、前列腺癌、恶性胶质瘤、骨肉瘤、大肠直肠 癌、胃癌、恶性间皮瘤、多发性骨髓瘤、卵巢癌、小细胞肺癌、非小细胞肺癌、滑膜肉瘤、甲状腺癌、或黑色素瘤。
本发明第五实施方式揭示一种第二实施方式的医药组合物的用途，其为用于制备治疗或预防血管新生导致的疾病或失调的医药。可投予制备的医药至个体，以抑制血管新生。亦即，可投予制备的医药至有治疗或预防血管新生导致的疾病或失调需求的个体，以透过抑制个体的血管新生来达到治疗或预防功效。
可采用不同的方式投药，如：口服给药、注射给药、吸入给药、或局部或经皮给药。
血管新生导致的疾病或失调可包含：自体免疫失调症、心脏失调症、视网膜病变、肾病、成血管细胞瘤、血管瘤、甲状腺增生、慢性炎症、梅格斯氏征候群、心包膜积水、肋膜积水、糖尿病、子宫内膜异位、不良纤维化、或癌症。较佳地，癌症包含：肾癌、胰腺癌、乳癌、头颈部癌、前列腺癌、恶性胶质瘤、骨肉瘤、大肠直肠癌、胃癌、恶性间皮瘤、多发性骨髓瘤、卵巢癌、小细胞肺癌、非小细胞肺癌、滑膜肉瘤、甲状腺癌、或黑色素瘤。
本发明第六实施方式揭示一种治疗或预防血管新生导致的疾病或失调的方法，其包含：投予第二实施方式的医药组合物至一有此治疗或预防需求的个体，借此结合至个体的第二型血管内皮生长因子受体以抑制血管新生。
可采用不同的方式投药，如：口服给药、注射给药、吸入给药、或局部或经皮给药。
血管新生导致的疾病或失调可包含：自体免疫失调症、心脏失调症、视网膜病变、肾病、成血管细胞瘤、血管瘤、甲状腺增生、慢性炎症、梅格斯氏征候群、心包膜积水、肋膜积水、糖尿病、子宫内膜异位、不良纤维化、或癌症。较佳地，癌症包含：肾癌、胰腺癌、乳癌、头颈部癌、前列腺癌、恶性胶质瘤、骨肉瘤、大肠直肠癌、胃癌、恶性间皮瘤、多发性骨髓瘤、卵巢癌、小细胞肺癌、非小细胞肺癌、滑膜肉瘤、甲状腺癌、或黑色素瘤。
五、其他事项
本发明第七实施方式揭示一种核酸，其包含用于编码第一实施方式的蛋白质的核苷酸序列。为调控蛋白质的表现，核酸更可含有一启动子，其为可操作地连接编码蛋白质的核苷酸序列。此处“可操作地连接”是指二个或二个以上核酸序列彼此间呈功能关系。
本发明第八实施方式揭示一种宿主细胞，其包含第七实施方式的核酸。由于本实施方式的宿主细胞含有编码蛋白质的核苷酸序列，故可透过培养宿主细胞来生成蛋白质。宿主细胞可为原核细胞或真核细胞。原核细胞的实例可为大肠杆菌，真核细胞的实例可为CHO细胞、COS细胞、或HEK293细胞。
本发明第九实施方式揭示一种制备第一实施方式的蛋白质的方法，其包含培养第八实施方式的宿主细胞使其表现蛋白质。可依启动子来选择适当的诱导子诱导细胞表现蛋白质。
兹以下列实施例，示范地说明本发明：
＜实施例1：引入双硫键的设计＞
如图1所示，蛋白质C7-NM具有8个β股，相邻的二个β股的间形成有一个环状结构。以蛋白质C7-NM为亲本蛋白质，并利用软件Disulfide by Design 2.0(DbD2)且参考B因子、χ3扭转角、与能量等参数来于亲本蛋白质的结构设计引入双硫键。如图2与表1所示，可设计双硫键的位置位于亲本蛋白质的二个β股之间、二个环之间、及一个β股与一个环之间。如表2所示，呈现带有双硫键的变异蛋白质的氨基酸序列，其以蛋白质C7的氨基酸序列为参考序列并依设计的双硫键位置进行氨基酸取代而取得。
表1、晶体学参数
Figure PCTCN2022129857-appb-000001
Figure PCTCN2022129857-appb-000002
表2、氨基酸序列
Figure PCTCN2022129857-appb-000003
Figure PCTCN2022129857-appb-000004
注1：方框内的字母符号为相对于蛋白质10Fn3-WT的突变序列
注2：底在线的字母符号为相对于蛋白质C7的突变序列
＜实施例2：蛋白质的制备＞
蛋白质C7及其变异蛋白质为于大肠杆菌内表现。简言之，蛋白质C7及其变异蛋白质为利用pET21a表现载体及BL21(DE3)pLysS菌株或SHuffle菌株制备取得的，其中BL21(DE3)pLysS菌株的培养基为LB培养基，SHuffle菌株的培养基为LLB培养基。
首先，BL21(DE3)pLysS菌株于37℃下培养于5mL培养基约16至18小时(SHuffle菌株于30℃培养)。将BL21(DE3)pLysS菌株转至500mL培养基后于37℃下培养约4小时(SHuffle菌株于30℃培养约6小时)。接着，添加500μL的IPTG(浓度1M)后于25℃下诱导BL21(DE3)pLysS菌株表现蛋白质约16至18小时(于16℃诱导SHuffle菌株约48小时)，然后离心收集细胞沉淀物。
将细菌溶解于binding Buffer A(50mM磷酸钠，300mM氯化钠，pH7.0)，并以法式压滤器破裂细胞(压力维持于1500psi)。透过高速离心取得上层液，并载入至经Buffer A预平衡的镍离子螯合管柱。利用提取缓冲液(300mM咪唑于Buffer A，pH7.0)的梯度提取目标蛋白质。提取后以甘胺酸 SDS-PAGE确认目标蛋白质。最后，以PBS透析得到的重组蛋白质并储存于-80℃直至使用。
＜实施例3：蛋白质特性的分析＞
利用差示扫描量热法(differential scanning calorimetry，DSC)测定蛋白质熔点温度。如表3与图3所示，蛋白质C7-SL1与蛋白质C7的熔点温度差为37.0℃，较其他具单个双硫键的蛋白质与蛋白质C7的熔点温度差高，表示蛋白质C7-SL1的热稳性佳。如表3与图3、4所示，以蛋白质C7-SL1为主体并引入其他双硫键取得的蛋白质C7-SL1-LL2与C7-SL1-LL3可进一步提升热稳性。
参照J Pharm Sci.2008 Oct；97(10):4155-66，利用硫酸铵沉淀方式测定蛋白质溶解度。如表3所示，蛋白质C7-SL1的PBS溶解度约为蛋白质C7的6倍，较其他具单个双硫键的蛋白质高，表示蛋白质C7-SL1的溶解度佳。如表3与图5所示，以蛋白质C7-SL1为主体并引入其他双硫键取得的蛋白质C7-SL1-LL2与C7-SL1-LL3可进一步提升PBS溶解度至约为蛋白质C7的7倍。
利用细胞增生实验分析蛋白质对人类脐静脉内皮细胞(human umbilical vein endothelial cell，HUVEC)增生的抑制活性。如表3所示，蛋白质C7-SL1对HUVEC增生的抑制活性与蛋白质C7无明显差异，表示蛋白质C7-SL1具有抑制血管新生的能力。如表3所示，以蛋白质C7-SL1为主体并引入其他双硫键取得的蛋白质C7-SL1-LL2与C7-SL1-LL3对HUVEC增生的抑制活性亦与蛋白质C7无明显差异，表示蛋白质C7-SL1-LL2与C7-SL1-LL3亦具有抑制血管新生的能力。
利用酵素结合免疫吸附分析法(enzyme-linked immunosorbent assay，ELISA)分析蛋白质结合至第二型血管内皮生长因子受体的亲和力。如表3所示，蛋白质C7-SL1结合至第二型血管内皮生长因子受体的亲和力与蛋白质C7无明显差异，表示蛋白质C7-SL1具有结合至第二型血管内皮生长因子受体的能力。如表3与图6所示，以蛋白质C7-SL1为主体并引入其他双硫键取得的蛋白质C7-SL1-LL2与C7-SL1-LL3结合至第二型血管内皮生长因子受体的亲和 力亦与蛋白质C7无明显差异，且蛋白质C7-SL1-LL2结合至第二型血管内皮生长因子受体的亲和力略优于蛋白质C7，表示蛋白质C7-SL1-LL2与C7-SL1-LL3亦具有结合至第二型血管内皮生长因子受体的能力。
表3、蛋白质的特性
Figure PCTCN2022129857-appb-000005
注1：ΔTm＝测试蛋白质的Tm值-蛋白质C7的Tm值
注2：ND表示无法测得，原因为形成于包涵体(inclusion body)或未进行
惟以上所述者，仅为本发明的较佳实施例，但不能以此限定本发明专利保护范围；故，凡依本发明权利要求书及说明书内容所作的简单的等效改变与修饰，皆仍属落入本发明专利保护范围内。
Figure PCTCN2022129857-appb-000006
Figure PCTCN2022129857-appb-000007
Figure PCTCN2022129857-appb-000008
Figure PCTCN2022129857-appb-000009
Figure PCTCN2022129857-appb-000010
Figure PCTCN2022129857-appb-000011
Figure PCTCN2022129857-appb-000012

Claims (10)

  1. 一种蛋白质，其特征在于，包括：第10人类纤维黏蛋白第III型结构域，且包含将一氨基酸位点取代为一半胱胺酸的第一突变及将另一氨基酸位点取代为另一半胱胺酸的第二突变，该第一突变与该第二突变发生于该第10人类纤维黏蛋白第III型结构域的环FG以外的其他区域，借此该第一突变取代成的半胱胺酸与该第二突变取代成的半胱胺酸形成双硫键。
  2. 根据权利要求1所述的蛋白质，其特征在于：更包含将环BC的氨基酸序列取代为RHPHFPTRY的突变、将环DE的氨基酸序列取代为PLQPPT的突变、以及将环FG的氨基酸序列取代为VTDGRNGRLLSIP的突变。
  3. 根据权利要求1所述的蛋白质，其特征在于：该第一突变包含：位置8的白胺酸取代为半胱胺酸，该第二突变包含：位置22的色胺酸取代为半胱胺酸；该第一突变包含：位置8的白胺酸取代为半胱胺酸，该第二突变包含：位置89的丝胺酸取代为半胱胺酸；该第一突变包含：位置17的丝胺酸取代为半胱胺酸，该第二突变包含：位置60的丝胺酸取代为半胱胺酸；该第一突变包含：位置19的白胺酸取代为半胱胺酸，该第二突变包含：位置58的苏胺酸取代为半胱胺酸；该第一突变包含：位置34的异白胺酸取代为半胱胺酸，该第二突变包含：位置48的苯丙胺酸取代为半胱胺酸；该第一突变包含：位置35的苏胺酸取代为半胱胺酸，该第二突变包含：位置45的缬胺酸取代为半胱胺酸；该第一突变包含：位置36的酪胺酸取代为半胱胺酸，该第二突变包含：位置70的异白胺酸取代为半胱胺酸；该第一突变包含：位置37的甘胺酸取代为半胱胺酸，该第二突变包含：位置45的缬胺酸取代为半胱胺酸；该第一突变包含：位置39的苏胺酸取代为半胱胺酸，该第二突变包含：位置67的天门冬胺酸取代为半胱胺酸；该第一突变包含：位置63的离胺酸取代为半胱胺酸，该第二突变包含：位置66的缬胺酸取代为半胱胺酸；或该第一突变包 含：位置67的天门冬胺酸取代为半胱胺酸，该第二突变包含：位置91的天门冬酰胺取代为半胱胺酸。
  4. 根据权利要求1所述的蛋白质，其特征在于：更包含将又一氨基酸位点取代为又一半胱胺酸的第三突变及将再一氨基酸位点取代为再一半胱胺酸的第四突变，该第三突变与该第四突变发生于该第10人类纤维黏蛋白第III型结构域的环FG以外的其他区域，借此该第三突变取代成的半胱胺酸与该第四突变取代成的半胱胺酸形成双硫键。
  5. 根据权利要求3所述的蛋白质，其特征在于：更包含将又一氨基酸位点取代为又一半胱胺酸的第三突变及将再一氨基酸位点取代为再一半胱胺酸的第四突变，该第三突变与该第四突变发生于该第10人类纤维黏蛋白第III型结构域的环FG以外的其他区域，借此该第三突变取代成的半胱胺酸与该第四突变取代成的半胱胺酸形成双硫键；该第一突变包含：位置8的白胺酸取代为半胱胺酸，该第二突变包含：位置89的丝胺酸取代为半胱胺酸，该第三突变包含：位置17的丝胺酸取代为半胱胺酸，该第四突变包含：位置60的丝胺酸取代为半胱胺酸；或该第一突变包含：位置8的白胺酸取代为半胱胺酸，该第二突变包含：位置89的丝胺酸取代为半胱胺酸，该第三突变包含：位置39的苏胺酸取代为半胱胺酸，该第四突变包含：位置67的天门冬胺酸取代为半胱胺酸。
  6. 根据权利要求1所述的蛋白质，其特征在于：包含一如SEQ ID NOs：4至16中任一所示的氨基酸序列。
  7. 根据权利要求1所述的蛋白质，其特征在于：包含一如SEQ ID NO：11、15、或16所示的氨基酸序列。
  8. 一种医药组合物，其特征在于，包括：
    一根据权利要求1所述的蛋白质；以及
    一医药上可接受的载体。
  9. 一种根据权利要求1所述的医药组合物的用途，其特征在于：用于制备治疗或预防第二型血管内皮生长因子受体活性或讯息传递导致或与其相关联的疾病或失调的医药。
  10. 一种根据权利要求1所述的医药组合物的用途，其特征在于：用于制备治疗或预防血管新生导致的疾病或失调的医药。
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