Fibronectin type III domain-derived proteins and their applications

Abstract

The present invention discloses a fibronectin type III domain-derived protein and its application. The protein comprises a 10th human fibronectin type III domain and a first mutation in which an amino acid residue is substituted with cysteine, and a second mutation in which another amino acid residue is substituted with another cysteine. The first mutation and the second mutation occur in regions other than the loop FG of the 10th human fibronectin type III domain, so that the cysteine ​​substituted by the first mutation and the cysteine ​​substituted by the second mutation form a disulfide bond.

Description

[Technical Field]

[0001] The present invention relates to protein engineering, and more particularly to a fibronectin type III domain-derived protein and its applications, including a method for treating or preventing a disease or disorder caused by or associated with the activity or signal transduction of vascular endothelial growth factor receptor 2 (VEGFR2), or a method for treating or preventing a disease or disorder caused by angiogenesis. [Background technology]

[0002] In recent years, efforts have been focused on developing molecular recognition modules based on scaffold proteins to replace immunoglobulin-based modules, in order to overcome the inherent limitations that arise when immunoglobulins are used as the main body of molecular recognition modules, such as excessive size, complex heterodimer structures, and the need for precise formation of disulfide bonds. The basic theory behind such protein engineering is to develop molecules that have high affinity and specificity for antibodies by using a binding interface composed of an appropriate non-antibody protein framework or scaffold molecule.

[0003] Currently, the 10th human fibronectin type III domain (10Fn3) is widely used to prepare novel non-antibody scaffolds for protein binding. Several 10Fn3-based molecules are currently in development and being tested for disease treatment, many of which are in clinical trials.

[0004] 10Fn3 has many advantages over immunoglobulin-derived systems. Based on a global β-sandwich fold, 10Fn3 belongs to the immunoglobulin superfamily. Its three surface loop regions near the N-terminus are structurally similar to the three antigen recognition loops or complementarity-determining regions (CDRs) of immunoglobulin variants. However, unlike typical regions of immunoglobulins, 10Fn3 does not contain disulfide bonds. Furthermore, its thermal transition temperature is above 80°C, making it structurally stable. Furthermore, 10Fn3 exhibits reversible and rapid unfolding and refolding properties. 10Fn3, with approximately 94 amino acid residues, is smaller than the antigen-binding unit (VHH) of a heavy-chain antibody. These properties make 10Fn3 compatible with different molecular display systems and simple and efficient production methods.

[0005] U.S. Patent Application No. 11 / 448,171 (titled "Inhibitors of Type 2 Vascular Endothelial Growth Factor Receptors") discloses a vascular endothelial growth factor receptor 2 binding protein, C7. Protein C7 has a structure mainly composed of 10Fn3 and has the sequence of the three surface loop regions of the wild type. 23 DAPAVTVRY, 51 PGSKST, and 75 VTGRGDSPASSKP, 23 RHPHFPTRY, 51 PLQPPT, and 75 It differs from the wild-type in that each of the three mutations is replaced by VTDGRNGRLLSIP. By using these three mutations as binding residues for vascular endothelial growth factor receptor 2, protein C7 can be used as a vascular endothelial growth factor receptor 2 antagonist and has the potential to become an anticancer drug. However, its poor thermal stability and low solubility hinder its development as a drug.

[0006] Therefore, the development of a scaffold protein whose main structure is 10Fn3, which has high thermal stability and solubility, has become a challenge that those in the technical field to which the present invention pertains are actively striving to solve. Summary of the Invention [Problem to be solved by the invention]

[0007] Conventionally, the mutant protein C7-NM has been obtained by improving the protein C7. The present invention is based on the following: Using Disulfide by Design 2.0 (DbD2) software, the B-factor, χ 3 Twist angle (χ 3 By designing the protein C7-NM to incorporate disulfide bonds into its structure by referring to parameters such as the molecular weight (Mn) and energy, we were able to obtain mutants with disulfide bonds. These mutants have the potential to be used as biopharmaceuticals, as they can increase thermal stability and solubility without affecting their ability to bind to specific proteins.

[0008] The protein of the present invention comprises a 10th human fibronectin type III domain and a first mutation in which an amino acid residue is substituted with cysteine ​​and a second mutation in which another amino acid residue is substituted with another cysteine, and since the first mutation and the second mutation occur in a region other than the loop FG of the 10th human fibronectin type III domain, the cysteine ​​substituted by the first mutation and the cysteine ​​substituted by the second mutation form a disulfide bond.

[0009] For example, the tenth human fibronectin type III domain comprises the amino acid sequence shown in SEQ ID NO:1.

[0010] The protein further comprises a mutation in which the amino acid sequence of loop BC is replaced with RHPHFPTRY, a mutation in which the amino acid sequence of loop DE is replaced with PLQPPT, and a mutation in which the amino acid sequence of loop FG is replaced with VTDGRNGRLLSIP.

[0011] For example, the first mutation occurs in any one of β strand A, β strand B, β strand C, β strand D, β strand E, β strand F, β strand G, loop AB, loop BC, loop CD, loop DE, and loop EF of the 10th human fibronectin type III domain, and the second mutation occurs in any one of β strand A, β strand B, β strand C, β strand D, β strand E, β strand F, β strand G, loop AB, loop BC, loop CD, loop DE, and loop EF of the 10th human fibronectin type III domain.

[0012] For example, the first mutation occurs in any one of β strand A, β strand B, β strand C, β strand D, β strand E, β strand F, β strand G, loop AB, loop CD, and loop EF of the 10th human fibronectin type III domain, and the second mutation occurs in any one of β strand A, β strand B, β strand C, β strand D, β strand E, β strand F, β strand G, loop AB, loop CD, and loop EF of the 10th human fibronectin type III domain.

[0013] For example, the first mutation and the second mutation may occur in the same region or in different regions.

[0014] For example, the first mutation may comprise a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of leucine at position 19 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine; Mutations include a tryptophan-for-cysteine ​​substitution at position 22, a valine-for-cysteine ​​substitution at position 45, a phenylalanine-for-cysteine ​​substitution at position 48, a threonine-for-cysteine ​​substitution at position 58, a serine-for-cysteine ​​substitution at position 60, a valine-for-cysteine ​​substitution at position 66, an aspartate-for-cysteine ​​substitution at position 67, a isoleucine-for-cysteine ​​substitution at position 70, a serine-for-cysteine ​​substitution at position 89, or an aspartic acid-for-cysteine ​​substitution at position 91.

[0015] For example, a first mutation comprises a substitution of leucine at position 8 with cysteine, a second mutation comprises a substitution of tryptophan at position 22 with cysteine, a first mutation comprises a substitution of leucine at position 8 with cysteine, a second mutation comprises a substitution of serine at position 89 with cysteine, a first mutation comprises a substitution of serine at position 17 with cysteine, a second mutation comprises a substitution of serine at position 60 with cysteine, a first mutation comprises a substitution of leucine at position 19 with cysteine, a second mutation comprises a substitution of threonine at position 58 with cysteine, a first mutation comprises a substitution of isoleucine at position 34 with cysteine, a second mutation comprises a substitution of phenylalanine at position 48 with cysteine, a first mutation comprises a substitution of threonine at position 35 with cysteine, two mutations include a substitution of valine at position 45 with cysteine, a first mutation includes a substitution of tyrosine at position 36 with cysteine, a second mutation includes a substitution of isoleucine at position 70 with cysteine, a first mutation includes a substitution of glycine at position 37 with cysteine, a second mutation includes a substitution of valine at position 45 with cysteine, a first mutation includes a substitution of threonine at position 39 with cysteine, a second mutation includes a substitution of aspartate at position 67 with cysteine, a first mutation includes a substitution of lysine at position 63 with cysteine, and a second mutation includes a substitution of valine at position 66 with cysteine; or a first mutation includes a substitution of aspartate at position 67 with cysteine, and a second mutation includes a substitution of aspartic acid at position 91 with cysteine.

[0016] For example, the protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 4-14.

[0017] For example, the protein comprises the amino acid sequence shown in SEQ ID NO:11.

[0018] For example, provided that the first mutation and the second mutation do not include a substitution of leucine at position 19 with cysteine, the protein includes 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine.

[0019] For example, the first mutation may include a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine; and the second mutation may include a substitution of tryptophan at position 22 with cysteine, a substitution of valine at position 45 with cysteine, a substitution of phenylalanine at position 48 with cysteine. a substitution of threonine at position 58 with cysteine, serine at position 60 with cysteine, valine at position 66 with cysteine, aspartate at position 67 with cysteine, isoleucine at position 70 with cysteine, serine at position 89 with cysteine, or aspartic acid at position 91 with cysteine; the protein also contains a mutation where alanine at position 12 is substituted with glutamic acid, a mutation where threonine at position 14 is substituted with serine, a mutation where leucine at position 18 is substituted with isoleucine, and a mutation where leucine at position 19 is substituted with glutamine.

[0020] For example, a first mutation comprises a substitution of leucine at position 8 with cysteine, a second mutation comprises a substitution of tryptophan at position 22 with cysteine, a first mutation comprises a substitution of leucine at position 8 with cysteine, a second mutation comprises a substitution of serine at position 89 with cysteine, a first mutation comprises a substitution of serine at position 17 with cysteine, a second mutation comprises a substitution of serine at position 60 with cysteine, a first mutation comprises a substitution of isoleucine at position 34 with cysteine, a second mutation comprises a substitution of phenylalanine at position 48 with cysteine, a first mutation comprises a substitution of threonine at position 35 with cysteine, a second mutation comprises a substitution of valine at position 45 with cysteine, a first mutation comprises a substitution of tyrosine at position 36 with cysteine, and a second mutation comprises a substitution of isoleucine at position 70 with cysteine. wherein the first mutation comprises a substitution of glycine at position 37 with cysteine, the second mutation comprises a substitution of valine at position 45 with cysteine, the first mutation comprises a substitution of threonine at position 39 with cysteine, and the second mutation comprises a substitution of aspartate at position 67 with cysteine, the first mutation comprises a substitution of lysine at position 63 with cysteine, and the second mutation comprises a substitution of valine at position 66 with cysteine; or the first mutation comprises a substitution of aspartate at position 67 with cysteine, and the second mutation comprises a substitution of aspartic acid at position 91 with cysteine;

[0021] For example, the protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17-26.

[0022] For example, the protein further includes a third mutation in which another amino acid residue is substituted with another cysteine ​​and a fourth mutation in which yet another amino acid residue is substituted with yet another cysteine, and the third and fourth mutations occur in regions other than the loop FG of the 10th human fibronectin type III domain, so that the cysteine ​​substituted by the third mutation and the cysteine ​​substituted by the fourth mutation form a disulfide bond.

[0023] For example, the third mutation occurs in any one of β strand A, β strand B, β strand C, β strand D, β strand E, β strand F, β strand G, loop AB, loop BC, loop CD, loop DE, and loop EF of the 10th human fibronectin type III domain, and the fourth mutation occurs in any one of β strand A, β strand B, β strand C, β strand D, β strand E, β strand F, β strand G, loop AB, loop BC, loop CD, loop DE, and loop EF of the 10th human fibronectin type III domain.

[0024] For example, the third mutation occurs in any one of β strand A, β strand B, β strand C, β strand D, β strand E, β strand F, β strand G, loop AB, loop CD, and loop EF of the 10th human fibronectin type III domain, and the fourth mutation occurs in any one of β strand A, β strand B, β strand C, β strand D, β strand E, β strand F, β strand G, loop AB, loop CD, and loop EF of the 10th human fibronectin type III domain.

[0025] For example, the first mutation, the second mutation, the third mutation, and the fourth mutation occur in different regions.

[0026] For example, the third mutation may include a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of leucine at position 19 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine; Mutations include a tryptophan-for-cysteine ​​substitution at position 22, a valine-for-cysteine ​​substitution at position 45, a phenylalanine-for-cysteine ​​substitution at position 48, a threonine-for-cysteine ​​substitution at position 58, a serine-for-cysteine ​​substitution at position 60, a valine-for-cysteine ​​substitution at position 66, an aspartate-for-cysteine ​​substitution at position 67, a isoleucine-for-cysteine ​​substitution at position 70, a serine-for-cysteine ​​substitution at position 89, or an aspartic acid-for-cysteine ​​substitution at position 91.

[0027] For example, the first mutation comprises a substitution of leucine at position 8 with cysteine, the second mutation comprises a substitution of serine at position 89 with cysteine, the third mutation comprises a substitution of serine at position 17 with cysteine, and the fourth mutation comprises a substitution of serine at position 60 with cysteine; or the first mutation comprises a substitution of leucine at position 8 with cysteine, the second mutation comprises a substitution of serine at position 89 with cysteine, the third mutation comprises a substitution of threonine at position 39 with cysteine, and the fourth mutation comprises a substitution of aspartate at position 67 with cysteine.

[0028] For example, the protein comprises the amino acid sequence shown in SEQ ID NO:15 or 16.

[0029] For example, provided that the first mutation, the second mutation, the third mutation, and the fourth mutation do not include a substitution of leucine at position 19 with cysteine, the protein includes 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine.

[0030] For example, the first mutation may include a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine, and the second mutation may include a substitution of tripeptide at position 22 with cysteine, a substitution of threonine at position 36 with cysteine, a substitution of tyrosine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine. a cysteine ​​substitution for threonine at position 45, a cysteine ​​substitution for valine at position 45, a cysteine ​​substitution for phenylalanine at position 48, a cysteine ​​substitution for threonine at position 58, a cysteine ​​substitution for serine at position 60, a cysteine ​​substitution for valine at position 66, a cysteine ​​substitution for aspartate at position 67, a cysteine ​​substitution for isoleucine at position 70, a cysteine ​​substitution for serine at position 89, or a cysteine ​​substitution for aspartic acid at position 91; The mutations include a leucine-to-cysteine ​​substitution at position 8, a serine-to-cysteine ​​substitution at position 17, a isoleucine-to-cysteine ​​substitution at position 34, a threonine-to-cysteine ​​substitution at position 35, a tyrosine-to-cysteine ​​substitution at position 36, a glycine-to-cysteine ​​substitution at position 37, a threonine-to-cysteine ​​substitution at position 39, a lysine-to-cysteine ​​substitution at position 63, or an aspartate-to-cysteine ​​substitution at position 67; and a fourth mutation is a tryptophan-to-cysteine ​​substitution at position 22. a substitution of valine at position 45 with cysteine, a substitution of phenylalanine at position 48 with cysteine, a substitution of threonine at position 58 with cysteine, a substitution of serine at position 60 with cysteine, a substitution of valine at position 66 with cysteine, a substitution of aspartate at position 67 with cysteine, a substitution of isoleucine at position 70 with cysteine, a substitution of serine at position 89 with cysteine, or a substitution of aspartic acid at position 91 with cysteine.The mutations include alanine at position 12 substituted with glutamic acid, threonine at position 14 substituted with serine, leucine at position 18 substituted with isoleucine, and leucine at position 19 substituted with glutamine.

[0031] For example, if the first mutation comprises a substitution of leucine at position 8 with cysteine, the second mutation comprises a substitution of serine at position 89 with cysteine, the third mutation comprises a substitution of serine at position 17 with cysteine, and the fourth mutation comprises a substitution of serine at position 60 with cysteine, or if the first mutation comprises a substitution of leucine at position 8 with cysteine, the second mutation comprises a substitution of serine at position 89 with cysteine, the third mutation comprises a substitution of threonine at position 39 with cysteine, and the fourth mutation comprises a substitution of aspartate at position 67 with cysteine, 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine.

[0032] The protein comprises the amino acid sequence shown in SEQ ID NO:27 or 28.

[0033] For example, proteins have been used to bind to DLL4, EGFR, VEGFR2, or IGF-1R.

[0034] The protein of the present invention is based on the tenth human fibronectin type III domain, and has a disulfide bond introduced at a specific position in the domain to enhance its thermal stability and solubility. The protein of the present invention further utilizes amino acid sequence substitutions to form a protein-binding interface, allowing it to bind to specific proteins such as DLL4, EGFR, VEGFR2, or IGF-1R, and has the potential to be used as a receptor antagonist and biologic.

[0035] The present invention further provides a pharmaceutical composition comprising the above-described protein and a pharmaceutically acceptable carrier.

[0036] For example, the pharmaceutical composition may be an oral dosage form, an injectable dosage form, an inhaled dosage form, or a topical or transdermal dosage form.

[0037] The present invention further provides use of the above-mentioned pharmaceutical composition for preparing a medicament for treating or preventing a disease or disorder caused by or associated with vascular endothelial growth factor receptor 2 activity or signal transduction.

[0038] For example, diseases or disorders caused by or associated with vascular endothelial growth factor receptor 2 activity or signal transduction include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer.

[0039] For example, the cancer includes kidney cancer, spleen 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.

[0040] The present invention further provides a method for treating or preventing a disease or disorder caused by or associated with vascular endothelial growth factor receptor 2 activity or signal transduction, comprising administering the above-mentioned pharmaceutical composition to an individual in need thereof, thereby binding to and inhibiting the activity of vascular endothelial growth factor receptor 2 in the individual.

[0041] For example, diseases or disorders caused by or associated with vascular endothelial growth factor receptor 2 activity or signal transduction include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer.

[0042] For example, the cancer includes kidney cancer, spleen 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.

[0043] The present invention further provides the use of the pharmaceutical composition for preparing a medicament for treating or preventing a disease or disorder caused by angiogenesis.

[0044] For example, diseases or disorders caused by angiogenesis include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer.

[0045] For example, the cancer includes kidney cancer, spleen 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.

[0046] The present invention further provides a method for treating or preventing a disease or disorder caused by angiogenesis, which comprises administering the above-mentioned pharmaceutical composition to an individual in need thereof, thereby binding to the individual's vascular endothelial growth factor receptor 2 and inhibiting angiogenesis.

[0047] For example, diseases or disorders caused by angiogenesis include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer.

[0048] For example, the cancer includes kidney cancer, spleen 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.

[0049] The present invention further provides a nucleic acid comprising a nucleotide sequence for encoding the above-described protein.

[0050] The present invention further provides a host cell comprising the above-described nucleic acid.

[0051] For example, the host cell can be a prokaryotic or eukaryotic cell.

[0052] For example, the prokaryotic cell is E. coli, and the eukaryotic cell is a CHO cell, a COS cell, or a HEK293 cell.

[0053] The present invention further provides a method for preparing the above-mentioned proteins, comprising culturing the above-mentioned host cells so as to express the proteins. [Brief explanation of the drawings]

[0054] [Figure 1] FIG. 1 is a protein structural diagram showing the three-dimensional structure of the protein C7-NM. [Figure 2] FIG. 1 is a structural diagram of the protein C7-NM, showing positions where disulfide bonds can be designed in the three-dimensional structure. [Figure 3] FIG. 1 is a diagram showing the results of differential scanning calorimetry comparing the melting points of protein C7 and its mutant proteins. [Figure 4] FIG. 1 is a diagram showing the results of differential scanning calorimetry comparing the melting point temperatures of proteins C7 and C7-SL1-LL2. [Figure 5] FIG. 1 is a solubility result diagram comparing the solubilities of proteins C7 and C7-SL1-LL2. [Figure 6] FIG. 1 shows the results of an enzyme-linked immunosorbent assay comparing the binding affinities of proteins C7 and C7-SL1-LL2 to vascular endothelial growth factor receptor 2. DETAILED DESCRIPTION OF THE INVENTION

[0055] In order to make the above and / or other objects, advantages and features of the present invention more clearly understandable, preferred embodiments are given below and described in detail. I. Definitions Unless otherwise specified, the term "protein" as used herein includes wild-type proteins expressed in natural cells, recombinant proteins expressed by genetic engineering techniques, and synthetic proteins obtained by chemical methods. At least one amino acid can be substituted, deleted, and / or inserted into the protein sequence without affecting the original activity. The term "amino acid" as used herein includes D-amino acids and L-amino acids unless otherwise specified. D- and L- refer to the absolute configuration of the amino acid, not a particular direction of rotation of plane-polarized light. Unless otherwise specified, this specification uses the abbreviations recommended by the IUPAC-IUB Biochemical Nomenclature Commission to represent amino acids. Protein sequences are represented by a string of characters consisting of multiple abbreviations, and the order of the abbreviations corresponds to the order of the amino acids from the N-terminus to the C-terminus of the protein. When a superscript number precedes an abbreviation, it indicates the order corresponding to the amino acids located at the position counted from the N-terminus of the protein. For example, 23 DAPAVTVRY indicates that aspartate is located at position 23 of the protein. The rest are similarly deduced by analogy, so we will not repeat the explanation.

[0056] Substitutions, deletions, and / or insertions in protein sequences can occur in non-skeleton regions of a protein and generally do not affect the original activity. Protein sequence substitutions can also include conservative amino acid substitutions, which refer to substitutions between amino acids with similar properties or related side chains. Substitutions between amino acids with similar properties include, for example, acidic amino acids, such as aspartate and glutamate, which can be substituted for each other. Alkaline amino acids, such as lysine, arginine, and histidine, can be substituted for each other. Nonpolar amino acids, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, can be substituted for each other. Polar, uncharged amino acids are interchangeable, i.e., glycine, aspartic acid, glutamine, cysteine, serine, threonine, and tyrosine. Substitutions between amino acids with related side chains are possible, for example, aliphatic hydroxy amino acids are interchangeable, i.e., serine and threonine. Amide-containing amino acids are interchangeable, i.e., aspartic acid and glutamine. Aliphatic amino acids are interchangeable, i.e., alanine, valine, leucine, and isoleucine. Aromatic amino acids are interchangeable, i.e., phenylalanine, tryptophan, and tyrosine.

[0057] Unless otherwise specified, the "10th human fibronectin type III domain" referred to in this specification includes, from the N-terminus to the C-terminus, the N-terminal region, β-strand A, loop AB, β-strand B, loop BC, β-strand C, loop CD, β-strand D, loop DE, β-strand E, loop EF, β-strand F, loop FG, β-strand G, and the C-terminal region, in that order, and has at least 94 amino acids without any disulfide bonds, such as SEQ ID NO:1. With reference to the specification of U.S. Patent Application No. 13 / 757,664, entitled "FIBRONECTIN BINDING DOMAINS WITH REDUCED IMMUNOGENICITY," the N-terminal region is defined as the amino acid fragment from positions 1 to 7, β-strand A is defined as the amino acid fragment from positions 8 to 13, loop AB is defined as the amino acid fragment from positions 14 to 17, β-strand B is defined as the amino acid fragment from positions 18 to 22, loop BC is defined as the amino acid fragment from positions 23 to 31, β-strand C is defined as the amino acid fragment from positions 32 to 36, loop CD is defined as the amino acid fragment from positions 37 to 47, and β-strand D is defined as the amino acid fragment from positions 18 to 22. is defined as the amino acid fragment from positions 48 to 50, loop DE is defined as the amino acid fragment from positions 51 to 56, β-strand E is defined as the amino acid fragment from positions 57 to 62, loop EF is defined as the amino acid fragment from positions 63 to 67, β-strand F is defined as the amino acid fragment from positions 68 to 74, loop FG is defined as the amino acid fragment from positions 75 to 87, β-strand G is defined as the amino acid fragment from positions 88 to 92, and the C-terminal region is defined as the amino acid fragments at positions 93 and 94. Loops BC, DE, and FG are located on one side of the molecule, and loops AB, CD, and EF are located on the other side of the molecule. For example, the N-terminal region is 1VSDVPRD, and β strand A is 8 LEVVAA, and loop AB contains 14 TPTS, and β-strand B is 18 The loop BC includes 23 DAPAVTVRY, and β-strand C is 32 Includes a loop CD 37 GETGGNSPVQE, and β strand D is 48 Including FTV, Loop DE 51 PGSKST, and β-strand E is 57 ATISGL, and the loop EF 63 KPGVD, and the β strand F is 68 Contains YTITVYA, loop FG 75 VTGRGDSPASSKP, and the β-strand G is 88 ISINY, and the C-terminal region 93 Includes RT.

[0058] Unless otherwise specified, the term "protein C7" as used herein refers to a mutant of the tenth human fibronectin type III domain, which can bind to and inhibit the activity of vascular endothelial growth factor receptor 2, e.g., SEQ ID NO: 2. The amino acid sequence corresponding to the wild-type loop BC is: 23 RHPHFPTRY, and the amino acid sequence corresponding to the wild-type loop DE is 51 PLQPPT, and the amino acid sequence corresponding to the wild-type loop FG is 75 The three mutation regions described above constitute the binding interface of vascular endothelial growth factor receptor 2.

[0059] Unless otherwise specified, the term "protein C7-NM" referred to herein refers to a variant of protein C7, such as SEQ ID NO: 3. Note that the amino acid corresponding to position 12 of the reference sequence is glutamic acid, the amino acid corresponding to position 14 of the reference sequence is serine, the amino acid corresponding to position 18 of the reference sequence is isoleucine, and the amino acid corresponding to position 19 of the reference sequence is glutamine.

[0060] Unless otherwise specified, the term "vascular endothelial growth factor receptor 2" used herein refers to a transmembrane receptor tyrosine kinase that can regulate angiogenesis induced by VEGF-A and VEGF-B. Vascular endothelial growth factor receptor 2 is synonymous with kinase insert domain receptor (KDR) and fetal liver kinase 1 (FLK-1), and can be used interchangeably.

[0061] As used herein, unless otherwise defined, "treatment" refers to a therapeutic intervention to cure or ameliorate a disease condition, ie, to a complete or partial cure or amelioration.

[0062] Unless otherwise defined, the term "prevention" as used herein refers to the complete or nearly complete prevention of a disease. For example, when a disease is absent or suspected but not yet manifested, preventive intervention can prevent the onset of the disease.

[0063] Unless otherwise defined, the term "pharmaceutically acceptable carrier" as used herein refers to an additive that is within the scope of sound medical judgment, is compatible with contact with individuals, and is free from excessive toxicity, irritation, allergic reaction, or other problems or complications, and has a reasonable benefit / risk ratio, such as a filler, diluent, flocculating agent, adhesive, lubricant, flow agent, stabilizer, colorant, moisturizer, or disintegrant.

[0064] 2. Proteins mainly consisting of the tenth human fibronectin type III domain The protein according to a first embodiment of the present invention is based on the tenth human fibronectin type III domain, and the thermal stability and solubility of the protein are improved by introducing disulfide bonds to replace amino acids at specific positions in the main body. Based on the high thermal stability and solubility described above and the inherent properties of the tenth human fibronectin type III domain, the amino acid substitutions can be further utilized to construct a protein-binding interface, allowing the protein to bind to specific proteins such as DLL4, EGFR, VEGFR2, or IGF-1R. In this way, the protein according to this embodiment can be used as a receptor antagonist or a biological drug.

[0065] The protein of this embodiment comprises a tenth human fibronectin type III domain, and a first mutation in which an amino acid residue is substituted with cysteine ​​and a second mutation in which another amino acid residue is substituted with another cysteine, wherein the first mutation and the second mutation occur in a region other than the loop FG of the tenth human fibronectin type III domain, so that the cysteine ​​substituted by the first mutation and the cysteine ​​substituted by the second mutation form a disulfide bond. Preferably, the tenth human fibronectin type III domain comprises the amino acid sequence shown in SEQ ID NO: 1.

[0066] In this region of the tenth human fibronectin type III domain, the first mutation may occur in β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loop AB, loop BC, loop CD, loop DE, or loop EF, and the second mutation may occur in β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loop AB, loop BC, loop CD, loop DE, or loop EF. Preferably, the first mutation occurs in β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loop AB, loop CD, or loop EF, and the second mutation occurs in β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loop AB, loop CD, or loop EF.

[0067] The first mutation and the second mutation may occur in the same or two different regions among the above groups. For example, the first mutation and the second mutation may both occur in loop CD or loop EF. For example, the first mutation may occur in β-strand A and the second mutation may occur in β-strand B. The first mutation may occur in β-strand A and the second mutation may occur in β-strand G. The first mutation may occur in loop AB and the second mutation may occur in β-strand E. The first mutation may occur in β-strand B and the second mutation may occur in β-strand E. The first mutation may occur in β-strand C and the second mutation may occur in β-strand D. The first mutation may occur in β-strand C and the second mutation may occur in loop CD. The first mutation may occur in β-strand C and the second mutation may occur in β-strand F. The first mutation can occur in loop CD and the second mutation can occur in loop EF, or the first mutation can occur in loop EF and the second mutation can occur in β-strand G.

[0068] According to the amino acid sequence of the 10th human fibronectin type III domain, the first mutation is a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of leucine at position 19 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine. The second mutation may include a tryptophan substitution at position 22 with cysteine, a valine substitution at position 45 with cysteine, a phenylalanine substitution at position 48 with cysteine, a threonine substitution at position 58 with cysteine, a serine substitution at position 60 with cysteine, a valine substitution at position 66 with cysteine, an aspartate substitution at position 67 with cysteine, a isoleucine substitution at position 70 with cysteine, a serine substitution at position 89 with cysteine, or an aspartate substitution at position 91 with cysteine.Specifically, the first mutation may comprise a substitution of leucine at position 8 with cysteine; the second mutation may comprise a substitution of tryptophan at position 22 with cysteine; the first mutation may comprise a substitution of leucine at position 8 with cysteine; the second mutation may comprise a substitution of serine at position 89 with cysteine; the first mutation may comprise a substitution of serine at position 17 with cysteine; the second mutation may comprise a substitution of serine at position 60 with cysteine; the first mutation may comprise a substitution of leucine at position 19 with cysteine; the second mutation may comprise a substitution of threonine at position 58 with cysteine; the first mutation may comprise a substitution of isoleucine at position 34 with cysteine; the second mutation may comprise a substitution of phenylalanine at position 48 with cysteine; the first mutation may comprise a substitution of threonine at position 35 with cysteine; The second mutation may comprise a substitution of valine at position 45 with cysteine; the first mutation may comprise a substitution of tyrosine at position 36 with cysteine; the second mutation may comprise a substitution of isoleucine at position 70 with cysteine; the first mutation may comprise a substitution of glycine at position 37 with cysteine; the second mutation may comprise a substitution of valine at position 45 with cysteine; the first mutation may comprise a substitution of threonine at position 39 with cysteine; the second mutation may comprise a substitution of aspartate at position 67 with cysteine; the first mutation may comprise a substitution of lysine at position 63 with cysteine; the second mutation may comprise a substitution of valine at position 66 with cysteine; or the first mutation may comprise a substitution of aspartate at position 67 with cysteine; and the second mutation may comprise a substitution of aspartate at position 91 with cysteine.

[0069] The protein of this embodiment may further include a third mutation in which another amino acid residue is substituted with another cysteine ​​and a fourth mutation in which yet another amino acid residue is substituted with yet another cysteine, and the third and fourth mutations occur in regions other than the loop FG of the tenth human fibronectin type III domain, so that the cysteines substituted by the third and fourth mutations form disulfide bonds. When the protein contains at least two disulfide bonds, the thermal stability and solubility of the protein can be further improved.

[0070] According to the region of the tenth human fibronectin type III domain, the third mutation may occur in β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loop AB, loop BC, loop CD, loop DE, or loop EF, and the fourth mutation may occur in β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loop AB, loop BC, loop CD, loop DE, or loop EF. Preferably, the third mutation occurs in β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loop AB, loop CD, or loop EF, and the fourth mutation occurs in β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loop AB, loop CD, or loop EF.

[0071] The first mutation, the second mutation, the third mutation, and the fourth mutation can occur in four different regions of the above group. For example, the first mutation can occur in β-strand A, the second mutation can occur in β-strand G, the third mutation can occur in loop AB, and the fourth mutation can occur in β-strand E; alternatively, the first mutation can occur in β-strand A, the second mutation can occur in β-strand G, the third mutation can occur in loop CD, and the fourth mutation can occur in loop EF.

[0072] According to the amino acid sequence of the 10th human fibronectin type III domain, the third mutation is a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of leucine at position 19 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine. The fourth mutation may include a tryptophan substitution at position 22 with cysteine, a valine substitution at position 45 with cysteine, a phenylalanine substitution at position 48 with cysteine, a threonine substitution at position 58 with cysteine, a serine substitution at position 60 with cysteine, a valine substitution at position 66 with cysteine, an aspartate substitution at position 67 with cysteine, a isoleucine substitution at position 70 with cysteine, a serine substitution at position 89 with cysteine, or an aspartate substitution at position 91 with cysteine. Specifically, the first mutation may comprise a substitution of leucine at position 8 with cysteine, the second mutation may comprise a substitution of serine at position 89 with cysteine, the third mutation may comprise a substitution of serine at position 17 with cysteine, and the fourth mutation may comprise a substitution of serine at position 60 with cysteine; alternatively, the first mutation may comprise a substitution of leucine at position 8 with cysteine, the second mutation may comprise a substitution of serine at position 89 with cysteine, the third mutation may comprise a substitution of threonine at position 39 with cysteine, and the fourth mutation may comprise a substitution of aspartate at position 67 with cysteine.

[0073] As described above, the protein according to this embodiment can bind to DLL4, EGFR, VEGFR2, or IGF-1R. To bind to VEGFR2, the protein according to this embodiment may further include a mutation in which the amino acid sequence of loop BC is substituted with RHPHFPTRY, a mutation in which the amino acid sequence of loop DE is substituted with PLQPPT, and a mutation in which the amino acid sequence of loop FG is substituted with VTDGRNGRLLSIP.

[0074] Preferably, the protein according to this embodiment comprises the amino acid sequence shown in SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. More preferably, the protein according to this embodiment comprises the amino acid sequence shown in SEQ ID NO: 11, 15, or 16.

[0075] It has previously been found that the C7-NM protein can improve its thermal stability and solubility. The protein in this example is modified with reference to the amino acid sequence of the C7-NM protein.

[0076] Provided that the first mutation and the second mutation do not include a substitution of leucine at position 19 with cysteine, the protein of this embodiment may further include 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine. Specifically, the first mutation includes a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine; and the second mutation includes a substitution of tryptophan at position 22 with cysteine, a substitution of valine at position 45 with cysteine, or a substitution of phenylalanine at position 48 with cysteine. Under conditions including the substitution of threonine at position 58 with cysteine, serine at position 60 with cysteine, valine at position 66 with cysteine, aspartate at position 67 with cysteine, isoleucine at position 70 with cysteine, serine at position 89 with cysteine, or aspartic acid at position 91 with cysteine, the protein of this example may further include 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine.More specifically, the first mutation comprises a substitution of leucine at position 8 with cysteine, the second mutation comprises a substitution of tryptophan at position 22 with cysteine, the first mutation comprises a substitution of leucine at position 8 with cysteine, the second mutation comprises a substitution of serine at position 89 with cysteine, the first mutation comprises a substitution of serine at position 17 with cysteine, and the second mutation comprises a substitution of serine at position 60 with cysteine; The first mutation comprises a substitution of isoleucine at position 34 with cysteine, the second mutation comprises a substitution of phenylalanine at position 48 with cysteine, the first mutation comprises a substitution of threonine at position 35 with cysteine, the second mutation comprises a substitution of valine at position 45 with cysteine, the first mutation comprises a substitution of tyrosine at position 36 with cysteine, and the second mutation comprises a substitution of isoleucine at position 70 with cysteine. wherein a first mutation comprises a substitution of glycine at position 37 with cysteine, a second mutation comprises a substitution of valine at position 45 with cysteine, a first mutation comprises a substitution of threonine at position 39 with cysteine, a second mutation comprises a substitution of aspartate at position 67 with cysteine, a first mutation comprises a substitution of lysine at position 63 with cysteine, and a second mutation comprises a substitution of valine at position 66 with cysteine; or Provided that the first mutation comprises substituting aspartate at position 67 with cysteine ​​and the second mutation comprises substituting aspartic acid at position 91 with cysteine, the protein of this embodiment may further comprise 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine.

[0077] Preferably, the protein according to this embodiment comprises the amino acid sequence shown in SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

[0078] Provided that the first mutation, the second mutation, the third mutation, and the fourth mutation do not include a substitution of leucine at position 19 with cysteine, the protein of this embodiment may further include 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine. Specifically, the first mutation includes a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine; and the second mutation includes a substitution of tryptophan at position 22 with cysteine, a substitution of valine at position 45 with cysteine, a substitution of phenylalanine at position 48 with cysteine, a substitution of threonine at position 58 with cysteine, a substitution of serine at position 60 with cysteine, a substitution of valine at position 66 with cysteine, or a substitution of aspartate at position 67 with cysteine. a third mutation comprising a substitution of leucine at position 8 with cysteine, a substitution of serine at position 17 with cysteine, a substitution of isoleucine at position 34 with cysteine, a substitution of threonine at position 35 with cysteine, a substitution of tyrosine at position 36 with cysteine, a substitution of glycine at position 37 with cysteine, a substitution of threonine at position 39 with cysteine, a substitution of lysine at position 63 with cysteine, or a substitution of aspartate at position 67 with cysteine; a fourth mutation comprising a substitution of tryptophan at position 22 with cysteine, a substitution of valine at position 45 with cysteine, or a substitution of phenylalanine at position 48 with cysteine;Under conditions including substitution of threonine at position 58 with cysteine, substitution of serine at position 60 with cysteine, substitution of valine at position 66 with cysteine, substitution of aspartate at position 67 with cysteine, substitution of isoleucine at position 70 with cysteine, substitution of serine at position 89 with cysteine, or substitution of aspartic acid at position 91 with cysteine, the protein of this embodiment may further include 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine. More specifically, provided that the first mutation comprises substituting leucine at position 8 with cysteine, the second mutation comprises substituting serine at position 89 with cysteine, the third mutation comprises substituting serine at position 17 with cysteine, and the fourth mutation comprises substituting serine at position 60 with cysteine, or the first mutation comprises substituting leucine at position 8 with cysteine, the second mutation comprises substituting serine at position 89 with cysteine, the third mutation comprises substituting threonine at position 39 with cysteine, and the fourth mutation comprises substituting aspartate at position 67 with cysteine, the protein of this embodiment may further comprise 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 position 18 is substituted with isoleucine, and a mutation in which leucine at position 19 is substituted with glutamine. ,

[0079] Preferably, the protein according to this embodiment comprises the amino acid sequence shown in SEQ ID NO:27 or 28.

[0080] The protein of this embodiment may be prepared by genetic engineering or chemical methods, such as solid-phase synthesis or solution synthesis, followed by isolation or purification of the protein of this embodiment by methods such as ammonium sulfate or ethanol precipitation, acid extraction, ion exchange chromatography, affinity chromatography, or lectin chromatography, preferably by high performance liquid chromatography.

[0081] The protein according to this embodiment may further have a hydrophilic group to enhance water solubility or circulatory half-life. The hydrophilic group may be linked to the N-terminus of the protein. Preferably, the hydrophilic group is polyethylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyvinyl alcohol, or dextran. More preferably, the hydrophilic group is polyethylene glycol composed of 2 to 40 overlapping ethylene glycol units.

[0082] The protein according to this embodiment may further comprise a purification tag to facilitate purification. The purification tag may be linked to the N-terminus or C-terminus of the protein. Preferably, 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 / antitermination protein (NusA-tag), or a small ubiquitin-related modification tag (SUMO-tag).

[0083] 3. Pharmaceutical Compositions The pharmaceutical composition according to the second embodiment of the present invention comprises the protein according to the first embodiment, and can be administered to an individual to bind to a specific protein, thereby acting as an antagonist of the specific protein and thereby suppressing the activity of the specific protein or inhibiting associated signal transduction. The pharmaceutical composition according to this embodiment comprises the protein according to the first embodiment and a pharmaceutically acceptable carrier.

[0084] The pharmaceutically acceptable carrier allows the pharmaceutical composition to be in different forms or to be administered by different routes. Preferably, the pharmaceutical composition is an oral dosage form, an injection dosage form, an inhalation dosage form, or a topical or transdermal dosage form, and is used for different routes of administration. Preferably, the pharmaceutical composition is a tablet, capsule, granule, powder, solution, syrup, suspension, or emulsion.

[0085] The pharmaceutically acceptable carrier may be an excipient, filler, diluent, flocculating agent, adhesive, lubricant, flow agent, stabilizer, colorant, moisturizer, or disintegrant. Examples of 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 adhesives 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, glycerol behenate, or hydrogenated vegetable oil; examples of flow agents may be aluminosilicate, calcium silicate, microcrystalline cellulose, corn starch, sodium benzoate, calcium carbonate, magnesium carbonate, talc, calcium stearate, magnesium stearate, zinc stearate, magnesium lauryl sulfate, or magnesium oxide; examples of stabilizers may be citric acid or ascorbic acid; examples of colorants may be titanium dioxide or iron oxide; and examples of moisturizers may be Pluronic F68 (Pluronic F68®), Polysorbate 20 (Tween 20), or Polysorbate 80 (Tween 80), and examples of disintegrants are potato starch, tapioca starch, or silicates.

[0086] 4. Medicinal Use A third embodiment of the present invention discloses the use of the pharmaceutical composition of the second embodiment for preparing a medicament for treating or preventing a disease or disorder caused by or related to the activity or signal transduction of vascular endothelial growth factor receptor 2. The prepared medicament can be administered to an individual to suppress the activity or inhibit the signal transduction of vascular endothelial growth factor receptor 2. That is, by administering the prepared medicament to an individual in need of treatment or prevention of a disease or disorder caused by or related to the activity or signal transduction of vascular endothelial growth factor receptor 2, the activity or signal transduction of vascular endothelial growth factor receptor 2 in the individual can be suppressed, thereby achieving a therapeutic or preventive effect.

[0087] Different modes of administration may be used, for example oral administration, injection administration, inhalation administration, or topical or transdermal administration.

[0088] Diseases or disorders caused by or associated with vascular endothelial growth factor receptor 2 activity or signaling may include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer. Preferably, the cancer includes kidney cancer, spleen 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.

[0089] A method for treating or preventing a disease or disorder caused by or associated with vascular endothelial growth factor receptor 2 activity or signal transduction according to a fourth embodiment of the present invention comprises administering the pharmaceutical composition according to the second embodiment to an individual in need of treatment or prevention, thereby causing the composition to bind to the individual's vascular endothelial growth factor receptor 2 and suppress its activity or inhibit its signal transduction.

[0090] Different modes of administration may be used, for example oral administration, injection administration, inhalation administration, or topical or transdermal administration.

[0091] Diseases or disorders caused by or associated with vascular endothelial growth factor receptor 2 activity or signaling may include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer. Preferably, the cancer includes kidney cancer, spleen 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.

[0092] The fifth embodiment of the present invention discloses the use of the pharmaceutical composition of the second embodiment for preparing a medicament for treating or preventing a disease or disorder caused by angiogenesis. The prepared medicament can be administered to an individual to inhibit angiogenesis. That is, the prepared medicament can be administered to an individual in need of treatment or prevention of a disease or disorder caused by angiogenesis, thereby inhibiting angiogenesis in the individual, thereby achieving a therapeutic or preventive effect.

[0093] Different modes of administration may be used, for example oral administration, injection administration, inhalation administration, or topical or transdermal administration.

[0094] The angiogenesis-induced disease or disorder may include autoimmune disease, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meigs' syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer. Preferably, the cancer includes kidney cancer, spleen 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.

[0095] A method for treating or preventing a disease or disorder caused by angiogenesis according to a sixth embodiment of the present invention comprises administering the pharmaceutical composition of the second embodiment to an individual in need of treatment or prevention, thereby causing the composition to bind to vascular endothelial growth factor receptor 2 in the individual and inhibiting angiogenesis.

[0096] Different modes of administration may be used, for example oral administration, injection administration, inhalation administration, or topical or transdermal administration.

[0097] The angiogenesis-induced disease or disorder may include autoimmune disease, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meigs' syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer. Preferably, the cancer includes kidney cancer, spleen 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.

[0098] V. Other matters A nucleic acid according to a seventh embodiment of the present invention comprises a nucleotide sequence encoding a protein according to the first embodiment. To regulate expression of the protein, the nucleic acid may further comprise a promoter, which is a nucleotide sequence operably linked to the encoded protein. As used herein, "operably linked" refers to a functional relationship between two or more nucleic acid sequences.

[0099] A host cell according to an eighth embodiment of the present invention comprises the nucleic acid according to the seventh embodiment. Because the host cell according to this embodiment comprises a nucleotide sequence encoding a protein, the protein can be produced by culturing the host cell. The host cell may be a prokaryotic cell or a eukaryotic cell. An example of a prokaryotic cell may be E. coli, and an example of a eukaryotic cell may be a CHO cell, a COS cell, or a HEK293 cell.

[0100] A ninth embodiment of the present invention provides a method for preparing the protein of the first embodiment, which comprises culturing a host cell according to the eighth embodiment so that the protein is expressed. Depending on the promoter, the cell can be induced to express the protein by selecting an appropriate inducer.

[0101] The following examples are provided to illustrate the invention. <Example 1: Design for introducing disulfide bonds> As shown in Figure 1, the C7-NM protein has eight β-strands, with one loop structure formed between two adjacent β-strands. Using the C7-NM protein as the parent protein and the Disulfide by Design 2.0 (DbD2) software, disulfide bonds were designed to be introduced into the parent protein structure by referring to parameters such as the B factor, χ3 torsion angle, and energy. As shown in Figure 2 and Table 1, disulfide bonds can be designed to be located between two β-strands, between two loops, and between one β-strand and one loop of the parent protein. As shown in Table 2, the amino acid sequences of mutant proteins with disulfide bonds are shown. These are obtained by substituting amino acids based on the designed disulfide bond positions using the amino acid sequence of the C7 protein as a reference sequence.

[0102] Table 1. Crystallographic parameters JPEG2026500602000002.jpg71170

[0103] Table 2, Amino acid sequences JPEG2026500602000003.jpg236170Note 1: The letters in the box indicate the sequence of the mutations relative to the protein 10Fn3-WT. Note 2: The underlined letters are the sequences of mutations to protein C7.

[0104] Example 2: Protein preparation Protein C7 and its mutant proteins are expressed in E. coli. In other words, protein C7 and its mutant proteins are obtained by expressing and preparing them in the BL21(DE3) pLysS strain or the SHuffle strain using pET21a as a carrier, with the BL21(DE3) pLysS strain culture medium being LB medium, and the SHuffle strain culture medium being LLB medium.

[0105] First, the BL21(DE3) pLysS strain is cultured in 5 mL of culture medium at 37°C for approximately 16-18 hours (the SHuffle strain is cultured at 30°C). The BL21(DE3) pLysS strain is transferred to 500 mL of culture medium and cultured at 37°C for approximately 4 hours (the SHuffle strain is cultured at 30°C for approximately 6 hours). Next, 500 μL of IPTG (1 M concentration) is added, and the BL21(DE3) pLysS strain is induced to express protein at 25°C for approximately 16-18 hours (the SHuffle strain is induced at 16°C for approximately 48 hours). The cell pellet is then collected by centrifugation.

[0106] The bacteria were lysed in binding buffer A (50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0) and the cells were ruptured using a French press (maintaining a pressure of 1500 psi). The supernatant was collected by high-speed centrifugation and loaded onto a nickel ion column pre-equilibrated with buffer A. The target protein was eluted using a gradient of elution buffer (300 mM imidazole in buffer A, pH 7.0). After elution, the target protein was confirmed by glycine SDS-PAGE. Finally, the recombinant protein was dialyzed against PBS and stored at -80°C until use.

[0107] Example 3: Analysis of protein properties The melting point temperature of the protein was measured by differential scanning calorimetry (DSC). As shown in Table 3 and Figure 3, the difference in melting point temperature between protein C7-SL1 and protein C7 was 37.0°C, which is higher than the difference in melting point temperature between protein C7 and other proteins having one disulfide bond, indicating the high thermal stability of protein C7-SL1. As shown in Table 3 and Figures 3 and 4, proteins C7-SL1-LL2 and C7-SL1-LL3, which were obtained by introducing another disulfide bond while mainly using protein C7-SL1, have even improved thermal stability.

[0108] See J Pharm Sci. 2008 Oct;97(10):4155-66, and measure the protein solubility using the ammonium sulfate precipitation method. As shown in Table 3, the PBS solubility of protein C7-SL1 is approximately six times that of protein C7, which is higher than that of other proteins with one disulfide bond, indicating the superior solubility of protein C7-SL1. As shown in Table 3 and Figure 5, proteins C7-SL1-LL2 and C7-SL1-LL3, which are based on protein C7-SL1 but have additional disulfide bonds introduced, have PBS solubility that is further improved to approximately seven times that of protein C7.

[0109] Using cell proliferation experiments, the inhibitory activity of proteins on the proliferation of human umbilical vein endothelial cells (HUVEC) was analyzed. 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 based on protein C7-SL1 but have been obtained by introducing other disulfide bonds, on HUVEC proliferation was 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.

[0110] The binding affinity of proteins to vascular endothelial growth factor receptor 2 was analyzed using enzyme-linked immunosorbent assay (ELISA). As shown in Table 3, the binding affinity of protein C7-SL1 to vascular endothelial growth factor receptor 2 was not significantly different from that of protein C7, indicating that protein C7-SL1 has the ability to bind to vascular endothelial growth factor receptor 2. As shown in Table 3 and Figure 6, the binding affinity of proteins C7-SL1-LL2 and C7-SL1-LL3, which are derived from protein C7-SL1 but have additional disulfide bonds introduced, was also not significantly different from that of protein C7. Furthermore, the binding affinity of protein C7-SL1-LL2 to vascular endothelial growth factor receptor 2 was slightly greater than that of protein C7, indicating that proteins C7-SL1-LL2 and C7-SL1-LL3 also have the ability to bind to vascular endothelial growth factor receptor 2.

[0111] Table 3. Protein characteristics JPEG2026500602000004.jpg88170Note 1: ΔTm = Tm value of test protein - Tm value of protein C7 Note 2: ND indicates that measurement was not possible, due to formation of inclusion bodies or measurement not being performed.

[0112] The above-described embodiments are intended to facilitate understanding of the present invention and are not intended to limit the present invention. The present invention may be modified or improved without departing from the spirit thereof, and it goes without saying that the present invention includes equivalents thereof.

Claims

1. A protein comprising a 10th human fibronectin type III domain, and a first mutation in which an amino acid residue is substituted with a cysteine ​​and a second mutation in which another amino acid residue is substituted with another cysteine, wherein the first mutation and the second mutation occur in a region other than the loop FG of the 10th human fibronectin type III domain, such that the cysteine ​​substituted by the first mutation and the cysteine ​​substituted by the second mutation form a disulfide bond.

2. The protein of claim 1, further comprising a mutation in which the amino acid sequence of loop BC is replaced with RHPHFPTRY, a mutation in which the amino acid sequence of loop DE is replaced with PLQPPT, and a mutation in which the amino acid sequence of loop FG is replaced with VTDGRNGRLLSIP.

3. the first mutation comprises a substitution of leucine at position 8 with cysteine, the second mutation comprises a substitution of tryptophan at position 22 with cysteine, the first mutation comprises a substitution of leucine at position 8 with cysteine, the second mutation comprises a substitution of serine at position 89 with cysteine, the first mutation comprises a substitution of serine at position 17 with cysteine, the second mutation comprises a substitution of serine at position 60 with cysteine, the first mutation comprises a substitution of leucine at position 19 with cysteine, the second mutation comprises a substitution of threonine at position 58 with cysteine, the first mutation comprises a substitution of isoleucine at position 34 with cysteine, the second mutation comprises a substitution of phenylalanine at position 48 with cysteine, the first mutation comprises a substitution of threonine at position 35 with cysteine, and the second mutation comprises a substitution of the first mutation comprises a substitution of tyrosine at position 36 with cysteine; the second mutation comprises a substitution of isoleucine at position 70 with cysteine; the first mutation comprises a substitution of glycine at position 37 with cysteine; the second mutation comprises a substitution of valine at position 45 with cysteine; the first mutation comprises a substitution of threonine at position 39 with cysteine; the second mutation comprises a substitution of aspartate at position 67 with cysteine; the first mutation comprises a substitution of lysine at position 63 with cysteine; the second mutation comprises a substitution of valine at position 66 with cysteine; or the first mutation comprises a substitution of aspartate at position 67 with cysteine; and the second mutation comprises a substitution of aspartic acid at position 91 with cysteine.

4. The protein described in claim 1, further comprising a third mutation in which another amino acid residue is substituted with another cysteine, and a fourth mutation in which yet another amino acid residue is substituted with yet another cysteine, wherein the third mutation and the fourth mutation occur in a region other than the loop FG of the 10th human fibronectin type III domain, so that the cysteine ​​substituted by the third mutation and the cysteine ​​substituted by the fourth mutation form a disulfide bond.

5. The human fibronectin type III domain further includes a third mutation in which another amino acid residue is substituted with another cysteine, and a fourth mutation in which yet another amino acid residue is substituted with yet another cysteine, and the third mutation and the fourth mutation occur in a region other than the loop FG of the tenth human fibronectin type III domain, so that the cysteine ​​substituted by the third mutation and the cysteine ​​substituted by the fourth mutation form a disulfide bond, the first mutation includes a substitution of leucine at position 8 with cysteine, and the second mutation includes a substitution of serine at position 89 with cysteine. the third mutation comprises substituting serine at position 17 with cysteine, and the fourth mutation comprises substituting serine at position 60 with cysteine; or the first mutation comprises substituting leucine at position 8 with cysteine, the second mutation comprises substituting serine at position 89 with cysteine, the third mutation comprises substituting threonine at position 39 with cysteine, and the fourth mutation comprises substituting aspartate at position 67 with cysteine.

6. The protein according to claim 1, characterized in that it comprises an amino acid sequence shown in any one of SEQ ID NOs: 4 to 16.

7. The protein of claim 1, comprising the amino acid sequence set forth in SEQ ID NO: 11, 15, or 16.

8. A protein according to claim 1; and a pharmaceutically acceptable carrier.

9. Use of the pharmaceutical composition described in claim 1, characterized in that it is used to prepare a medicine for treating or preventing diseases or disorders caused by or associated with the activity or signal transduction of vascular endothelial growth factor receptor 2.

10. 10. Use of the pharmaceutical composition according to claim 1, characterized in that it is used for preparing a medicament for treating or preventing a disease or disorder caused by angiogenesis.

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