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 fibronectin type III domain-derived proteins and their applications, including methods for treating or preventing diseases or disorders caused by or related to the activity or signaling of vascular endothelial growth factor receptor 2 (VEGFR2), or methods for treating or preventing diseases or disorders caused by angiogenesis. [Background technology]

[0002] In recent years, in order to overcome the inherent limitations of using immunoglobulins as the main component of molecular identification modules, such as their excessive size, complex heterodimer structure, and the need for precise disulfide bond formation, efforts have been focused on developing scaffold protein-based molecular identification modules to replace those composed of immunoglobulins. The fundamental theory of such protein engineering is to develop molecules with high affinity and specificity to antibodies through 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 actively under development and being tested for disease treatment, although many are still in clinical trials.

[0004] 10Fn3 possesses many features superior to those of systems derived from immunoglobulins. Based on a global β-sandwich fold, 10Fn3 belongs to the immunoglobulin superfamily, and its three surface loop regions near the N-terminus are structurally similar to the three antigen-recognition loop regions or complementarity-determining regions (CDRs) of immunoglobulin variants. However, unlike typical immunoglobulin regions, 10Fn3 does not contain disulfide bonds. Furthermore, 10Fn3 has a thermal transition temperature above 80°C and is structurally stable. In addition, 10Fn3 is reversible and rapidly unfolds and refolds. 10Fn3 has approximately 94 amino acid residues, which is smaller than the antigen-binding unit (VHH) of heavy chain antibodies. Due to the above characteristics, 10Fn3 is suitable for different molecular display systems and simple and effective production methods.

[0005] U.S. Patent Application No. 11 / 448, 171 (titled "Inventors of Type 2 Vascular Endothelial Growth Factor Receptors") discloses the vascular endothelial growth factor receptor 2 binding protein C7. Protein C7 is primarily composed of 10Fn3 and has a sequence of three wild-type surface loop regions. 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 mutant regions is substituted by VTDGRNGRLLSIP. By making the three mutant regions mentioned above the binding residues of vascular endothelial growth factor receptor 2, protein C7 can be made an antagonist of vascular endothelial growth factor receptor 2 and has the potential to become an anticancer drug. However, its poor thermal stability and low solubility are hindering its development as a drug.

[0006] Therefore, developing a scaffold protein whose structure is primarily composed of 10Fn3, which has high thermal stability and solubility, is a problem that those in the technical field to which this invention belongs are actively aiming to solve. [Overview of the project] [Problems that the invention aims to solve]

[0007] Conventionally, mutant protein C7-NM has been obtained by modifying protein C7. This invention is completed based on the following: Disulfide by Design 2.0 (DbD2) software is used, along with B factor and χ². 3 Twist angle (χ 3 By designing a system that introduces a disulfide bond into the structure of protein C7-NM by referencing parameters such as angle and energy, it is possible to obtain a mutant with a disulfide bond. This allows for increased thermal stability and solubility without affecting the activity of binding to specific proteins, thus giving it the potential to become a biopharmaceutical.

[0008] The protein according to the present invention includes a 10th human fibronectin type III domain and includes 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. Since the first and second mutations occur in regions 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 10th human fibronectin type III domain contains the amino acid sequence shown in SEQ ID NO:1.

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

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

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

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

[0014] For example, the first mutation includes substitution of leucine at position 8 with cysteine, serine at position 17 with cysteine, leucine at position 19 with cysteine, isoleucine at position 34 with cysteine, threonine at position 35 with cysteine, tyrosine at position 36 with cysteine, glycine at position 37 with cysteine, threonine at position 39 with cysteine, lysine at position 63 with cysteine, or aspartate at position 67 with cysteine, and the second Mutations include substitution of tryptophan at position 22 with cysteine, valine at position 45 with cysteine, phenylalanine at position 48 with cysteine, 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.

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

[0016] For example, a protein contains an amino acid sequence shown in one of the following SEQ ID NOs:4-14.

[0017] For example, the protein contains 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 substitution of leucine at position 19 with cysteine, the protein comprises a mutation of substitution of alanine at position 12 with glutamic acid, 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 glutamine.

[0019] For example, provided that 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 aspartate at position 67 with cysteine, and 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 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 comprises a mutation of substitution of alanine at position 12 with glutamic acid, 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 glutamine.

[0020] For example, the first mutation comprises substitution of leucine at position 8 with cysteine, the second mutation comprises substitution of tryptophan at position 22 with 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 first mutation comprises substitution of serine at position 17 with cysteine, the second mutation comprises substitution of serine at position 60 with cysteine, the first mutation comprises substitution of isoleucine at position 34 with cysteine, the second mutation comprises substitution of phenylalanine at position 48 with cysteine, the first mutation comprises substitution of threonine at position 35 with cysteine, the second mutation comprises substitution of valine at position 45 with cysteine, the first mutation comprises substitution of tyrosine at position 36 with cysteine, the second mutation comprises substitution of isoleucine at position 70 with cysteine, the first mutation comprises substitution of glycine at position 37 with cysteine, the second mutation comprises substitution of valine at position 45 with cysteine, the first mutation comprises substitution of threonine at position 39 with cysteine, the second mutation comprises substitution of aspartate at position 67 with cysteine, the first mutation comprises substitution of lysine at position 63 with cysteine, the second mutation comprises substitution of valine at position 66 with cysteine, or under the condition that the first mutation comprises substitution of aspartate at position 67 with cysteine and the second mutation comprises substitution of aspartic acid at position 91 with cysteine, the protein comprises a mutation of substitution of alanine at position 12 with glutamic acid, 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 glutamine.

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

[0022] For example, the protein further contains a third mutation in which other amino acid residues are substituted with other cysteines, and a fourth mutation in which yet another amino acid residues are substituted with yet another cysteine. Since the third and fourth mutations occur in regions other than the loop FG of the 10th human fibronectin type III domain, 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 one of the following β-strands of the 10th human fibronectin type III domain: β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loops AB, loops BC, loops CD, loops DE, and loops EF. The fourth mutation occurs in one of the following β-strands of the 10th human fibronectin type III domain: β-strand A, β-strand B, β-strand C, β-strand D, β-strand E, β-strand F, β-strand G, loops AB, loops BC, loops CD, loops DE, and loops EF.

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

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

[0026] For example, the third mutation includes substitution of leucine at position 8 with cysteine, serine at position 17 with cysteine, leucine at position 19 with cysteine, isoleucine at position 34 with cysteine, threonine at position 35 with cysteine, tyrosine at position 36 with cysteine, glycine at position 37 with cysteine, threonine at position 39 with cysteine, lysine at position 63 with cysteine, or aspartate at position 67 with cysteine, and the fourth mutation. Mutations include substitution of tryptophan at position 22 with cysteine, valine at position 45 with cysteine, phenylalanine at position 48 with cysteine, 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.

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

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

[0029] For example, under conditions where the first, second, third, and fourth mutations do not involve the 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 includes substitution of leucine at position 8 with cysteine, serine at position 17 with cysteine, isoleucine at position 34 with cysteine, threonine at position 35 with cysteine, tyrosine at position 36 with cysteine, glycine at position 37 with cysteine, threonine at position 39 with cysteine, lysine at position 63 with cysteine, or aspartate at position 67 with cysteine, and the second mutation includes substitution of a triple at position 22. This includes substitution of tophan with cysteine, substitution of valine at position 45 with cysteine, substitution of phenylalanine at position 48 with cysteine, 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, and the third accretionary mutation. The mutations include substitution of leucine at position 8 with cysteine, serine at position 17 with cysteine, isoleucine at position 34 with cysteine, threonine at position 35 with cysteine, tyrosine at position 36 with cysteine, glycine at position 37 with cysteine, threonine at position 39 with cysteine, lysine at position 63 with cysteine, or aspartate at position 67 with cysteine, and the fourth mutation is that tryptophan at position 22 is replaced with cysteine Under conditions including substitution with , substitution of valine at position 45 with cysteine, substitution of phenylalanine at position 48 with cysteine, 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 isThis includes mutations where alanine at position 12 is replaced by glutamate, where threonine at position 14 is replaced by serine, where leucine at position 18 is replaced by isoleucine, and where leucine at position 19 is replaced by glutamine.

[0031] For example, under the condition that the first mutation includes the substitution of leucine at position 8 with cysteine, the second mutation includes the substitution of serine at position 89 with cysteine, the third mutation includes the substitution of serine at position 17 with cysteine, and the fourth mutation includes the substitution of serine at position 60 with cysteine, or under the condition that the first mutation includes the substitution of leucine at position 8 with cysteine, the second mutation includes the substitution of serine at position 89 with cysteine, the third mutation includes the substitution of threonine at position 39 with cysteine, and the fourth mutation includes the substitution of aspartate at position 67 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.

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

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

[0034] The protein according to the present invention is primarily composed of the 10th human fibronectin type III domain, and its thermal stability and solubility are enhanced by introducing a disulfide bond at a specific position in the main body. The protein according to the present invention has the potential to be used as a receptor antagonist and a biopharmaceutical by further utilizing amino acid sequence substitutions to form a protein binding interface and bind to specific proteins such as DLL4, EGFR, VEGFR2, or IGF-1R.

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

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

[0037] The present invention further provides the use of the above-described pharmaceutical compositions for preparing a medicament for treating or preventing a disease or disorder caused by or related to the activity or signaling of vascular endothelial growth factor receptor 2.

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

[0039] For example, 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 diseases or disorders caused by or related to the activity or signaling of vascular endothelial growth factor receptor 2, comprising administering the above-described pharmaceutical composition to an individual in need, thereby binding to the individual's vascular endothelial growth factor receptor 2 and suppressing its activity.

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

[0042] For example, 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 a pharmaceutical composition used to prepare a medicine for treating or preventing a disease or disorder caused by angiogenesis.

[0044] For example, diseases or disorders resulting from neovascularization include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meggs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer.

[0045] For example, 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 diseases or disorders caused by angiogenesis, comprising administering the above-described pharmaceutical composition to an individual in need, thereby binding to the individual's vascular endothelial growth factor receptor 2 and suppressing angiogenesis.

[0047] For example, diseases or disorders resulting from neovascularization include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meggs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, malignant fibrosis, or cancer.

[0048] For example, 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 nucleic acids comprising a nucleotide sequence for encoding the aforementioned protein.

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

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

[0052] For example, prokaryotic cells include E. coli, while eukaryotic cells include CHO cells, COS cells, or HEK293 cells.

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

[0054] [Figure 1] This is a structural diagram of the protein C7-NM, showing its three-dimensional structure. [Figure 2] This is a structural diagram of the protein C7-NM, showing the positions in its three-dimensional structure where disulfide bonds can be designed. [Figure 3] This is a differential scanning calorimetry (DSC) graph comparing the melting points of protein C7 and its variant proteins. [Figure 4] This is a differential scanning calorimetry (DSC) result chart comparing the melting points of proteins C7 and C7-SL1-LL2. [Figure 5] This is a solubility chart comparing the solubility of proteins C7 and C7-SL1-LL2. [Figure 6] This graph shows the results of an enzyme-linked immunosorbent assay comparing the affinity of proteins C7 and C7-SL1-LL2 to vascular endothelial growth factor receptor 2. MODES FOR CARRYING OUT THE INVENTION

[0055] In order to further clearly understand the above and / or other objects, effects and features of the present invention, preferred embodiments will be given and described in detail below. 1. Definition of Terms Unless otherwise specifically defined, the term "protein" as used herein includes wild-type proteins expressed in natural cells, recombinant proteins expressed by genetic engineering techniques, or 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. Unless otherwise specifically defined, the term "amino acid" as used herein includes D-amino acids or L-amino acids. D- and L- indicate the absolute configuration of an amino acid, and do not refer to a specific rotation direction of plane-polarized light. Unless otherwise specified, amino acids are represented herein by abbreviations recommended by the IUPAC-IUB Biochemical Nomenclature Commission. A protein sequence is represented by a character string composed of a plurality of abbreviations, and the order of the abbreviations corresponds to the order from the N-terminus to the C-terminus of the amino acids in the protein. When a superscript number precedes an abbreviation, it indicates the order corresponding to the position of the amino acid 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 can be inferred by analogy, so the description will not be repeated.

[0056] Substitutions, deletions, and / or insertions of protein sequences can occur in non-skeletal regions of a protein and usually do not affect its original activity. Furthermore, protein sequence substitutions may include substitutions of conserved amino acids, which are substitutions between amino acids having similar properties or related side chains. Examples of substitutions between amino acids with similar properties include: acidic amino acids (aspartate, glutamate); alkaline amino acids (lysine, arginine, and histidine); and nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan). Polar uncharged amino acids are mutually substitutable, namely glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Substitutions between amino acids with related side chains include, for example, aliphatic hydroxyl group amino acids are mutually substitutable, namely serine and threonine. Amino acids with amide-containing groups are mutually substitutable, namely aspartic acid and glutamine. Aliphatic amino acids are mutually substitutable, namely alanine, valine, leucine, and isoleucine. Aromatic amino acids are mutually substitutable, namely phenylalanine, tryptophan, and tyrosine.

[0057] In this specification, the "10th human fibronectin type III domain" refers to a domain between the N-terminus and the C-terminus that sequentially includes the N-terminus 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-terminus region, having at least 94 amino acids, and lacking any disulfide bonds; for example, having SEQ ID NO:1. Referring to U.S. Patent Application No. 13 / 757, 664, with the invention title "FIBRONECTIN BINDING DOMAINS WITH REDUCED IMMUNOGENICITY", the N-terminal region is defined as an amino acid fragment from position 1 to 7, β-strand A is defined as an amino acid fragment from position 8 to 13, loop AB is defined as an amino acid fragment from position 14 to 17, β-strand B is defined as an amino acid fragment from position 18 to 22, loop BC is defined as an amino acid fragment from position 23 to 31, β-strand C is defined as an amino acid fragment from position 32 to 36, loop CD is defined as an amino acid fragment from position 37 to 47, and β-strand D Loop BC is defined as the amino acid fragment from position 48 to 50, loop DE is defined as the amino acid fragment from position 51 to 56, β-strand E is defined as the amino acid fragment from position 57 to 62, loop EF is defined as the amino acid fragment from position 63 to 67, β-strand F is defined as the amino acid fragment from position 68 to 74, loop FG is defined as the amino acid fragment from position 75 to 87, β-strand G is defined as the amino acid fragment from position 88 to 92, and the C-terminal region is defined as the amino acid fragment at positions 93 and 94. Loops BC, DE, and FG are located on one side of the molecule, while loops AB, CD, and EF are located on the other side of the molecule. For example, the N-terminal region is 1It contains VSDVPRD, and β-strand A 8 Including LEVVAA, loop AB is 14 Including TPTS, β-strand B is 18 Including LLISW, loop BC is 23 It contains DAPAVTVRY, and β-strand C. 32 The loop CD includes YRITY. 37 GETGGNSPVQE is included, and β-strand D is, 48 Including FTV, Loop DE is 51 PGSKST is included, and β-strand E is, 57 Including ATISGL, the loop EF is, 63 It contains KPGVD, and β-strand F is 68 Including YTITVYA, Loop FG, 75 VTGRGDSPASSKP is included, and β-strand G is also included. 88 Including ISINY, the C-end region is 93 Includes RTs.

[0058] In this specification, "protein C7" refers to a variant of the 10th human fibronectin type III domain, unless otherwise specified, that can bind to and inhibit the activity of vascular endothelial growth factor receptor 2, for example, SEQ ID NO:2. Incidentally, the amino acid sequence corresponding to the wild-type loop BC is: 23 The amino acid sequence corresponding to the wild-type loop DE is RHPHFPTRY, 51 PLQPPT, and the amino acid sequence corresponding to the wild-type loop FG is: 75 This is VTDGRNGRLLSIP. The three mutant regions mentioned above constitute the binding interface for vascular endothelial growth factor receptor 2.

[0059] In this specification, "protein C7-NM" refers to a variant of protein C7 unless otherwise specified, for example, SEQ ID NO:3. Incidentally, the amino acid corresponding to position 12 of the reference sequence is glutamic acid, the amino acid corresponding to position 14 is serine, the amino acid corresponding to position 18 is isoleucine, and the amino acid corresponding to position 19 is glutamine.

[0060] In this specification, "vascular endothelial growth factor receptor 2" refers to transmembrane receptor tyrosine kinase unless otherwise specified, and is capable of regulating angiogenesis caused 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, “treatment” means, unless otherwise specified, any therapeutic intervention to treat or improve a disease, including treating or improving it completely or partially.

[0062] In this specification, "prevention" means completely or nearly completely preventing a disease, unless otherwise specified. For example, if a disease is absent or suspected but has not yet manifested, preventive intervention can prevent the onset of the disease.

[0063] As used herein, “medically acceptable carrier” means, unless otherwise specified, an additive that is within the bounds of sound medical judgment, suitable for contact with individuals, free from excessive toxicity, irritation, allergic reactions, or other problems or complications, and possessing a reasonable benefit / risk ratio, such as fillers, diluents, flocculants, adhesives, lubricants, fluidizers, stabilizers, colorants, humectants, or disintegrants.

[0064] 2. Proteins primarily composed of the 10th human fibronectin type III domain. The protein according to the first embodiment of the present invention is primarily composed of the 10th human fibronectin type III domain, and its thermal stability and solubility are improved by introducing disulfide bonds to substitute amino acids at specific positions in the main body. Based on the above-mentioned high thermal stability and high solubility, as well as the inherent properties of the 10th human fibronectin type III domain, a protein binding interface can be constructed by further utilizing amino acid substitutions, for example, 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 biopharmaceutical.

[0065] The protein according to this embodiment contains the 10th human fibronectin type III domain and includes 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. Since the first and second mutations occur in regions 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. Preferably, the 10th human fibronectin type III domain contains the amino acid sequence shown in SEQ ID NO:1.

[0066] Regarding this tenth human fibronectin type III domain region, 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 and second mutations can occur in the same one or two different regions from the above group. For example, both the first and second mutations can occur in loops CD or loops EF. For example, the first mutation can occur in β-strand A and the second mutation in β-strand B. The first mutation can occur in β-strand A and the second mutation in β-strand G. The first mutation can occur in loops AB and the second mutation in β-strand E. The first mutation can occur in β-strand B and the second mutation in β-strand E. The first mutation can occur in β-strand C and the second mutation in β-strand D. The first mutation can occur in β-strand C and the second mutation in loops CD. The first mutation can occur in β-strand C and the second mutation in β-strand F. The first mutation may occur in loop CD, and the second mutation may occur in loop EF. Alternatively, the first mutation may occur in loop EF, and the second mutation may occur in β-strand G.

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

[0069] The protein according to this embodiment may further include a third mutation in which other amino acid residues are substituted with other cysteines, and a fourth mutation in which yet another amino acid residues are substituted with yet another cysteine. Since the third and fourth mutations occur in regions other than the loop FG of the 10th human fibronectin type III domain, the cysteine ​​substituted by the third mutation and the cysteine ​​substituted by the fourth mutation form a disulfide bond. Under conditions containing at least two disulfide bonds, the protein can further improve its thermal stability and solubility.

[0070] According to the region of the 10th 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, second, third, and fourth mutations can occur in four different regions from the above group. For example, the first mutation may occur in β-strand A, the second mutation in β-strand G, the third mutation in loops AB, and the fourth mutation in β-strand E; or the first mutation may occur in β-strand A, the second mutation in β-strand G, the third mutation in loops CD, and the fourth mutation in loops EF.

[0072] According to the amino acid sequence of the 10th human fibronectin type III domain, the third mutation involves the substitution of leucine at position 8 with cysteine, serine at position 17 with cysteine, leucine at position 19 with cysteine, isoleucine at position 34 with cysteine, threonine at position 35 with cysteine, tyrosine at position 36 with cysteine, glycine at position 37 with cysteine, threonine at position 39 with cysteine, lysine at position 63 with cysteine, or aspartate at position 67 with cysteine. The fourth mutation may include substitution, and may include substitution of tryptophan at position 22 with cysteine, valine at position 45 with cysteine, phenylalanine at position 48 with cysteine, 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. Specifically, the first mutation may include the substitution of leucine at position 8 with cysteine, the second mutation may include the substitution of serine at position 89 with cysteine, the third mutation may include the substitution of serine at position 17 with cysteine, the fourth mutation may include the substitution of serine at position 60 with cysteine, or the first mutation may include the substitution of leucine at position 8 with cysteine, the second mutation may include the substitution of serine at position 89 with cysteine, the third mutation may include the substitution of threonine at position 39 with cysteine, and the fourth mutation may include the 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. In order to bind to VEGFR2, the protein according to this embodiment may further include mutations in which the amino acid sequence of loop BC is replaced with RHPHFPTRY, the amino acid sequence of loop DE is replaced with PLQPPT, and the amino acid sequence of loop FG is replaced with VTDGRNGRLLSIP.

[0074] Preferably, the protein according to this embodiment contains 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 contains the amino acid sequence shown in SEQ ID NO: 11, 15, or 16.

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

[0076] Under conditions where the first and second mutations do not involve the substitution of leucine at position 19 with cysteine, the protein according to 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 substitution of leucine at position 8 with cysteine, serine at position 17 with cysteine, isoleucine at position 34 with cysteine, threonine at position 35 with cysteine, tyrosine at position 36 with cysteine, glycine at position 37 with cysteine, threonine at position 39 with cysteine, lysine at position 63 with cysteine, or aspartate at position 67 with cysteine. The second mutation includes substitution of tryptophan at position 22 with cysteine, valine at position 45 with cysteine, and phenylalanine at position 48 with cysteine. In addition, 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 according to 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, the first mutation includes the substitution of leucine at position 8 with cysteine, the second mutation includes the substitution of tryptophan at position 22 with cysteine, the first mutation includes the substitution of leucine at position 8 with cysteine, the second mutation includes the substitution of serine at position 89 with cysteine, the first mutation includes the substitution of serine at position 17 with cysteine, the second mutation includes the substitution of serine at position 60 with cysteine, The first mutation includes substitution of isoleucine at position 34 with cysteine, the second mutation includes substitution of phenylalanine at position 48 with cysteine, the first mutation includes substitution of threonine at position 35 with cysteine, the second mutation includes substitution of valine at position 45 with cysteine, the first mutation includes substitution of tyrosine at position 36 with cysteine, and the second mutation includes substitution of isoleucine at position 70 with cysteine. The first mutation includes substitution of glycine at position 37 with cysteine, the second mutation includes substitution of valine at position 45 with cysteine, the first mutation includes substitution of threonine at position 39 with cysteine, the second mutation includes substitution of aspartate at position 67 with cysteine, the first mutation includes substitution of lysine at position 63 with cysteine, the second mutation includes substitution of valine at position 66 with cysteine, or Under the condition that the first mutation includes the substitution of aspartate at position 67 with cysteine, and the second mutation includes the substitution of aspartic acid at position 91 with cysteine, the protein according to 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.

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

[0078] Under conditions where the first, second, third, and fourth mutations do not involve the substitution of leucine at position 19 with cysteine, the protein according to 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 substitution of leucine at position 8 with cysteine, serine at position 17 with cysteine, isoleucine at position 34 with cysteine, threonine at position 35 with cysteine, tyrosine at position 36 with cysteine, glycine at position 37 with cysteine, threonine at position 39 with cysteine, lysine at position 63 with cysteine, or aspartate at position 67 with cysteine. The second mutation includes substitution of tryptophan at position 22 with cysteine, valine at position 45 with cysteine, phenylalanine at position 48 with cysteine, threonine at position 58 with cysteine, serine at position 60 with cysteine, valine at position 66 with cysteine, or aspartate at position 67 with cysteine. The third mutation includes substitution 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, and the fourth mutation includes substitution of tryptophan at position 22 with cysteine, substitution of valine at position 45 with cysteine, or substitution of phenylalanine at position 48 with cysteine.In the condition that the threonine at position 58 is replaced by cysteine, the serine at position 60 is replaced by cysteine, the valine at position 66 is replaced by cysteine, the aspartate at position 67 is replaced by cysteine, the isoleucine at position 70 is replaced by cysteine, the serine at position 89 is replaced by cysteine, or the aspartic acid at position 91 is replaced by cysteine, the protein according to this example may further include a mutation in which alanine at position 12 is replaced by glutamic acid, a mutation in which threonine at position 14 is replaced by serine, a mutation in which leucine at position 18 is replaced by isoleucine, and a mutation in which leucine at position 19 is replaced by glutamine. More specifically, the protein according to this embodiment may further include a mutation in which alanine at position 12 is replaced by glutamic acid, a mutation in which threonine at position 14 is replaced by serine, a mutation in which leucine at position 18 is replaced by isoleucine, and a mutation in which leucine at position 19 is replaced by glutamine. ,

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

[0080] The protein according to this embodiment may be prepared by genetic engineering techniques or chemical methods, such as solid-phase synthesis or solution synthesis. Subsequently, the protein according to this embodiment is separated and obtained or purified 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 have hydrophilic groups to further enhance its water solubility or cyclic half-life. The hydrophilic groups may be linked to the N-terminus of the protein. Preferably, the hydrophilic groups are polyethylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyvinyl alcohol, or dextran. More preferably, the hydrophilic groups are polyethylene glycol composed of 2 to 40 overlapping ethylene glycol units.

[0082] The protein according to this embodiment may further contain a purification tag to aid in purification. The purification tag may be ligated 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 / anti-termination protein (NusA-tag), or a low molecular weight ubiquitin-related modifier tag (SUMO-tag).

[0083] 3. Pharmaceutical Compositions The pharmaceutical composition according to the second embodiment of the present invention contains 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 to a specific protein, suppressing the activity of that specific protein or inhibiting related signaling. The pharmaceutical composition according to this embodiment contains the protein according to the first embodiment and a pharmaceutically acceptable carrier.

[0084] Pharmacopoecitable carriers can be used to make the entire pharmaceutical composition into different forms or to apply to different routes of administration. Preferably, the pharmaceutical composition is an oral formulation, an injectable formulation, an inhaled formulation, or a topical or transdermal formulation used for different routes of administration. Preferably, the pharmaceutical composition is a tablet, capsule, granule, powder, solvent, syrup, suspension, or emulsion.

[0085] Pharmacopoecitable carriers may include excipients, fillers, diluents, flocculants, adhesives, lubricants, fluidizers, stabilizers, colorants, humectants, or disintegrants. 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 fluidizers 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; examples of humectants may be Pluronic F68. It may also be polysorbate 20 (Tween 20) or polysorbate 80 (Tween 80), and examples of disintegrants may be potato starch, tapioca starch, or silicates.

[0086] 4. Use of medicine A third embodiment of the present invention illustrates the use of the pharmaceutical composition of the second embodiment, which is used to prepare a medicine for treating or preventing diseases or disorders caused by or related to the activity or signaling of vascular endothelial growth factor receptor 2. By administering the prepared medicine to an individual, the activity of vascular endothelial growth factor receptor 2 can be suppressed or its signaling inhibited. That is, by administering the prepared medicine to an individual who needs to treat or prevent diseases or disorders caused by or related to the activity or signaling of vascular endothelial growth factor receptor 2, a therapeutic or preventive effect can be achieved by suppressing the activity of the individual's vascular endothelial growth factor receptor 2 or inhibiting its signaling.

[0087] Different methods of administration may be used, such as oral administration, injection, inhalation, or topical or transdermal administration.

[0088] Diseases or disorders resulting from 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, Meggs syndrome, pericardial effusion, pleural effusion, diabetes mellitus, endometriosis, malignant fibrosis, or cancer. Preferably, cancer includes renal cancer, splenic 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 related to the activity or signaling of vascular endothelial growth factor receptor 2 according to the 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 binding to the individual's vascular endothelial growth factor receptor 2 and suppressing its activity or inhibiting its signaling.

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

[0091] Diseases or disorders resulting from or associated with the activity or signaling of vascular endothelial growth factor receptor 2 may include autoimmune diseases, heart disease, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meggs syndrome, pericardial effusion, pleural effusion, diabetes mellitus, endometriosis, malignant fibrosis, or cancer. Preferably, cancer includes renal cancer, splenic 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] A fifth embodiment of the present invention illustrates the use of the pharmaceutical composition of the second embodiment, which is used to prepare a medicine for treating or preventing diseases or disorders caused by angiogenesis. By administering the prepared medicine to an individual, angiogenesis can be suppressed. That is, by administering the prepared medicine to an individual in need of treatment or prevention of diseases or disorders caused by angiogenesis, a therapeutic or preventive effect can be achieved by suppressing angiogenesis in the individual.

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

[0094] Diseases or disorders resulting from angiogenesis may include autoimmune diseases, heart diseases, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meggs syndrome, pericardial effusion, pleural effusion, diabetes mellitus, endometriosis, malignant fibrosis, or cancer. Preferably, cancer includes renal cancer, splenic 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 the sixth embodiment of the present invention includes administering the pharmaceutical composition of the second embodiment to an individual in need of treatment or prevention, thereby binding to the individual's vascular endothelial growth factor receptor 2 and suppressing angiogenesis.

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

[0097] Diseases or disorders resulting from angiogenesis may include autoimmune diseases, heart diseases, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid enlargement, chronic inflammation, Meggs syndrome, pericardial effusion, pleural effusion, diabetes mellitus, endometriosis, malignant fibrosis, or cancer. Preferably, cancer includes renal cancer, splenic 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] 5. Other matters The nucleic acid according to the seventh embodiment of the present invention comprises a nucleotide sequence for encoding the protein according to the first embodiment. To regulate protein expression, the nucleic acid may further contain a promoter, which is a nucleotide sequence that is operably linked to the coding protein. "Operablely linked" as used herein means that two or more nucleic acid sequences exhibit a functional relationship.

[0099] The host cells according to the eighth embodiment of the present invention contain the nucleic acid according to the seventh embodiment. Since the host cells according to this embodiment contain a nucleotide sequence that codes for a protein, a protein can be produced by culturing the host cells. The host cells may be prokaryotic cells or eukaryotic cells. An example of a prokaryotic cell may be Escherichia coli, and an example of a eukaryotic cell may be CHO cells, COS cells, or HEK293 cells.

[0100] A ninth embodiment of the present invention describes a method for preparing the protein of the first embodiment, which includes culturing host cells according to the eighth embodiment so that the protein is expressed. By selecting an appropriate inducer using a promoter, the cells can be induced to express the protein.

[0101] The present invention will be described with reference to the following examples. <Example 1: Design incorporating a disulfide bond> As shown in Figure 1, protein C7-NM has eight β-strands, with one loop-like structure formed between two adjacent β-strands. Using protein C7-NM as the parent protein and utilizing Disulfide by Design 2.0 (DbD2) software, we design the introduction of disulfide bonds into the parent protein's structure by referencing parameters such as factor B, χ3 torsion angle, and energy. As shown in Figure 2 and Table 1, the disulfide bond can be designed to be located between two β-strands, between two loops, and between one β-strand and one loop in the parent protein. As shown in Table 2, the amino acid sequence of the mutant protein with disulfide bonds is shown, and it is obtained by using the amino acid sequence of protein C7 as the reference sequence and making amino acid substitutions based on the designed disulfide bond position.

[0102] Table 1, Crystallographic Parameters JPEG0007917758000001.jpg71170

[0103] Table 2, Amino Acid Sequences JPEG0007917758000002.jpg236170 Note 1: The letters and symbols within the box represent the mutation sequences for protein 10Fn3-WT. Note 2: The underlined letters represent the mutation sequences for 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 them in a carrier and the BL21(DE3) pLysS strain or the SHuffle strain using pET21a, and the culture medium for the BL21(DE3) pLysS strain is an LB culture medium, and the culture medium for the SHuffle strain is an LLB culture medium.

[0105] First, the BL21(DE3) pLysS strain is cultured at 37°C in 5 mL of culture medium for approximately 16-18 hours (the SHuffle strain is cultured at 30°C). The BL21(DE3) pLysS strain is then 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). Then, the cells are centrifuged and the cell precipitate is collected.

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

[0107] <Example 3: Analysis of Protein Characteristics> The melting point of the protein was measured using 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 with one disulfide bond, indicating that protein C7-SL1 has high thermal stability. As shown in Table 3 and Figures 3 and 4, proteins C7-SL1-LL2 and C7-SL1-LL3, which were obtained by introducing other disulfide bonds into protein C7-SL1 as the main component, showed further improved thermal stability.

[0108] Referring to J Pharm Sci. 2008 Oct;97(10):4155-66, the solubility of the proteins was measured using the ammonium sulfate precipitation method. As shown in Table 3, the PBS solubility of protein C7-SL1 was approximately 6 times that of protein C7, and 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 were obtained by introducing other disulfide bonds into protein C7-SL1, showed further improved PBS solubility to approximately 7 times that of protein C7.

[0109] Using cell proliferation experiments, we analyzed the inhibitory activity of proteins on the proliferation of human umbilical vein endothelial cells (HUVECs). As shown in Table 3, the inhibitory activity of protein C7-SL1 on HUVEC proliferation did not differ significantly from that of protein C7, indicating that protein C7-SL1 has angiogenesis-inhibiting ability. As shown in Table 3, the inhibitory activity of proteins C7-SL1-LL2 and C7-SL1-LL3, obtained by introducing other disulfide bonds in addition to protein C7-SL1, also did not differ significantly from that of protein C7, indicating that proteins C7-SL1-LL2 and C7-SL1-LL3 also have angiogenesis-inhibiting ability.

[0110] The affinity of proteins to vascular endothelial growth factor receptor 2 (C2) was analyzed using enzyme-linked immunosorbent assay (ELISA). As shown in Table 3, the affinity of protein C7-SL1 to C2 was not significantly different from that of protein C7, indicating that protein C7-SL1 has the ability to bind to C2. As shown in Table 3 and Figure 6, the affinity of proteins C7-SL1-LL2 and C7-SL1-LL3, obtained by introducing other disulfide bonds into protein C7-SL1, was also not significantly different from that of protein C7. Furthermore, the affinity of protein C7-SL1-LL2 to C2 was slightly superior to that of protein C7, indicating that proteins C7-SL1-LL2 and C7-SL1-LL3 also have the ability to bind to C2.

[0111] Table 3, Protein Characteristics JPEG0007917758000003.jpg88170 Note 1: ΔTm = Tm value of test protein - Tm value of protein C7 Note 2: ND indicates that measurement was not possible, either due to formation in an inclusion body or because the measurement was not performed.

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

Claims

1. A protein characterized by containing the amino acid sequence shown in SEQ ID NO: 11, 13, 15, or 16.

2. The protein according to claim 1, A pharmaceutical composition characterized by comprising a pharmaceutically acceptable carrier.

3. The use of the pharmaceutical composition according to claim 2, characterized in that it is used to prepare a medicine for treating or preventing a disease or disorder caused by or related to the activity or signaling of vascular endothelial growth factor receptor 2.

4. The use of the pharmaceutical composition according to claim 2, characterized in that it is used to prepare a medicine for treating or preventing a disease or disorder caused by angiogenesis.

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