Protein derived from III-type domain of fibromucin and application thereof

Abstract

Provided are a protein derived from a Type III domain of a fibromucin, the protein comprising a Type III domain of a 10th human fibromucin, and comprising a first mutation in which an amino acid site is substituted with a cysteine and a second mutation in which another amino acid site is substituted with another cysteine, and a use thereof, the protein comprising a Type III domain of a 10th human fibromucin, the Type III domain of the 10th human fibromucin, the Type III domain of the 10th human fibromucin, and the Type III domain of the 10th human fibromucin, the first mutation and the second mutation occur in other regions except the ring FG of the III type domain of the tenth human fibromucin, and cysteine substituted by the first mutation and cysteine substituted by the second mutation form a disulfide bond.

Description

Proteins derived from fibronectin type III domain and their applications Technical Field

[0001] The present invention relates to protein engineering, and more particularly to a protein derived from the type III domain of fibronectin and its applications, including methods for treating or preventing diseases or disorders caused by or associated with type II vascular endothelial growth factor receptor 2 (VEGFR2) activity or signaling, or methods for treating or preventing diseases or disorders caused by angiogenesis. Background Art

[0002] In recent years, efforts have focused on developing molecular recognition modules based on protein scaffolds to replace those constructed using immunoglobulins. This approach aims to overcome the limitations inherent in using immunoglobulins as the primary molecular recognition modules, such as their large size, complex heterodimer structure, and the need for proper disulfide bond formation. The fundamental principle of this protein engineering approach is to develop molecules with affinity and specificity comparable to antibodies by constructing binding interfaces within appropriate non-antibody protein frameworks or molecular scaffolds.

[0003] The 10th human fibronectin type III domain (10Fn3) is one of the most widely used non-antibody scaffolds for the development of novel protein-binding molecules. Several 10Fn3-based molecules are being developed and tested for disease treatment, though most remain in clinical trials.

[0004] 10Fn3 possesses numerous advantages over immunoglobulin-derived systems. Based on its overall β-sandwich fold, 10Fn3 belongs to the immunoglobulin superfamily. The three surface loops near the N-terminus are structurally similar to the three antigen-recognition loops, or complementarity-determining regions (CDRs), of the immunoglobulin variable region. However, unlike typical immunoglobulin regions, 10Fn3 lacks disulfide bonds. Furthermore, 10Fn3 exhibits a thermal transition temperature exceeding 80°C, demonstrating a stable conformation. Furthermore, 10Fn3 exhibits reversible and rapid unfolding and refolding properties.

[0005] 10Fn3 has approximately 94 amino acid residues, which is smaller than the antigen-binding unit (VHH) of a heavy-chain antibody. These properties make 10Fn3 compatible with various molecular expression systems and simple and efficient production methods.

[0006] US patent application No. 11 / 448,171, entitled "INHIBITORS OF TYPE 2 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS", proposes a type 2 vascular endothelial growth factor receptor binding protein C7. Protein C7 is based on 10Fn3 and differs from the wild type in that the three surface loop regions of the wild type are sequenced in the same way. 23 DAPAVTVRY, 51 PGSKST, and 75 VTGRGDSPASSKP each 23 RHPHFPTRY, 51 PLQPPT, and 75 By replacing VTDGRNGRLLSIP with the three mutated regions described above, protein C7 acts as a VEGF receptor type II binding site, potentially enabling it to act as a VEGF receptor type II antagonist and potentially become an anti-cancer drug. However, its thermal stability and low solubility have hindered its development as a drug.

[0007] Therefore, developing a protein scaffold with high thermal stability and high solubility using 10Fn3 as the main structural component is indeed one of the issues that people in the technical field of the present invention are actively trying to solve.

[0008] Summary of the Invention

[0009] The present invention is based on the following: using the software Disulfide by Design 2.0 (DbD2) and referring to the B-factor, χ 3 Torsion angle (χ 3 Parameters such as angle, and energy are derived from the structural design of the protein C7-NM. The introduction of disulfide bonds creates a variant with disulfide bonds, which improves thermal stability and solubility without affecting its binding activity to specific proteins, thus possessing potential as a biopharmaceutical.

[0010] The present invention provides a protein comprising a tenth human fibronectin type III domain, comprising a first mutation substituting one amino acid position with a cysteine ​​and a second mutation substituting another amino acid position with another cysteine. The first mutation and the second mutation occur in regions other than the FG loop of the tenth human fibronectin type III domain, whereby the cysteine ​​substituted by the first mutation and the cysteine ​​substituted by the second mutation form a disulfide bond.

[0011] For example, the tenth human fibronectin type III domain comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0012] For example, the protein further comprises a mutation that replaces the amino acid sequence of loop BC with RHPHFPTRY, a mutation that replaces the amino acid sequence of loop DE with PLQPPT, and a mutation that replaces the amino acid sequence of loop FG with VTDGRNGRLLSIP.

[0013] 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 tenth 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 tenth human fibronectin type III domain.

[0014] 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 tenth 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 tenth human fibronectin type III domain.

[0015] Illustratively, the first mutation and the second mutation occur in the same region or in different regions.

[0016] For example, the first mutation comprises: a substitution of leucine at position 8 to cysteine, a substitution of serine at position 17 to cysteine, a substitution of leucine at position 19 to cysteine, a substitution of isoleucine at position 34 to cysteine, a substitution of threonine at position 35 to cysteine, a substitution of tyrosine at position 36 to cysteine, a substitution of glycine at position 37 to cysteine, a substitution of threonine at position 39 to cysteine, a substitution of lysine at position 63 to cysteine, or a substitution of aspartic acid at position 67 to cysteine, The second mutation comprises: a substitution of tryptophan at position 22 for cysteine, a substitution of valine at position 45 for cysteine, a substitution of phenylalanine at position 48 for cysteine, a substitution of threonine at position 58 for cysteine, a substitution of serine at position 60 for cysteine, a substitution of valine at position 66 for cysteine, a substitution of aspartic acid at position 67 for cysteine, a substitution of isoleucine at position 70 for cysteine, a substitution of serine at position 89 for cysteine, or a substitution of asparagine at position 91 for cysteine.

[0017] For example, the first mutation comprises: substitution of leucine at position 8 for cysteine, and the second mutation comprises: substitution of tryptophan at position 22 for cysteine; the first mutation comprises: substitution of leucine at position 8 for cysteine, and the second mutation comprises: substitution of serine at position 89 for cysteine; the first mutation comprises: substitution of serine at position 17 for cysteine, and the second mutation comprises: substitution of serine at position 60 for cysteine; the first mutation comprises: substitution of leucine at position 19 for cysteine, and the second mutation comprises: substitution of threonine at position 58 for cysteine; the first mutation comprises: substitution of isoleucine at position 34 for cysteine, and the second mutation comprises: substitution of phenylalanine at position 48 for cysteine; the first mutation comprises: substitution of threonine at position 35 for cysteine, and the second mutation comprises The mutation comprises: substitution of valine at position 45 to cysteine; the first mutation comprises: substitution of tyrosine at position 36 to cysteine, and the second mutation comprises: substitution of isoleucine at position 70 to cysteine; the first mutation comprises: substitution of glycine at position 37 to cysteine, and the second mutation comprises: substitution of valine at position 45 to cysteine; the first mutation comprises: substitution of threonine at position 39 to cysteine, and the second mutation comprises: substitution of aspartic acid at position 67 to cysteine; the first mutation comprises: substitution of lysine at position 63 to cysteine, and the second mutation comprises: substitution of valine at position 66 to cysteine; or the first mutation comprises: substitution of aspartic acid at position 67 to cysteine, and the second mutation comprises: substitution of asparagine at position 91 to cysteine.

[0018] Illustratively, the protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 14.

[0019] Illustratively, the protein comprises an amino acid sequence as shown in SEQ ID NO:11.

[0020] For example, under the condition that the first mutation and the second mutation do not include a substitution of leucine at position 19 to cysteine, the protein includes a mutation that substitutes alanine at position 12 to glutamate, a mutation that substitutes threonine at position 14 to serine, a mutation that substitutes leucine at position 18 to isoleucine, and a mutation that substitutes leucine at position 19 to glutamate.

[0021] For example, the first mutation comprises: substitution of leucine at position 8 to cysteine, substitution of serine at position 17 to cysteine, substitution of isoleucine at position 34 to cysteine, substitution of threonine at position 35 to cysteine, substitution of tyrosine at position 36 to cysteine, substitution of glycine at position 37 to cysteine, substitution of threonine at position 39 to cysteine, substitution of lysine at position 63 to cysteine, or substitution of aspartic acid at position 67 to cysteine; the second mutation comprises: substitution of tryptophan at position 22 to cysteine, substitution of valine at position 45 to cysteine, substitution of phenylalanine at position 48 to cysteine. Under the conditions that the protein comprises a mutation in which an alanine at position 12 is substituted with glutamine, 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, the protein comprises a mutation in which an alanine at position 12 is substituted with glutamine, 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.

[0022] For example, the first mutation comprises: substitution of leucine at position 8 to cysteine, and the second mutation comprises: substitution of tryptophan at position 22 to cysteine; the first mutation comprises: substitution of leucine at position 8 to cysteine, and the second mutation comprises: substitution of serine at position 89 to cysteine; the first mutation comprises: substitution of serine at position 17 to cysteine, and the second mutation comprises: substitution of serine at position 60 to cysteine; the first mutation comprises: substitution of isoleucine at position 34 to cysteine, and the second mutation comprises: substitution of phenylalanine at position 48 to cysteine; the first mutation comprises: substitution of threonine at position 35 to cysteine, and the second mutation comprises: substitution of valine at position 45 to cysteine; the first mutation comprises: substitution of tyrosine at position 36 to cysteine, and the second mutation comprises: substitution of isoleucine at position 70 to cysteine ; the first mutation comprises: a substitution of glycine at position 37 to cysteine, and the second mutation comprises: a substitution of valine at position 45 to cysteine; the first mutation comprises: a substitution of threonine at position 39 to cysteine, and the second mutation comprises: a substitution of aspartic acid at position 67 to cysteine; the first mutation comprises: a substitution of lysine at position 63 to cysteine, and the second mutation comprises: a substitution of valine at position 66 to cysteine; or the first mutation comprises: a substitution of aspartic acid at position 67 to cysteine, and the second mutation comprises: a substitution of asparagine at position 91 to cysteine, under the conditions, the protein comprises a mutation that substitutes alanine at position 12 to glutamate, a mutation that substitutes threonine at position 14 to serine, a mutation that substitutes leucine at position 18 to isoleucine, and a mutation that substitutes leucine at position 19 to glutamate.

[0023] Illustratively, the protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 17 to 26.

[0024] For example, the protein further comprises a third mutation that substitutes another amino acid position with another cysteine ​​and a fourth mutation that substitutes another amino acid position with another cysteine, wherein the third mutation and the fourth mutation occur in regions other than the FG loop of the tenth human fibronectin type III domain, whereby the cysteine ​​substituted by the third mutation and the cysteine ​​substituted by the fourth mutation form a disulfide bond.

[0025] 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 tenth 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 tenth human fibronectin type III domain.

[0026] 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 tenth 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 tenth human fibronectin type III domain.

[0027] For example, the first mutation, the second mutation, the third mutation, and the fourth mutation occur in different regions.

[0028] For example, the third mutation comprises: a substitution of leucine at position 8 to cysteine, a substitution of serine at position 17 to cysteine, a substitution of leucine at position 19 to cysteine, a substitution of isoleucine at position 34 to cysteine, a substitution of threonine at position 35 to cysteine, a substitution of tyrosine at position 36 to cysteine, a substitution of glycine at position 37 to cysteine, a substitution of threonine at position 39 to cysteine, a substitution of lysine at position 63 to cysteine, or a substitution of aspartic acid at position 67 to cysteine. The fourth mutation comprises: a substitution of tryptophan at position 22 for cysteine, a substitution of valine at position 45 for cysteine, a substitution of phenylalanine at position 48 for cysteine, a substitution of threonine at position 58 for cysteine, a substitution of serine at position 60 for cysteine, a substitution of valine at position 66 for cysteine, a substitution of aspartic acid at position 67 for cysteine, a substitution of isoleucine at position 70 for cysteine, a substitution of serine at position 89 for cysteine, or a substitution of asparagine at position 91 for cysteine.

[0029] For example, the first mutation comprises: substitution of leucine at position 8 to cysteine, the second mutation comprises: substitution of serine at position 89 to cysteine, the third mutation comprises: substitution of serine at position 17 to cysteine, and the fourth mutation comprises: substitution of serine at position 60 to cysteine; or the first mutation comprises: substitution of leucine at position 8 to cysteine, the second mutation comprises: substitution of serine at position 89 to cysteine, the third mutation comprises: substitution of threonine at position 39 to cysteine, and the fourth mutation comprises: substitution of aspartic acid at position 67 to cysteine.

[0030] Illustratively, the protein comprises an amino acid sequence as shown in SEQ ID NO: 15 or 16.

[0031] For example, under the condition that the first mutation, the second mutation, the third mutation, and the fourth mutation do not include a substitution of leucine at position 19 to cysteine, the protein includes a mutation that substitutes alanine at position 12 to glutamate, a mutation that substitutes threonine at position 14 to serine, a mutation that substitutes leucine at position 18 to isoleucine, and a mutation that substitutes leucine at position 19 to glutamate.

[0032] For example, the first mutation comprises: substitution of leucine at position 8 to cysteine, substitution of serine at position 17 to cysteine, substitution of isoleucine at position 34 to cysteine, substitution of threonine at position 35 to cysteine, substitution of tyrosine at position 36 to cysteine, substitution of glycine at position 37 to cysteine, substitution of threonine at position 39 to cysteine, substitution of lysine at position 63 to cysteine, or substitution of aspartic acid at position 67 to cysteine, and the second mutation comprises: substitution of tryptophan at position 22 to cysteine, substitution of tyrosine at position 45 to cysteine, substitution of threonine at position 39 to cysteine, substitution of lysine at position 63 to cysteine, or substitution of tyrosine at position 45 to cysteine. The first mutation comprises a substitution of valine for cysteine, a substitution of phenylalanine at position 48 for cysteine, a substitution of threonine at position 58 for cysteine, a substitution of serine at position 60 for cysteine, a substitution of valine at position 66 for cysteine, a substitution of aspartic acid at position 67 for cysteine, a substitution of isoleucine at position 70 for cysteine, a substitution of serine at position 89 for cysteine, or a substitution of asparagine at position 91 for cysteine. The third mutation comprises a substitution of leucine at position 8 for cysteine, a substitution of serine at position 17 for cysteine, a substitution of valine at position 66 for cysteine, a substitution of aspartic acid at position 67 for cysteine, a substitution of isoleucine at position 70 for cysteine, a substitution of serine at position 89 for cysteine, or a substitution of asparagine at position 91 for cysteine. The fourth mutation comprises: substitution of isoleucine at position 22 for cysteine, substitution of valine at position 45 for cysteine, substitution of phenylalanine at position 48 for cysteine, substitution of threonine at position 58 for cysteine, substitution of glycine at position 37 for cysteine, substitution of threonine at position 39 for cysteine, substitution of lysine at position 63 for cysteine, or substitution of aspartic acid at position 67 for cysteine; the fourth mutation comprises: substitution of tryptophan at position 22 for cysteine, substitution of valine at position 45 for cysteine, substitution of phenylalanine at position 48 for cysteine, substitution of threonine at position 58 for cysteine, substitution of threonine at position 6 Under the conditions that the serine at position 0 is substituted with cysteine, the valine at position 66 is substituted with cysteine, the aspartic acid at position 67 is substituted with cysteine, the isoleucine at position 70 is substituted with cysteine, the serine at position 89 is substituted with cysteine, or the asparagine at position 91 is substituted with cysteine, the protein comprises a mutation that substitutes an alanine at position 12 with glutamate, a mutation that substitutes threonine at position 14 with serine, a mutation that substitutes leucine at position 18 with isoleucine, and a mutation that substitutes leucine at position 19 with glutamate.

[0033] For example, under the conditions where the first mutation comprises a substitution of leucine at position 8 to cysteine, the second mutation comprises a substitution of serine at position 89 to cysteine, the third mutation comprises a substitution of serine at position 17 to cysteine, and the fourth mutation comprises a substitution of serine at position 60 to cysteine; or where the first mutation comprises a substitution of leucine at position 8 to cysteine, the second mutation comprises a substitution of serine at position 89 to cysteine, the third mutation comprises a substitution of threonine at position 39 to cysteine, and the fourth mutation comprises a substitution of aspartic acid at position 67 to cysteine, the protein comprises a mutation where alanine at position 12 is substituted with glutamate, 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 glutamate.

[0034] Illustratively, the protein comprises an amino acid sequence as shown in SEQ ID NO: 27 or 28.

[0035] Illustratively, the protein is used to bind to DLL4, EGFR, VEGFR2, or IGF-1R.

[0036] The protein of this invention is based on the tenth human fibronectin type III domain, with disulfide bonds introduced at specific positions to enhance thermal stability and solubility. The protein of this invention can further utilize amino acid sequence substitutions to construct protein-binding interfaces, enabling it to bind to specific proteins such as DLL4, EGFR, VEGFR2, or IGF-1R, acting as a receptor antagonist and possessing potential as a biopharmaceutical.

[0037] The present invention further provides a pharmaceutical composition comprising: the protein as described above; and a pharmaceutically acceptable carrier.

[0038] Illustratively, the pharmaceutical composition is an oral administration formulation, an injectable administration formulation, an inhalation administration formulation, or a topical or transdermal administration formulation.

[0039] The present invention further provides a use of the pharmaceutical composition, which is for preparing a medicine for treating or preventing diseases or disorders caused by or associated with VEGF receptor type II activity or signaling.

[0040] Illustratively, diseases or disorders caused by or associated with VEGF receptor type 2 activity or signaling include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs syndrome, pericardial effusion, pleural effusion, diabetes, endometriosis, dysfibrosis, or cancer.

[0041] Illustratively, the cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

[0042] The present invention further provides a method for treating or preventing diseases or disorders caused by or associated with VEGF receptor type II activity or signaling, comprising administering the pharmaceutical composition described above to a subject in need thereof, thereby binding to the subject's VEGF receptor type II to inhibit its activity.

[0043] Illustratively, diseases or disorders caused by or associated with VEGF receptor type 2 activity or signaling include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, pleural effusion, diabetes, endometriosis, dysfibrosis, or cancer.

[0044] Illustratively, the cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

[0045] The present invention further provides a use of the pharmaceutical composition for preparing a medicine for treating or preventing diseases or disorders caused by angiogenesis.

[0046] Illustratively, diseases or disorders caused by angiogenesis include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, hydrothorax, diabetes, endometriosis, dysfibrosis, or cancer.

[0047] Illustratively, the cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

[0048] The present invention further provides a method for treating or preventing diseases or disorders caused by angiogenesis, comprising administering the pharmaceutical composition described above to a subject in need thereof, thereby binding to the subject's type II vascular endothelial growth factor receptor to inhibit angiogenesis.

[0049] Illustratively, diseases or disorders caused by angiogenesis include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, hydrothorax, diabetes, endometriosis, dysfibrosis, or cancer.

[0050] Illustratively, the cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

[0051] The present invention further provides a nucleic acid comprising a nucleotide sequence encoding the protein as described above.

[0052] The present invention further provides a host cell comprising the nucleic acid described above.

[0053] Illustratively, the host cell is a prokaryotic cell or a eukaryotic cell.

[0054] For example, the prokaryotic cell is Escherichia coli, and the eukaryotic cell is CHO cell, COS cell, or HEK293 cell.

[0055] The present invention further provides a method for preparing the protein as described above, which comprises culturing the host cell as described above to allow it to express the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 is a protein structure diagram showing the three-dimensional structure of protein C7-NM;

[0057] FIG2 is a protein structure diagram showing the position of the disulfide bond designed in the three-dimensional structure of protein C7-NM;

[0058] FIG3 is a graph showing differential scanning calorimetry results, comparing the melting points of protein C7 and its variant proteins;

[0059] FIG4 is a graph showing differential scanning calorimetry results, comparing the melting temperatures of proteins C7 and C7-SL1-LL2;

[0060] FIG5 is a graph showing solubility results, comparing the solubility of proteins C7 and C7-SL1-LL2;

[0061] FIG6 is a graph showing the results of enzyme-bound immunosorbent assay, comparing the binding affinities of proteins C7 and C7-SL1-LL2 to VEGF receptor type II. DETAILED DESCRIPTION

[0062] To make the above and / or other purposes, effects, and features of the present invention more clearly understood, preferred embodiments are described in detail below:

[0063] I. Definition of Terms

[0064] Unless otherwise specified, "protein" herein includes wild-type proteins expressed in natural cells, recombinant proteins expressed using genetic engineering techniques, and synthetic proteins obtained through chemical means. At least one amino acid may be substituted, deleted, and / or inserted into the protein sequence without affecting the original activity.

[0065] Unless otherwise specified, the "amino acids" referred to in this article include D-amino acids or L-amino acids. D- and L- represent the absolute configuration of the amino acid, rather than the specific rotation direction of plane polarization. Unless otherwise specified, this article uses the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission to represent amino acids. Protein sequences are represented by a string of multiple single-letter symbols, and the order of the single-letter symbols corresponds to the order of amino acids from the N-terminus to the C-terminus of the protein. If there is a superscript number before the single-letter symbol, it indicates the position order of the corresponding amino acid in the protein starting from the N-terminus; for example, 23 DAPAVTVRY indicates that aspartic acid is located at position 23 of the protein; the rest are similar and will not be repeated here.

[0066] Protein sequence substitutions, deletions, and / or insertions can occur in non-framework regions of the protein, which generally do not affect the original activity. In addition, protein sequence substitutions can include conservative amino acid substitutions, which are substitutions between amino acids with similar properties or related side chains. Substitutions between amino acids with similar properties, for example: acidic amino acids can substitute for each other, i.e., aspartate and glutamate; basic amino acids can substitute for each other, i.e., lysine, arginine, and histidine; non-polar amino acids can substitute for each other, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and uncharged polar amino acids can substitute for each other, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Substitutions between amino acids with related side chains, for example: aliphatic-hydroxy amino acids can substitute for each other, i.e., serine and threonine; amide-containing amino acids can substitute for each other, i.e., asparagine and glutamine; aliphatic amino acids can substitute for each other, i.e., alanine, valine, leucine, and isoleucine; and aromatic amino acids can substitute for each other, i.e., phenylalanine, tryptophan, and tyrosine.

[0067] Unless otherwise specified, the "10th human fibronectin type III domain" referred to herein comprises, from N-terminus to 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, and has at least 94 amino acids without any disulfide bonds, as shown in SEQ ID NO: 1. Referring to U.S. patent application Ser. No. 13 / 757,664, entitled "FIBRONECTIN BINDING DOMAINS WITH REDUCED IMMUNOGENICITY," the N-terminal region is defined as the amino acid segment from positions 1 to 7, β-strand A is defined as the amino acid segment from positions 8 to 13, loop AB is defined as the amino acid segment from positions 14 to 17, and β-strand B is defined as the amino acid segment from positions 18 to 22. The amino acid segment of the molecule is defined as loop BC, loop BC is defined as the amino acid segment at positions 23 to 31, beta strand C is defined as the amino acid segment at positions 32 to 36, loop CD is defined as the amino acid segment at positions 37 to 47, beta strand D is defined as the amino acid segment at positions 48 to 50, loop DE is defined as the amino acid segment at positions 51 to 56, beta strand E is defined as the amino acid segment at positions 57 to 62, loop EF is defined as the amino acid segment at positions 63 to 67, beta strand F is defined as the amino acid segment at positions 68 to 74, loop FG is defined as the amino acid segment at positions 75 to 87, beta strand G is defined as the amino acid segment at positions 88 to 92, and the C-terminal region is defined as the amino acid segment at positions 93 and 94; loop BC, loop DE, and loop FG are located on one side of the molecule, and loop AB, loop CD, and loop EF are located on the other side of the molecule. For example, the N-terminal region comprises 1 VSDVPRD, beta strand A contains 8 LEVVAA, ring AB contains 14 TPTS, beta strand B contains 18 LLISW, ring BC contains 23 DAPAVTVRY, beta strand C contains 32 YRITY, Ring CD contains 37 GETGGNSPVQE, beta strand D contains 48 FTV, Ring DE included 51 PGSKST, beta strand E contains 57 ATISGL, Ring EF contains 63 KPGVD, beta strand F contains 68YTITVYA, Ring FG contains 75 VTGRGDSPASSKP, β strand G contains 88 ISINY, the C-terminal region contains 93 RT.

[0068] Unless otherwise specified, the term "protein C7" herein refers to a variant of the type III domain of human fibronectin 10, which can bind to the type II vascular endothelial growth factor receptor to inhibit its activity, such as SEQ ID NO: 2. Specifically, the amino acid sequence corresponding to the wild-type loop BC is 23 RHPHFPTRY, corresponding to the wild-type loop DE amino acid sequence is 51 PLQPPT, the amino acid sequence corresponding to the wild-type loop FG is 75 VTDGRNGRLLSIP, through the three mutation regions mentioned above, constitutes the binding interface of type II vascular endothelial growth factor receptor.

[0069] Unless otherwise specified, "protein C7-NM" herein refers to a variant of protein C7, such as SEQ ID NO: 3. Specifically, the amino acid corresponding to position 12 of the reference sequence is glutamine, 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 glutamate.

[0070] Unless otherwise specified, "VEGF receptor type II" herein refers to a transmembrane receptor tyrosine kinase that regulates angiogenesis induced by VEGF-A and VEGF-B. VEGF receptor type II is synonymous with kinase insert domain receptor (KDR) and fetal liver kinase 1 (FLK-1) and is used interchangeably.

[0071] Unless otherwise specified, the term "treatment" as used herein refers to therapeutic intervention to cure or improve a disease, including complete or partial cure or improvement.

[0072] As used herein, "prevention" unless otherwise specified refers to the complete or near-complete prevention of a disease. For example, preventive intervention can be used to prevent the onset of a disease when the disease is not present or suspected but has not yet occurred.

[0073] Unless otherwise specified, the term "pharmaceutically acceptable carrier" as used herein refers to an additive that is suitable for contact with an individual, without excessive toxicity, irritation, allergic reaction, or other problems or complications, and has a reasonable benefit-risk ratio, within the scope of sound medical judgment, such as a filler, diluent, agglutinating agent, binder, lubricant, glidant, stabilizer, colorant, wetting agent, or disintegrant.

[0074] 2. Proteins based on the human fibronectin type III domain

[0075] The first embodiment of the present invention discloses a protein based on the tenth human fibronectin type III domain. Amino acid substitutions at specific positions within the protein are used to introduce disulfide bonds, thereby enhancing the protein's thermal stability and solubility. Based on this high thermal stability and solubility, as well as the inherent properties of the tenth human fibronectin type III domain, amino acid substitutions can be further used to construct protein-binding interfaces for binding to specific proteins, such as DLL4, EGFR, VEGFR2, or IGF-1R. Consequently, the protein of this embodiment can be used as a receptor antagonist and a biopharmaceutical.

[0076] The protein of this embodiment comprises the tenth human fibronectin type III domain and includes a first mutation that substitutes one amino acid position with a cysteine ​​and a second mutation that substitutes another amino acid position with another cysteine. The first and second mutations occur in regions other than the FG loop of the tenth human fibronectin type III domain, whereby 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 set forth in SEQ ID NO: 1.

[0077] With respect to 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.

[0078] The first mutation and the second mutation can occur in one of the same or two different regions within the above groups. For example, both the first mutation and the second mutation can occur in loop CD or loop EF. For another example, the first mutation can occur in β strand A, and the second mutation can occur in β strand B; the first mutation can occur in β strand A, and the second mutation can occur in β strand G; the first mutation can occur in loop AB, and the second mutation can occur in β strand E; the first mutation can occur in β strand B, and the second mutation can occur in β strand E; the first mutation can occur in β strand C, and the second mutation can occur in β strand D; the first mutation can occur in β strand C, and the second mutation can occur in loop CD; the first mutation can occur in β strand C, and the second mutation can 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.

[0079] According to the amino acid sequence of the 10th human fibronectin type III domain, the first mutation may include: substitution of leucine at position 8 to cysteine, substitution of serine at position 17 to cysteine, substitution of leucine at position 19 to cysteine, substitution of isoleucine at position 34 to cysteine, substitution of threonine at position 35 to cysteine, substitution of tyrosine at position 36 to cysteine, substitution of glycine at position 37 to cysteine, substitution of threonine at position 39 to cysteine, substitution of lysine at position 63 to cysteine, or substitution of lysine at position 67 to cysteine. The second mutation may comprise: a substitution of tryptophan at position 22 for cysteine, a substitution of valine at position 45 for cysteine, a substitution of phenylalanine at position 48 for cysteine, a substitution of threonine at position 58 for cysteine, a substitution of serine at position 60 for cysteine, a substitution of valine at position 66 for cysteine, a substitution of aspartic acid at position 67 for cysteine, a substitution of isoleucine at position 70 for cysteine, a substitution of serine at position 89 for cysteine, or a substitution of asparagine at position 91 for cysteine. Specifically, the first mutation may include: substitution of leucine at position 8 to cysteine, and the second mutation may include: substitution of tryptophan at position 22 to cysteine; the first mutation may include: substitution of leucine at position 8 to cysteine, and the second mutation may include: substitution of serine at position 89 to cysteine; the first mutation may include: substitution of tryptophan at position 22 to cysteine; the second mutation may include: substitution of serine at position 89 to cysteine; the first mutation may include: substitution of tryptophan at position 22 to cysteine; the second mutation may include: substitution of tryptophan at position 2 ... first mutation may include: substitution of tryptophan at position 22 to cysteine The first mutation may comprise: the serine at position is substituted for cysteine, and the second mutation may comprise: the serine at position 60 is substituted for cysteine; the first mutation may comprise: the leucine at position 19 is substituted for cysteine, and the second mutation may comprise: the threonine at position 58 is substituted for cysteine; the first mutation may comprise: the isoleucine at position 34 is substituted for cysteine, and the second mutation may comprise: the phenylalanine at position 48 is substituted for cysteine; the first mutation may comprise: the threonine at position 35 is substituted for cysteine, and the second mutation may comprise: the valine at position 45 is substituted for cysteine; the first mutation may comprise: the tyrosine at position 36 is substituted for cysteine, and the second mutation may comprise: the tyrosine at position 37 is substituted for cysteine The first mutation may comprise: substitution of isoleucine at position 70 with cysteine; the first mutation may comprise: substitution of glycine at position 37 with cysteine, and the second mutation may comprise: substitution of valine at position 45 with cysteine; the first mutation may comprise: substitution of threonine at position 39 with cysteine, and the second mutation may comprise: substitution of aspartic acid at position 67 with cysteine; the first mutation may comprise: substitution of lysine at position 63 with cysteine, and the second mutation may comprise: substitution of valine at position 66 with cysteine; or the first mutation may comprise: substitution of aspartic acid at position 67 with cysteine, and the second mutation may comprise: substitution of asparagine at position 91 with cysteine.

[0080] The protein of this embodiment may further comprise a third mutation that substitutes another amino acid position with another cysteine ​​and a fourth mutation that substitutes yet another amino acid position with yet another cysteine. The third and fourth mutations occur in regions other than the FG loop of the tenth human fibronectin type III domain. The cysteine ​​substituted by the third mutation forms a disulfide bond with the cysteine ​​substituted by the fourth mutation. The presence of at least two disulfide bonds further enhances the thermal stability and solubility of the protein.

[0081] With respect to 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.

[0082] The first mutation, the second mutation, the third mutation, and the fourth mutation can occur in four different regions within the above groupings. 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; or 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.

[0083] According to the amino acid sequence of the tenth human fibronectin type III domain, the third mutation may include: substitution of leucine at position 8 to cysteine, substitution of serine at position 17 to cysteine, substitution of leucine at position 19 to cysteine, substitution of isoleucine at position 34 to cysteine, substitution of threonine at position 35 to cysteine, substitution of tyrosine at position 36 to cysteine, substitution of glycine at position 37 to cysteine, substitution of threonine at position 39 to cysteine, substitution of lysine at position 63 to cysteine, or substitution of lysine at position 67 to cysteine. The fourth mutation may comprise: a substitution of tryptophan at position 22 for cysteine, a substitution of valine at position 45 for cysteine, a substitution of phenylalanine at position 48 for cysteine, a substitution of threonine at position 58 for cysteine, a substitution of serine at position 60 for cysteine, a substitution of valine at position 66 for cysteine, a substitution of aspartic acid at position 67 for cysteine, a substitution of isoleucine at position 70 for cysteine, a substitution of serine at position 89 for cysteine, or a substitution of asparagine at position 91 for cysteine. Specifically, the first mutation may include: substitution of leucine at position 8 with cysteine, the second mutation may include: substitution of serine at position 89 with cysteine, the third mutation may include: substitution of serine at position 17 with cysteine, and the fourth mutation may include: substitution of serine at position 60 with cysteine; or the first mutation may include: substitution of leucine at position 8 with cysteine, the second mutation may include: substitution of serine at position 89 with cysteine, the third mutation may include: substitution of threonine at position 39 with cysteine, and the fourth mutation may include: substitution of aspartic acid at position 67 with cysteine.

[0084] As described above, the protein of this embodiment can bind to DLL4, EGFR, VEGFR2, or IGF-1R. To bind to VEGFR2, the protein of this embodiment can further comprise a mutation that replaces the amino acid sequence of the BC loop with RHPHFPTRY, a mutation that replaces the amino acid sequence of the DE loop with PLQPPT, and a mutation that replaces the amino acid sequence of the FG loop with VTDGRNGRLLSIP.

[0085] Preferably, the protein of this embodiment comprises an amino acid sequence as shown in SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. More preferably, the protein of this embodiment comprises an amino acid sequence as shown in SEQ ID NO: 11, 15, or 16.

[0086] It has been previously discovered that protein C7-NM can improve thermal stability and solubility. The protein of this embodiment can be modified with reference to the amino acid sequence of protein C7-NM.

[0087] Under the condition that the first mutation and the second mutation do not include a substitution of leucine at position 19 to cysteine, the protein of this embodiment may further include a mutation substituting alanine at position 12 to glutamate, a mutation substituting threonine at position 14 to serine, a mutation substituting leucine at position 18 to isoleucine, and a mutation substituting leucine at position 19 to glutamate. Specifically, the first mutation comprises: substitution of leucine at position 8 for cysteine, substitution of serine at position 17 for cysteine, substitution of isoleucine at position 34 for cysteine, substitution of threonine at position 35 for cysteine, substitution of tyrosine at position 36 for cysteine, substitution of glycine at position 37 for cysteine, substitution of threonine at position 39 for cysteine, substitution of lysine at position 63 for cysteine, or substitution of aspartic acid at position 67 for cysteine; the second mutation comprises: substitution of tryptophan at position 22 for cysteine, substitution of valine at position 45 for cysteine, substitution of phenylalanine at position 48 for cysteine, substitution of tyrosine at position 36 for cysteine, substitution of glycine at position 37 for cysteine, substitution of threonine at position 39 for cysteine, substitution of lysine at position 63 for cysteine, or substitution of tyrosine at position 37 for cysteine. Under the conditions that the threonine at position 58 is substituted by cysteine, the serine at position 60 is substituted by cysteine, the valine at position 66 is substituted by cysteine, the aspartic acid at position 67 is substituted by cysteine, the isoleucine at position 70 is substituted by cysteine, the serine at position 89 is substituted by cysteine, or the asparagine at position 91 is substituted by cysteine, the protein of this embodiment may further include a mutation that replaces the alanine at position 12 with glutamate, a mutation that replaces the threonine at position 14 with serine, a mutation that replaces the leucine at position 18 with isoleucine, and a mutation that replaces the leucine at position 19 with glutamate.More specifically, the first mutation comprises: substitution of leucine at position 8 to cysteine, and the second mutation comprises: substitution of tryptophan at position 22 to cysteine; the first mutation comprises: substitution of leucine at position 8 to cysteine, and the second mutation comprises: substitution of serine at position 89 to cysteine; the first mutation comprises: substitution of serine at position 17 to cysteine, and the second mutation comprises: substitution of serine at position 60 to cysteine; the first mutation comprises: substitution of isoleucine at position 34 to cysteine, and the second mutation comprises: substitution of phenylalanine at position 48 to cysteine; the first mutation comprises: substitution of threonine at position 35 to cysteine, and the second mutation comprises: substitution of valine at position 45 to cysteine; the first mutation comprises: substitution of tyrosine at position 36 to cysteine, and the second mutation comprises: substitution of isoleucine at position 70 to cysteine; the first mutation comprises: substitution of tyrosine at position 36 to cysteine, and the second mutation comprises: substitution of isoleucine at position 70 to cysteine; Under the conditions that the first mutation comprises: substitution of glycine at position 37 to cysteine, and the second mutation comprises: substitution of valine at position 45 to cysteine; the first mutation comprises: substitution of threonine at position 39 to cysteine, and the second mutation comprises: substitution of aspartic acid at position 67 to cysteine; the first mutation comprises: substitution of lysine at position 63 to cysteine, and the second mutation comprises: substitution of valine at position 66 to cysteine; or the first mutation comprises: substitution of aspartic acid at position 67 to cysteine, and the second mutation comprises: substitution of asparagine at position 91 to cysteine, the protein of this embodiment may further comprise a mutation that substitutes alanine at position 12 to glutamate, a mutation that substitutes threonine at position 14 to serine, a mutation that substitutes leucine at position 18 to isoleucine, and a mutation that substitutes leucine at position 19 to glutamate.

[0088] Preferably, the protein of this embodiment comprises an amino acid sequence as shown in SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

[0089] Under the condition 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 substituting alanine at position 12 with glutamate, a mutation substituting threonine at position 14 with serine, a mutation substituting leucine at position 18 with isoleucine, and a mutation substituting leucine at position 19 with glutamate. Specifically, the first mutation comprises: substitution of leucine at position 8 to cysteine, substitution of serine at position 17 to cysteine, substitution of isoleucine at position 34 to cysteine, substitution of threonine at position 35 to cysteine, substitution of tyrosine at position 36 to cysteine, substitution of glycine at position 37 to cysteine, substitution of threonine at position 39 to cysteine, substitution of lysine at position 63 to cysteine, or substitution of aspartic acid at position 67 to cysteine; the second mutation comprises: substitution of tryptophan at position 22 to cysteine, substitution of valine at position 45 to cysteine. The first mutation comprises a substitution of leucine at position 8 for cysteine, a substitution of phenylalanine at position 48 for cysteine, a substitution of threonine at position 58 for cysteine, a substitution of serine at position 60 for cysteine, a substitution of valine at position 66 for cysteine, a substitution of aspartic acid at position 67 for cysteine, a substitution of isoleucine at position 70 for cysteine, a substitution of serine at position 89 for cysteine, or a substitution of asparagine at position 91 for cysteine. The third mutation comprises a substitution of leucine at position 8 for cysteine, a substitution of serine at position 17 for cysteine, a substitution of isoleucine at position 34 for cysteine, and a substitution of valine at position 66 for cysteine. The first mutation comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 Under the conditions that valine at position 66 is substituted with cysteine, aspartic acid at position 67 is substituted with cysteine, isoleucine at position 70 is substituted with cysteine, serine at position 89 is substituted with cysteine, or asparagine at position 91 is substituted with cysteine, the protein of this embodiment may further include a mutation that replaces alanine at position 12 with glutamate, a mutation that replaces threonine at position 14 with serine, a mutation that replaces leucine at position 18 with isoleucine, and a mutation that replaces leucine at position 19 with glutamate.More specifically, under the conditions where the first mutation comprises a substitution of leucine at position 8 to cysteine, the second mutation comprises a substitution of serine at position 89 to cysteine, the third mutation comprises a substitution of serine at position 17 to cysteine, and the fourth mutation comprises a substitution of serine at position 60 to cysteine; or where the first mutation comprises a substitution of leucine at position 8 to cysteine, the second mutation comprises a substitution of serine at position 89 to cysteine, the third mutation comprises a substitution of threonine at position 39 to cysteine, and the fourth mutation comprises a substitution of aspartic acid at position 67 to cysteine, the protein of this embodiment may further comprise a mutation substituting alanine at position 12 to glutamate, a mutation substituting threonine at position 14 to serine, a mutation substituting leucine at position 18 to isoleucine, and a mutation substituting leucine at position 19 to glutamate.

[0090] Preferably, the protein of this embodiment comprises an amino acid sequence as shown in SEQ ID NO: 27 or 28.

[0091] The protein of this embodiment can be prepared through genetic engineering techniques or chemical methods, such as solid-phase synthesis or solution synthesis. The protein of this embodiment can subsequently be isolated or purified using methods such as ammonium sulfate or ethanol precipitation, acid extraction, ion exchange chromatography, affinity chromatography, or lectin chromatography, preferably using high-performance liquid chromatography.

[0092] The protein of this embodiment may further comprise a hydrophilic group to enhance water solubility or circulation half-life. The hydrophilic group may be attached to the N-terminus of the protein. Preferably, the hydrophilic group is polyethylene glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyvinyl alcohol, or polyglucose. More preferably, the hydrophilic group is polyethylene glycol composed of 2 to 40 repeating units of ethylene glycol.

[0093] The protein of this embodiment may further contain a purification tag to facilitate purification. The purification tag may be attached 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 terminator / anti-terminator protein (NusA-tag), or a small ubiquitin-related modifier tag (SUMO-tag).

[0094] 3. Pharmaceutical Compositions

[0095] A second embodiment of the present invention discloses a pharmaceutical composition containing the protein of the first embodiment. This composition can be administered to a subject to bind to a specific protein, acting as an antagonist of the specific protein, thereby inhibiting the activity of the specific protein or blocking related signaling. The pharmaceutical composition of this embodiment comprises: the protein of the first embodiment; and a pharmaceutically acceptable carrier.

[0096] Pharmaceutically acceptable carriers can allow the pharmaceutical composition to take on different forms or be suitable for different routes of administration. Preferably, the pharmaceutical composition is an oral formulation, an injectable formulation, an inhaled formulation, or a topical or transdermal formulation, so as to be suitable for different routes of administration. Preferably, the pharmaceutical composition is a tablet, capsule, granule, powder, solution, syrup, suspension, or emulsion.

[0097] A pharmaceutically acceptable carrier can be an excipient, filler, diluent, agglutinating agent, binder, lubricant, glidant, stabilizer, colorant, wetting agent, or disintegrant. Examples of excipients include sodium citrate, calcium carbonate, or calcium phosphate; examples of fillers include lactose or high molecular weight polyethylene glycol; examples of diluents include water, ethanol, propylene glycol, or glycerol; examples of binders include sucrose, gelatin, or gum arabic; examples of lubricants include magnesium stearate, calcium stearate, zinc stearate, sodium stearate, stearic acid, aluminum stearate, leucine, glyceryl behenate, or hydrogenated vegetable oil; examples of glidants include sodium 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 include citric acid or ascorbic acid; examples of colorants include titanium dioxide or iron oxide; examples of wetting agents include Pluronic F68, Tween 20, or the like. 20), or Tween 80, and examples of disintegrants may be potato starch, tapioca starch, or silicates.

[0098] 4. Medical Use

[0099] A third embodiment of the present invention discloses a use of the pharmaceutical composition of the second embodiment for preparing a medicament for treating or preventing a disease or disorder caused by or associated with type II vascular endothelial growth factor receptor activity or signaling. The prepared medicament can be administered to a subject to inhibit type II vascular endothelial growth factor receptor activity or block its signaling. Specifically, the prepared medicament can be administered to a subject in need of treating or preventing a disease or disorder caused by or associated with type II vascular endothelial growth factor receptor activity or signaling, thereby achieving a therapeutic or preventive effect by inhibiting the subject's type II vascular endothelial growth factor receptor activity or blocking its signaling.

[0100] The drug can be administered in different ways, such as orally, by injection, by inhalation, or topically or transdermally.

[0101] Diseases or disorders caused by or associated with VEGF receptor type 2 activity or signaling may include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, hydropleural effusion, diabetes, endometriosis, dysfibrosis, or cancer. Preferably, the cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

[0102] A fourth embodiment of the present invention discloses a method for treating or preventing a disease or disorder caused by or associated with VEGF receptor type II activity or signaling, comprising administering the pharmaceutical composition of the second embodiment to a subject in need of such treatment or prevention, thereby binding to the subject's VEGF receptor type II to inhibit its activity or block its signaling.

[0103] The drug can be administered in different ways, such as orally, by injection, by inhalation, or topically or transdermally.

[0104] Diseases or disorders caused by or associated with VEGF receptor type 2 activity or signaling may include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, hydropleural effusion, diabetes, endometriosis, dysfibrosis, or cancer. Preferably, the cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

[0105] A fifth embodiment of the present invention discloses a 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 medicament can be administered to a subject to inhibit angiogenesis. Specifically, the medicament can be administered to a subject in need of treating or preventing a disease or disorder caused by angiogenesis, thereby achieving a therapeutic or preventive effect by inhibiting angiogenesis in the subject.

[0106] The drug can be administered in different ways, such as orally, by injection, by inhalation, or topically or transdermally.

[0107] Diseases or disorders caused by angiogenesis may include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, hydropleural effusion, diabetes, endometriosis, dysfibrosis, or cancer. Preferably, the cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

[0108] A sixth embodiment of the present invention discloses a method for treating or preventing a disease or disorder caused by angiogenesis, comprising administering the pharmaceutical composition of the second embodiment to a subject in need of such treatment or prevention, thereby binding to the subject's type II vascular endothelial growth factor receptor to inhibit angiogenesis.

[0109] The drug can be administered in different ways, such as orally, by injection, by inhalation, or topically or transdermally.

[0110] Diseases or disorders caused by angiogenesis may include autoimmune disorders, cardiac disorders, retinopathy, kidney disease, hemangioblastoma, hemangioma, thyroid hyperplasia, chronic inflammation, Meigs' syndrome, hydropericardium, hydropleural effusion, diabetes, endometriosis, dysfibrosis, or cancer. Preferably, the cancer includes renal cancer, pancreatic cancer, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma.

[0111] V. Other matters

[0112] A seventh embodiment of the present invention discloses a nucleic acid comprising a nucleotide sequence encoding the protein of the first embodiment. To regulate protein expression, the nucleic acid may further comprise a promoter operably linked to the protein-encoding nucleotide sequence. "Operably linked" herein means that two or more nucleic acid sequences are in a functional relationship with each other.

[0113] The eighth embodiment of the present invention discloses a host cell comprising the nucleic acid of the seventh embodiment. Because the host cell of this embodiment contains a nucleotide sequence encoding a protein, the protein can be produced by culturing the host cell. The host cell can be a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells include Escherichia coli, and examples of eukaryotic cells include CHO cells, COS cells, or HEK293 cells.

[0114] The ninth embodiment of the present invention discloses a method for producing the protein of the first embodiment, comprising culturing the host cell of the eighth embodiment to express the protein. An appropriate inducer can be selected based on the promoter to induce the cell to express the protein.

[0115] The present invention is now described by way of example with reference to the following embodiments:

[0116] <Example 1: Design of introducing disulfide bonds>

[0117] As shown in Figure 1, protein C7-NM has eight β-strands, with a loop structure formed between two adjacent β-strands. Using protein C7-NM as the parent protein, the Disulfide by Design 2.0 (DbD2) software was used to design disulfide bonds within the parent protein, taking into account parameters such as B-factors, χ3 torsion angles, 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 a β-strand and a loop. Table 2 shows the amino acid sequences of variant proteins with disulfide bonds, obtained by using the amino acid sequence of protein C7 as the reference sequence and making amino acid substitutions at the designed disulfide bond positions.

[0118] Table 1. Crystallographic parameters

[0119]

[0120]

[0121] Table 2. Amino acid sequence

[0122]

[0123]

[0124] Note 1: The letters in the box represent the mutant sequence relative to the protein 10Fn3-WT

[0125] Note 2: The letters on the bottom line are the mutation sequences relative to protein C7

[0126] <Example 2: Preparation of protein>

[0127] Protein C7 and its variants were expressed in E. coli. Briefly, protein C7 and its variants were produced using the pET21a expression vector and the BL21(DE3)pLysS strain or the SHuffle strain. The BL21(DE3)pLysS strain was grown in LB medium, while the SHuffle strain was grown in LLB medium.

[0128] First, the BL21(DE3)pLysS strain was cultured in 5 mL of culture medium at 37°C for approximately 16 to 18 hours (the SHuffle strain was cultured at 30°C). The BL21(DE3)pLysS strain was transferred to 500 mL of culture medium and cultured at 37°C for approximately 4 hours (the SHuffle strain was cultured at 30°C for approximately 6 hours). Next, 500 μL of 1 M IPTG was added and the BL21(DE3)pLysS strain was induced for protein expression at 25°C for approximately 16 to 18 hours (the SHuffle strain was induced at 16°C for approximately 48 hours), and the cell pellet was collected by centrifugation.

[0129] Bacteria were dissolved in binding buffer A (50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0) and disrupted using a French filter press (maintained at 1500 psi). The supernatant was collected by high-speed centrifugation and loaded onto a nickel ion chelating column pre-equilibrated with buffer A. The target protein was extracted using a gradient of extraction buffer (300 mM imidazole in buffer A, pH 7.0). After extraction, the target protein was confirmed by glycine SDS-PAGE. Finally, the recombinant protein was dialyzed against PBS and stored at -80°C until use.

[0130] <Example 3: Analysis of Protein Properties>

[0131] Protein melting temperatures were measured using differential scanning calorimetry (DSC). As shown in Table 3 and Figure 3, the melting point difference between protein C7-SL1 and protein C7 is 37.0°C, which is higher than the melting point difference between other proteins with a single disulfide bond and protein C7, indicating that protein C7-SL1 exhibits superior thermal stability. As shown in Table 3 and Figures 3 and 4, proteins C7-SL1-LL2 and C7-SL1-LL3, which are based on protein C7-SL1 and incorporate additional disulfide bonds, further enhance thermal stability.

[0132] Protein solubility was determined using ammonium sulfate precipitation, referring to J Pharm Sci. 2008 Oct;97(10):4155-66. As shown in Table 3, the PBS solubility of protein C7-SL1 was approximately 6 times that of protein C7, which was higher than that of other proteins with a single disulfide bond, indicating that protein C7-SL1 had excellent solubility. As shown in Table 3 and Figure 5, proteins C7-SL1-LL2 and C7-SL1-LL3, which were based on protein C7-SL1 and introduced with additional disulfide bonds, further increased their PBS solubility to approximately 7 times that of protein C7.

[0133] The inhibitory activity of the proteins on human umbilical vein endothelial cell (HUVEC) proliferation was analyzed using a cell proliferation assay. 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 derived from protein C7-SL1 with additional disulfide bonds, on HUVEC proliferation was also not significantly different from that of protein C7, indicating that proteins C7-SL1-LL2 and C7-SL1-LL3 also have the ability to inhibit angiogenesis.

[0134] The protein binding affinity to VEGF receptor type II was analyzed using an enzyme-linked immunosorbent assay (ELISA). As shown in Table 3, the binding affinity of protein C7-SL1 to VEGF receptor type II was not significantly different from that of protein C7, indicating that protein C7-SL1 has the ability to bind to VEGF receptor type II. 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 with additional disulfide bonds introduced, to VEGF receptor type II was also not significantly different from that of protein C7. In addition, the binding affinity of protein C7-SL1-LL2 to VEGF receptor type II was slightly higher than that of protein C7, indicating that proteins C7-SL1-LL2 and C7-SL1-LL3 also have the ability to bind to VEGF receptor type II.

[0135] Table 3. Protein properties

[0136]

[0137] Note 1: ΔTm = Tm value of test protein - Tm value of protein C7

[0138] Note 2: ND means not measurable, because it is formed in inclusion bodies or not tested.

[0139] However, the above is only a preferred embodiment of the present invention, and it cannot be used to limit the scope of protection of the patent of the present invention; therefore, any simple equivalent changes and modifications made according to the claims and description of the present invention still fall within the scope of protection of the patent of the present invention.

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

Claims

1. A protein, characterized in that include: The tenth human fibronectin type III domain comprises a first mutation that substitutes one amino acid position with cysteine ​​and a second mutation that substitutes another amino acid position with another cysteine, wherein the first mutation and the second mutation occur in regions other than the FG loop of the tenth human fibronectin type III domain, whereby the cysteine ​​substituted by the first mutation and the cysteine ​​substituted by the second mutation form a disulfide bond.

2. The protein according to claim 1, characterized in that: It further includes a mutation that replaces the amino acid sequence of loop BC with RHPHFPTRY, a mutation that replaces the amino acid sequence of loop DE with PLQPPT, and a mutation that replaces the amino acid sequence of loop FG with VTDGRNGRLLSIP.

3. The protein according to claim 1, characterized in that: The first mutation comprises: substitution of leucine at position 8 for cysteine, and the second mutation comprises: substitution of tryptophan at position 22 for cysteine; the first mutation comprises: substitution of leucine at position 8 for cysteine, and the second mutation comprises: substitution of serine at position 89 for cysteine; the first mutation comprises: substitution of serine at position 17 for cysteine, and the second mutation comprises: substitution of serine at position 60 for cysteine; the first mutation comprises: substitution of leucine at position 19 for cysteine, and the second mutation comprises: substitution of threonine at position 58 for cysteine; the first mutation comprises: substitution of isoleucine at position 34 for cysteine, and the second mutation comprises: substitution of phenylalanine at position 48 for cysteine; the first mutation comprises: substitution of threonine at position 35 for cysteine, and the second mutation comprises: substitution of The mutation comprises: substitution of valine at position 45 to cysteine; the first mutation comprises: substitution of tyrosine at position 36 to cysteine, and the second mutation comprises: substitution of isoleucine at position 70 to cysteine; the first mutation comprises: substitution of glycine at position 37 to cysteine, and the second mutation comprises: substitution of valine at position 45 to cysteine; the first mutation comprises: substitution of threonine at position 39 to cysteine, and the second mutation comprises: substitution of aspartic acid at position 67 to cysteine; the first mutation comprises: substitution of lysine at position 63 to cysteine, and the second mutation comprises: substitution of valine at position 66 to cysteine; or the first mutation comprises: substitution of aspartic acid at position 67 to cysteine, and the second mutation comprises: substitution of asparagine at position 91 to cysteine.

4. The protein according to claim 1, characterized in that: The invention further comprises a third mutation that substitutes another amino acid position with another cysteine ​​and a fourth mutation that substitutes another amino acid position with another cysteine, wherein the third mutation and the fourth mutation occur in regions other than the FG loop of the tenth human fibronectin type III domain, whereby the cysteine ​​substituted by the third mutation and the cysteine ​​substituted by the fourth mutation form a disulfide bond.

5. The protein according to claim 3, characterized in that: The invention further comprises a third mutation that substitutes another amino acid position with another cysteine ​​and a fourth mutation that substitutes another amino acid position with another cysteine, wherein the third mutation and the fourth mutation occur in a region other than the FG loop of the tenth human fibronectin type III domain, whereby the cysteine ​​substituted by the third mutation and the cysteine ​​substituted by the fourth mutation form a disulfide bond; the 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 aspartic acid at position 67 with cysteine.

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

7. The protein according to claim 1, characterized in that: The amino acid sequence comprises an amino acid sequence as shown in SEQ ID NO: 11, 15, or 16.

8. A pharmaceutical composition, characterized in that include: A protein according to claim 1; and A pharmaceutically acceptable carrier.

9. A use of the pharmaceutical composition according to claim 1, characterized in that: For preparing medicines for treating or preventing diseases or disorders caused by or associated with type II vascular endothelial growth factor receptor activity or signal transmission.

10. A use of the pharmaceutical composition according to claim 1, characterized in that: Used for preparing medicines for treating or preventing diseases or disorders caused by angiogenesis.

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