Angiogenesis inhibiting polypeptide and application thereof

Abstract

The invention relates to the field of biological medicines, in particular to an angiogenesis inhibition polypeptide specifically bound with an ITB3 target or pharmaceutically acceptable salt thereof, which is characterized in that the polypeptide is a) or b) or c) as follows: a) a polypeptide shown in any one amino acid sequence in SEQ ID NO: 1-7, b) a polypeptide shown in any one amino acid sequence in SEQ ID NO: 2-7, and c) a polypeptide shown in any one amino acid sequence in SEQ ID NO: 3-7; b) a derivative polypeptide obtained by adding more than one amino acid residue to the amino terminal and / or carboxyl terminal of the polypeptide shown in any one amino acid sequence in SEQ ID NO: 1-7; and c) a derivative polypeptide obtained by adding or replacing more than one amino acid at any site of the polypeptide shown as any one amino acid sequence in SEQ ID NO: 1-7 except the amino terminal and the carboxyl terminal. The angiogenesis inhibiting polypeptide provided by the invention can specifically recognize and bind ITB3, can effectively inhibit angiogenesis, endothelial cell invasion and migration and target transfer in in-vivo and in-vitro experiments, is efficient and low in toxicity, and has potential effects of treating individual tumors, diabetic vascular proliferative diseases, rheumatoid arthritis and neovascular eye diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an angiogenesis-inhibiting polypeptide and its application. Background Art

[0002] Angiogenesis is a process of new blood vessel formation, which involves the migration, growth, and differentiation of endothelial cells on the inner wall of blood vessels and plays an important role in the occurrence of various diseases. For example, diabetic angiogenic diseases, or it is a prerequisite for providing oxygen and nutrients to proliferating tumors. The angiogenesis process may contribute to tumor progression, invasion, and metastasis and is generally considered an indicator of tumor prognosis. At the same time, angiogenesis plays an important role in the pathogenesis of rheumatoid arthritis, leading to the chronic inflammation and joint damage characteristics of this autoimmune disease. Angiogenesis in rheumatoid arthritis is driven by inflammatory cytokines, especially vascular endothelial growth factor (VEGF) and other angiogenic factors. The new blood vessels provide nutrients and oxygen to the inflamed synovium, promote the influx of immune cells, and promote the persistence of inflammation (Dudley AC, Angiogenesis. 2023; 26(3): 313-347.).

[0003] Integrin is a heterodimeric transmembrane glycoprotein formed by two subunits, α (120 - 185 kD) and β (90 - 110 kD). Integrin β3 (also known as CD61, GP3A, ITGB3, or ITB3) is one of the most extensively studied components of the integrin family. As a heterodimer, ITB3 mainly exists in two forms, namely αIIβ3 integrin formed with αIIb and αVβ3 integrin formed with αV. Among them, αIIβ3 integrin is highly expressed in platelets, is related to the pathogenesis of thrombocytopenia, and may be involved in the occurrence of platelet tumors (Nurden AT, Blood. 2011; 118(23):5996 - 6005.); αVβ3 integrin is overexpressed in angiogenic endothelial cells and tumor cells, thus promoting the invasion and migration of various malignant tumors (Zhang N, J Cancer. 2017; 8(18):3742 - 3754.). And both can selectively distinguish ligands containing the RGD (arginine - glycine - aspartic acid) tripeptide active site, such as vitronectin and fibronectin. When ITB3 binds to these extracellular ligands, it initiates inside - out signaling. This leads to conformational changes in integrin, triggering the recruitment and activation of intracellular signaling molecules. The downstream signaling pathways activated by ITB3 include FAK / PI3K / AKT, MEK / ERK, Akt, YAP / TAZ, KRAS / RalB / NF - κb, and these signal cascades regulate and affect the biological behaviors of tumor cells such as proliferation, gene transduction, and apoptosis (Gao YY, Cell Death Dis. 2018; 9(9):845.).

[0004] Angiogenesis inhibitors may become a means to inhibit tumor growth and metastasis and may also have potential effects in treating rheumatoid arthritis. However, current angiogenesis inhibitors have unclear action targets, and their specificity and selectivity for blood vessels are not strong enough, so their dosage exceeds the normal administration dosage, which leads to relatively strong toxic and side effects of such drugs, greatly limiting their clinical application.

[0005] Polypeptides are compounds of three or more amino acid molecules connected by peptide bonds. As one of the important substances of life, peptide substances widely exist in living organisms to regulate the functional activities of various systems, organs, and cells in the body. In recent years, polypeptide drugs have attracted much attention in the development of new drugs due to their advantages such as high targeting, low immunogenicity, high tissue permeability, and safety.

[0006] Therefore, the development of a polypeptide drug with strong selective action on neovascularization marker molecules (such as ITB3) can achieve the therapeutic effect of targeted inhibition of angiogenesis, improve the inhibitory effect on angiogenesis as a whole, and can be safe and effective at low doses, with strong patient compliance and good tolerance. It has broad application prospects in the treatment of solid tumors, rheumatoid arthritis or diabetic angioproliferative diseases. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the purpose of this application is to provide a new angiogenesis-inhibiting polypeptide and its application. The polypeptide can effectively bind to the ITB3 target and can effectively inhibit angiogenesis, endothelial cell invasion and migration in in vitro and in vivo experiments.

[0008] To solve the above technical problems, this application proposes the following technical solutions:

[0009] In the first aspect of this application, there is provided an angiogenesis-inhibiting polypeptide that specifically binds to the ITB3 target or a pharmaceutically acceptable salt thereof, characterized in that the polypeptide is one of the following a), b), or c):

[0010] a) A polypeptide shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7;

[0011] b) A derivative polypeptide obtained by adding one or more amino acid residues to the amino terminus and / or carboxyl terminus of the polypeptide shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7;

[0012] c) A derivative polypeptide obtained by adding or substituting one or more amino acids at any site other than the amino terminus and carboxyl terminus of the polypeptide shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7.

[0013] In some embodiments, the polypeptide is further modified.

[0014] In the second aspect of this application, there is provided a biological material, which is selected from any one of the following (1)-(3):

[0015] (1) A polynucleotide encoding the aforementioned polypeptide or a pharmaceutically acceptable salt thereof; or

[0016] (2) An expression vector containing the polynucleotide described in (1); or

[0017] (3) A host cell containing the polynucleotide described in (1) or the expression vector described in (2).

[0018] In the third aspect of this application, there is provided a multimer formed by the aforementioned polypeptide or a pharmaceutically acceptable salt thereof.

[0019] The fourth aspect of the present application provides a pharmaceutical composition, which comprises the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, as well as a pharmaceutically acceptable adjuvant, carrier or targeting compound.

[0020] The fifth aspect of the present application provides an angiogenesis-inhibiting reagent or kit, which contains the aforementioned polypeptide or a pharmaceutically acceptable salt thereof or the aforementioned polymer.

[0021] The sixth aspect of the present application provides the use of the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned biomaterial, the aforementioned polymer and / or the aforementioned pharmaceutical composition in the preparation of a drug for treating angiogenesis-related diseases.

[0022] In some embodiments, the angiogenesis-related diseases are selected from one or more of tumors, diabetic angioproliferative diseases, rheumatoid arthritis, and neovascular eye diseases.

[0023] The seventh aspect of the present application provides the use of the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned biomaterial, the aforementioned polymer and / or the aforementioned pharmaceutical composition in the preparation of a drug for inhibiting angiogenesis, and the drug has one or more of the following functions:

[0024] (a) inhibiting ITB3-induced angiogenesis;

[0025] (b) inhibiting the migration of ITB3-induced vascular endothelial cells;

[0026] (c) inhibiting the invasion of ITB3-induced vascular endothelial cells.

[0027] The eighth aspect of the present application provides a method for treating angiogenesis-related diseases, which inhibits angiogenesis by using a therapeutically effective amount of the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned polymer and / or the aforementioned pharmaceutical composition in an individual in need.

[0028] In some embodiments, the angiogenesis-related diseases are selected from one or more of tumors, diabetic angioproliferative diseases, rheumatoid arthritis, and neovascular eye diseases.

[0029] In some embodiments, the tumors are selected from at least one of lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, uterine cancer, ovarian cancer, liposarcoma, breast cancer, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymic cancer, and brain or central nervous system cancer.

[0030] Compared with the prior art, the present disclosure has at least the following beneficial effects:

[0031] The polypeptide provided by the present application can specifically recognize and bind to ITB3, and can effectively inhibit angiogenesis, endothelial cell invasion and migration in in vitro and in vivo experiments. Considering the potential therapeutic effects of angiogenesis inhibitors in angiogenesis-related diseases (such as tumors and rheumatoid arthritis, etc.), therefore, the angiogenesis-inhibiting polypeptide provided by the present application also has the potential to treat individual tumors, diabetic angioproliferative diseases, rheumatoid arthritis, and neovascular eye diseases. In addition, the polypeptide provided by the present application has a specific target and the characteristics of high efficiency and low toxicity. The 22a polypeptide can effectively inhibit angiogenesis in the chick embryo chorioallantoic membrane model at a low concentration of 1 mg / mL, which makes up for the deficiencies of current treatment methods to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figures 1A - 1H It is the SPR experimental results of 8 polypeptides of SEQ ID NO: 1-8 and the ITB3 target in Example 4;

[0033] Figures 2A - 2B It is the anti-angiogenesis experimental results of 8 polypeptides of SEQ ID NO: 1-8 and fruquintinib in Example 5;

[0034] Figures 3A - 3F It is the anti-cell migration experimental results of 3 polypeptides of SEQ ID NO: 4, 6, 8 and fruquintinib in Example 6;

[0035] Figures 4A - 4C It is the anti-cell invasion experimental results of 3 polypeptides of SEQ ID NO: 4, 6, 8 and fruquintinib in Example 7;

[0036] Figure 5 It is the experimental results of inhibiting angiogenesis in the chick embryo chorioallantoic membrane of 2 polypeptides of SEQ ID NO: 4, 6 and fruquintinib, RGD-ED positive control in Example 8. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only one implementation manner of the present application. For those of ordinary skill in the art, other implementation manners can also be obtained according to these drawings.

[0038] Definition

[0039] As used herein, the terms "a" and "an" and "the" and similar referents denote singular and plural unless otherwise specified herein or the context is clearly contradictory.

[0040] As used herein, the terms "about", "substantially", and "similar to" refer to an acceptable error range of a specific value determined by a person of ordinary skill in the art, and the error range may depend in part on the manner in which the value is measured or determined, or on the limitations of the measurement system.

[0041] The term "polypeptide" as used herein refers to a linear polymer formed by three or more amino acids linked by peptide bonds.

[0042] The term "polynucleotide" as used herein refers to a linear polymer of more than 10 nucleotides linked by 3',5'-phosphodiester bonds.

[0043] The term "gene" as used herein refers to a genetic unit that occupies a specific locus on a chromosome.

[0044] The term "fragment" means a polypeptide in which one or more (several) amino acids are deleted from the amino and / or carboxyl termini of the polypeptide.

[0045] The term "variant" means a polypeptide that contains amino acid changes at one or more (several) positions, i.e., a polypeptide having the activity of a parental or wild-type polypeptide with one or more (several) amino acid residue substitutions, insertions, and / or deletions. "Substitution" means replacing the amino acid occupying a position with a different amino acid; "deletion" means removing the amino acid occupying a position; and "insertion" means adding one or several amino acids adjacent to the amino acid occupying a position.

[0046] The term "polypeptide-carrier protein or drug conjugate" means a conjugate formed by coupling a polypeptide of the present application with a carrier protein or a drug, wherein one carrier protein or drug can be coupled to one or more polypeptides, and when multiple polypeptides are coupled, the multiple polypeptides have the same amino acid sequence. Depending on the differences in the physicochemical properties of the specific polypeptide sequences coupled, the types of the specific carrier proteins or drugs, and the different coupling methods, the number of polypeptides coupled to each carrier protein or drug varies. In the present application, 2 to 50 are preferred, more preferably 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, 15 to 30; or, more preferably, any one of 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, and 10 to 15.

[0047] The term "diabetic angioproliferative disease" refers to the relevant indications involving microangiopathy among the common complications of diabetes, which is specific. Its main characteristics are the thickening of the microvascular basement membrane and the deposition of hyaline substances, and it can manifest as diabetic retinopathy, diabetic nephropathy, extensive sclerosis of the blood vessels nourishing nerves, microvascular lesions of the heart, etc. Among them, retinopathy is the most common, often leading to visual impairment and ultimately blindness.

[0048] The first aspect of the present application provides an angiogenesis-inhibiting polypeptide that specifically binds to the ITB3 target or a pharmaceutically acceptable salt thereof, and the polypeptide is one of the following a), b), or c):

[0049] a) a polypeptide shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7;

[0050] b) a derivative polypeptide obtained by adding more than 1 amino acid residue to the amino terminus and / or carboxyl terminus of the polypeptide shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7;

[0051] c) a derivative polypeptide obtained by adding or substituting more than 1 amino acid at any site except the amino terminus and carboxyl terminus of the polypeptide shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7.

[0052] In some embodiments, the present application relates to a polypeptide whose amino acid sequence is shown by any one of SEQ ID NO. 1 to 7. The inventors predicted through computer prediction software and experimentally verified that the polypeptide sequence has a high affinity for ITB3. Further, through an anti-angiogenesis experiment, it was verified that the peptide segment has an anti-angiogenesis effect.

[0053] In some embodiments, the present application also relates to variants of the polypeptide. The variant contains amino acid changes at one or more (several) positions, that is, substitution, insertion, and / or deletion of one or more (several) amino acid residues, but still has the activity of the polypeptide, that is, has a high affinity for ITB3 and at least has the ability to inhibit angiogenesis. In a specific embodiment, "several" means 5 or less, more preferably 3 or less, and most preferably 2 or less. For example, the variant of the polypeptide may have substitution, insertion, and / or deletion of 5, 4, 3, 2, or 1 amino acid residue compared with the polypeptide shown by any one of SEQ ID NO. 1 to 7.

[0054] In some embodiments, the amino acid change is a conservative substitution. Examples of conservative substitutions are within the following groups: basic amino acid group (arginine, lysine, and histidine), acidic amino acid group (glutamic acid and aspartic acid), polar amino acid group (glutamine and asparagine), hydrophobic amino acid group (leucine, isoleucine, and valine), aromatic amino acid group (phenylalanine, tryptophan, and tyrosine), and small amino acid group (glycine, alanine, serine, threonine, and methionine).

[0055] In addition to the 20 standard amino acids, amino acid residues in the polypeptide can be replaced with non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine). A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids can replace amino acid residues. Unnatural amino acids can be synthesized by chemical methods and are preferably commercially available, including, for example, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0056] Furthermore, essential amino acids in the parent polypeptide can be identified according to methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced into each residue in the molecule, and the biological activity of the resulting mutant molecule is tested to identify the amino acid residues critical for the activity of the molecule. See Hilton et al., 1996, J. Biol. Chem. 271:4699-4708.

[0057] The polypeptides of the present application can be prepared using well-known techniques. For example, the polypeptides can be prepared using recombinant DNA technology or by chemical synthesis methods. The polypeptides of the present application can also be expressed by viral vectors or bacterial vectors. Examples of suitable expression vectors include, but are not limited to: viral vectors, such as lentivirus, vaccinia virus, fowlpox virus, adenovirus, and adeno-associated virus vectors, retroviral vectors; BCG (Bacille Calmette Guerin) vectors; bacterial vectors, such as Salmonella typhi vectors. Preferably, the viral vector is a replication-incompetent and non-pathogenic vector.

[0058] The polypeptide of the present application is preferably an isolated polypeptide. The term "isolated polypeptide" as used herein refers to a polypeptide isolated from its source. For example, the polypeptide is at least 60% pure, preferably at least 80% pure, more preferably at least 90% pure, and most preferably at least 95% pure, as determined by SDS-PAGE and HPLC. Preferably, the "isolated polypeptide" is a substantially pure polypeptide. "Substantially pure polypeptide" as used herein refers to a polypeptide preparation that contains, by weight, at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of other polypeptide substances that are naturally or recombinantly associated with it.

[0059] In some embodiments, the polypeptide is further modified. The modification is a chemical group modification, an amino acid modification, or a nucleic acid modification. In some embodiments, the chemical modification is selected from one or more of N-methylation modification, phosphorylation modification, fatty acid acylation modification, glycosylation modification, PEG modification, or fluorescent labeling modification.

[0060] In some embodiments, in order to better achieve the directional coupling of the polypeptide, the polypeptide can be a cysteine-modified peptide segment. Preferably, the cysteine modification is the addition of cysteine at any of the following positions of the polypeptide: N-terminus, C-terminus, both N- and C-termini, side chain, or backbone modification; more preferably, the addition of cysteine in the middle of the peptide chain includes inserting one or more cysteines in the middle of the peptide chain, or one or more cysteines are connected to the middle of the peptide chain in a branched form.

[0061] In some embodiments, it contains the polypeptide shown in SEQ ID NO.4 or a pharmaceutically acceptable salt thereof.

[0062] In some embodiments, the polypeptide composition contains the polypeptide shown in SEQ ID NO.6 or a pharmaceutically acceptable salt thereof.

[0063] The second aspect of the present application provides a biomaterial selected from any one of the following (1)-(3):

[0064] (1) A polynucleotide encoding the aforementioned polypeptide or a pharmaceutically acceptable salt thereof; or

[0065] (2) An expression vector containing the polynucleotide of (1); or

[0066] (3) A host cell containing the polynucleotide of (1) or the expression vector of (2).

[0067] In some embodiments, the polynucleotides provided in the second aspect (1) of the present application can be constructed into vectors, such as viral vectors, bacterial vectors, eukaryotic vectors or BCG vectors. Examples of viral vectors include but are not limited to lentivirus, vaccinia virus, fowlpox virus, adenovirus and adeno-associated virus vectors, retroviral vectors, preferably lentiviral vectors. More preferably, the viral vector is an attenuated or detoxified vector. Examples of bacterial vectors include but are not limited to Salmonella typhi vectors, Bacillus subtilis vectors, Escherichia coli vectors, etc. Examples of eukaryotic vectors include but are not limited to yeast vectors, such as Saccharomyces cerevisiae vectors.

[0068] In some embodiments, the expression vector provided in the second aspect (2) of the present application can be transformed or transfected into a host cell such as a host cell. Suitable host cells can be any host cells suitable for the expression of the vector.

[0069] The third aspect of the present application provides a multimer formed from the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, which comprises at least two polypeptides or pharmaceutically acceptable salts thereof provided in the first aspect of the present application.

[0070] In some embodiments, the multimer comprises the polypeptide shown in SEQ ID NO.1 or a pharmaceutically acceptable salt thereof, and at least one of the polypeptides shown in SEQ ID NOs. 2-7 or pharmaceutically acceptable salts thereof.

[0071] In some embodiments, the multimer comprises the polypeptide shown in SEQ ID NO.2 or a pharmaceutically acceptable salt thereof, and at least one of the polypeptides shown in SEQ ID NO.1 or SEQ ID NOs. 3-7 or pharmaceutically acceptable salts thereof.

[0072] In some embodiments, the multimer comprises the polypeptide shown in SEQ ID NO.3 or a pharmaceutically acceptable salt thereof, and at least one of the polypeptides shown in SEQ ID NOs. 1-2 or SEQ ID NOs. 4-7 or pharmaceutically acceptable salts thereof.

[0073] In some embodiments, the multimer comprises the polypeptide shown in SEQ ID NO.4 or a pharmaceutically acceptable salt thereof, and at least one of the polypeptides shown in SEQ ID NOs. 1-3 or SEQ ID NOs. 5-7 or pharmaceutically acceptable salts thereof.

[0074] In some embodiments, the multimer comprises the polypeptide shown in SEQ ID NO.5 or a pharmaceutically acceptable salt thereof, and at least one of the polypeptides shown in SEQ ID NOs. 1-4 or SEQ ID NOs. 6-7 or pharmaceutically acceptable salts thereof.

[0075] In some embodiments, the multimer comprises the polypeptide shown in SEQ ID NO.6 or a pharmaceutically acceptable salt thereof, and at least one of the polypeptides shown in SEQ ID NOs. 1-5 or SEQ ID NO.7 or a pharmaceutically acceptable salt thereof.

[0076] In some embodiments, the multimer comprises the polypeptide shown in SEQ ID NO.7 or a pharmaceutically acceptable salt thereof, and at least one of the polypeptides shown in SEQ ID NOs. 1-6 or a pharmaceutically acceptable salt thereof.

[0077] The fourth aspect of the present application provides a pharmaceutical composition, which comprises the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, carrier or pharmaceutical targeting compound. In some embodiments, the carrier comprises a carrier protein or a carrier drug. In some embodiments, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, and casein. In some embodiments, the carrier drug is selected from cytotoxic drugs such as alkylating agents, methotrexate, cisplatin, cytarabine, fluorouracil, etc. In some embodiments, the polypeptide is conjugated to the carrier protein or drug through a linking sequence. In some embodiments, 5-50 polypeptides are conjugated to each carrier protein or drug; in some embodiments, 5-30 polypeptides are conjugated to each carrier protein or drug.

[0078] According to the requirements for the preparation of the pharmaceutical composition, a specific and suitable carrier protein or drug can be selected to form the pharmaceutical composition. The carrier proteins or drugs in the present application include but are not limited to BSA (bovine serum albumin), OVA (ovalbumin), KLH (keyhole limpet hemocyanin) or CS (casein). According to the amino acid sequence composition of different polypeptides, in order to facilitate conjugation with carrier proteins or drugs, it is necessary to conjugate with carrier proteins or drugs through a linking sequence (also called a linker or linker).

[0079] According to the physicochemical properties of the polypeptide amino acids, the differences in the carrier proteins or drugs used, and the differences in the conjugation methods, the number of polypeptides that can be conjugated to each carrier protein or drug also varies. Considering the conjugation efficiency and the ability of antibodies to recognize and bind, it is preferred that 2-50 polypeptides are conjugated to each carrier protein or drug, more preferably 3-45, 5-40, 5-35, 5-30, 8-30, 10-30, 12-30, 15-30; or, more preferably, any one of 6-36, 8-32, 10-28, 10-26, 10-24, 10-22, 10-20, 10-18, 10-16, and 10-15.

[0080] Exemplary adjuvants include, but are not limited to, aluminum phosphate, aluminum hydroxide, and alum; Exemplarily, the carrier is selected from at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant, or a lubricant.

[0081] In some embodiments, the pharmaceutical composition may contain one or a combination of the polypeptides provided in the first aspect of the present application or their pharmaceutically acceptable salts.

[0082] In some embodiments, the pharmaceutical composition may further include a polypeptide stabilizer; Any reagent or component capable of maintaining the storage stability of the polypeptide can be used as the polypeptide stabilizer of the present application.

[0083] In some embodiments, the polypeptide stabilizer is selected from nanoparticle materials, such as poly(lactic-co-glycolic acid) (PLGA) nanoparticles.

[0084] In some embodiments, the polypeptide stabilizer comprises 150 - 180 mM NaCl, 100 - 140 mM polylysine hydrochloride, and water. More preferably, the polypeptide stabilizer comprises 153 - 158 mM NaCl, 110 - 130 mM polylysine hydrochloride, and water; Further preferably, the polypeptide stabilizer comprises 154 mM NaCl, 126.4 mM polylysine hydrochloride, and water. Specifically, in the polypeptide stabilizer, the concentration of NaCl can be 150 mM, 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, 156 mM, 157 mM, 158 mM, 159 mM, 160 mM, 161 mM, 162 mM, 163 mM, 164 mM, 165 mM, 166 mM, 167 mM, 168 mM, 169 mM, 170 mM, 171 mM, 172 mM, 173 mM, 174 mM, 175 mM, 176 mM, 177 mM, 178 mM, 179 mM, or 180 mM; the concentration of polylysine hydrochloride can be 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, 120 mM, 121 mM, 122 mM, 123 mM, 124 mM, 125 mM, 126 mM, 127 mM, 128 mM, 129 mM, 130 mM, 131 mM, 132 mM, 133 mM, 134 mM, 135 mM, 136 mM, 137 mM, 138 mM, 139 mM, or 140 mM.

[0085] In some embodiments, in the pharmaceutical composition, the effective amount of the polypeptide can be from 0.01 μM to 0.1 M; preferably, from 0.01 μM to 100 μM; preferably, from 0.1 mM to 100 mM; preferably, from 10 μM to 10 mM; preferably from 10 μM to 1000 μM. More specifically, it can be 0.1 μM, 0.2 μM, 0.5 μM, 0.75 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 200 μM, 500 μM, 750 μM, 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, 75 mM, 100 mM, etc., or a range composed of any value therebetween.

[0086] In some embodiments, in the pharmaceutical composition, in addition to the polypeptide of the present application, other active ingredients may also be included, such as anticancer drugs. Suitable anticancer drugs include but are not limited to abarelix, aclarubicin, aminopurine, aldesleukin, alemtuzumab, aishutan, altretamine, amifostine, aminoglutethimide, anakinra, anastrozole, azacitidine, bleomycin, bortezomib, busulfan, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dasatinib, decitabine, docetaxel, exemestane, gemtuzumab, rituximab, vincristine, vindesine, vorinostat, zoledronic acid.

[0087] In some embodiments, the pharmaceutical composition of the present application may also be in the form of comprising the polynucleotide, expression vector, and host cell of the present application. Those skilled in the art can formulate the pharmaceutical composition into a suitable dosage form according to needs, such as liposomes, particles, powders, etc. The composition can be administered systemically or locally to the target tumor by, for example, oral, intradermal, subcutaneous, intravenous injection, etc.

[0088] In some embodiments, the dosage form of the pharmaceutical composition includes oral preparations, external preparations, or injections. In some embodiments, the oral preparations include granules, tablets, pastes, or oral liquids. In some embodiments, the external preparations include ointments, gels, suppositories, medicated bath solutions, or sprays.

[0089] The pharmaceutical composition can be used to treat diseases related to overexpression of ITB3, such as cancer. The cancer includes but is not limited to carcinoma, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, uterine cancer, ovarian cancer, liposarcoma, breast cancer, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymoma, and brain or central nervous system cancer.

[0090] The fifth aspect of the present application provides a reagent or kit for inhibiting angiogenesis, which contains the aforementioned polypeptide or its pharmaceutically acceptable salt, the aforementioned polymer or the aforementioned pharmaceutical composition.

[0091] The sixth aspect of the present application provides the use of the aforementioned polypeptide or its pharmaceutically acceptable salt, the aforementioned biological material, the aforementioned polymer and / or the aforementioned pharmaceutical composition in the preparation of a drug for treating angiogenesis-related diseases.

[0092] In some embodiments, the angiogenesis-related diseases are selected from one or more of tumors, diabetic angioproliferative diseases, rheumatoid arthritis, and neovascular eye diseases.

[0093] In some embodiments, the tumor is selected from ITB3-related tumors or cancers.

[0094] In some embodiments, the polypeptide of the present application treats ITB3-related tumors or cancers by inhibiting the growth or proliferation of ITB3-related tumor or cancer cells.

[0095] In some embodiments, the tumor or cancer is selected from at least one of lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, uterine cancer, ovarian cancer, liposarcoma, breast cancer, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymic cancer, and brain or central nervous system cancer.

[0096] Angiogenesis is an extremely complex process, which includes the dilation of existing blood vessels, the increase in vascular permeability, the degradation of the perivascular matrix, the activation and proliferation of endothelial cells, migration, and the formation of new capillary-like lumens. Angiogenesis plays a key role in wound healing and the occurrence of various diseases. In the present invention, the angiogenesis-related diseases are not particularly limited and include various angiogenesis-related diseases known in the art. Representative examples of angiogenesis-related diseases include (but are not limited to): neovascular eye diseases, tumors, ischemic heart disease, non-inflammatory cardiomyopathy, coronary arteriosclerosis, obstructive arteriosclerosis, arterial embolism, arterial thrombosis, Berger's disease, chronic inflammation, inflammatory bowel disease, ulcers, rheumatoid arthritis, scleroderma, psoriasis, infertility or sarcoidosis, etc.

[0097] Diabetic angioproliferative diseases refer to the relevant indications involved in microvascular lesions among the common complications of diabetes, which are specific. Their main feature is the thickening of the microvascular basement membrane and the deposition of hyaline substances, and they can manifest as diabetic retinopathy, diabetic nephropathy, extensive sclerosis of the blood vessels nourishing nerves, microvascular lesions of the heart, etc. Among them, retinopathy is the most common, often leading to visual impairment and ultimately blindness.

[0098] In some embodiments, the neovascular eye diseases include (but are not limited to): involving the choroid, retina, cornea or iris, including age-related macular degeneration, retinal vascular occlusive diseases, retinopathy of prematurity, corneal infection, neovascular glaucoma, etc.

[0099] The seventh aspect of the present application provides the use of the aforementioned polypeptide or its pharmaceutically acceptable salt, the aforementioned biomaterial, the aforementioned polymer and / or the aforementioned pharmaceutical composition in the preparation of a drug for inhibiting angiogenesis, and the drug has one or more of the following functions:

[0100] (a) Inhibiting ITB3-induced angiogenesis;

[0101] (b) Inhibiting the migration of ITB3-induced vascular endothelial cells;

[0102] (c) Inhibiting the invasion of ITB3-induced vascular endothelial cells.

[0103] The eighth aspect of the present application provides a method for treating angiogenesis-related diseases, by using a therapeutically effective amount of the aforementioned polypeptide or its pharmaceutically acceptable salt, the aforementioned polymer and / or the aforementioned pharmaceutical composition in an individual in need to inhibit angiogenesis.

[0104] In some embodiments, the individual in need is a mammal, preferably a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow. In a specific embodiment, the individual in need is a human; more specifically, a human suffering from the aforementioned tumor or cancer.

[0105] Those skilled in the art can formulate the drug into a suitable dosage form as needed, such as liposomes, granules, powders, etc. The composition can be administered systemically or locally to the target tumor by, for example, oral, intradermal, subcutaneous, intravenous injection, etc.

[0106] Those skilled in the art can appropriately adjust the dosage of the polypeptide, polynucleotide, nucleic acid construct, vector, host cell, polypeptide composition, polypeptide-carrier protein or drug conjugate, or pharmaceutical composition in the drug of the present application according to the disease to be prevented or treated, the age, weight, administration method, etc. of the subject, because the selection and optimization of these dosages are within the capabilities of those skilled in the art.

[0107] In some embodiments, the effective amount of the polypeptide can be from 0.1 μM to 0.1 M; preferably, from 0.1 μM to 100 μM; preferably, from 0.1 mM to 100 mM; preferably, from 10 μM to 10 mM; preferably from 10 μM to 1000 μM. More specifically, it can be 0.1 μM, 0.2 μM, 0.5 μM, 0.75 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 200 μM, 500 μM, 750 μM, 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, 75 mM, 100 mM, etc., or a range composed of any value therebetween.

[0108] In some embodiments, the effective amount of the polypeptide can be from 0.001 mg to 1000 mg, preferably from 0.01 mg to 100 mg, more preferably from 0.1 mg to 10 mg, such as 3 mg.

[0109] In the following examples, various aspects of the present application will be described. These examples are for illustrative purposes only and do not limit the scope of the present application in any way.

[0110] Example 1: Detection of polypeptide chip and construction of learning model

[0111] After protein fluorescence labeling, polypeptide chip detection is performed to obtain the binding force signal between the polypeptide and the protein. Based on this binding force data, a deep learning model is constructed to obtain the binding force prediction pattern between the polypeptide and the protein.

[0112] 1. Select the candidate ITB3 protein (Beijing Sino Biological Inc., product number: CT098-H08H) and perform fluorescence labeling on it (kit: ThermoFisher, Alexa Fluor TM 555 Microscale Protein Labeling Kit, product number: A30007).

[0113] 2. Perform high-throughput short polypeptide array chip (Zhuhai Carboncloud Intelligent Technology Co., Ltd., V16 chip) detection on the fluorescently labeled ITB3 protein:

[0114] a. Prepare the fluorescently labeled sample into a stock solution of 0.2 mg / ml, and dilute it into solutions of 50 ng / ml and 20 ng / ml for detection. The diluent is PBSTP solution, and the chip is assembled into an assay cassette according to the positions designed in the experiment.

[0115] b. Add the diluted sample to the assembled chip at 600 μL / well, and place it on a thermostatic shaker for shaking incubation for 1 hour.

[0116] c. Place the assay cassette in a plate washer for cleaning.

[0117] d. Disassemble, clean, and dry the chip in the assay cassette, then assemble it into an imaging cassette, and put it into the ImageXpress micro 4 imager of Molecular Device for scanning imaging. Finally, a TIFF picture file is obtained for each test sample, which is the original data.

[0118] e. Data normalization

[0119] Extract the fluorescence intensity values of the features, and output 1 GPR5 data file and 1 corner images file. Among them, the GPR5 file contains all the information of a sample and the fluorescence intensity information of all features.

[0120] Extract the fluorescence intensity information of the features from the GPR5 data files of all samples to generate the original fluorescence intensity (FG, foreground) data matrix, then perform logarithmic transformation on the data of each sample respectively to obtain the LFG (log - transferred foreground) data matrix, and perform z - score normalization to obtain the NLFG (normalized and log - transferred foreground) data matrix; this step will also generate a sample chip information file, which includes information such as the sample array position and the chip number used.

[0121] 3. Randomly split the ~3200000 data on the V16 chip according to the ratio of 7:2:1. Among them, 7 is used as the training set, 2 is used as the validation set, and 1 is used as the test set. Construct a neural network with a multi - layer perceptron and residuals, and build a deep learning model using one - hot encoded polypeptide sequences.

[0122] The polypeptide chip used in this application is the V16 chip of Zhuhai Carboncloud Intelligent Technology Co., Ltd. There are 4 repeated polypeptide arrays on this chip, and there are approximately 3,200,000 kinds of polypeptides on each array. These 3,200,000 kinds of polypeptides are polypeptide sequences formed by 5 - 16 unbiased random amino acid combinations respectively, and the diverse coverage rate of pentamers (5 - peptides) can reach 99.9%.

[0123] Example 2: Screening of candidate polypeptides

[0124] First, use the data of the binding of the ITB3 target protein to the chip, and at the same time combine the existing database to construct the search space of polypeptide sequences, and use a deep learning model to synthesize polypeptide sequences. The synthesized polypeptide sequences are derived from the deep learning model and belong to brand - new synthesized polypeptide sequences. Then, predict the binding affinity of the synthesized polypeptide sequences to the target protein ITB3, and select the polypeptides with high predicted binding for subsequent performance verification:

[0125] 1) Construction of the polypeptide search space

[0126] a. Polypeptide sequences on the chip, approximately 3,200,000;

[0127] b. Polypeptides recorded in the data that are on the market or have therapeutic effects, approximately 1,000;

[0128] c. Endogenous or naturally - sourced polypeptides recorded in the data, approximately 25,000,000;

[0129] d. Polypeptides designed based on the spatial structure calculation of ITB3, approximately 20,000;

[0130] 2) Construction of polypeptides based on the deep learning model

[0131] Use the deep learning model constructed with chip data to simulate and synthesize candidate polypeptides.

[0132] 3) Prediction and selection of polypeptide binding affinity

[0133] Use the deep learning model constructed with chip data to predict the binding affinity of the simulated and synthesized candidate polypeptide sequences, and sort them according to the predicted binding affinity, and select the top 30 with high signals for synthesis.

[0134] Example 3: Polypeptide synthesis

[0135] Entrust Genscript to synthesize candidate peptide segments and conduct HPLC analysis (ultraviolet detector, detection wavelength 220 nm).

[0136] Example 4: SPR evaluation of the affinity between polypeptides and the ITB3 target

[0137] This example further verified the affinity between the candidate polypeptide and the ITB3 target through SPR experiments. The specific experimental procedure is as follows:

[0138] 1) Preparation of running buffer (1x PBS-P buffer containing 0.5% DMSO)

[0139] 2) Protein coupling

[0140] a. Take 60 μL of ITB3 protein (200 μg / mL) and add it to 240 μL of sodium acetate buffer (10 mM sodium acetate, pH 4.5), mix well. The final protein concentration is about 40 μg / mL;

[0141] b. Load the CM5 chip onto the device;

[0142] c. According to the sample position information displayed on the screen, place the protein and sodium hydroxide (50 mM) in the corresponding designated positions;

[0143] d. Run the pH scouting program for pre-enrichment;

[0144] e. Ligand coupling: Protein immobilization is carried out by the amino coupling method. Apply the coupling (Immobiliztion) program in the Biacore 8K control software to complete ligand coupling;

[0145] 3) Preparation and detection of polypeptide gradient solutions

[0146] A series of polypeptide DMSO solutions with different concentration gradients are prepared by the two-fold dilution method;

[0147] a. Add the corresponding volume of DMSO to the polypeptide powder and mix well to obtain a polypeptide stock solution with a concentration of 100 mM;

[0148] b. Take a clean 96-well plate and add 5 μL of DMSO to each well from the first column to the fifth column;

[0149] c. Take 5 μL of the polypeptide stock solution and add it to the first column of the 96-well plate respectively. Add different types of polypeptide stock solutions to each row, and gently pipette 7 - 10 times with a pipette to mix well. At this time, the polypeptide concentration is diluted to 50 mM;

[0150] d. Use the two-fold dilution method for dilution. Pipette 5 μL of the solution from the first column and add it to the second column, mix well. Pipette 5 μL of the solution from the second column and add it to the third column, mix well, and repeat this until the fifth column. That is, each polypeptide is diluted into 5 polypeptide gradient solutions. Seal the 96-well plate with a sealing film and store it at 4°C;

[0151] e. Take another clean 96-well plate, add 199 μL of 1×PBSP solution to each well, and take 1 μL of the diluted polypeptide gradient solution and add it to the 96-well plate respectively, and mix well. At this time, the highest concentration of the diluted polypeptide is 250 μM (the highest concentration is adjusted according to the experimental purpose), and the concentration of DMSO is 0.5%;

[0152] f. Use the Multi-cycle Kinetics / Affinity program in Biacore 8K control software to complete the detection and fitting of affinity and kinetic data, and obtain the KD value.

[0153] 4) Select polypeptides according to the KD value.

[0154] Among the 30 synthesized polypeptides, 23 polypeptides did not show obvious binding to the target protein ITB3, and 7 polypeptides had good affinity for the target protein ITB3. The specific information can be seen in Table 1, and their nucleotide sequences are shown in SEQ ID NO: 1-7. In addition, iCXOncIt3a shown in SEQ ID NO: 8 was selected as the positive control in the subsequent physiological performance verification experiment.

[0155] Table 1 Polypeptide Information

[0156]

[0157] Example 5: Anti-angiogenesis experiment

[0158] The inventors further verified the anti-angiogenic performance in vitro of the polypeptides screened in the foregoing examples. The specific experimental process is as follows:

[0159] 1) Select HUVEC cells, use the standard subculture cell procedure, use cells in a better state of 3-5 generations, and make them reach about 80% confluence within 24 hours. Then perform starvation treatment on HUVEC cells: replace the complete culture medium with DMEM culture medium containing 0.2% FBS and culture for 24 hours.

[0160] 2) Thaw Matrigel, take out Matrigel from the -20°C or -80°C refrigerator, put it into the 4°C refrigerator until thawed, and use a pre-cooled pipette or pipette tip to mix Matrigel until it is in a uniform state.

[0161] 3) Dilute Matrigel, dilute Matrigel with culture medium to 6 mg / mL, and it can be adjusted according to the actual situation.

[0162] 4) Place the completely thawed Matrigel on ice, invert it several times to mix the contents, and then add 50 μL to each well.

[0163] 5) Transfer the 96-well plate to an incubator and incubate at 37 °C for 30 min to allow the basement membrane to form a gel.

[0164] 6) Sample preparation: The final concentrations of the 8 polypeptides of SEQ ID NOs: 1-8 are set to 0.25 μg / mL, 1 μg / mL, 4 μg / mL, 16 μg / mL, 64 μg / mL, and 256 μg / mL; the concentrations of fruquintinib are set to 0.001 μM, 0.005 μM, 0.019 μM, 0.075 μM, 0.3 μM, and 1.2 μM.

[0165] 7) Group setting: Set the 7 polypeptides of SEQ ID NOs: 1-7 as the test groups, SEQ ID NO: 8 polypeptide and fruquintinib as the positive control group, and the blank control group, with 2 replicates for each concentration.

[0166] 8) Digest the HUVEC cells that have reached 80% confluence in the T25 flask, centrifuge, and count.

[0167] 9) Resuspend the cells in a medium containing 10% FBS to make a single-cell suspension with a concentration of 500,000 cells per milliliter.

[0168] 10) Add 100 μL of the cell suspension (containing 50,000 cells) containing the sample to be tested to each well of the 96-well plate.

[0169] 11) Incubate the 96-well plate at 37 °C, 5% CO2, and 90% humidity, and take pictures and observe at the 2, 4, and 6 h time points. The formation of a vascular network can be seen at 3-12 hours, and the tube formation time is closely related to the cell state.

[0170] 12) Use the Angiogenesis Analyzer plug-in of ImageJ to quantitatively analyze the taken pictures, and process the analysis results with Microsoft Office Excel 2016 and GraphPad Prism.

[0171] Figures 2A - 2B The anti-angiogenic experimental results of the above 8 polypeptides of SEQ ID NOs: 1-8 and fruquintinib are shown. It can be seen that except for the positive control group 3a, all polypeptides have varying degrees of anti-angiogenic effects at high concentrations. Among them, the inhibition rate of 22a has reached 77.8% at 4 μg / mL, which is better than other polypeptides. The polypeptide in the 3a group reproduced the results reported in the article (see DOI: 10.1074 / jbc.M312921200), and its inhibitory effect at high concentrations is actually inferior to that at medium concentrations.

[0172] Example 6: Cell scratch assay

[0173] The applicant further verified the anti-cell migration performance of polypeptides 18a and 22a in vitro. The specific experimental procedure is as follows:

[0174] 1) First, use a marker pen to draw horizontal lines evenly on the back of a 24-well plate with the help of a ruler. Draw a line approximately every 0.6 cm, crossing the wells horizontally, and draw 2 lines through each well;

[0175] 2) Take umbilical vein endothelial cells in the logarithmic growth phase, digest them into a single-cell suspension, count the cells, and inoculate 7w / well into a 24-well plate. The final total volume of the culture medium in each well is 2 mL;

[0176] 3) Incubate at 37 °C, 5% CO2, and 90% humidity for 24 h;

[0177] 4) The next day, use a 10 μL long pipette tip to draw a scratch parallel to the horizontal lines on the back of the 24-well plate with the help of a ruler. The pipette tip should be perpendicular and not tilted;

[0178] 5) Sample preparation: The final concentrations of the 3 polypeptides of SEQ ID NO: 4, 6, and 8 are set to 0.3 μg / mL, 1 μg / mL, 3 μg / mL, 9 μg / mL, 27 μg / mL, and 81 μg / mL; the concentration of fruquintinib is set to 0.001 μM, 0.005 μM, 0.019 μM, 0.075 μM, 0.3 μM, and 1.2 μM;

[0179] 6) Group setting: Set the 2 polypeptides of SEQ ID NO: 4 and 6 as the test groups, the polypeptide of SEQ ID NO: 8 and fruquintinib as the two positive control groups, and the blank control group. There are two replicates for each concentration, and each well contains 2 mL of the drug-containing culture medium;

[0180] 7) Photograph: Wipe off the marker horizontal line scratches on the back of the 24-well plate. Take photos under a 4-fold microscope at 0 h, 24 h, and 48 h, ensuring that the scratches are centered and perpendicular, and pay attention to the consistent background.

[0181] Figures 3A - 3F The anti-cell migration experimental results of polypeptides 3a, 18a, 22a, and fruquintinib are shown. The results indicate that 18a and 22a both have the effect of inhibiting cell migration within 48 h at concentrations of 3 μg / mL - 81 μg / mL, and the inhibitory effect shows a positive correlation with the sample concentration. Among them, at a high concentration (81 μg / mL), the inhibitory migration effect of 22a within 48 h is significantly greater than that of the positive controls 3a and fruquintinib.

[0182] Example 7: Anti-cell invasion experiment (Trans well experiment)

[0183] The applicant further verified the anti-cell invasion performance of polypeptides 18a and 22a in vitro. The specific experimental procedure is as follows:

[0184] 1) Observe the growth status of HUVEC cells. Remove the culture medium from cells with good growth status, add serum-free medium, and starve for 24 h.

[0185] 2) Place the Matrigel on ice and melt it overnight at 4°C. Place the pipette or pipette tip in a 4°C pre-cooled environment overnight.

[0186] 3) Use a pre-cooled pipette or pipette tip to mix the Matrigel until it is in a uniform state.

[0187] 4) On ice, dilute the Matrigel with serum-free medium to 1 mg / mL, and use a pre-cooled pipette tip to mix it until it is in a uniform state.

[0188] 5) Take 60 μL of the above mixed solution and vertically add it to the Transwell chamber, and evenly spread it on the bottom. Pay attention to evenly spreading the gel without generating bubbles. Then incubate at 37°C for 1 hour.

[0189] 6) Carefully aspirate the unbound Matrigel.

[0190] 7) Add 100 μL of serum-free culture medium, place the culture plate in an incubator at 37°C, and incubate for 30 minutes for hydration.

[0191] 8) Remove the liquid in the chamber, check whether there is liquid passing through the chamber into the lower chamber. If not, it can be used for cell seeding.

[0192] 9) Sample preparation: Dilute the three polypeptides of SEQ ID NO: 4, 6, and 8 with serum-free medium to 4 μg / mL, 16 μg / mL, and 64 μg / mL (this concentration is twice the final concentration); set the concentrations of fruquintinib to 0.006 μM, 0.06 μM, and 0.6 μM (this concentration is twice the final concentration), and at the same time set a blank control group with only medium added.

[0193] 10) Add 500 μL of complete medium containing 10% FBS to the lower chamber of a 24-well plate, and place the Transwell chamber in the 24-well plate with tweezers.

[0194] 11) Digest the starved cells, resuspend them with serum-free medium, and adjust the cell density to 200,000 cells / mL.

[0195] 12) Add to the chamber according to the ratio of sample: cell suspension = 1:1. First add 100 μL of the sample working solution, and then add 100 μL of the cell suspension. At this time, the sample concentration is diluted by 2 times to be the final concentration.

[0196] 13) Place the 24-well plate in an incubator at 37°C, 5% CO2, and 90% humidity for 24 - 48 hours.

[0197] 14) Remove the Transwell chamber, discard the culture medium, and gently wipe the Matrigel and cells inside the chamber with a cotton swab or cotton soaked in PBS.

[0198] 15) Add 600 μl of 4% paraformaldehyde fixative to a clean well of a 24-well plate, and place the chamber into it for fixation for 30 minutes.

[0199] 16) Discard the fixative, and wash the inside and outside of the chamber once with PBS.

[0200] 17) Add 600 μL of crystal violet staining solution to a clean well of a 24-well plate, and place the chamber into it for staining for 10 minutes.

[0201] 18) Take out the chamber, and wash the inside and outside of the chamber three times with PBS.

[0202] 19) After appropriate air drying, observe under a microscope for qualitative research; take 3 - 5 fields of view for photographing, and then use ImageJ to count and take the average value for quantitative research.

[0203] Figures 4A - 4C The anti - cell invasion experimental results of the three polypeptides of SEQ ID NO:4, 6, 8 and fruquintinib are shown above. The results show that with the increase of the use concentration, the inhibitory effects of 18a and 22a on cell invasion are continuously enhanced, and the inhibitory effects are significantly better than those of the positive control 3a and fruquintinib.

[0204] Example 8: Chicken embryo chorioallantoic membrane angiogenesis inhibition experiment

[0205] In order to further study the angiogenesis - inhibiting properties of 18a and 22a polypeptides, the inventors verified it through the chicken embryo chorioallantoic membrane angiogenesis experiment in this example. The specific experimental process is as follows:

[0206] 1) Purchase 90 0 - day - old chicken embryo eggs. After disinfection, incubate them at a constant temperature in an incubator at 37.5 °C. Turn the eggs until the 7th day, and then candling to remove dead embryos. Select the chicken embryo eggs with good development and mark the air chambers.

[0207] 2) Grouping: At 8 - 9 days old, select 70 chicken embryo eggs with good development. Randomly divide the chicken embryo eggs into 5 groups according to weight, with 10 chicken embryo eggs in each group: negative control group (A), positive (fruquintinib) control group (B), iCXOncIt18a dose group (C), iCXOncIt22a dose group (D), RGD - ED positive control group (E). See Table 2 for details.

[0208] 3) Administration: Use a 14-mm diameter silicone administration ring, sterilize it by high-pressure steam, and dry it in a laminar flow hood for later use. Transfer the chicken embryo eggs into the laminar flow hood. After disinfecting the eggshells, mark the air chamber under an egg candler, make a hole above the air chamber by twisting it with your fingers, wipe off the powder on the eggshell with an alcohol swab, enlarge the aperture with forceps, avoiding damage to the shell membrane. Blow off the eggshell powder on the shell membrane with a pipette, drip 200 μL of sterile saline to moisten it, remove the saline, then remove the shell membrane. After taking a photo, administer the drugs. Group A is given PBS, Group B is given 1 nM fruquintinib, Group C is given 1 mg / mL iCXOncIt18a, Group D is given 1 mg / mL iCXOncIt22a, and Group E is given 1 mg / mL RGD-ED (reference DOI: 10.1021 / bc900070r). Take 40 μL of the test substance solution and add it into the administration ring, place it on the area with dense blood vessels on the chorioallantoic membrane of the chicken embryo, seal the egg opening with plastic wrap, and transfer the chicken embryo egg horizontally to the incubator for continued incubation for 48 h, then end the experiment.

[0209] Table 2 Administration Table for the Chicken Embryo Chorioallantoic Membrane Model

[0210]

[0211] 4) After 48 h, add 1 - 2 mL of 4% paraformaldehyde to each chicken embryo egg for fixation for 20 - 30 min. Cut open the chicken embryo along the long axis of the eggshell, carefully remove the contents. During the operation, ensure that the chorioallantoic membrane of the chicken embryo at the drug administration end adheres to the wall. Centering on the drug administration opening, take a photo with a fluorescence stereomicroscope and perform vascular quantitative analysis.

[0212] 5) Use the Image-Pro Plus 6.0 image analysis system to analyze the vascular areas of each drug administration group and the control group. Calculate the angiogenesis rate: vascular area at the drug administration site / total area of the chorioallantoic membrane of the chicken embryo at the drug administration site. All data are processed using the GraphPad Prism 5.0 statistical software. Perform a t-test on each drug administration group and the blank control group. P < 0.05 indicates that the difference is statistically significant.

[0213] Figure 5 The experimental results of inhibiting angiogenesis in the chicken embryo chorioallantoic membrane by the above-mentioned 18a and 22a polypeptides and fruquintinib are shown. The blood vessels in the negative control group grew well and the capillaries were clearly visible; the blood vessel diameters in the positive control group decreased and the number of capillaries decreased. The blood vessel diameters in the 22a-dose group decreased significantly, and the number of capillaries was small, and it was significantly better than the positive control RGD-ED; the blood vessel diameters in the 18a-dose group were significantly reduced. The number of capillaries in the RGD-ED group decreased.

Claims

1. An angiogenesis inhibitory polypeptide that specifically binds to the ITB3 target or a pharmaceutically acceptable salt thereof, characterized in that, The polypeptide is any one of the following a), b), or c): a) A polypeptide as shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7; b) A derivative polypeptide obtained by adding one or more amino acid residues to the amino terminus and / or carboxyl terminus of the polypeptide as shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7; c) A derivative polypeptide obtained by adding or substituting one or more amino acids at any site other than the amino terminus and carboxyl terminus of the polypeptide as shown by the amino acid sequence of any one of SEQ ID NO: 1 to 7.

2. The polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The polypeptide is further modified; Preferably, the modification is chemical group modification, amino acid modification, or nucleic acid modification; Preferably, the chemical modification is selected from one or more of N-methylation modification, phosphorylation modification, fatty acid acylation modification, glycosylation modification, PEG modification, or fluorescent labeling modification; Preferably, the modification site is selected from one or more of N-terminal modification, C-terminal modification, N- and C-terminal modification, side chain modification, or backbone modification.

3. The polypeptide according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, characterized in that, The amino acid sequence of the polypeptide is as shown by SEQ ID NO: 4 or SEQ ID NO:

6.

4. A biological material, which is selected from any one of the following (1)-(3): (1) A polynucleotide encoding the polypeptide according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof; or (2) An expression vector containing the polynucleotide described in (1); or (3) A host cell containing the polynucleotide described in (1) or the expression vector described in (2).

5. A multimer formed by the polypeptide according to any one of claims 1 or 2 or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition, which comprises the polypeptide according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, carrier, or pharmaceutical targeting compound.

7. The pharmaceutical composition according to claim 6, wherein, The polypeptide stabilizer contains PLGA; Preferably, the polypeptide stabilizer further includes sodium chloride, polylysine hydrochloride, and water; Preferably, the amount of substance of sodium chloride is 150-180 mM; Preferably, the amount of substance of polylysine hydrochloride is 100-140 mM.

8. The pharmaceutical composition according to claim 6 or 7, wherein, The dosage form of the pharmaceutical composition includes oral preparations, topical preparations, or injections; Preferably, the oral preparations include granules, tablets, pastes, or oral liquids; Preferably, the topical preparations include ointments, gels, suppositories, medicated bath solutions, or sprays.

9. An angiogenesis-inhibiting reagent or kit, which contains the polypeptide according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, the multimer according to claim 5, or the pharmaceutical composition according to any one of claims 6-8.

10. Use of the polypeptide according to claims 1-3 or a pharmaceutically acceptable salt thereof, the biological material according to claim 4, the multimer according to claim 5, and / or the pharmaceutical composition according to claims 6-8 in the preparation of a drug for treating angiogenesis-related diseases; Preferably, the angiogenesis-related diseases are selected from one or more of tumors, diabetic angioproliferative diseases, rheumatoid arthritis, and neovascular eye diseases.

11. Use of the polypeptide according to claims 1-3 or a pharmaceutically acceptable salt thereof, the biomaterial according to claim 4, the polymer according to claim 5 and / or the pharmaceutical composition according to claims 6-8 in the preparation of a medicament for inhibiting angiogenesis, wherein the medicament has one or more of the following functions: (a) inhibiting ITB3-induced angiogenesis; (b) inhibiting the migration of vascular endothelial cells induced by ITB3; (c) inhibiting the invasion of vascular endothelial cells induced by ITB3.

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