Polypeptide capable of effectively killing tumor cells and causing immunogenic death of tumor cells as well as preparation method, drug delivery system and application of polypeptide

Abstract

The invention relates to the technical field of biological medicine, in particular to a polypeptide molecule LCX17 capable of killing tumor cells or inhibiting tumor cell growth and causing tumor cell immunogenicity death, a design and preparation method of a mutant of the polypeptide molecule LCX17, a drug delivery system and application of the polypeptide molecule LCX17. The polypeptide disclosed by the invention shows a good anti-tumor effect on a cellular level and an animal tumor model, has the potential of being used as a medicine for treating, adjunctively treating and preventing tumors, and is particularly used for treating, adjunctively treating and preventing solid tumors such as breast cancer, lung cancer, colon cancer and the like. The anti-tumor immune polypeptide provided by the invention is a drug delivery system (such as injection, micelle, liposome and the like) which is used for treating solid tumors and takes the anti-tumor immune polypeptide as an effective component, and is used for treating or preventing tumors.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a polypeptide capable of effectively killing tumor cells and inducing immunogenic death of tumor cells, a preparation method thereof, a drug delivery system and applications thereof. Background Art

[0002] At present, cancer has become the second leading cause of death globally and is one of the major public health problems threatening human health. According to the latest data of the International Agency for Research on Cancer (IARC) of the World Health Organization, the number of cancer patients diagnosed globally in 2020 reached 19.29 million, and the number of people who died from cancer increased to 9.96 million. It is estimated that compared with 2020, the cancer burden will increase by 47% in 2040, and the number of newly diagnosed cancer cases will reach 28.4 million at that time. The economic and social burden caused by malignant tumors is increasing day by day, yet there has been no substantial progress in the treatment methods for malignant tumors in recent years.

[0003] Currently, the clinical treatment methods for malignant solid tumors include traditional surgical resection, chemotherapy, radiotherapy and immunotherapy. Traditional cancer treatment methods can directly remove the diseased tissue or directly kill tumor cells, and can produce obvious curative effects on most patients in the early stage of treatment, but the cure rate is extremely low. Moreover, in the late stage of treatment, patients often present with metastasis, recurrence and drug resistance. Chemotherapy drugs have defects such as low selectivity, large adverse reactions and drug resistance, which greatly limit their use. Although monoclonal antibody-based tumor-targeted therapies have solved the problem of targeting, their protein molecular weight is large, immunogenicity is high, and they are prone to allergic reactions and immune cross-reactions. In addition, due to high R & D costs, great R & D difficulties, limited production capacity and other reasons, their selling prices are high, and it is difficult for ordinary cancer patients to popularize their use. At the same time, monoclonal antibody drugs have problems such as relatively single dosage forms, mostly intravenous injection or drip for administration, and inability to be taken orally, making drug application very inconvenient.

[0004] Inducing immunogenic cancer cell death (ICCD) can not only directly kill tumor cells, but also activate specific anti-tumor immune responses, further eliminate residual lesions or metastatic lesions, and prevent tumor recurrence. A polypeptide is a compound formed by one or more amino acids linked together by peptide bonds, usually formed by dehydration condensation of 10 - 100 amino acid molecules, and their molecular weight < 10,000 Da. Polypeptides are the active groups for proteins to exert their functions, are bioactive substances involved in various cell functions in organisms, and participate in regulating various physiological functions. Therefore, they have very important development value in clinical applications.

[0005] As a new strategy for tumor immunotherapy, being able to effectively kill tumor cells and induce immunogenic death of tumor cells has the effects of treating, adjuvant treating, and / or preventing cancer (recurrence). Antitumor immune polypeptides also have their unique advantages: compared with small molecule chemical drugs, they have higher affinity for target tumors, lower adverse reactions, and can also increase the sensitivity of tumors to other treatment methods. Compared with monoclonal antibody drugs, due to their tiny size, they are more likely to penetrate into solid tumors, and inducing ICDD while killing tumor cells is not possessed by monoclonal antibody drugs. Antitumor immune polypeptides can be chemically synthesized or / and expressed by genetic engineering, and can be chemically modified by various means, which helps in the design and research of novel active polypeptides.

[0006] Therefore, it is particularly important to develop and design a polypeptide that can effectively kill tumor cells and induce immunogenic death of tumor cells as a drug for treating malignant tumors (solid tumors). Summary of the Invention

[0007] In view of this, the present invention provides a polypeptide SEQ ID NO: 1 that can cause immunogenic death of tumor cells, its preparation method, pharmaceutical preparation, and application. The technical solution provided by the present invention is to design a fusion protein by combining the sumo enzyme cleavage site sequence with the polypeptide by means of genetic engineering, so that the polypeptide is expressed in a soluble form, while increasing the expression level of the polypeptide, and the polypeptide obtained after removing the tag by sumo enzyme cleavage has no extra amino acids, which is beneficial to the purification of the polypeptide.

[0008] In order to achieve the above invention object, the present invention provides the following technical solutions:

[0009] In the first aspect, the present invention provides a polypeptide, which has:

[0010] (Ⅰ) The amino acid sequence shown in SEQ ID No.1; or

[0011] (Ⅱ) A sequence in which 1 or more amino acids are substituted, deleted, added, replaced, and / or modified on the basis of the amino acid sequence shown in (Ⅰ); or

[0012] (Ⅲ) An amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence similarity to the amino acid sequence shown in (Ⅰ);

[0013] (IV) An amino acid sequence having the same or similar function as the amino acid sequence shown in (Ⅰ).

[0014] In some specific embodiments of the invention, the multiple amino acids include but are not limited to 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0015] In some specific embodiments of the invention, the modification includes, but is not limited to, one or more of amidation, phosphorylation, methylation, acetylation, ubiquitination, glycosylation, PEG (polyethylene glycol) group modification, PEG dithiolate group modification, or maleimide group modification.

[0016] In some specific embodiments of the invention, the polypeptide has the amino acid sequence shown in any one of SEQ ID No. 5-7.

[0017] In a second aspect, the present invention provides a method for preparing a polypeptide, which is prepared by chemical synthesis or bioengineering methods.

[0018] In a third aspect, the present invention provides the polypeptide prepared by the above preparation method.

[0019] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the polypeptide, and the nucleic acid molecule has:

[0020] (I) The nucleotide sequence shown in SEQ ID No. 2; or

[0021] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0022] (III) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0023] (IV) A nucleotide sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to the nucleotide sequence described in any one of (I)-(III).

[0024] In some specific embodiments of the invention, the plurality of nucleotides includes, but is not limited to, 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 or 40 nucleotides.

[0025] In a fifth aspect, the present invention provides a gene element, including a sumo protease cleavage site and the above nucleic acid molecule.

[0026] In some specific embodiments of the invention, the gene element has:

[0027] (I) a nucleotide sequence as shown in SEQ ID No. 3; or

[0028] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0029] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0030] (IV) a nucleotide sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to the nucleotide sequence described in any one of (I) to (III).

[0031] In some specific embodiments of the invention, the plurality of nucleotides include, but are not limited to, 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 or 40 nucleotides.

[0032] In a sixth aspect, the invention provides a recombinant vector, including any one of the following:

[0033] (I) the nucleic acid molecule described above; or

[0034] (II) the gene element described above.

[0035] In some specific embodiments of the invention, the recombinant vector has:

[0036] (I) a nucleotide sequence as shown in SEQ ID No. 4; or

[0037] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0038] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0039] (IV) A nucleotide sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to the nucleotide sequence described in any one of (I) to (III).

[0040] In some specific embodiments of the invention, the plurality of nucleotides include, but are not limited to, 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 or 40 nucleotides.

[0041] In some specific embodiments of the invention, the source of the backbone vector of the recombinant vector includes, but is not limited to, plants, animals, bacteria, fungi, phages or viruses.

[0042] In a seventh aspect, the present invention provides a host:

[0043] (I) Integrated with the nucleic acid molecule described above; or

[0044] (II) Integrated with the gene element described above; or

[0045] (III) Transfected or transformed with the recombinant vector described above.

[0046] In some specific embodiments of the invention, the host includes, but is not limited to, prokaryotes or eukaryotes; the prokaryotes include, but are not limited to, Escherichia coli.

[0047] In an eighth aspect, the present invention provides a recombinant polypeptide, which is obtained by preparing any one of the following;

[0048] (I) The gene element described above; or

[0049] (II) The recombinant vector described above; or

[0050] (III) The host described above.

[0051] In some specific embodiments of the invention, the preparation method of the recombinant polypeptide includes the following steps:

[0052] Step 1: Construction of a genetically engineered strain: Construct a genetically engineered bacterial strain expressing the recombinant polypeptide;

[0053] Step 2. Expression and harvest of the fusion protein: Inoculate the genetically engineered strain described in step (1) into LB medium for culture, add an inducer (such as IPTG) to induce the expression of the fusion protein, collect the bacterial cells, suspend them in a buffer, lyse the cells by ultrasonic treatment and centrifuge, and harvest the supernatant containing the recombinant fusion protein;

[0054] Step 3. Isolation and purification of the target polypeptide: Purify and isolate the supernatant containing the recombinant fusion protein obtained in step (2) to obtain the recombinant polypeptide.

[0055] In some specific embodiments of the invention, the purity of the recombinant polypeptide obtained by purification is higher than 95%.

[0056] In some specific embodiments of the invention, the purification includes one or more of affinity chromatography, desalting, enzymatic cleavage, or high performance liquid chromatography.

[0057] In some specific embodiments of the invention, the affinity chromatography is nickel column affinity chromatography.

[0058] In some specific embodiments of the invention, the protease selected for enzymatic cleavage is sumo enzyme, and the enzymatic cleavage conditions are that the ratio of sumo enzyme to the recombinant fusion protein is (50 - 200 U): 1 mg, and it is left standing at 4 - 25 °C for 4 - 24 h. The preferred conditions are that the ratio of sumo enzyme to the recombinant fusion protein is 200 U: 1 mg, and it is left standing at 4 °C for 18 h.

[0059] In the ninth aspect, the invention provides the recombinant polypeptide prepared by the preparation method.

[0060] In the tenth aspect, the invention provides the use of any one of the following in the preparation of products for preventing, improving, adjuvantly treating, and / or treating tumors;

[0061] (I), the polypeptide described above; or

[0062] (II), the polypeptide prepared by the preparation method described above; or

[0063] (III), the nucleic acid molecule; or

[0064] (IV), the gene element; or

[0065] (V), the recombinant vector; or

[0066] (VI), the host; or

[0067] (VII), the recombinant polypeptide or the recombinant polypeptide prepared by the preparation method.

[0068] In some specific embodiments of the invention, the products include but are not limited to vaccines, drugs, and / or drug combinations.

[0069] In some specific embodiments of the invention, the tumor is selected from one or more of the following: lung cancer, breast cancer, cervical cancer, glioma, endometrial cancer, melanoma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer or colorectal cancer; preferably breast cancer.

[0070] In a tenth aspect, the present invention provides a vaccine comprising any of the following and an acceptable immunological adjuvant or excipient:

[0071] (I), the polypeptide described above; or

[0072] (II), the polypeptide prepared by the preparation method described above; or

[0073] (III), the nucleic acid molecule described above; or

[0074] (IV), the gene element described above; or

[0075] (V), the recombinant vector described above; or

[0076] (VI), the host described above; or

[0077] (VII), the recombinant polypeptide described above or the recombinant polypeptide prepared by the preparation method described above.

[0078] In some specific embodiments of the invention, the immunological adjuvant includes one or more of Freund's adjuvant, aluminum hydroxide adjuvant, monophosphoryl lipid A (MPLA) adjuvant, preferably monophosphoryl lipid A adjuvant.

[0079] In an eleventh aspect, the present invention provides the use of the vaccine described above in the prevention of tumors.

[0080] In some specific embodiments of the invention, the tumor is selected from one or more of the following: lung cancer, breast cancer, cervical cancer, glioma, endometrial cancer, melanoma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer or colorectal cancer; preferably breast cancer.

[0081] In a twelfth aspect, the present invention provides a method for preventing tumors, which is to inoculate the vaccine described above.

[0082] In some specific embodiments of the invention, the tumor is selected from one or more of the following: lung cancer, breast cancer, cervical cancer, glioma, endometrial cancer, melanoma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer or colorectal cancer; preferably breast cancer.

[0083] In a thirteenth aspect, the present invention provides a drug comprising any of the following and a pharmaceutically acceptable carrier molecule, immunological adjuvant and / or pharmaceutically acceptable excipient:

[0084] (I), the polypeptide described above; or

[0085] (II), the polypeptide prepared by the preparation method; or

[0086] (III), the nucleic acid molecule described above; or

[0087] (IV), the gene element described above; or

[0088] (V), the recombinant vector described above; or

[0089] (VI), the host described above; or

[0090] (VII), the recombinant polypeptide described above or the recombinant polypeptide prepared by the preparation method.

[0091] In some specific embodiments of the invention, the carrier molecule includes but is not limited to one or more of nanomaterials, liposomes, polyethylene glycol modifications or oily compounds, or a mixture composed of a variety of oily compounds; preferably, it is pegylated phosphatidylethanolamine (PEG-DSPE); or

[0092] The immune adjuvant includes one or more of Freund's adjuvant, aluminum hydroxide adjuvant or monophosphoryl lipid A (MPLA) adjuvant; preferably, it is monophosphoryl lipid A adjuvant.

[0093] In some specific embodiments of the invention, the dosage form of the drug includes but is not limited to injection.

[0094] In some specific embodiments of the invention, the injection includes one or more of micelle injection, liposome injection or nanomaterial injection, and preferably, it is micelle injection.

[0095] In some specific embodiments of the invention, the injection is a lyophilized powder, which is obtained by adding a lyoprotectant and then freeze-drying; preferably, the lyoprotectant is mannitol at 0.05 g / ml.

[0096] In some specific embodiments of the invention, the molar ratio of the recombinant polypeptide, the carrier molecule and the immune adjuvant is (4 - 160):720:(3 - 80);

[0097] Preferably, the molar ratio of the recombinant polypeptide, the carrier molecule PEG-DSPE and the immune adjuvant MPLA is 4:100:3.

[0098] In the fifteenth aspect, the present invention provides a drug combination, including the drug according to any one of claims 33 to 38 and any other active ingredient.

[0099] In the sixteenth aspect, the present invention provides the use of the drug or the drug combination in adjuvant treatment and / or treatment of tumors.

[0100] In some specific embodiments of the invention, the tumor is selected from one or more of the following: lung cancer, breast cancer, cervical cancer, glioma, endometrial cancer, melanoma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer or colorectal cancer; preferably breast cancer.

[0101] In a seventeenth aspect, the present invention provides a method for adjuvant treatment of tumors, comprising administering any one of the following:

[0102] (I), the drug described above; or

[0103] (II), the drug combination described above.

[0104] The recombinant anti-tumor polypeptide provided by the present invention has the following characteristics:

[0105] 1), the obtained recombinant fusion protein is in a soluble non-inclusion body state.

[0106] 2), the prepared recombinant anti-tumor polypeptide does not contain extra amino acids, and its polypeptide sequence is consistent with the amino acid sequence obtained after cDNA translation.

[0107] 3), the prepared anti-tumor polypeptide has high anti-tumor activity and the ability to induce immunogenic cell death of tumor cells both in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0109] Figure 1 Showing the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against breast cancer 4T1 cells;

[0110] Figure 2 Showing the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against lung cancer A549 cells;

[0111] Figure 3 Showing the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against breast cancer MCF7 cells;

[0112] Figure 4 Showing the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against colorectal cancer HT29 cells;

[0113] Figure 5 Showing the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against uterine cancer HEC-1-B cells;

[0114] Figure 6Show the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against prostate cancer PC3 cells;

[0115] Figure 7 Show the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against liver cancer hepG2 cells;

[0116] Figure 8 Show the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against renal cancer 786 - O cells;

[0117] Figure 9 Show the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against liver cancer LM3 cells;

[0118] Figure 10 Show the cytotoxicity test results of the polypeptide SeqID NO.1 of the present invention against glioma U251MG cells;

[0119] Figure 11 Show the morphological observation results of the polypeptide Seq ID NO.1 of the present invention against breast cancer 4T1 cells;

[0120] Figure 12 Show the tumor - free results of the polypeptide Seq ID NO.1 of the present invention in a mouse tumor immunotherapy model;

[0121] Figure 13 Show the tumor - free results of the polypeptide Seq ID NO.1 of the present invention in a mouse tumor intratumoral administration treatment model;

[0122] Figure 14 Show the cytotoxicity test results of the polypeptides Seq ID NO.5 to Seq ID NO.7 of the present invention against breast cancer MCF7 cells. Detailed implementation manners

[0123] The present invention discloses a polypeptide that can effectively kill tumor cells and cause immunogenic death of tumor cells, and its preparation method, drug delivery system and application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0124] The purpose of the present invention is to design a polypeptide with broad - spectrum anti - tumor immune activity.

[0125] The object of the present invention is to design a polypeptide capable of inducing immunogenic death of tumor cells.

[0126] The object of the present invention is to design a recombinant plasmid capable of highly expressing an anti-tumor polypeptide for the expression of the anti-tumor polypeptide.

[0127] The object of the present invention is to provide a recombinant Escherichia coli capable of solubilizing and highly producing a bioactive anti-tumor polypeptide.

[0128] The object of the present invention is to provide a method for expressing and purifying an anti-tumor polypeptide, which method has high safety, low production cost, simple polypeptide purification steps, high product purity and the obtained polypeptide has biological activity.

[0129] The object of the present invention is to provide a drug delivery system for the treatment of solid tumors with an anti-tumor immune polypeptide as an active ingredient.

[0130] The object of the present invention is the application of an anti-tumor immune polypeptide in the treatment, adjuvant treatment and / or prevention of cancer.

[0131] In order to achieve the above object, the present invention describes a production method, a drug delivery system and their applications of an anti-tumor polypeptide, including polypeptide genetic engineering expression and purification (including codon optimization, construction of recombinant expression plasmid, construction of recombinant genetic engineering bacteria, expression of fusion protein, purification of anti-tumor polypeptide), in vitro anti-tumor activity detection, microscopic cell morphology observation, animal model tumor prevention experiment, and solid tumor treatment animal model experiment, preparation of micelle injection, etc.

[0132] The characteristics of the present invention are as follows: according to the codon preference of the host expression bacteria, the gene sequence of the anti-tumor polypeptide Seq ID NO.1 is optimized, and after the target gene sequence is ligated with the expression vector pET-32a(+), it is transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21(DE3) containing the recombinant plasmid. This recombinant Escherichia coli BL21(DE3) is used to produce a recombinant anti-tumor polypeptide with activity. The anti-tumor polypeptide produced by the method of the present invention has the effects of inhibiting tumor growth and inducing immunogenic death of tumor cells in in vivo and in vitro experiments. The steps of the present invention are as follows:

[0133] 1) According to the codon preference of Escherichia coli BL21(DE3), using the amino acid sequence of the anti-tumor polypeptide Seq ID NO.1 as a template, the optimized DNA sequence of the anti-tumor polypeptide is obtained, hereinafter referred to as LCX17-DNA-seq, and the DNA sequence is SEQ ID NO.2;

[0134] 2) According to the codon preference of Escherichia coli BL21(DE3), using the amino acid sequence of the sumo protease recognition site as a template, an optimized DNA sequence was obtained, hereinafter referred to as sumo-DNA-seq, and the DNA sequence is SEQ ID NO.3;

[0135] 3) According to the types of restriction enzymes in the multiple cloning site of the expression vector pET-32a(+), the restriction enzymes Kpn1 (GGTACC) and Xho1 (CTCGAG) were selected, and in the order of GGTACC+sumo-DNA-seq+LCX17-DNA-seq+TAA+CTCGAG, the entire DNA sequence was ligated with the expression vector pET-32a(+) to obtain the recombinant expression vector pET-32a(+)-LCX17, and the DNA sequence is hereinafter referred to as Sumo-LCX17-DNA-seq, and the DNA sequence is SEQ ID NO.4.

[0136] 4) The recombinant expression vector pET-32a(+)-LCX17 was transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21(DE3), hereinafter referred to as BL21-LCX17.

[0137] 5) IPTG was used to induce the recombinant Escherichia coli BL21-LCX17 to express the fusion protein of the anti-tumor polypeptide with a tag.

[0138] 6) The fusion protein was purified by nickel column affinity chromatography, and the fusion protein was digested with sumo protease to separate the tag protein and the anti-tumor polypeptide.

[0139] 7) The digestion products were treated by purification methods such as nickel column affinity chromatography, ion exchange, and high performance liquid chromatography to obtain the anti-tumor polypeptide with high purity.

[0140] 8) The purified anti-tumor polypeptide was aliquoted according to a certain specification and then freeze-dried and stored at -80°C.

[0141] 9) The freeze-dried polypeptide was used for cytological experiments and animal experiments to detect the anti-tumor activity of the polypeptide.

[0142] Term Explanation

[0143] The terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, "a plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0144] The term "optionally" is for descriptive purposes only and should not be construed as indicating or implying relative importance. Thus, a feature qualified with "optionally" may or may not include that feature, either explicitly or implicitly.

[0145] The term "polypeptide" is used in its normal sense to denote a series of residues (usually L-amino acids) that are typically linked to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The term includes modified peptides and synthetic peptide analogues.

[0146] The polypeptides of the present invention can be manufactured using chemical methods (Peptide Chemistry, A practical Textbook. Mikos Bodansky, Springer-Verlag, Berlin.). For example, the polypeptides can be synthesized by solid-phase techniques (Roberge JY et al., (1995) Science 269:202 - 204), cleaved from the resin, and purified by preparative high-performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). Automated synthesis can be achieved, for example, using a 431A peptide synthesizer (PerkinElmer) in accordance with the manufacturer's instructions.

[0147] Alternatively, the polypeptides can be generated by recombinant means or by cleavage from a longer polypeptide. For example, the polypeptides can be obtained by cleavage from the thyrotropin receptor protein, which can then be modified at one or both termini. The composition of the polypeptides can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure).

[0148] For practical purposes, the polypeptides can exhibit various other characteristics. For example, importantly, the peptides are stable enough in vivo to be therapeutically useful. The in vivo half-life of the peptides can be at least 10 minutes, 30 minutes, 4 hours, or 24 hours.

[0149] The polypeptides can also exhibit good in vivo bioavailability. The peptides can maintain their in vivo conformation, enabling them to bind to targets on the cell surface without excessive hindrance.

[0150] In addition, the polypeptides of the present invention also include functionally equivalent derivatives, variants, or fragments thereof.

[0151] "Functionally equivalent" derivatives, variants or fragments thereof refer to peptides related to or derived from the polypeptides of the present invention, wherein the amino acid sequence has been modified, for example, using modified amino acids or by substitution, addition and / or deletion of one or more amino acids (e.g., 1 to 10, e.g., 1 to 5, especially 1 or 2 residues), but still retain functional activity. One such method is described in international patent application PCT / GB2006 / 002390, published as WO2007 / 00601 on January 4, 2007, the content of which is incorporated herein by reference in its entirety for all purposes.

[0152] Within the meaning of "addition", it includes variants of amino- and / or carboxy-terminal fusion proteins or polypeptides that comprise an additional protein or polypeptide fused to the peptide sequence.

[0153] As described above, the peptide can preferably be replaced at the N- or C-terminus by another moiety. These moieties can be added to assist in the function of the peptide, its targeting or its synthesis, capture or identification, e.g., tags (such as biotin) or lipid molecules. Alternatively, such moieties can be found within the peptide itself. For example, a moiety such as a tag can be attached to an amino acid located within the peptide.

[0154] "Substituted" variants preferably involve replacing one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, an amino acid can be replaced with an alternative amino acid having similar properties, such as another basic amino acid, another charged amino acid, another hydrophilic amino acid or another aliphatic amino acid.

[0155] Suitably, "derivatives" or "variants" include those in which the amino acids that replace the naturally occurring amino acids in the sequence are structural analogs thereof. The amino acids used in the sequence can also be derivatized or modified, e.g., labeled, provided that there is no significant adverse effect on the function of the peptide.

[0156] Derivatives and variants as described above can be prepared during the synthesis of the peptide or by post-production modification, or when the peptide is in recombinant form, using known techniques such as site-directed mutagenesis, random mutagenesis or enzymatic cleavage and / or nucleic acid ligation.

[0157] Functionally equivalent "fragments" according to the present invention can be made by truncation (e.g., by removing one or more amino acids from the N- and / or C-terminus).

[0158] Suitably, functionally variant amino acid sequences according to the present invention have at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% homology with the sequences listed in the present invention.

[0159] With respect to amino acid sequences, "sequence identity" refers to sequences having the stated value when evaluated using ClustalW (Thompson et al., 1994, supra), which ClustalW has the following parameters:

[0160] Pairwise alignment parameters - Method: Exact, Matrix: PAM, Gap opening penalty: 10.00, Gap extension penalty: 0.10;

[0161] Multiple alignment parameters - Matrix: PAM, Gap opening penalty: 10.00, Delayed divergence percentage: 30, Penalize end gaps: Yes, Gap separation distance: 0, Negative matrix: No, Gap extension penalty: 0.20, Residue specific gap penalty: Yes, Hydrophilic gap penalty: Yes, Hydrophilic residues: GPSNDQEKR. Sequence identity of specific residues is intended to include simply derived identical residues.

[0162] The peptides of the invention as defined herein can be chemically modified, such as post-translationally modified. For example, they can be glycosylated or contain modified amino acid residues. They can be various forms of polypeptide derivatives, including amides and conjugates with polypeptides.

[0163] Chemically modified peptides also include peptides having one or more residues chemically derivatized by reaction of functional side groups. Such derivatized side groups include those groups derivatized to form amine hydrochlorides, tosyl, benzyloxycarbonyl, tert-butoxycarbonyl, chloroacetyl, and formyl. Free carboxyl groups can be derivatized to form salts, methyl esters, and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.

[0164] Chemically modified peptides also include cyclic peptides, i.e., peptides of the invention, which are joined by covalent bonds to produce a ring. Typically, the amino terminus and the carboxyl terminus (so-called head-to-tail cyclization), the amino terminus and a side chain (so-called head-to-side chain cyclization), the carboxyl terminus and a side chain (so-called side chain-to-tail cyclization), or side chains and side chains (so-called side chain-to-side chain cyclization) can be joined by covalent bonds to form a cyclic peptide. Head-to-tail cyclic peptides can generally be formed by amide bond formation. Side chain-to-side chain rings can generally be formed by formation of a Cys-Cys disulfide bond bridge within the cyclic peptide or by amide bond formation. Alternatively, the amino terminus, carboxyl terminus, or side chain can be joined by covalent bonds to the peptide backbone to form a cyclic peptide.

[0165] Chemically modified peptides also include those peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can replace proline, or homoserine can replace serine.

[0166] The peptides of the present invention can carry a display label. Suitable labels include radioisotopes, fluorescent labels, enzyme labels, or other protein labels such as biotin.

[0167] Any formula given herein is also intended to represent both the unlabeled form and the isotopically labeled form of the peptide. The isotopically labeled peptides have the structure described by the formula given herein, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the peptides of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36Cl, 125I. The present invention includes various isotopically labeled peptides as defined herein, such as those in which radioisotopes such as 3H and 14C are present. These isotopically labeled peptides can be used for metabolic studies (with 14C), reaction kinetics studies (with, for example, 2H or 3H), detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), which include determination of drug or substrate tissue distribution, or for radioactive treatment of patients. In particular, for PET or SPECT studies, 18F or labeled peptides may be particularly desirable. The isotopically labeled peptides of the present invention and their prodrugs can generally be prepared by replacing the non-isotopically labeled reagents with readily available isotopically labeled reagents by implementing the protocols or methods disclosed in the examples and preparations described below.

[0168] The isotopically labeled peptides of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described herein, using suitable isotopically labeled reagents in place of the previously used unlabeled reagents.

[0169] Pharmaceutically acceptable solvates according to the present invention include those in which the crystallization solvent can be replaced by an isotope, such as D2O, d6-acetone, d6-DMSO.

[0170] The above peptides used according to the present invention can be prepared by conventional synthetic means including genetic or chemical means.

[0171] For reasons of purity, antigenic specificity, freedom from unwanted by-products, and ease of production, synthetic techniques (such as solid-phase Merrifield-type synthesis) may be preferred. Suitable techniques for solid-phase peptide synthesis are well known to those skilled in the art (see, for example, Merrifield et al., 1969, Adv. Enzymol 32, 221-96 and Fields et al., 1990, Int. J. Phaptide Protein Res, 35, 161-214). Chemical synthesis can be carried out by methods well known in the art, including a cyclic set of reactions of selective deprotection of the functional groups of the terminal amino acids and coupling of selectively protected amino acid residues, followed finally by complete deprotection of all functional groups.

[0172] Synthesis can be carried out in solution or on a solid support using a suitable solid phase known in the art.

[0173] Since the polypeptides of the present invention are intended for use in pharmaceutical compositions, it is readily understood that they are each preferably provided in a substantially pure form, for example at least 60% pure, more suitably at least 75% pure, and preferably at least 85%, especially at least 98% pure (% by weight based on weight). Impure preparations of the polypeptide can be used to prepare the more pure forms used in pharmaceutical compositions; these less pure compound preparations should contain at least 1%, more suitably at least 5%, preferably 10-59% of the polypeptide provided by the present invention.

[0174] In alternative embodiments, the polypeptides of the present invention can be produced or delivered in the form of polynucleotides that encode and are capable of expressing it. Such polynucleotides can be synthesized according to methods well known in the art, as described, for example, in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). Such polynucleotides can be used in vitro or in vivo to produce the peptides of the present invention. Such polynucleotides can thus be administered or used to treat the cancers or another disease / condition described herein.

[0175] The present invention also includes expression vectors containing such polynucleotide sequences. Such expression vectors are typically constructed in the field of molecular biology and can, for example, involve the use of plasmid DNA and suitable initiators, promoters, enhancers, and other elements that may be necessary and which are in the correct orientation, such as polyadenylation signals, in order to permit the expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, reference is made to Sambrook et al. (ibid).

[0176] Thus, the peptide can be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Suitably, the polynucleotide of the invention or the polynucleotide for use in the invention in a vector is operably linked to a control sequence capable of providing for expression of the coding sequence in a host cell, i.e., the vector is an expression vector. The term "operably linked" refers to juxtaposition wherein the components described are in a relationship that allows them to function in their intended manner. A regulatory sequence (such as a promoter) that is "operably linked" to a coding sequence is positioned so that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

[0177] The vector can be, for example, a plasmid, virus or phage vector provided with an origin of replication, optionally a promoter for expression of the polynucleotide and optionally a regulator of the promoter. The vector can contain one or more selectable marker genes, such as the ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. The vector can be used in vitro, for example for production of DNA or RNA or for transfection or transformation of a host cell, such as a mammalian host cell. The vector can also be suitable for in vivo use, for example allowing in vivo expression of a peptide.

[0178] The promoter and other expression control signals can be selected to be compatible with the host cell in which expression is designed. For example, yeast promoters include the S. cerevisiae GAL4 and ADH promoters, the S. pombe nmt1 and adh promoters. Mammalian promoters can be used, such as the β-actin promoter. Tissue-specific promoters are particularly preferred. Mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium. Viral promoters can also be used, such as the SV40 large T antigen promoter, adenovirus promoters, Moloney murine leukemia virus long terminal repeat (MMLV LTR), Rous sarcoma virus (RSV) LTR promoter, SV40 promoter, human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSY IE promoter) or HPV promoters, especially the HPV upstream regulatory region (URR). All of these promoters are readily available in the prior art.

[0179] The present invention also includes cells that have been modified to express the peptides of the present invention. Such cells include transient or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells such as yeast, or prokaryotic cells such as bacterial cells. Specific examples of cells that can be modified by inserting a vector encoding the peptides of the present invention include mammalian HEK293T, CHO, HeLa, and COS cells. Suitably, the selected cell line should be not only stable but also allow for the mature glycosylation of the polypeptide and cell surface expression. Expression can be achieved in transformed oocytes. Suitable peptides can be expressed in the cells of transgenic non-human animals (especially mice). Transgenic non-human animals expressing the peptides of the present invention are included within the scope of the present invention. The peptides of the present invention can also be expressed in Xenopus laevis oocytes or melanocytes.

[0180] The present invention also extends to antibodies (monoclonal or polyclonal) against the peptides as defined above and their antigen-binding fragments (e.g., F(ab)2, Fab, and Fv fragments, i.e., fragments comprising the "variable" regions of the antibody that contain the antigen-binding site), which bind to the epitopes present on the peptides and thus selectively and specifically bind to these peptides and can be used in the methods of the present invention.

[0181] The present invention also relates to compositions, such as pharmaceutical compositions comprising the polypeptides or recombinant polypeptides according to the present invention.

[0182] The peptides may comprise multiple peptides, for example, two, three, four, five, or more than six polypeptides.

[0183] The compositions of the present invention can be used for prophylactic or therapeutic purposes.

[0184] When administered for prophylactic use, the compositions can reduce or prevent the growth of tumor or cancer cells. The term "reduce" indicates a reduction in the observed tumor or cancer cells, such as a 60%, 70%, 80%, 90%, 95%, or 99% reduction in the number of tumor or cancer cells as observed before treating the patient with the composition (or the number of tumor or cancer cells observed in an untreated patient over the same time period). The term "prevent" indicates that no perceptible growth of tumor or cancer cells is observed.

[0185] When administered for therapeutic use, the compositions can inhibit the growth of tumor or cancer cells. The term "inhibit" indicates a decrease compared to the level before polypeptide treatment or compared to the level observed at the same time point in the absence of treatment.

[0186] When there are two or more polypeptides, the pharmaceutical composition can be in the form of a kit, where some or each polypeptide is provided separately for simultaneous administration, separate administration, or sequential administration.

[0187] Alternatively (or additionally), if the pharmaceutical composition (or any part thereof) is administered in multiple-dose form, each dose may be separately packaged.

[0188] Similarly, in the pharmaceutical compositions of the present invention, each polypeptide may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s).

[0189] The compositions may be administered together in the form of a combined composition or mixture. However, there may be instances where it is preferred to separately provide the polypeptides in the form of a kit for simultaneous, separate, sequential or combined administration.

[0190] The kit may also comprise means for mixing and / or administering (such as a vapouriser for intranasal administration, or a syringe or needle for subcutaneous / intradermal dosing). The kit may also contain instructions for use.

[0191] The pharmaceutical compositions or kits of the present invention may be used for treating and / or preventing diseases.

[0192] The polypeptides of the present invention, including also pharmaceutically acceptable salts, may also be obtained in the form of their hydrates or include other solvents used for their crystallization.

[0193] As used herein, the term "pharmaceutically acceptable salts" refers to salts which retain the biological effectiveness and properties of the compounds of the present invention and which are generally not biologically or otherwise undesirable. Due to the presence of amino and / or carboxyl groups or similar groups, the peptides of the present invention are capable of forming acid and / or base salts.

[0194] Pharmaceutically acceptable acid addition salts may be formed using inorganic and organic acids, such as acetates, aspartates, benzoates, benzenesulfonates, bromides / hydrobromides, bicarbonates / carbonates, bisulfates / sulfates, camphorsulfonates, chlorides / hydrochlorides, chlortheophyllonates, citrates, edisylates, fumarates, glucoheptonates, gluconates, glucuronates, hippurates, hydroiodides / iodides, hydroxyethylsulfonates, lactates, lactobionates, lauryl sulfates, malates, maleates, malonates, mandelates, mesylates, methyl sulfates, naphthoates, naphthalenesulfonates, nicotinates, nitrates, octadecanoates, oleates, oxalates, palmitates, pamoates, phosphates / phosphatemonohydrogenates / dihydrogen phosphates, polygalacturonates, propionates, stearates, succinates, sulfosalicyclates, tartrates, toluenesulfonates, trifluoroacetates and trifluoromethylsulfonates.

[0195] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, trifluoromethylsulfonic acid, sulfosalicylic acid, etc.

[0196] Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.

[0197] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from Groups I - XII of the periodic table. In some embodiments, the salts can be derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

[0198] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Some organic amines include isopropylamine, benzathine penicillin, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0199] In addition, the polypeptides of the present invention, i.e., peptides containing groups capable of acting as hydrogen bond donors and / or acceptors, may be capable of forming co - crystals with suitable co - crystal formers. These co - crystals can be prepared by known co - crystal formation procedures for peptides. Such procedures include grinding, heating, co - sublimation, co - melting under crystallization conditions, or contacting the peptide with the co - crystal former in solution and separating the co - crystal thus formed. Suitable co - crystal formers include those described in WO 2004 / 078163.

[0200] Those skilled in the art will understand that when basic and acidic groups are present, the peptides of the present invention can also form internal salts, such as zwitterions.

[0201] In another aspect, the present invention provides a method of reducing abnormal cell division, wherein the cells are administered the peptides of the present invention.

[0202] As described herein, "abnormal cell division" refers to cell division above the normal level considered appropriate under the conditions in which it occurs (i.e., abnormal cell division). Markers of abnormal cell division are well known to those skilled in the art and can be used to determine whether a particular cell has been affected. For example, cells undergoing abnormal cell division may show atypical cytology, such as cell pleomorphism, nuclear pleomorphism, hyperchromasia, or an increased nuclear - cytoplasmic ratio. Cells undergoing abnormal cell division may show a failure of cell differentiation. More particularly, such abnormal cell division can be present in certain conditions or diseases / disorders as described below, such as cancer.

[0203] "Reducing" cell division refers to decreasing the rate of cell growth. Suitably, over the same time period (where control growth = 1), relative to control growth (without the active agent), cell growth is reduced to less than 0.5, particularly less than 0.25, such as less than 0.1. Suitably, the reduced cell division includes cell death / lack of viability that may additionally occur, or as an alternative to the reduced cell growth. When cell death occurs, suitably, more than 50% of the existing cells, particularly more than 75% of the cells are destroyed.

[0204] By adjusting the dose of the active agent used, it may also be possible to completely eliminate some malignant tumors. Thus, the polypeptides of the present invention can be used to slow the growth of cancer cells or completely destroy cancer cells. As explained in more detail below, the appropriate dose will depend on many factors and can be determined by a skilled practitioner.

[0205] Treatment according to the present invention can be symptomatic or prophylactic.

[0206] Thus, in another aspect, the present invention includes the active agents of the present invention for use as a medicament.

[0207] Thus, according to another aspect, the present invention provides the active agents of the present invention for the treatment or prophylaxis of conditions or disorders in which abnormal cell division occurs.

[0208] Thus, according to another aspect, the present invention provides the use of the active agents of the present invention in the manufacture of a medicament for the prophylaxis or treatment of conditions or disorders in which abnormal cell division occurs.

[0209] Thus, according to another aspect, the present invention provides a method for the prophylaxis or treatment of a condition or disorder in which abnormal cell division occurs, which comprises administering to a subject in need thereof a therapeutically effective amount of the active agent of the present invention.

[0210] In accordance with the foregoing, as another aspect, the present invention also provides a method for the prophylaxis or treatment of a condition or disorder in which abnormal cell division occurs, particularly cancer, which comprises administering to a subject in need thereof, particularly a human subject, a therapeutically effective amount of the active agent of the present invention.

[0211] In another aspect, the present invention provides the active agents of the present invention for the prophylaxis or treatment of conditions or disorders in which abnormal cell division occurs, particularly cancer.

[0212] In another aspect, the present invention provides the use of the active agents of the present invention in the manufacture of a medicament for the prophylaxis or treatment of conditions or disorders in which abnormal cell division occurs, particularly cancer.

[0213] As mentioned herein, a "condition" or "disease" refers to a potential pathological disorder of a symptomatic or asymptomatic organism relative to a normal organism, which may be caused by, for example, an infection or an acquired or congenital genetic defect.

[0214] A "medical condition" refers to the mental or physical state of an organism that has not undergone disease development, such as, for example, the presence of a substance such as a toxin, drug, or pollutant in the body.

[0215] As used herein, the terms "treat (verb)", "treating (gerund)", or "treatment (noun)" for any disease / medical condition refer in one embodiment to ameliorating a disease or condition (i.e., slowing or halting or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treat (verb)", "treating (gerund)", or "treatment (noun)" refer to alleviating or ameliorating at least one physical parameter, including those that may not be discernible by the patient. In another embodiment, "treat (verb)", "treating (gerund)", or "treatment (noun)" refer to modulating the disease or condition physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both. In yet another embodiment, "treat (verb)", "treating (gerund)", or "treatment (noun)" refer to preventing or delaying the onset or development or progression of a disease or condition. For example, symptoms that may be affected include tumor size or the number of cancer cells in a given sample.

[0216] "Preventing" a medical condition or disease refers to delaying or preventing the onset of or reducing the severity of the medical condition or disease, as evaluated based on the appearance or degree of one or more symptoms of the medical condition or disease.

[0217] As used herein, the term "subject" refers to an animal. Typically, the animal is a mammal. The subject also refers to, for example, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0218] As used herein, a subject "is in need of" treatment if such subject would benefit, in terms of biology, medicine, or quality of life, from such treatment.

[0219] The term "therapeutically effective amount" of the active agent of the present invention refers to the amount of the active agent of the present invention that will elicit a biological or medical response in a subject, such as reducing or inhibiting enzyme or protein activity, or improving symptoms, alleviating a condition, slowing or delaying disease progression, or preventing a disease, etc. In one non-limiting embodiment, the term "therapeutically effective amount" refers to the active agent of the present invention being effective in at least partially alleviating, inhibiting, preventing, and / or improving a condition or disorder in which abnormal cell division occurs when administered to a subject. In another non-limiting embodiment, the term "therapeutically effective amount" refers to the amount of the active agent of the present invention being effective in at least partially reducing abnormal cell division when administered to a cell or tissue or non-cellular biological material or medium.

[0220] As an alternative method of performing the method in vivo, these methods can be carried out, for example, in vitro to reduce cell division in a sample or eliminate cells undergoing abnormal cell growth. Suitable culture conditions are as described for other methods of the present invention below.

[0221] This is particularly useful in cell samples containing normal and abnormal cells (where the abnormal cells can be controlled / removed), as well as samples containing normal cells for subsequent processes (e.g., returning to the donor body). This can be useful, for example, in eliminating abnormal hematopoietic cells, such as leukemia cells, in a patient's blood sample, and the remaining cells can then be returned to the patient's body.

[0222] In yet another aspect, the present invention provides a method for reducing abnormal cell division in a sample (particularly reducing cancer cell growth, more particularly involving cancer cell death and thus reducing the number of cancer cells), wherein the active agent of the present invention as described above is administered to the sample. In a method for treating a patient suffering from (or preventing) a condition or disorder characterized by abnormal cell division, the sample can be collected from the patient and then returned to the patient as described below. In this context, a "sample" refers to any material obtained from a human or non-human animal (including embryonic, fetal, immature, and adult stages of the animal) that contains cells undergoing abnormal cell division and includes tissues and body fluids.

[0223] In this case, "body fluids" particularly include blood, cerebrospinal fluid, and lymph fluid, and "tissues" include tissues obtained by surgery or other means.

[0224] Suitably, abnormal cell division occurs in cells from eukaryotes, which can be any eukaryote such as humans, other mammals and animals, birds, insects, and fish.

[0225] Non-human animals from which cells can be obtained or in which the methods of the present invention can be performed include, but are not limited to, mammals, particularly primates, domestic animals, livestock, and laboratory animals. Thus, animals include mice, rats, chickens, frogs, guinea pigs, cats, dogs, pigs, cows, goats, sheep, and horses. Suitably, the cells are derived from humans, and the method is for the treatment or prevention in humans.

[0226] In particular, the cells undergoing abnormal cell division are cancer cells, and the condition to be treated or prevented is cancer. The peptides of the present invention block the growth of cancer cells, reduce their proliferation, or directly cause their death.

[0227] In another embodiment, the peptides of the present invention can act on cancer cells to transfer them from a quiescent state into the cell cycle and thus make them more susceptible to other (e.g.) cytotoxic, anti-cancer treatments.

[0228] The cancer is a malignant or pre-malignant or benign tumor and includes carcinomas, sarcomas, gliomas, melanomas, and lymphomas, including bladder cancer, kidney cancer, pancreatic cancer, brain cancer, head and neck cancer, breast cancer, bowel cancer, prostate cancer, lung cancer, and ovarian cancer, and leukemia and lymphoma. In particular, colorectal, pancreatic, bladder, prostate, cervical, ovarian, gastric, and non-small cell lung cancer.

[0229] A condition or disorder characterized by abnormal cell division is cancer, including but not limited to mesothelioma, hepatobiliary (liver and bile duct), primary or secondary CNS tumors, primary or secondary brain tumors, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, melanoma and non-melanoma skin cancer, head or neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal area cancer, gastric cancer, gastrointestinal (stomach, colorectal, and duodenum), gastrointestinal stromal tumors, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, cartilage or bone, urethral cancer, penile cancer, prostate cancer, testicular cancer, testicular lymphoma, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brainstem gliomas, pituitary adenomas, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

[0230] In one embodiment of the invention, the cancer is lung cancer (NSCLC and SCLC), melanoma, head or neck cancer, ovarian cancer, colon cancer, rectal cancer, anal area cancer, gastric cancer, breast cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, thyroid cancer, parathyroid cancer, pancreatic cancer, prostate cancer, central nervous system (CNS) tumors, primary CNS lymphoma, non-Hodgkin's lymphoma or spinal tumors, or a combination of one or more of the foregoing cancers.

[0231] In a specific embodiment, the cancer is lung cancer (NSCLC and SCLC), melanoma, head or neck cancer, ovarian cancer, breast cancer, prostate cancer, colon cancer or renal cell carcinoma.

[0232] In some cancers such as acute myeloid leukemia (AML), the peptides of the invention can block the proliferation of cancer cells, but can also stimulate those cells to leave the GO / G1 quiescent state and enter the cell cycle. These two effects are seen in the same cells under the same conditions. This may be because the cells are triggered by the peptide to leave GO / G1 (i.e., enter the cell cycle), but then cannot divide, but rather differentiate or undergo apoptosis.

[0233] As used herein, "sample" refers to any material obtained from a donor, for example, including embryos, fetuses, immature and adult stages of human or non-human animals of said animal, which contain stem cells and include tissues and body fluids.

[0234] "Body fluid" includes blood and cerebrospinal fluid.

[0235] "Tissue sample" includes tissues obtained by surgical intervention (such as bone marrow or liver) or by other means such as placenta and umbilical cord. The cells are preferably from the source or the animal to which the method is applied as described above for the method of reducing abnormal cell division.

[0236] Formulations: The compositions can be prepared as injectable solutions (as liquid solutions or suspensions); they can also be prepared in solid form suitable for dissolution or suspension in a liquid before injection. Emulsion formulations can also be prepared, or the peptides can be encapsulated in liposomes. The active ingredient can be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline (such as phosphate buffered saline), dextrose, glycerol, ethanol, etc. and combinations thereof.

[0237] Additionally, if desired, the compositions can contain small amounts of auxiliary substances such as wetting or emulsifying agents and / or pH buffering reagents. Buffer salts include phosphates, citrates, acetates, hydrochloric acid and / or pH adjustment can be carried out using sodium hydroxide. For stabilization, disaccharides such as sucrose or trehalose can be used.

[0238] If the composition comprises a plurality of peptides, then the relative ratios of the peptides can be approximately equal. Alternatively, the relative proportion of each peptide can be varied.

[0239] After formulation, the composition can be incorporated into a sterile container which is then sealed and stored at a low temperature, such as 4 °C or it can be lyophilized.

[0240] Conveniently, the composition is prepared as a lyophilized (freeze-dried) powder. Lyophilization allows for long-term storage in a stable form. The lyophilization step is well known in the art, see for example http: / / www.devicelink.com / ivdt / archive / 97 / 01 / 006.html. Bulking agents, such as mannitol, dextran or glycine, are typically used prior to freeze-drying.

[0241] The composition can be administered by convenient means, such as orally, intravenously (in the case of water solubility), intramuscularly, subcutaneously, sublingually, intranasally, intradermally or by the rectal route or implantation (e.g., using slow release molecules).

[0242] The composition can advantageously be administered by the intranasal, subcutaneous or intradermal route.

[0243] The peptides and compositions of the present invention can be used to treat human subjects.

[0244] Typically, a physician will determine the actual dosage most suitable for an individual subject and it will vary with the specific patient's age, weight and response.

[0245] In a preferred embodiment, a "dose escalation" protocol can be followed, where multiple doses are administered to the patient at increasing concentrations.

[0246] As used herein, "pharmaceutically acceptable" refers to ingredients that are compatible with the other ingredients of the composition and physiologically acceptable to the recipient.

[0247] As used herein, pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweetening agents, flavoring agents, dyes, etc. and combinations thereof, as would be known to those of ordinary skill in the art (see for example Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pp. 1289-1329). Any conventional carrier is contemplated for use in the therapeutic or pharmaceutical composition, except those that are incompatible with the active ingredient.

[0248] The pharmaceutical compositions according to the invention can be formulated by conventional means using readily available ingredients. Thus, the active ingredient (i.e., the peptide), optionally together with other active substances, can be mixed with one or more conventional carriers, diluents and / or excipients to produce conventional galenical preparations such as tablets, pills, powders, lozenges, cachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, etc.

[0249] The pharmaceutical compositions can be formulated for specific routes of administration, such as oral administration, parenteral administration, rectal administration, etc. In addition, the pharmaceutical compositions of the present invention can be prepared in solid form (including but not limited to capsules, tablets, pills, granules, powders or suppositories) or in liquid form (including but not limited to solutions, suspensions or emulsions). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations, such as sterilization and / or can contain conventional inert diluents, lubricants or buffering agents, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifying agents and buffering agents, etc.

[0250] Typically, the pharmaceutical composition is a tablet or gelatin capsule containing the active ingredient (the polypeptide or recombinant polypeptide provided by the present invention) and the following ingredients

[0251] a) diluents such as lactose, polyolactone, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine;

[0252] b) lubricants such as silica, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol;

[0253] c) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone;

[0254] d) disintegrants such as starch, agar, alginic acid or its sodium salt or effervescent mixtures; and / or

[0255] e) absorbents, colorants, flavors and sweeteners.

[0256] Tablets can be film-coated or enteric-coated according to methods known in the art.

[0257] Suitable compositions for oral administration include the compounds of the invention in the form of an effective amount of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft gelatin capsules, or syrups or elixirs. Compositions intended for oral use are prepared by any method known in the art for the preparation of pharmaceutical compositions, and such compositions may contain one or more substances selected from sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide a pharmaceutically elegant and palatable preparation. Tablets may contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin, or acacia; and lubricating agents such as magnesium stearate, stearic acid, or talc. The tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, delayed release materials such as glyceryl monostearate or glyceryl distearate may be used. Preparations for oral use may be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0258] Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and / or contain adjuvants such as preserving agents, stabilizing agents, wetting agents or emulsifying agents, solution promoters, salts regulating osmotic pressure, and / or buffering agents. Additionally, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating, or coating methods and contain from about 0.1 - 75% or contain from about 1 - 50% of the active ingredient.

[0259] Suitable compositions for transdermal application contain an effective amount of the active agent of the invention and a suitable vehicle. Vehicles suitable for transdermal delivery include absorbable pharmaceutically acceptable solvents that assist in passing through the skin of the host. For example, a transdermal device is in the form of a patch that includes a backing layer, a reservoir containing the compound and optionally a vehicle, a rate control barrier that optionally delivers the compound to the skin of the host at a controlled and predetermined rate over an extended period of time, and means for securing the device to the skin.

[0260] Suitable compositions for topical application (such as to the skin and eyes) include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, for example for delivery by aerosol etc. Such topical delivery systems will be particularly suitable for skin application, for example for treating skin cancer, for example for prophylactic application in sunscreens, lotions, sprays etc. They are thus particularly suitable for topical formulations known in the art, including cosmetic formulations. These may contain solubilizers, stabilizers, tonicity enhancers, buffers and preservatives.

[0261] As used herein, topical application may also involve inhalation or intranasal application. They can be conveniently delivered from a dry powder inhaler in dry powder form (alone, as a mixture, such as a dry blend with lactose, or mixed component particles, such as with phospholipids) or from a pressurized container, pump, sprayer, atomizer or nebuliser, in the form of an aerosol spray product with or without a suitable propellant.

[0262] The dose of the polypeptide or recombinant polypeptide provided by the present invention employed to practice the present invention will of course vary depending on, for example, the particular condition to be treated, the desired effect and the mode of administration. Generally, a suitable daily dose for administration by inhalation is in the order of 0.0001 to 30 mg / kg per patient, typically 0.01 to 10 mg, while for oral administration, a suitable daily dose is in the order of 0.01 to 100 mg / kg.

[0263] The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the polypeptide or recombinant polypeptide provided by the present invention as an active ingredient, since water can promote the degradation of certain compounds.

[0264] The anhydrous pharmaceutical compositions and dosage forms of the present invention can be prepared using anhydrous or low moisture content ingredients and low moisture or low humidity conditions. The anhydrous pharmaceutical compositions can be prepared and stored such that they maintain their anhydrous nature. Thus, anhydrous compositions are packaged using materials known to prevent exposure to water so that they can be included in a suitable formulation kit. Examples of suitable packaging include but are not limited to sealed foils, plastics, unit dose containers (such as vials), blister packs and strip packs.

[0265] The present invention further provides pharmaceutical compositions and dosage forms comprising one or more substances that reduce the rate of decomposition of the polypeptide or recombinant polypeptide of the present invention as an active ingredient. Such substances, referred to herein as "stabilizers", include but are not limited to antioxidants such as ascorbic acid, pH buffers or salt buffers etc.

[0266] The active agent of the present invention can be administered simultaneously with, before, or after one or more other therapeutic agents. The active agent of the present invention can be administered separately by the same or different routes of administration, or administered together with other active agents in the same pharmaceutical composition.

[0267] In one embodiment, the present invention provides a product comprising a polypeptide or recombinant polypeptide of the present invention and at least one other therapeutic agent for use simultaneously, separately, or sequentially in therapy as a combination product. In one embodiment, the therapy is the treatment of a condition or disorder in which abnormal cell division occurs. The product provided as a combination product includes a composition comprising the active agent of the present invention and other therapeutic agents in the same pharmaceutical composition, or the active agent of the present invention and other therapeutic agents in separate forms (e.g., in the form of a kit).

[0268] In one embodiment, the present invention provides a pharmaceutical composition comprising a polypeptide or recombinant polypeptide of the present invention and another therapeutic agent. Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient as described above.

[0269] Those skilled in the art will understand that the polypeptide or recombinant polypeptide of the present invention can be administered to a subject, particularly a human subject, in which the subject is being treated with surgery or radiotherapy for a condition or disorder in which abnormal cell division occurs. The compounds of the present invention can also be administered to a subject, particularly a human subject, in which the subject has previously (e.g., within 24 hours) been treated with surgery or radiotherapy for a condition or disorder in which abnormal cell division occurs. The subject, particularly a human subject, can also be treated with surgery or radiotherapy for a condition or disorder in which abnormal cell division occurs, in which the compound of the present invention has been previously (e.g., within 24 hours) administered to the subject.

[0270] In one embodiment, the present invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a polypeptide or recombinant polypeptide of the present invention. In one embodiment, the kit comprises means for separately storing the compositions, such as containers, separate bottles, or separate foil packages. An example of such a kit is a blister pack commonly used for packaging tablets, capsules, etc.

[0271] The kit of the present invention can be used to administer different dosage forms, such as oral and parenteral, to administer separate compositions at different dosage intervals, or to titrate separate compositions against each other. To assist compliance, the kit of the present invention typically includes dosing instructions.

[0272] In the combination therapies of the present invention, the polypeptides of the present invention and other therapeutic agents can be manufactured and / or formulated by the same or different manufacturers. Additionally, the polypeptides and other therapeutic agents provided by the present invention can be introduced into the combination therapy together: (i) before the combined product is released to the physician (e.g., in the case of a kit containing the active agent of the present invention and another therapeutic agent); (ii) by the physician himself / herself (or under the physician's guidance) shortly before administration; (iii) by the patient himself / herself, e.g., during the sequential administration of the active agent of the present invention and other therapeutic agents.

[0273] Accordingly, the present invention provides the use of the polypeptides or recombinant polypeptides provided for the treatment of a condition or disorder in which abnormal cell division occurs, wherein the medicament is prepared for administration together with another therapeutic agent. The present invention also provides the use of another therapeutic agent for the treatment of a condition or disorder in which abnormal cell division occurs, wherein the medicament is administered together with the active agent of the present invention.

[0274] The present invention also provides the polypeptides or recombinant polypeptides provided by the present invention for use in a method for treating a condition or disorder in which abnormal cell division occurs, wherein the polypeptides or recombinant polypeptides provided by the present invention are prepared for administration together with another therapeutic agent. The present invention also provides another therapeutic agent for use in a method for treating a condition or disorder in which abnormal cell division occurs, wherein the other therapeutic agent is prepared for administration together with the polypeptides or recombinant polypeptides provided by the present invention.

[0275] The present invention also provides the use of the active agent of the present invention for the treatment of a condition or disorder in which abnormal cell division occurs, wherein the subject has been previously treated (e.g., within 24 hours) with another therapeutic agent. The present invention also provides the use of another therapeutic agent for the treatment of a condition or disorder in which abnormal cell division occurs, wherein the subject has been previously treated (e.g., within 24 hours) with the active agent of the present invention.

[0276] The composition can additionally contain molecules that assist or enhance the action of the active agent of the present invention, e.g., cytotoxic agents such as antimetabolites, alkylating agents, cytotoxic antibiotics, topoisomerase I and / or II inhibitors, vinca alkaloids, and monoclonal antibodies.

[0277] If desired, the composition can also contain a targeting moiety attached to the active ingredient, e.g., a ligand that specifically and selectively binds to an endogenous receptor to allow targeting of a specific cell type or location, e.g., lymphocytes, monocytes, macrophages, endothelial cells, epithelial cells, blood cells, red blood cells, platelets, eosinophils, neutrophils, natural killer cells, dendritic cells, brain cells, heart cells, lung cells, pancreatic islet cells, kidney cells, cancer cells, hormone gland cells, skin, bone, joints, bone marrow, gastric mucosa, lymph nodes, Peyer's patches, omentum, and other suitable tissues.

[0278] The peptides of the present invention can be used to assist or enhance the action of active agents used in conventional therapies, such as cytotoxic agents, to reduce their side effects.

[0279] In one embodiment, the polypeptide or recombinant polypeptide provided by the present invention is administered together with one or more other therapeutic active agents. For example, the polypeptide or recombinant polypeptide provided by the present invention can be used as a combined chemotherapeutic agent. The polypeptide or recombinant polypeptide provided by the present invention can induce some cancer cells.

[0280] The polypeptide or recombinant polypeptide provided by the present invention can also be used in combination with other anti-cancer therapies to achieve the purpose of treating or preventing cancer. This reduction in potential side effects can also allow the dose or level of conventional therapy to be used by the patient to be higher than might otherwise be possible or safe.

[0281] The present invention includes combinations of the polypeptide or recombinant polypeptide provided by the present invention with anti-tumor agents selected from the group consisting of anti-proliferative agents, kinase inhibitors, angiogenesis inhibitors, growth factor inhibitors, cox-I inhibitors, cox-II inhibitors, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, radiation, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxic agents, anti-hormones, statins, anti-androgens, and photochemical therapeutic agents.

[0282] In one embodiment of the present invention, the anti-tumor agent used in combination with the composition of the present invention is an anti-angiogenic agent, a kinase inhibitor, a pan kinase inhibitor, or a growth factor inhibitor.

[0283] Suitable pan kinase inhibitors include SU-11248 (sutinib malate) as described in U.S. Patent No. 6,573,293 (Pfizer Inc).

[0284] Anti-angiogenic agents include, but are not limited to, the following active agents, such as EGF inhibitors, EGFR inhibitors, VEGF inhibitors, VEGFR inhibitors, TIE2 inhibitors, IGF1R inhibitors, COX-II (cyclooxygenase II) inhibitors, MMP-2 (matrix-metalloproteinase 2) inhibitors, and MMP-9 (matrix metalloproteinase 9) inhibitors. Suitable VEGF inhibitors include, for example, Avastin (bevacizumab), an anti-VEGF monoclonal antibody of Genentech, Inc. of South San Francisco, California.

[0285] Other VEGF inhibitors include CP-547,632 (Pfizer Inc.), AG13736 (axitinib, Pfizer Inc.), ZD-6474 (AstraZeneca), AEE788 (Novartis), AZD-2171, VEGF Trap (Regeneron / Aventis), vatalanib (also known as PTK-787, ZK-222584; Novartis & Schering AG), Macugen (pegaptanib octasodium, NX-1838, EYE-001, Pfizer Inc. / Gilead Eyetech), IM862 (Cytran Inc. of Kirkland, Washington, USA); and the synthetic ribozyme Angiozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California) and combinations thereof. VEGF inhibitors useful in practicing the present invention are disclosed in U.S. Patent Nos. 6,534,524 and 6,235,764, the contents of which are incorporated herein by reference in their entirety for all purposes. Particularly suitable VEGF inhibitors include CP-547,632, axitinib, vatalanib, Macugen and combinations thereof.

[0286] Other antiproliferative agents that can be used in combination with the polypeptides or recombinant polypeptides provided by the present invention include inhibitors of the enzyme farnesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFr. PDGFr inhibitors include, but are not limited to, those disclosed in International Patent Application Publication No. WO01 / 40217 published on July 7, 2001 and International Patent Application Publication No. WO2004 / 020431 published on March 11, 2004, the contents of which are incorporated herein by reference in their entirety for all purposes. Suitable PDGFr inhibitors include CP-673,451 and CP-868,596 from Pfizer and their pharmaceutically acceptable salts.

[0287] Suitable GARF inhibitors include AG-2037 (pelitrexol and its pharmaceutically acceptable salts) from Pfizer. GARF inhibitors useful in practicing the present invention are disclosed in U.S. Patent No. 5,608,082, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0288] Other useful inhibitors as anti-tumor agents for use in combination with the polypeptides or recombinant polypeptides provided by the present invention include aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit the enzymes that produce prostaglandins (cyclooxygenase I and II), resulting in lower levels of prostaglandins, including but not limited to the following: salsalate (Amigesic), diflunisal (Dolobid), ibuprofen (Motrin), ketoprofen (Orudis), nabumetone (Relafen), piroxicam (Feldene), naproxen (Aleve, Naprosyn), diclofenac (Voltaren), indomethacin (Indocin), sulindac (Clinoril), tolmetin (Tolectin), etodolac (Lodine), ketorolac (Toradol), oxaprozin (Daypro), and combinations thereof.

[0289] Targeted drugs used in combination with the polypeptides or recombinant polypeptides provided by the present invention include EGFr inhibitors, such as Iressa (gefitinib, AstraZeneca), Tarceva (erlotinib or OSI-774, OSI Pharmaceuticals Inc.), Erbitux (cetuximab, Immone Pharmaceuticals.Inc.), EMD-7200 (MerckAG), ABX-EGF (Amgen Inc. and AbgeniXInc.), HR3 (Cuban government), IgA antibody (University of Erlangen-Nuremberg), TP-38 (IVAX), EGFR fusion protein, EGF-vaccine, anti-EGFr immunoliposome (Hermes Biosciences Inc.) and combinations thereof. Suitable EGFr inhibitors include Iressa, Erbitux, Tarceva and combinations thereof. Other anti-tumor agents include those selected from pan erb receptor inhibitors or ErbB2 receptor inhibitors, such as CP-724,714 (Pfizer, Inc.), CM 033 (canertinib, Pfizer.Inc.), Herceptin (trastuzumab, Genentech Inc.), Omitarg (2C4, pertuzumab, Genentech Inc.), TAK-165 (Takeda), GW-572016 (lonafamib, GlaxoSmithKline), GW-282974 (GlaxoSmithKline), EKB-569 (Wyeth), PKM66 (Novartis), dHER2 (HER2 vaccine, Corixa and GlaxoSmithKline), APC8024 (HER2 vaccine, Dendreon), anti-HER2 / neu bispecific antibody (DecofCancer Center), B7.her2.1gG3 (Agensys), AS HER2 (Radiology&;Research Institutefor Rad Biology&Medicine), trifunctional bispecific antibody (University of Munich) and mAB AR-209 (AroneX Pharmaceuticals Inc) and mAB 2B-1 (Chiron) and combinations thereof. Specific erb-selective anti-tumor agents include Herceptin, TAK-165, CP-724,714, ABX-EGF, HER3 and combinations thereof. Suitable pan erb receptor inhibitors include GW572016, CM 033, EKB-569 and Omitarg and combinations thereof.

[0290] In addition, other anti-tumor agents may be selected from the following active agents: BAY-43-9006 (Onyx Pharmaceuticals Inc.), Genasense (augmerosen, Genta), panitumumab (Abgenix / Amgen), Zevalin (Schering), Bexxar (Corixa / GlaxoSmithKline), abarelix, Alimta, EPO 906 (Novartis), discodermolide (XAA-296), ABT-510 (Abbott), Neovastat (Aeterna), enzastaurin (Eli Lilly), Combrestatin A4P (Oxigene), ZD-6126 (AstraZeneca), flavopiridol (Aventis), CYC-202 (Cyclacel), AVE-8062 (Aventis), DMXAA (Roche / Antisoma), Thymitaq (Eximias), Temodar (temozolomide, Schering Plough), and Revlimid (Celgene) and combinations thereof.

[0291] Other anti-tumor agents may be selected from the following drugs: CyPat (cyproterone acetate), Histerelin (histrelin acetate), Plenaixis (abarelix depot), Atrasentan (ABT-627), satraplatin (JM-216), thalomid (Thalidomide), Theratope, Temilifene (DPPE)1ABI-007 (paclitaxel), Evista (raloxifene), Atamestane (Biomed-777), Xyotax (polyglutamate paclitaxel), Targetin (bexarotine) and combinations thereof.

[0292] In addition, other anti-tumor agents may also be selected from the following active agents: Trizaone (tiazamine), Aposyn (ecisulindac), Nevastat (AE-941), Ceplene (histamine dihydrochloride), Orathecin (rubitecan), verulizine, Gastrimmune (G17DT), DX_8951f (exatecan mesylate), Onconase (ranpirnase), BEC2 (mitumoab), Xcytrin (motexafin gadolinium), and their combinations. Other anti-tumor agents may be selected from the following active agents: CeaVac (CEA), NeuTrexin (trimetrexate glucuronate), and their combinations. Other anti-tumor agents may be selected from the following active agents: OvaRex (oregovomab), Osidem (IDM-I), and their combinations.

[0293] Additional anti-tumor agents may be selected from the following active agents: Advexin (ING 201), Tirazone (tiazamine), and their combinations. Additional anti-tumor agents may be selected from the following active agents: RSR13 (efaproxiral), Cotara (131I-chTNT 1 / b), NBI-3001 (IL-4), and their combinations. Additional anti-tumor agents may be selected from the following active agents: Canvaxin, GMK vaccine, PEG Interferon A, Taxoprexin (DHA / paclitaxel), and their combinations.

[0294] Other anti-tumor agents include Pfizer's MEK1 / 2 inhibitor PD325901, Array Biopharm's MEK inhibitor ARRY-142886, Bristol Myers' CDK2 inhibitor BMS-387,032, Pfizer's CDK inhibitor PD0332991, and AstraZeneca's AXD-5438, and their combinations.

[0295] In addition, mTOR inhibitors such as CCI-779 (Wyeth), rapamycin derivatives RAD001 (Novartis), and AP-23573 (Ariad), HDAC inhibitor SAHA (Merck Inc / Aton Pharmaceuticals), and their combinations may also be used. Additional anti-tumor agents include aurora 2 inhibitor VX-680 (Vertex), Chk1 / 2 inhibitor XL844 (Exelixis).

[0296] One or more of the following cytotoxic agents, such as those selected from epirubicin (Ellence), docetaxel (Taxotere), paclitaxel, dexrazoxane (Zinecard, dexrazoxane), rituximab (Rituxan), imatinib mesylate (Glivec), and combinations thereof, can be used in combination with the inventive compositions described herein.

[0297] The present invention also contemplates the use of the polypeptides or recombinant polypeptides provided by the present invention in combination with hormone therapy, including but not limited to exemestane (Aromasin, Pfizer Inc.), leuprolide (Lupron or Leuplin, TAP / Abbott / Takeda), anastrozole (Arimidex, Astrazeneca), goserelin (Zoladex, AstraZeneca), doxercalciferol, fadrozole, formestane, tamoxifen citrate (tamoxifen, Nolvadex, AstraZeneca), casodex (AstraZeneca), abarelix (Praecis), Trelstar, and combinations thereof.

[0298] The present invention also relates to hormone therapeutic agents, such as anti-estrogens, including but not limited to fulvestrant, toremifene, raloxifene, lasofoxifene, letrozole (Femara, Novartis), anti-androgens such as bicalutamide, flutamide, mifepristone, nilutamide, casodex (R) (4'-cyano-3-(4-fluorobenzenesulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide, bicalutamide), and combinations thereof.

[0299] The following topoisomerase I inhibitors can be used as anti-tumor agents: camptothecin; irinotecan hydrochloride (Camptosar); edotecarin; orathecin (Supergen); esatidecin (Daiichi); BN-80915 (Roche); and combinations thereof. Particularly preferred topoisomerase II inhibitors include epirubicin (Ellence).

[0300] Alkylating agents include, but are not limited to, nitrogen mustard N-oxides, cyclophosphamide, ifosfamide, melphalan, busulfan, dibromomannitol, carboquone, thiotepa, ranimustine, nimustine, temozolomide, AMD-473, altretamine, AP-5280, apaziquone, brostallicin, bendamustine, carmustine, estramustine phosphate, fotemustine, glucophosphamide, ifosfamide, KW-2170, maphosphamide, and dibromodulcitol; platinum coordinated alkylating agents include, but are not limited to, cisplatin, carboplatin (Paraplatin), heptaplatin, lobaplatin, nedaplatin, oxaliplatin (Eloxatin, Sanofi), or satraplatin and combinations thereof. Particularly preferred alkylating agents include oxaliplatin (Eloxatin).

[0301] Antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil (5-FU) alone or in combination with leucovorin, tegafur, LIFT, doxifluridine, fluorouracil hexylamine, cytarabine, cytarabine ocfosfate, enocitabine, S-1, pemetrexed disodium (LY231514, MTA), gemcitabine (Gemzar, Eli Lilly), fludarabine, 5-azacytidine, capecitabine, cladribine, clofarabine, decitabine, elonixibat, ethynylcytidine, cytosine arabinoside, hydroxyurea, TS-1, melphalan, nelarabine, nolatrexed, ocfosfate, disodium premetrexed, pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, vinorelbine; or, for example, one of the preferred antimetabolites disclosed in European Patent Application 239362, such as N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thiophenecarbonyl)-L-glutamic acid and combinations thereof.

[0302] Antibiotics include intercalating antibiotics but are not limited to: aclarubicin, actinomycin D, amrubicin, annamycin, doxorubicin, bleomycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, galarubicin, idarubicin, mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebeccamycin, stimalamer, streptozocin, valrubicin, zinostatin and combinations thereof.

[0303] Antitumor substances of plant origin include, for example, those selected from mitotic inhibitors such as vinblastine, docetaxel (Taxotere), paclitaxel, and combinations thereof.

[0304] Cytotoxic topoisomerase inhibitors include one or more active agents selected from aclarubicn, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, irinotecan hydrochloride (Camptosar), edotecarin, epirubicin (Eilence), etoposide, esaintecone, gemotecan, leotecan, mitoxantrone, pirarubicin, pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide, topotecan, and combinations thereof. Preferred cytotoxic topoisomerase inhibitors include one or more active agents selected from camptothecin, 10-hydroxycamptothecin, 9-aminoanthracene, irinotecan hydrochloride (Camptosar), edotecarin, epirubicin (Eilence), etoposide, SN-38, topotecan, and combinations thereof.

[0305] Immunology includes interferons and many other immunopotentiators. Interferons include interferon α, interferon α-2a, interferon α-2b, interferon β, interferon γ-1a, interferon γ-1b (Actimmune), or interferon γ-n1, and combinations thereof. Other active agents include filgrastim, ientinan, schizophyllan, TheraCys, ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab, imiquimod, lenograstim, Ientinan, Corixa, molgramostim, OncoVAX-CL, sargramostim, tasocitinib, tecleukin, thymalasin, tositumomab, velurecitabine, 2-100, epratuzumab, mitotane, oregovomab, pemtumomab (Y-muHMFGl), Provenge (Dendreon), and combinations thereof.

[0306] Biological response modifiers are active agents that alter the defense mechanisms or biological responses of living organisms (such as the survival, growth, or differentiation of tissue cells) to direct them to have antitumor activity. Such active agents include polysaccharide-K, Ientinan, sizofiran, Picibanil, ubenimex, and combinations thereof.

[0307] Other anti-cancer agents include alitretinoin, polyinosinic acid, atrasentan, bexarotene, bortezomib, bosentan, calcitriol, exisulind, finasteride, fotemustine, ibandronic acid, miltefosine, mitoxantrone, L-asparaginase, procarbazine, dacarbazine, hydroxyurea, pegaspargase, pentostatin, tazarotene, Telcyta (TLK-286, Telik Inc.), Velcade (bortemazib, Millennium), retinoic acid, and combinations thereof.

[0308] Other anti-angiogenic compounds include acitretin, fenretinide, thalidomide, zoledronic acid, angiostatin, aplidine, cilengtide, combretastatin A-4, endostatin, halofuginone, rebimastat, removab, squalamine, ukrain, Vitaxin, and combinations thereof. Platinum coordination compounds include, but are not limited to, cisplatin, carboplatin, nedaplatin, oxaliplatin, and combinations thereof.

[0309] Camptothecin derivatives include, but are not limited to, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, SN-38, edotecarin, topotecan, and combinations thereof. Other anti-tumor agents include mitoxantrone, L-asparaginase, procarbazine, dacarbazine, hydroxyurea, pentostatin, retinoic acid, and combinations thereof.

[0310] Anti-tumor agents that can enhance the anti-tumor immune response can also be used, such as CTLA4 (cytotoxic T lymphocyte antigen 4) antibodies and other active agents that can block CTLA4, such as MDX-010 (Medarex) and CTLA-4 compounds disclosed in U.S. Patent No. 6,682,736; and anti-proliferative agents such as other farnesyl protein transferase inhibitors, such as farnesyl protein transferase inhibitors. In addition, specific CTLA4 antibodies useful in the present invention include those described in U.S. Provisional Application 60 / 113,647 (filed December 23, 1998) and U.S. Patent No. 6,682,736, the entire contents of which are incorporated herein by reference.

[0311] Gene therapy agents can also be used as anti-tumor agents, such as TNFerade (GeneVec), which expresses TNFα in response to radiotherapy.

[0312] Statins can also be used in combination with the polypeptides or recombinant polypeptides provided by the present invention. Statins (HMG-CoA reductase inhibitors) can be selected from atorvastatin (Lipitor, Pfizer Inc.), pravastatin (Pravachol, Bristol-Myers Squibb), lovastatin (Mevacor, Merck Inc.), simvastatin (Zocor, Merck Inc), fluvastatin (Lescol, Novartis), cerivastatin (Baycol, Bayer), rosuvastatin (Crestor, AstraZeneca), Lovostatin and Niacin (Advicor, Kos Pharmaceuticals) and their derivatives and combinations. In a preferred embodiment, the statins are selected from atorvastatin and lovastatin, their derivatives and combinations. Other active agents that can be used as anti-tumor agents include Caduet.

[0313] The polypeptides or recombinant polypeptides provided by the present invention can be used in combination with photochemical therapeutic agents for locally generating reactive oxygen species. Examples of photochemical therapeutic agents include palladium for photodynamic therapy

[0314] Bacteriophephorbide (TOOKAD); psoralen, 8-methoxypsoralen / methoxsalen, 4,5,8-trimethylpsoralen / trisoralen, UVAR or XTSTM Photopheresis System (Therakos, Inc., Exton, PA) used in PUVA (Psoralen Ultra Violet A light): (Macopharma); Cobe Spectra + Photo Immune System UVA PIT (Med Tech Solution); photosensitizers such as calcipotriene, tazarotene, chrysarobin and its synthetic derivative anthralin / 1,8-dihydroxy-9-anthrone / dithranol (BioLuminescence Activated Destruction (BLADe)) luciferase of Photinus pyralis; erythrosin B (EB); erythrosine sodium; m-tetrahydroxyphenylchlorin (m-THPC) / temoporfin (Biolitec AG); porphyrins such as δ-aminolevulinic acid (δ-ALA) (Levulan DUSA Pharmaceuticals, Inc.), 5-ALA methylesther (MLA / M-ALA) (PhotoCure ASA), 5-ALA benzylesther, 5-ALA hexylesther, tin ethyletiopurpurin (SnET2) / Snetiopurpurin / rostaporfin (Miravant Medical Technologies), boronated protoporphyrin, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) (Rosewell Park Cancer Institute), texaphyrin, including europium texaphyrin (Eu-Tex), dysprosium texaphyrin (Dy-Tex), manganese texaphyrin (Mn-Tex), lutetium texaphyrin / PCI-0123 (Luu), motexafin lutetium (MLu) / lutetium(III) texaphyrin (Lu-Tex) (Pharmacyclics Inc.), motexafin gadolinium (MGd) / PCI-0120 (source: Pharmacyclics Inc.)Phthalocyanine-4 (Pc 4), taporfin sodium / NPe6 / mono-L-aspartyl chlorin e6 / taporfin sodium / LS11 (Light Science Corporation), benzoporphyrin derivative-monocarboxylic acid ring A (BPD-MA) / verteporfin) (Novartis Pharmaceuticals), partially purified hematoporphyrin derivative (HpD), porfimer sodium (Axcan Pharma, Inc.), dihematoporphyrin ether (DHE), photosan-3 (PS-3), phytochrome-II, meso-tetraphenylporphyrin (TPP), and tetraphenylporphyrin sulfonate (TPPS4).

[0315] The following are the preferred embodiments of the present invention. It should be noted that for those skilled in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also regarded as the protection scope of the present invention.

[0316] Unless otherwise specified, the tumor cells such as human lung cancer cell A549, breast cancer cell MCF7, and cervical cancer cell HeLa used in the following examples are all purchased from the China Center for Type Culture Collection (CCTCC), Wuhan University.

[0317] Unless otherwise specified, the polypeptides used in the following examples are all produced by Wuhan Moore Biotechnology Co., Ltd., and the purity is ≥95%.

[0318] In the polypeptide provided by the present invention that can effectively kill tumor cells and cause immunogenic death of tumor cells, its preparation method, drug delivery system and application, the raw materials and reagents used can all be purchased from the market.

[0319] The present invention will be further described below through specific examples. However, it should be understood that these examples are only used for more detailed and specific description, and should not be construed as limiting the present invention in any form.

[0320] Example 1: Polypeptide genetic engineering expression and purification

[0321] Using the amino acid sequence of the anti-tumor polypeptide SeqID NO.1 and the amino acid sequence of the sumo enzyme recognition site as templates, the nucleic acid sequence was optimized according to the codon preference of Escherichia coli BL21(DE3). The DNA sequence is SEQ ID NO.4- / , the vector used is pET-32a(+), and the gene synthesis company was sent to prepare the recombinant expression vector pET-32a(+)-LCX17.

[0322] The obtained recombinant expression plasmid pET-32a(+)-LCX17 was transformed into Escherichia coli BL21(DE3). Take 2 μL of the recombinant expression plasmid pET-32a(+)-LCX17 and slowly add it to the competent cells of the host bacterium BL21(DE3). Mix well, incubate on ice for 30 minutes, place in a 42°C water bath for heat shock for 90 seconds and quickly transfer to an ice bath for 2 minutes. Add 500 μL of SOC medium solution, culture in a shaker at 37°C (150 rpm) for 1 hour, mix the bacterial solution well, take 100 μL of the bacterial solution and spread it on an LB agar plate containing 100 μg / mL ampicillin. After the bacterial solution is absorbed, place it in an incubator at 37°C for 16 - 24 h and observe the results. Pick a single colony from the agar plate cultured overnight and culture it overnight in 10 mL of LB liquid medium containing 100 μg / mL ampicillin in a shaker at 37°C (200 rpm). Take 500 μL of the bacterial solution and add 500 μL of 30% glycerol. Mix well and store in liquid nitrogen to obtain recombinant Escherichia coli BL21-LCX17.

[0323] After thawing the stored bacterial strain, take 10 μL of the bacterial solution and inoculate it into 50 mL of LB liquid medium (containing 100 μg / mL ampicillin), and culture it at 37°C and 200 rpm for 16 h - 20 h. Take the bacterial solution cultured overnight and inoculate it into 500 mL of LB liquid medium (containing 100 μg / mL ampicillin) at a ratio of 2% (V / V), and culture it at 37°C and 230 rpm until OD 600 is about 0.8. Add IPTG with a final concentration of 1 mM and induce expression at 28°C and 200 rpm. After 4 h of culture, the induction ends. Centrifuge at 10000 rpm and 4°C for 10 min to collect the bacterial cells.

[0324] Take the wet bacterial cells collected after the induction ends and add Binding Buffer (pH 8.0) at a ratio of 1:10, and stir to suspend the bacterial cells. Under ice bath conditions, lyse the bacteria by ultrasonic wave. The power of ultrasonic wave is 400 W (60%), the working time is 3 s, the interval time is 3 s, and the lysis time is 25 min. Centrifuge the lysate at 12000 rpm and 4°C for 30 min, collect the supernatant, filter the supernatant with a 0.45 μm mixed membrane, and collect the filtered supernatant for purification.

[0325] Nickel column affinity purification, the parameters are as follows (5 mL pre-packed column, the instrument used is AKTA primer):

[0326] Column equilibration: The flow rate is 5 mL / min, and rinse the nickel column with Binding Buffer for 10 CV until the reading is stable.

[0327] Sample loading: Take the filtered solution and load the sample at a flow rate of 2.5 mL / min.

[0328] Washing and Miscellaneous Operations: Equilibrate the nickel column with Binding Buffer at a flow rate of 5 mL / min until the reading stabilizes.

[0329] Elution: Elute with Elution Buffer at a flow rate of 5 mL / min, collect the eluate (containing the fusion protein), and start collecting from the moment the reading starts to rise until the reading drops to a stable level and then stop collecting.

[0330] Take the purified fusion protein and perform enzymatic digestion at 4°C for 16 h at a ratio of fusion protein: sumo enzyme = 1 mg: 200 U. Take the enzymatic digestion product, perform nickel column affinity chromatography, and collect the flow-through to obtain a crude purified solution containing the recombinant anti-tumor polypeptide.

[0331] Take the crude purified solution for HPLC purification. The parameters of HPLC are as follows: separation column C18: 10×250 mm; flow rate 5 mL / min; mobile phase A is water containing 0.1% trifluoroacetic acid (TFA); mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid (TFA) and 80%; the ultraviolet detection wavelength is 214 nm. The elution program is shown in Table 1.

[0332] Table 1. HPLC Purification Program.

[0333] Time Mobile Phase A Mobile Phase B 0.0 min 95％ 5％ 45 min 5 95％

[0334] Manually collect the polypeptide peak of SEQ ID NO.1 to obtain a higher purity anti-tumor polypeptide. Purify again by HPLC to make the purity of the polypeptide ≥ 95%. After 3 times of vacuum freeze-drying, store at -80°C and use for subsequent experimental studies.

[0335] The preparation method of polypeptide Seq ID No.5 to polypeptide Seq ID No.7 is the same as that of polypeptide Seq ID No.1.

[0336] Dissolve the polypeptide lyophilized powder in sterile PBS buffer or sterilized water to an appropriate concentration to prepare a polypeptide solution (i.e., polypeptide Seq ID NO.1 solution, polypeptide Seq ID NO.2 solution, polypeptide Seq ID NO.3 solution, polypeptide Seq ID NO.4 solution, polypeptide Seq ID NO.5 solution, polypeptide Seq ID NO.6 solution, polypeptide Seq ID NO.7 solution in the following examples) for experimental studies.

[0337] Example 2: Toxicity Detection of Polypeptide SEQ ID NO.1 on Various Tumor Cells

[0338] Experiment 1: Toxicity of Polypeptides with Different Concentrations to Murine 4T1 Tumor Cell Line

[0339] The 4T1 tumor cells were used as the model cells for studying the anti-tumor polypeptides on the murine breast cancer cell line. Cells in the logarithmic growth phase were digested with trypsin, counted, and diluted with RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to 1×10 5 cells / mL of medium. The cell suspension was added to a 96-well cell culture plate, 100 μL / well, that is, each well contained 10,000 cells. The 96-well cell plate was placed in a carbon dioxide incubator and cultured overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, the supernatant was discarded. In the sample group, 90 μL of cell complete medium and 10 μL of polypeptide SeqID NO.1 solution at different concentrations were added to each well, so that the final concentration of the polypeptide was 60 μM, 20 μM, 6.7 μM, 2.2 μM, 0.7 μM, and 0.2 μM. The blank control group and the background control group were added with 90 μL of cell complete medium and 10 μL of sterilized water. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, 10 μL of CCK8 solution was added to each well. After incubating in the cell incubator for 1 - 4 h, the absorbance (OD value) was measured at 450 nm with an enzyme-linked immunosorbent assay reader. Calculate the cell mortality rate (Cell mortality rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results showed that, as 450 shown, compared with the 4T1 cells without polypeptide treatment, after adding different concentrations of polypeptide to the 4T1 cells and incubating, with the increase of the polypeptide concentration, the cell mortality rate gradually increased. It indicated that the polypeptide Seq ID NO.1 could significantly inhibit the growth of the breast cancer cell line 4T1 cells. The half-lethal concentration (IC50) of the polypeptide SEQ ID NO.1 was 3.6 μM. The specific data are shown in Table 1. 450 value) / (OD 450 value of the blank control group - OD 450 value of the background control group)) × 100%. The experimental results showed that, as Figure 1 shown, compared with the 4T1 cells without polypeptide treatment, after adding different concentrations of polypeptide to the 4T1 cells and incubating, with the increase of the polypeptide concentration, the cell mortality rate gradually increased. It indicated that the polypeptide Seq ID NO.1 could significantly inhibit the growth of the breast cancer cell line 4T1 cells. The half-lethal concentration (IC50) of the polypeptide SEQ ID NO.1 was 3.6 μM. The specific data are shown in Table 1.

[0340] Table 1. Cytotoxicity of polypeptide SEQ ID NO.1 on 4T1 cells

[0341] Concentration / μM 60 30 15 7.5 3.75 1.875 Cell Death Rate / % 93.8 94.5 75.4 33 0 6.3

[0342] Experiment 2: Toxicity of Polypeptides with Different Concentrations to A549 Tumor Cells

[0343] The A549 tumor cells were used as the model cells for studying the anti-tumor polypeptides on the non-small cell lung cancer cell line. Cells in the logarithmic growth phase were digested with trypsin, counted, and diluted with F-12K complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to 5×10 4Cells / mL of culture medium, add the cell suspension to a 96-well cell culture plate, 100 μL / well, that is, each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. Add 90 μL of complete cell medium and 10 μL of polypeptide solutions with different concentrations of Seq ID NO.1 to each well of the sample group, so that the final concentration of the polypeptide is 100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM and 0.4 μM. The blank control group and the background control group add 90 μL of complete cell medium and 10 μL of sterilized water. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. After incubating in the cell incubator for 1 - 4 h, measure the absorbance (OD value) at 450 nm with an enzyme-linked immunosorbent assay reader. Calculate the cell death rate (Cell death rate (%) = ((1 - (OD value of the polypeptide treatment group - OD value of the background control group)) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as shown in 450 value - OD value of the background control group 450 value) / (OD value of the blank control group 450 value - OD value of the background control group 450 value)) × 100%. The experimental results show that, as Figure 2 shown, compared with A549 cells without polypeptide treatment, after adding different concentrations of polypeptide to A549 cells and incubating, with the increase of polypeptide concentration, the cell death rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of the non-small cell lung cancer cell line A549, and its half-lethal concentration (IC 50 ) is 18.3 μM, and the specific data are shown in Table 2.

[0344] Table 2. Cytotoxicity of polypeptide SEQ ID NO.1 on A549 cells

[0345] Concentration / μM 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 91.8 82.3 26.2 15.4 17.7 7.7

[0346] Experiment 3: Toxicity of Polypeptides with Different Concentrations to MCF7 Tumor Cells

[0347] Use MCF7 tumor cells as model cells to study the anti-tumor polypeptide on breast cancer cell lines. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells with MEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to 5×10 4cells / mL of the culture medium. Add the cell suspension to a 96-well cell culture plate, 100 μL per well, that is, each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. Add 90 μL of complete cell culture medium and 10 μL of polypeptide solutions with different concentrations of Seq ID NO.1 to each well of the sample group, so that the final concentration of the polypeptide is 60 μM, 20 μM, 6.7 μM, 2.2 μM, 0.7 μM, and 0.2 μM. The blank control group and the background control group are added with 90 μL of complete cell culture medium and 10 μL of sterilized water. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. After incubating in the cell incubator for 1 - 4 h, measure the absorbance (OD value) at 450 nm with an enzyme-linked immunosorbent assay reader. Calculate the cell mortality rate (cell mortality rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group)) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as shown in 450 value - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value)) × 100%. The experimental results show that, as Figure 3 shown, compared with MCF7 cells without polypeptide treatment, after adding different concentrations of polypeptides to incubate in MCF7 cells, with the increase of polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of breast cancer cell line MCF7, and its half-lethal concentration (IC 50 ) is 6.1 μM. The specific data are shown in Table 3.

[0348] Table 3. Cytotoxicity of polypeptide SEQ ID NO.1 against MCF7 cells

[0349] Concentration / μM 60 30 15 7.5 3.75 1.875 Cell Death Rate / % 84.6 92.6 69.7 42.6 39.2 39.8

[0350] Experiment 4: Toxicity of Polypeptides with Different Concentrations to HT29 Tumor Cells

[0351] Use HT29 tumor cells as the model cells for studying the anti-tumor polypeptide against colorectal cancer cell lines. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells with McCoy's 5A complete culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to 5 × 10 4cells / mL of culture medium, add the cell suspension to a 96-well cell culture plate, 100 μL per well, that is, each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. Add 90 μL of complete cell culture medium and 10 μL of polypeptide solutions with different concentrations of Seq ID NO.1 to each well of the sample group, so that the final concentration of the polypeptide is 60 μM, 20 μM, 6.7 μM, 2.2 μM, 0.7 μM, and 0.2 μM. The blank control group and the background control group add 90 μL of complete cell culture medium and 10 μL of sterile water. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. After incubating in the cell culture incubator for 1 - 4 h, measure the absorbance (OD value) at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the cell death rate (cell death rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as shown in 450 value - background control group OD 450 value) / (blank control group OD 450 value - background control group OD 450 value)) × 100%. The experimental results show that, as Figure 4 shown, compared with HT29 cells without polypeptide treatment, after adding different concentrations of polypeptide to HT29 cells and incubating, with the increase of polypeptide concentration, the cell death rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of the colorectal cancer cell line HT29, and its half-lethal dose (IC50) is 7.0 μM. The specific data are shown in Table 4.

[0352] Table 4. Cytotoxicity of polypeptide SEQ ID NO.1 on HT29 cells

[0353] Concentration / μM 60 30 15 7.5 3.75 1.875 Cell Death Rate / % 88.8 93.6 56.7 20.7 25.4 18.9

[0354] Experiment 5: Toxicity of Polypeptides with Different Concentrations to HEC-1-B Tumor Cells

[0355] Use HEC-1-B tumor cells as model cells for studying anti-tumor polypeptides on endometrial cancer cell lines. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells with MEM complete medium (containing 10% fetal bovine serum and 1% streptomycin) to 5 × 10 4Cells / mL of culture medium. Add the cell suspension to a 96-well cell culture plate, 100 μL per well, i.e., each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. Add 90 μL of complete cell culture medium and 10 μL of polypeptide solutions with different concentrations of Seq ID NO.1 to each well in the sample group, so that the final concentration of the polypeptide is 60 μM, 30 μM, 15 μM, 7.5 μM, 3.75 μM, and 1.875 μM. Add 90 μL of complete cell culture medium and 10 μL of sterilized water to the blank control group and the background control group. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. After incubating in the cell culture incubator for 1 - 4 h, measure the absorbance (OD value) at 450 nm using a microplate reader. Calculate the cell mortality rate (Cell mortality rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group)) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as shown in 450 value - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value)) × 100%. The experimental results show that, as Figure 5 shown, compared with HEC-1-B cells without polypeptide treatment, after adding different concentrations of polypeptide to HEC-1-B cells and incubating, with the increase of polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of endometrial cancer cell line HEC-1-B, and its half-lethal concentration (IC 50 ) is 12.8 μM. The specific data are shown in Table 5.

[0356] Table 5. Cytotoxicity of polypeptide SEQ ID NO.1 on HEC-1-B cells

[0357] Concentration / μM 60 30 15 7.5 3.75 1.875 Cell Death Rate / % 96 93 54.4 -4.7 -15.2 -22.6

[0358] Experiment 6: Toxicity of Polypeptides with Different Concentrations to PC3 Tumor Cell Line

[0359] Use PC3 tumor cells as model cells for studying the anti-tumor polypeptide on human prostate cancer cell line. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells with Ham's F-12K complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to 5 × 10 4Cells / mL of culture medium. Add the cell suspension to a 96-well cell culture plate, 100 μL per well, i.e., each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. In the sample group, add 90 μL of complete cell culture medium and 10 μL of polypeptide solutions with different concentrations of SeqID NO.1 to each well, so that the final concentration of the polypeptide is 100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, and 0.4 μM. The blank control group and the background control group add 90 μL of complete cell culture medium and 10 μL of sterile water. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. After incubating in the cell culture incubator for 1 - 4 h, measure the absorbance (OD value) at 450 nm using an enzyme-linked immunosorbent assay reader. Calculate the cell death rate (Cell death rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group)) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as shown, compared with PC3 cells without polypeptide treatment, after adding different concentrations of polypeptide to PC3 cells and incubating, with the increase of the polypeptide concentration, the cell death rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of the human malignant melanoma cell line PC3, and its half-lethal concentration (IC 450 value - OD value of the background control group 450 value) / (OD value of the blank control group 450 value - OD value of the background control group 450 value)) × 100%. The experimental results show that, as shown, compared with PC3 cells without polypeptide treatment, after adding different concentrations of polypeptide to PC3 cells and incubating, with the increase of the polypeptide concentration, the cell death rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of the human malignant melanoma cell line PC3, and its half-lethal concentration (IC Figure 6 is 4.9 μM, and the specific data are shown in Table 6. 50 ) is 4.9 μM, and the specific data are shown in Table 6.

[0360] Table 6. Cytotoxicity of polypeptide SEQ ID NO.1 on PC3 cells

[0361] Concentration / μM 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 73 90.4 73.8 47.1 34.3 32.4

[0362] Experiment 7: Toxicity of Polypeptides with Different Concentrations to hepG2 Tumor Cell Line

[0363] Use hepG2 tumor cells as model cells to study the anti-tumor polypeptide on the liver cancer cell line. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells with MEM complete medium (containing 10% fetal bovine serum and 1% streptomycin) to 5 × 10 4Cells / mL culture medium, add cell suspension to 96-well cell culture plates, 100 μL / well, that is, each well contains 5000 cells. The 96-well cell plate was placed in a carbon dioxide incubator and cultured overnight at 37°C and 5% CO2 to allow the cells to adhere to the wall. The next day, the supernatant was discarded, and 90 μL of complete cell culture medium and 10 μL of SeqID NO.1 peptide solution of different concentrations were added to each well of the sample group, so that the final concentration of the peptide was 100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM and 0.4 μM. 90 μL of complete cell culture medium and 10 μL of sterile water were added to the blank control group and the background control group. After the 96-well cell plate was placed in a carbon dioxide incubator and cultured at 37°C and 5% CO2 for 24 hours, 10 μL of CCK8 solution was added to each well. After incubation in the cell culture incubator for 1 to 4 hours, the absorbance (OD value) was measured at 450nm using an enzyme marker. Calculate the cell death rate (cell death rate (%) = (1-(OD 450 Value-background control group OD 450 value) / (blank control group OD 450 Value-background control group OD 450 value))×100%. The experimental results show that Figure 7 As shown in the figure, compared with hepG2 cells not treated with polypeptides, after adding different concentrations of polypeptides to hepG2 cells for incubation, the cell death rate gradually increased with the increase of polypeptide concentration. This shows that the polypeptide can significantly inhibit the growth of hepG2 liver cancer cell line, and its half lethality (IC 50 ) is 12.14 μM, and the specific data are shown in Table 7.

[0364] Table 7. Cytotoxicity of polypeptide SEQ ID NO.1 to hepG2 cells

[0365] Concentration / μM 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 106.4 113 11.5 -5.4 -22.8 -33.4

[0366] Experiment 8: Toxicity of Polypeptides with Different Concentrations to 786-O Tumor Cell Line

[0367] 786-O tumor cells were used as model cells to study the effects of anti-tumor peptides on renal cancer cell lines. Cells in the logarithmic growth phase were obtained, trypsinized, and the cells were counted. The cells were diluted to 5×10 with RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% streptavidin). 4Cells / mL of the culture medium, add the cell suspension to a 96-well cell culture plate, 100 μL per well, that is, each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. Add 90 μL of complete cell culture medium and 10 μL of polypeptide solutions with different concentrations of Seq ID NO.1 to each well in the sample group, so that the final concentration of the polypeptide is 100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, and 0.4 μM. Add 90 μL of complete cell culture medium and 10 μL of sterile water to the blank control group and the background control group. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. After incubating in the cell culture incubator for 1 - 4 h, measure the absorbance (OD value) at 450 nm using an enzyme-linked immunosorbent assay reader. Calculate the cell mortality rate (Cell mortality rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group)) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as shown in 450 value - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value)) × 100%. The experimental results show that, as Figure 8 shown, compared with 786-O cells without polypeptide treatment, after adding different concentrations of the polypeptide to 786-O cells and incubating, with the increase in the polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of the renal cancer cell line 786-O, and its half-lethal concentration (IC 50 ) is 8.7 μM. The specific data are shown in Table 8.

[0368] Table 8. Cytotoxicity of polypeptide SEQ ID NO.1 against 786-O cells

[0369] Concentration / μM 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 90.9 95.9 77.2 28.7 26.4 26.4

[0370] Experiment 9: Toxicity of Polypeptides with Different Concentrations to LM3 Tumor Cell Line

[0371] Using LM3 tumor cells as model cells for studying anti-tumor polypeptides against human liver cancer cell lines. Take cells in the logarithmic growth phase, digest them with trypsin, count the cells, and dilute the cells with DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to 5 × 10 4Cells / mL of culture medium. Add the cell suspension to a 96-well cell culture plate, 100 μL per well, i.e., each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. Add 90 μL of complete cell culture medium and 10 μL of polypeptide solutions with different concentrations of SeqID NO.1 to each well of the sample group, so that the final concentration of the polypeptide is 100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, and 0.4 μM. Add 90 μL of complete cell culture medium and 10 μL of sterilized water to the blank control group and the background control group. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. After incubating in the cell culture incubator for 1 - 4 h, measure the absorbance (OD value) at 450 nm with an enzyme-linked immunosorbent assay reader. Calculate the cell death rate (Cell death rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as shown, compared with LM3 cells without polypeptide treatment, after adding polypeptides with different concentrations to LM3 cells and incubating, with the increase in polypeptide concentration, the cell death rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of human liver cancer cell line LM3, and its half-lethal dose (IC 450 value - OD value of the background control group 450 value) / (OD value of the blank control group 450 value - OD value of the background control group 450 value)) × 100%. The experimental results show that, as Figure 9 shown, compared with LM3 cells without polypeptide treatment, after adding polypeptides with different concentrations to LM3 cells and incubating, with the increase in polypeptide concentration, the cell death rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of human liver cancer cell line LM3, and its half-lethal dose (IC 50 ) is 14.0 μM, and the specific data are shown in Table 9.

[0372] Table 9. Cytotoxicity of polypeptide SEQ ID NO.1 on LM3 cells

[0373]

[0374]

[0375] Experiment 10: Toxicity of Polypeptides with Different Concentrations to U251MG Tumor Cell Line

[0376] Use U251MG tumor cells as model cells for studying the anti-tumor polypeptide on glioma cell lines. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells with MEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to 5 × 10 4cells / mL of culture medium. Add the cell suspension to a 96-well cell culture plate, 100 μL per well, that is, each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. In the sample group, add 90 μL of complete cell culture medium and 10 μL of polypeptide solutions with different concentrations of Seq ID NO.1 to each well, so that the final concentration of the polypeptide is 60 μM, 30 μM, 15 μM, 7.5 μM, 3.75 μM, and 1.875 μM. The blank control group and the background control group add 90 μL of complete cell culture medium and 10 μL of sterilized water. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. Incubate in the cell incubator for 1 - 4 h, and then measure the absorbance (OD value) at 450 nm using an enzyme-linked immunosorbent assay reader. Calculate the cell death rate (cell death rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as shown in 450 value - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value of the polypeptide treatment group - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value of the polypeptide treatment group - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in 450 value of the polypeptide treatment group - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%. The experimental results show that, as shown in Figure 10 shown, compared with U251MG cells without polypeptide treatment, after adding different concentrations of polypeptide to U251MG cells and incubating, with the increase of polypeptide concentration, the cell death rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of glioma cell line U251MG, and its half-lethal concentration (IC 50 ) is 21.6 μM. The specific data are shown in Table 10.

[0377] Table 10. Cytotoxicity of polypeptide SEQ ID NO.1 on U251MG cells

[0378] Concentration / μM 60 30 15 7.5 3.75 1.875 Cell Death Rate / % 84.4 76.9 2.2 -2.9 -4 -4.2

[0379] Example 3: Microscopic morphological observation of tumor cells

[0380] Use 4T1 tumor cells as model cells for studying the anti-tumor polypeptide on mouse breast cancer cell lines. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, suspend the cells with RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% streptomycin), add the cell suspension to a T25 culture flask, 2.0×10 6cells / bottle. Place the cell culture flask in a carbon dioxide incubator and culture overnight at 37°C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant, wash twice with 5 ml of PBS buffer, and add 5 ml of RPMI-1640 basal medium containing the polypeptide Seq ID No.1 prepared in Example 1 (final concentration 21.2 μM). Place it in a carbon dioxide incubator and culture at 37°C and 5% CO2. Observe the morphological changes of the cells under a microscope at 0 min, 10 min, 12 h, 24 h, and 36 h. As can be seen from the experimental results, as Figure 11 shown, the polypeptide Seq ID No.1 causes cell death by disrupting the cell membrane, and with the prolongation of time, the degree of damage to the cell membrane gradually increases, the cell contents are released, and finally a naked nucleus is formed.

[0381] Example 4: Tumor immunity experiment in animals

[0382] Use 4T1 tumor cells as model cells for studying the anti-tumor polypeptide on the mouse breast cancer cell line. Rapidly resuscitate breast cancer 4T1 cells under a 37°C water bath condition, and then transfer them to RPMI-1640 medium containing 10% fetal bovine serum and 1% streptomycin, and culture in a cell incubator at 37°C and 5% CO2. When the cells grow to the logarithmic phase, passage them to obtain a sufficient number of cells. Add 1.5×10 7 cells to each T175 cell culture flask, and culture overnight in a cell incubator at 37°C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant and wash twice with PBS. The experimental group was added with 20 mL of basal medium (RPMI-1640 medium, servicebio, product number: G4535-500ml) containing the polypeptide Seq ID NO.1 prepared in Example 1 (final concentration 5.3 μM); the control group was added with 20 mL of basal medium (RPMI-1640 medium) containing doxorubicin (final concentration 5.3 μM), and cultured in a cell incubator at 37°C and 5% CO2 for 24 h. The freeze-thaw group collected the normally cultured cells, adjusted the cell concentration to 7.5×10 6 cells / ml with basal medium, and freeze-thawed them 3 times at -80°C for 15 min and 37°C for 15 min. Subcutaneously inoculate these treated tumor cells under the left scapula of the mice, and inoculate 1.5×10 6 cells per mouse. On the 15th day after the first inoculation (day 0), count the normally passaged and cultured 4T1 cells (untreated), dilute them with basal medium to 3×10 6 cells / mL, and subcutaneously inoculate 4T1 cells under the right scapula of the mice, and inoculate 3×10 6cells. The growth of tumors in mice was observed, and the number of tumor-free and tumor-bearing mice in each group was dynamically observed. The tumor diameter was measured with a vernier caliper. The formula for calculating the tumor volume (TV) was: TV = 0.5 × a × b × b, where a and b represent the major axis and minor axis of the tumor mass, respectively. The formula for calculating the ratio of tumor-free mice (TF) was: TF = (number of mice without tumors / total number of mice) × 100%. As can be seen from the experimental results, as Figure 12 shown, at the end of the experiment, compared with the freeze-thaw group (TF = 0%) and the control group (treated with doxorubicin, TF = 17%), it can be seen that the Seq ID NO.1 experimental group (TF = 83%) had a higher proportion of tumor-free, indicating that the necrotic tumor cells after treatment with Seq ID NO.1 could induce anti-tumor immune protection and activate specific anti-tumor immune responses. The specific data are shown in Table 11.

[0383] Table 11. Tumor-free data of tumor immunity test

[0384] Tumor-free Freeze-thaw Group Control Group Seq ID NO.1 Group Day 0 100.0％ 100.0％ 100.0％ Day 4 100.0％ 100.0％ 100.0％ Day 6 8.3％ 58.3％ 100.0％ Day 8 8.3％ 58.3％ 100.0％ Day 10 0.0％ 50.0％ 100.0％ Day 13 0.0％ 50.0％ 100.0％ Day 16 0.0％ 16.7％ 83.3％

[0385] Example 5: Intratumoral administration treatment experiment for orthotopic tumors

[0386] MCF7 tumor cells were used as model cells to study the anti-tumor polypeptide on breast cancer cell lines. Breast cancer MCF7 cells were rapidly resuscitated under a 37 °C water bath condition, and then transferred to a special MCF7 medium (Wuhan Punosai, product number CM-0149), and passaged in a cell culture incubator at 37 °C and 5% CO2 until sufficient cell numbers were obtained. One day before inoculating cells in the fat pad, a slow-release estrogen implant for mice (0.36 mg / tablet / mouse) was subcutaneously inoculated. Injection was carried out according to the following parameters: 5 × 10 6 cells / mouse, the injection volume was 100 μl, and the injection site was the right inguinal mammary fat pad (the 4th mammary gland) of the mouse. After the tumor grew to 90 - 110 mm 3 , mice with basically the same tumor size were selected for intratumoral administration treatment. The formula for calculating the drug dose of the experimental group (intratumoral injection of polypeptide Seq ID NO.1) was m (mg) = 5.3 × 150 × V × M × 10 -9 , where V is the arithmetic mean of the tumor volumes of the mice in each group (mm 3)、M is the molecular weight of the polypeptide (Da). The positive control group was injected with doxorubicin intratumorally at a dose of 5 mg / kg. The negative control group was injected with PBS buffer intratumorally. The drug injection volume was 70 μl, and the drug was administered three times at an interval of three days each time. The dosage for each administration was the initial dosage. The experiment ended on the 7th day after the third administration. The tumor growth of the mice was observed, and the number of tumor-free and tumor-bearing mice in each group was dynamically observed. The tumor diameter was measured with a vernier caliper. The formula for calculating the tumor volume (TV) is: TV = 0.5 × a × b × b, where a and b represent the long diameter and short diameter of the tumor mass, respectively. The formula for calculating the ratio of tumor-free mice (TF) is: TF = (number of mice without tumors / total number of mice) × 100%. As can be seen from the experimental results, as Figure 13 shown, by the end of the experiment, the TF of the mice in the experimental group of SeqID NO.1 was 66.7%, indicating that after intratumoral injection of the polypeptide Seq ID NO.1, it could kill tumor cells and inhibit tumor growth. The specific data are shown in Table 12.

[0387] Table 12. Tumor-free data of intratumoral administration treatment for orthotopic tumors

[0388] Tumor-free Experimental Group Positive Control Group Negative Control Group Day 1 0.0％ 0.0％ 0.0％ Day 5 16.7％ 33.3％ 0.0％ Day 9 33.3％ 80.0％ 20.0％ Day 12 50％ 100.0％ 40.0％ Day 15 66.7％ 100.0％ 60.0％

[0389] Example 6: Preparation of micellar injection solution

[0390] To prepare the micellar injection solution of polypeptide Seq ID NO.1, the steps are as follows:

[0391] Step (1): Preparation of volatile organic solvent mixture. Absolute ethanol and chloroform are mixed evenly at a volume ratio of 1:2.

[0392] Step (2): Preparation of MPLA mother liquor. Dissolve MPLA with the organic solvent mixture obtained in step (1).

[0393] Step (3): Preparation of PEG-DSPE mother liquor. Dissolve PEG-DSPE with the volatile organic solvent chloroform.

[0394] Step (4): According to a molar ratio of 100:3, take the MPLA mother liquor obtained in step (3) and the PEG-DSPE mother liquor obtained in step (2) into a rotary evaporation flask, and gently shake to mix evenly.

[0395] Step (5): Remove all organic solvents from the mixed solution obtained in step (4) so that the carrier molecules (PEG-DSPE) and adjuvant molecules (MPLA) form a uniformly distributed mixed lipid membrane. Remove the organic solvents using a rotary evaporator under water bath heating conditions (water bath temperature: 40°C, condenser water temperature: -2°C, rotation speed: 32 rpm / min, rotary evaporation time: 3 - 5 min). After film formation, place it in a vacuum drying oven overnight to completely remove the residual organic solvents (temperature setting: 40°C, vacuum degree: 0.1 MPa) to obtain a lipid membrane;

[0396] Step (6): Prepare a physiological saline solution of polypeptide Seq ID NO.1. According to the molar ratio of PEG-DSPE:polypeptide Seq ID NO.1 = 100:4, dissolve polypeptide Seq ID NO.1 in physiological saline to prepare a 0.149 mg / ml solution;

[0397] Step (7): Hydrate the lipid membrane. Take the polypeptide Seq ID NO.1 physiological saline solution obtained in step (6) and hydrate the lipid membrane obtained in step 5 at 45°C for 30 min, then let it stand at room temperature for 2 h to form a micellar injection solution of polypeptide Seq ID NO.1 that is uniform, colorless, and transparent in appearance.

[0398] Step (8): Take the micellar injection solution of polypeptide Seq ID NO.1 in step (7), filter and sterilize it through a 0.22 μm filter membrane, and store it at 4°C for later use (it can be stored for two weeks).

[0399] The preparation method of the micellar injection solutions of polypeptide Seq ID No.5 to polypeptide Seq ID No.7 is the same as that of polypeptide Seq ID No.1.

[0400] Example 7: Preparation of freeze-dried micellar injection

[0401] Prepare the freeze-dried powder of the micellar injection of polypeptide Seq ID NO.1. The steps are as follows:

[0402] Prepare the micellar injection solution of polypeptide Seq ID NO.1 according to steps (1) to (7) of Example 6. Take the solution after standing at room temperature for 2 h in step (7), accurately weigh mannitol and add it to the sample to make the final concentration of mannitol 0.5 mg / ml. After complete dissolution, filter and sterilize it through a 0.22 μm filter membrane, and dispense it into 2 ml sterile vials at 1 ml per vial, and place it at -80°C for pre-freezing overnight. Vacuum freeze-dry the pre-frozen sample for 36 h (vacuum degree 0.1 mbar) to obtain a freeze-dried preparation.

[0403] The preparation method of the freeze-dried powders of the micellar injections of polypeptide Seq ID No.5 to polypeptide Seq ID No.7 is the same as that of polypeptide Seq ID No.1.

[0404] Example 8: Toxicity of Polypeptides Seq ID NO.5 to Seq ID NO.7 to MCF7 Tumor Cells

[0405] Using MCF7 tumor cells as model cells for studying the anti-tumor polypeptides against breast cancer cell lines. Take cells in the logarithmic growth phase, digest them with trypsin, count the cells, and dilute the cells with MEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to 5×10 4 cells / mL medium. Add the cell suspension to a 96-well cell culture plate, 100 μL / well, that is, each well contains 5000 cells. Place the 96-well cell plate in a carbon dioxide incubator and culture overnight at 37 °C and 5% CO2 to allow the cells to adhere. The next day, discard the supernatant. In the sample group, add 90 μL of cell complete medium and 10 μL of polypeptide solutions with different concentrations of Seq ID NO.5 to Seq ID NO.7 to each well, so that the final concentration of the polypeptide is 60 μM, 20 μM, 6.7 μM, 2.2 μM, 0.7 μM, and 0.2 μM. The blank control group and the background control group add 90 μL of cell complete medium and 10 μL of sterilized water. After culturing the 96-well cell plate in a carbon dioxide incubator at 37 °C and 5% CO2 for 24 h, add 10 μL of CCK8 solution to each well. After incubating in the cell incubator for 1 - 4 h, measure the absorbance (OD value) at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the cell mortality rate (Cell mortality rate (%) = (1 - (OD value of the polypeptide treatment group - OD value of the background control group) / (OD value of the blank control group - OD value of the background control group)) × 100%). The experimental results show that, as 450 shown, compared with MCF7 cells without polypeptide treatment, after adding polypeptides with different concentrations to MCF7 cells and incubating, with the increase of the polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of the breast cancer cell line MCF7, and its half lethal concentration (IC 450 ) are 4.3 μM, 2.8 μM, and 3.5 μM respectively. Compared with polypeptide Seq ID NO.1 (IC 450 is 3.6 μM, data from Experiment 3 in Example 2), the IC 450 of polypeptides Seq ID NO.5 to Seq ID NO.7 has increased, indicating that after amino acid substitution, deletion, and addition of polypeptide Seq ID NO.1, its toxicity to MCF7 tumor cells has increased. The specific data are shown in Table 13. Figure 14 shown, compared with MCF7 cells without polypeptide treatment, after adding polypeptides with different concentrations to MCF7 cells and incubating, with the increase of the polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of the breast cancer cell line MCF7, and its half lethal concentration (IC 50 ) are 4.3 μM, 2.8 μM, and 3.5 μM respectively. Compared with polypeptide Seq ID NO.1 (IC 50 is 3.6 μM, data from Experiment 3 in Example 2), the IC 50 of polypeptides Seq ID NO.5 to Seq ID NO.7 has increased, indicating that after amino acid substitution, deletion, and addition of polypeptide Seq ID NO.1, its toxicity to MCF7 tumor cells has increased. The specific data are shown in Table 13.

[0406] Table 13. Cytotoxicity of Polypeptides SEQ ID NO.5 to SEQ ID NO.7 to MCF7

[0407] Concentration / Cell Death Rate SEQ ID NO.5 SEQ ID NO.6 SEQ ID NO.7 60 μM 94.6％ 99.5％ 98.8％ 20 μM 92.6％ 91.2％ 93.6％ 6.7 μM 69.7％ 78.5％ 77.3％ 2.2 μM 42.6％ 51.2％ 48.5％ 0.7 μM 22.7％ 21.5％ 25.4％ 0.2 μM 23.6％ 18.5％ 23.9％ IC50 / μM 4.3 2.8 3.5

[0408] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide has: (Ⅰ) an amino acid sequence as shown in SEQ ID No.1; or (Ⅱ) a sequence in which one or more amino acids are substituted, deleted, added, replaced and / or modified on the basis of the amino acid sequence as shown in (Ⅰ); or (Ⅲ) an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to the amino acid sequence as shown in (Ⅰ); (IV) an amino acid sequence having the same or similar function as the amino acid sequence as shown in (Ⅰ).

2. The polypeptide according to claim 1, wherein The plurality of amino acids includes but is not limited to 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

3. The polypeptide according to claim 1 or 2, characterized in that The modification includes but is not limited to one or more of amidation, phosphorylation, methylation, acetylation, ubiquitination, glycosylation, PEG (polyethylene glycol) group modification, PEG dithiol acid group modification or maleimide group modification.

4. The polypeptide according to any one of claims 1 to 3, characterized in that, The polypeptide has an amino acid sequence as shown in any one of SEQ ID No.5 to 7.

5. The preparation method of the polypeptide according to any one of claims 1 to 4, characterized in that, It is prepared by chemical synthesis or biotechnological methods.

6. A polypeptide prepared by the preparation method according to claim 5.

7. A nucleic acid molecule encoding the polypeptide according to claim 1 or 6, characterized in that, The nucleic acid molecule has: (I) a nucleotide sequence as shown in SEQ ID No.2; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) a nucleotide sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to the nucleotide sequence described in any one of (I) to (III).

8. The nucleic acid molecule according to claim 7, wherein The plurality of nucleotides includes but is not limited to 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 or 40 nucleotides.

9. A genetic element, characterized in that, It includes a sumo protease cleavage site and the nucleic acid molecule according to claim 7 or 8.

10. The gene element according to claim 9, characterized in that, The gene element has: (I) a nucleotide sequence as shown in SEQ ID No.3; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) A nucleotide sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to the nucleotide sequence described in any one of (I) to (III).

11. The gene element according to claim 10, wherein The multiple nucleotides include, but are not limited to, 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 or 40 nucleotides.

12. Recombinant vector, characterized in that, (I) The nucleic acid molecule according to claim 7 or 8; or (II) The gene element according to any one of claims 9 to 11. (I) The nucleotide sequence shown in SEQ ID No. 4; or 13. The recombinant vector according to claim 12, characterized in that, (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) A nucleotide sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to the nucleotide sequence described in any one of (I) to (III). (III) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or (IV) A nucleotide sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to the nucleotide sequence described in any one of (I) to (III).

14. The recombinant vector according to claim 13, wherein, The multiple nucleotides include, but are not limited to, 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 or 40 nucleotides.

15. The recombinant vector according to any one of claims 12 to 14, characterized in that, (I) Integrating the nucleic acid molecule according to claim 7 or 8; or 16. A host, characterized in that, (II) Integrating the gene element according to any one of claims 9 to 11; or (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15.

17. The host according to claim 16, characterized in that, (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15.

18. Recombinant polypeptide, characterized in that, (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15.

19. The method for preparing the recombinant polypeptide according to claim 18, characterized in that, (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. (III) Transfecting or transforming the recombinant vector according to any one of claims 12 to 15. Step 2. Expression and harvest of the fusion protein: Inoculate the genetically engineered strain described in step (1) into LB medium for culture, add an inducer to induce the expression of the fusion protein, collect the bacterial cells, suspend them in a buffer, disrupt the cells by ultrasonic wave and centrifuge, and harvest the supernatant containing the recombinant fusion protein. Step 3. Isolation and purification of the target polypeptide: Purify and isolate the supernatant containing the recombinant fusion protein obtained in step (2) to obtain the recombinant polypeptide.

20. The preparation method according to claim 19, characterized in that, The purification includes one or more of affinity chromatography, desalting, enzymatic cleavage or high performance liquid chromatography.

21. The preparation method according to claim 20, characterized in that, The affinity chromatography is nickel column affinity chromatography.

22. The preparation method according to claim 20, characterized in that, The protease selected for the enzymatic cleavage is sumo enzyme, and the enzymatic cleavage condition is that the ratio of sumo enzyme to the recombinant fusion protein is (50 - 200 U): 1 mg, and it is allowed to stand at 4 - 25 °C for 4 - 24 h. The preferred condition is that the ratio of sumo enzyme to the recombinant fusion protein is 200 U: 1 mg, and it is allowed to stand at 4 °C for 18 h.

23. The recombinant polypeptide prepared by the preparation method according to any one of claims 19 to 22.

24. Use of any of the following in the preparation of a product for preventing, improving, adjuvantly treating and / or treating tumors; (I) The polypeptide according to any one of claims 1 to 4; or (II) The polypeptide according to claim 6; or (III) The nucleic acid molecule according to claim 7 or 8; or (IV) The gene element according to any one of claims 9 to 11; or (V) The recombinant vector according to any one of claims 12 to 15; or (VI) The host according to claim 16 or 17; or (VII) The recombinant polypeptide according to claim 18 or 23.

25. The application according to claim 24, wherein The product includes but is not limited to vaccines, drugs and / or drug combinations.

26. The application according to claim 24 or 25, characterized in that, The tumor is selected from one or more of the following: lung cancer, breast cancer, cervical cancer, glioma, endometrial cancer, melanoma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer or colorectal cancer; preferably breast cancer.

27. A vaccine, characterized in that, Comprising any of the following and an acceptable immunoadjuvant or excipient: (I) The polypeptide according to any one of claims 1 to 4; or (II) The polypeptide according to claim 6; or (III) The nucleic acid molecule according to claim 7 or 8; or (IV) The gene element according to any one of claims 9 to 11; or (V) The recombinant vector according to any one of claims 12 to 15; or (VI) The host according to claim 16 or 17; or (VII) The recombinant polypeptide according to claim 18 or 23.

28. The vaccine according to claim 27, wherein, The immunoadjuvant includes one or more of Freund's adjuvant, aluminum hydroxide adjuvant, monophosphoryl lipid A adjuvant, preferably monophosphoryl lipid A adjuvant.

29. Use of the vaccine according to claim 27 or 28 in the prevention of tumors.

30. The application according to claim 29, wherein The tumor is selected from one or more of the following: lung cancer, breast cancer, cervical cancer, glioma, endometrial cancer, melanoma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer or colorectal cancer; preferably breast cancer.

31. A method for preventing tumors, characterized in that, Administer the vaccine according to claim 27 or 28.

32. The method according to claim 31, wherein The tumor is selected from one or more of the following: lung cancer, breast cancer, cervical cancer, glioma, endometrial cancer, melanoma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer or colorectal cancer; preferably breast cancer.

33. A drug, characterized in that, Comprising any of the following and a pharmaceutically acceptable carrier molecule, immunoadjuvant and / or pharmaceutically acceptable excipient: (I), the polypeptide according to any one of claims 1 to 4; or (II), the polypeptide according to claim 6; or (III), the nucleic acid molecule according to claim 7 or 8; or (IV), the genetic element according to any one of claims 9 to 11; or (V), the recombinant vector according to any one of claims 12 to 15; or (VI), the host according to claim 16 or 17; or (VII), the recombinant polypeptide according to claim 18 or 23.

34. The drug according to claim 33, wherein, The carrier molecule includes but is not limited to one or more of nanomaterials, liposomes, polyethylene glycol modification or oily compounds, or a mixture composed of various oily compounds; preferably pegylated phosphatidylethanolamine; or The immunoadjuvant includes one or more of Freund's adjuvant, aluminum hydroxide adjuvant or monophosphoryl lipid A adjuvant; preferably monophosphoryl lipid A adjuvant.

35. The medicament according to claim 33 or 34, characterized in that, The dosage form of the drug includes but is not limited to injections.

36. The drug according to claim 35, characterized in that, The injection includes one or more of micelle injections, liposome injections or nanomaterial injections, preferably micelle injections.

37. The medicament according to claim 35 or 36, characterized in that, The injection is a freeze-dried powder, which is freeze-dried after adding a freeze-drying protectant to obtain a freeze-dried powder; preferably the freeze-drying protectant is mannitol at 0.05 g / ml.

38. The medicament according to any one of claims 33 to 37, characterized in that, The molar ratio of the recombinant polypeptide, the carrier molecule and the immunoadjuvant according to claim 18 or 23 is (4 to 160):720:(3 to 80); Preferably, the molar ratio of the recombinant polypeptide, the carrier molecule PEG-DSPE and the immunoadjuvant MPLA according to claim 18 or 23 is 4:100:

3.

39. A pharmaceutical combination, characterized in that, Comprising the drug according to any one of claims 33 to 38 and any other active ingredient.

40. Use of the drug according to any one of claims 33 to 38 or the drug combination according to claim 39 in adjuvant treatment and / or treatment of tumors.

41. The application according to claim 40, characterized in that, The tumor is selected from one or more of the following: lung cancer, breast cancer, cervical cancer, glioma, endometrial cancer, melanoma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer or colorectal cancer; preferably breast cancer.

42. A method for adjuvant treatment and / or treating tumors, characterized in that, Administer any of the following: (I), the drug according to any one of claims 33 to 38; or (II), the drug combination according to claim 39.

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