Polypeptide capable of causing immunogenic death of tumor cells and killing tumor cells in broad spectrum 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 LCX18 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 LCX18, a drug delivery system and application of the polypeptide molecule LCX18. 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 biomedicine technology, and particularly relates to a polypeptide that can cause immunogenic death of tumor cells and kill tumor cells in a broad spectrum, and a preparation method, a drug delivery system and an application 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 the cancer burden will increase by 47% in 2040 compared with 2020, 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 advanced stage of treatment, patients often experience 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 and immune cross-reactions. In addition, due to high R & D costs, large 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 infusion for drug 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 play their roles, are bioactive substances involved in various cell functions in organisms, and participate in regulating various physiological functions, so they have very important development value in clinical applications.

[0005] As a new strategy for cancer 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 small 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] To solve the above problems, 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 genetic engineering method with the sumo enzyme cleavage site sequence and the polypeptide, so that the polypeptide is soluble expressed, 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] 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) an amino acid sequence as shown in SEQ ID No.1; or

[0011] (Ⅱ) a sequence obtained by substituting, deleting, adding, replacing, and / or modifying one or more amino acids 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 with 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 present invention, the plurality of 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 present 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 dithiol acid group modification, or maleimide group modification.

[0016] In some specific embodiments of the present 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 the polypeptide, which is prepared by chemical synthesis or bioengineering methods.

[0018] In a third aspect, the present invention provides the polypeptide prepared by the 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) a nucleotide sequence as 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 1 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 present 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.

[0025] Fifth aspect, the present invention provides a gene element, which comprises a SUMO protease cleavage site and the nucleic acid molecule described above.

[0026] In some specific embodiments of the present 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 present 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.

[0032] Sixth aspect, the present 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 present 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 a nucleotide sequence 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 present 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 present 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 the 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 present invention, the host includes, but is not limited to, prokaryotes or eukaryotes; the prokaryotes include, but are not limited to, Escherichia coli.

[0047] In the 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 the ninth aspect, the present invention provides a method for preparing the recombinant polypeptide, comprising the following steps:[[]]

[0052] Step 1, construction of a genetically engineered strain: constructing 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, disrupt the cells by ultrasonic wave and centrifuge, and harvest the supernatant containing the recombinant fusion protein.

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

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

[0056] In some specific embodiments of the present 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 present invention, the affinity chromatography is nickel column affinity chromatography.

[0058] In some specific embodiments of the present 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 allowed to stand 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 allowed to stand at 4 °C for 18 h.

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

[0060] In the eleventh aspect, the present invention provides the use of any one of the following in the preparation of products for preventing, improving, adjuvant 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 described above; or

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

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

[0066] (VI), the host described above; or

[0067] (VII), the recombinant polypeptide described above.

[0068] In some specific embodiments of the present invention, the product includes, but is not limited to, vaccines, drugs, and / or drug combinations.

[0069] In some specific embodiments of the present 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 twelfth 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.

[0078] In some specific embodiments of the present 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 a thirteenth aspect, the present invention provides the use of the vaccine described above in the prevention of tumors.

[0080] In some specific embodiments of the present 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 fourteenth aspect, the present invention provides a method for preventing tumors, by inoculating the vaccine as claimed in claim 27 or 28.

[0082] In some specific embodiments of the present 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 fifteenth 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 described above; 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.

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

[0092] The immunoadjuvant 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 present invention, the dosage form of the drug includes, but is not limited to, injections.

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

[0095] In some specific embodiments of the present 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 present invention, the molar ratio of the recombinant polypeptide, the vector molecule, and the immunoadjuvant is (4 - 160):720:(3 - 80);

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

[0098] In the sixteenth aspect, the present invention provides a drug combination, including the drug described above and any other active ingredient.

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

[0100] In some specific embodiments of the present 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 an eighteenth aspect, the present invention provides a method for adjuvant treatment and / or the treatment of tumors, comprising administering any one of the following:

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

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

[0104] According to the production method of the present invention, the obtained recombinant anti-tumor polypeptide 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 by translating its cDNA.

[0107] 3), the prepared anti-tumor polypeptide has high anti-tumor activity both in vivo and in vitro and the ability to induce immunogenic cell death of tumor cells. 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 Shows the cytotoxicity test results of the polypeptide Seq ID NO.1 of the present invention against breast cancer 4T1 cells;

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

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

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

[0113] Figure 5Show the toxicity test results of the polypeptide Seq ID NO.1 of the present invention against cervical cancer HeLa cells;

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

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

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

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

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

[0119] Figure 11 Show the toxicity test results of the polypeptide Seq ID NO.1 of the present invention against uterine cancer HEC - 1 - B cells;

[0120] Figure 12 Show the morphological observation results of the polypeptide Seq ID NO.1 of the present invention against breast cancer 4T1 cells;

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

[0122] Figure 14 Show the tumor - free results of the polypeptide Seq ID NO.1 of the present invention in the mouse intratumoral administration treatment model;

[0123] Figure 15 Show the toxicity 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 mode

[0124] The present invention discloses a polypeptide SEQ ID NO.1 that can induce immunogenic death of tumor cells, its preparation method, pharmaceutical preparation and application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. 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 relevant personnel can obviously make changes or appropriate modifications 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.

[0125] The object of the present invention is to design a polypeptide with broad-spectrum anti-tumor immune activity.

[0126] The object of the present invention is to design a polypeptide that can induce immunogenic death of tumor cells.

[0127] The object of the present invention is to design a recombinant plasmid that can efficiently express anti-tumor polypeptides for the expression of anti-tumor polypeptides.

[0128] The object of the present invention is to provide a recombinant Escherichia coli that can express bioactive anti-tumor polypeptides solubly and in high yield.

[0129] The object of the present invention is to provide a method for expressing and purifying anti-tumor polypeptides. This method has high safety, low production cost, simple polypeptide purification steps, high product purity and the obtained polypeptides have biological activity.

[0130] The object of the invention is to provide a drug delivery system for the treatment of solid tumors with anti-tumor immune polypeptides as the active ingredient.

[0131] The object of the present invention is the application of anti-tumor immune polypeptides in the treatment, adjuvant treatment and / or prevention of cancer.

[0132] In order to achieve the above object, the present invention elaborates on the production method, drug delivery system and application of anti-tumor polypeptides, including polypeptide genetic engineering expression and purification (including codon optimization, construction of recombinant expression plasmids, construction of recombinant genetic engineering bacteria, expression of fusion proteins, purification of anti-tumor polypeptides), in vitro anti-tumor activity detection, microscopic cell morphology observation, animal model tumor prevention experiments, solid tumor treatment animal model experiments, preparation of micellar injections, etc.

[0133] The features of the present invention are as follows: According to the codon preference of the host expression bacterium, the gene sequence of the anti-tumor polypeptide Seq ID NO.1 is optimized. After ligating the target gene sequence 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 the active recombinant anti-tumor polypeptide. 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 both in vivo and in vitro experiments. The steps of the present invention are as follows:

[0134] 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, hereinafter referred to as LCX18-DNA-seq, is obtained. The DNA sequence is shown as SEQ ID NO.2;

[0135] 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, the optimized DNA sequence, hereinafter referred to as sumo-DNA-seq, is obtained. The DNA sequence is shown as SEQ ID NO.3;

[0136] 3) According to the types of restriction enzymes in the multiple cloning site of the expression vector pET-32a(+), the restriction enzymes BamHⅠ(GGATCC) and XhoⅠ(CTCGAG) are selected. In the order of GGATCC+sumo-DNA-seq+LCX18-DNA-seq+TAA+CTCGAG, the entire DNA sequence is ligated with the expression vector pET-32a(+) to obtain the recombinant expression vector pET-32a(+)-LCX18. The DNA sequence is hereinafter referred to as Sumo-LCX18-DNA-seq, and the DNA sequence is shown as SEQ ID NO.4.

[0137] 4) The recombinant expression vector pET-32a(+)-LCX18 is transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21(DE3), hereinafter referred to as BL21-LCX18.

[0138] 5) IPTG is used to induce the recombinant Escherichia coli BL21-LCX18 to express the fusion protein of the anti-tumor polypeptide with a tag.

[0139] 6) The fusion protein is purified by nickel column affinity chromatography, and the fusion protein is digested with sumo protease to separate the tag protein and the anti-tumor polypeptide.

[0140] 7) The digested products are processed by purification methods such as nickel column affinity chromatography, ion exchange, and high performance liquid chromatography to obtain anti-tumor polypeptides with relatively high purity.

[0141] 8) The purified anti-tumor polypeptides are aliquoted according to certain specifications and then freeze-dried and stored at -80 °C.

[0142] 9) The freeze-dried polypeptides are taken for cytological experiments and animal experiments to detect the anti-tumor activities of the polypeptides.

[0143] Glossary of Terms

[0144] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0145] The term "optionally" is used for descriptive purposes only and should not be construed as indicating or implying relative importance. Thus, features defined with "optionally" may explicitly or implicitly include or exclude such feature.

[0146] 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 analogs.

[0147] 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) according to the instructions provided by the manufacturer.

[0148] Alternatively, the polypeptide may be generated by recombinant means or by cleavage from a longer polypeptide. For example, the polypeptide may be obtained by cleavage from a thyrotropin receptor protein, which may then be modified at one or both termini. The composition of the polypeptide may be confirmed by amino acid analysis or sequencing (e.g., Edman degradation procedure).

[0149] For practical purposes, the polypeptide may exhibit various other properties. For example, it is important that the peptide is stable enough in vivo to be therapeutically useful. The in vivo half-life of the peptide may be at least 10 minutes, 30 minutes, 4 hours, or 24 hours.

[0150] The polypeptide may also exhibit good in vivo bioavailability. The peptide may maintain its conformation in vivo, enabling it to bind to targets on the cell surface without excessive hindrance.

[0151] In addition, the polypeptides of the present invention also include functionally equivalent derivatives, variants, or fragments thereof.

[0152] "Functionally equivalent" derivatives, variants, or fragments thereof refer to peptides related to or derived from the polypeptides of the present invention, in which the amino acid sequence has been modified, e.g., 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.

[0153] Within the meaning of "addition", it includes amino- and / or carboxy-terminal fusion proteins or variants of polypeptides that contain additional proteins or polypeptides fused to the peptide sequence.

[0154] As described above, the peptide may preferably be replaced at the N- or C-terminus by another moiety. These moieties may be added to assist in the function of the peptide, its targeting, or its synthesis, capture, or identification, e.g., tags (e.g., biotin) or lipid molecules. Alternatively, such moieties may be found within the peptide itself. For example, a moiety such as a tag may be attached to an amino acid located within the peptide.

[0155] "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may 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.

[0156] Suitably, "derivatives" or "variants" include those in which the amino acids that occur in the sequence in place of the naturally occurring amino acids are structural analogues thereof. The amino acids used in the sequence may also be derivatized or modified, for example, labeled, provided that the function of the peptide is not significantly adversely affected.

[0157] Derivatives and variants as described above can be prepared during peptide synthesis or by post-production modification, or when the peptide is in recombinant form, using known techniques of site-directed mutagenesis, random mutagenesis or enzymatic cleavage and / or nucleic acid ligation.

[0158] 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).

[0159] 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.

[0160] With respect to amino acid sequences, "sequence identity" means a sequence having the stated value when evaluated using ClustalW (Thompson et al., 1994, supra), which ClustalW has the following parameters:

[0161] Pairwise alignment parameters - Method: exact, Matrix: PAM, Gap opening penalty: 10.00, Gap extension penalty: 0.10;

[0162] Multiple alignment parameters - Matrix: PAM, Gap opening penalty: 10.00, Delay divergent percentage: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue specific gap penalty: on, Hydrophilic gap penalty: on, Hydrophilic residues: GPSNDQEKR. Sequence identity of specific residues is intended to include simply derivatized identical residues.

[0163] The peptides of the present 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.

[0164] 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 groups. 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.

[0165] Chemically modified peptides also include cyclic peptides, i.e., the peptides of the present invention, which are covalently linked 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 covalently linked 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, the carboxyl terminus, or a side chain can be covalently linked to the peptide backbone to form a cyclic peptide.

[0166] 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.

[0167] The peptides of the present invention can carry display labels. Suitable labels include radioisotopes, fluorescent labels, enzyme labels, or other protein labels such as biotin.

[0168] Any formula given herein is also intended to represent both unlabeled and isotopically labeled forms of a peptide. The isotopically labeled peptide has 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 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, respectively. The invention includes various isotopically labeled peptides as defined herein, such as those in which radioactive isotopes 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 and prodrugs thereof of the invention can generally be prepared by carrying out the protocols or methods disclosed in the Examples and Preparations described below, by substituting non-isotopically labeled reagents with readily available isotopically labeled reagents.

[0169] The isotopically labeled peptides of the invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described herein, using appropriate isotopically labeled reagents in place of the previously used unlabeled reagents.

[0170] Pharmaceutically acceptable solvates according to the invention include those in which the crystallization solvent can be replaced by an isotope, such as D2O, d6-acetone, d6-DMSO.

[0171] The above-mentioned peptides used according to the invention can be prepared by conventional synthetic means including genetic or chemical means.

[0172] 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.

[0173] Synthesis can be carried out in solution or on a solid support using suitable solid phases known in the art.

[0174] 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, such as 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 polypeptides can be used to prepare the more pure forms used in pharmaceutical compositions; such less pure preparations of the compounds should contain at least 1%, more suitably at least 5%, preferably 10 - 59% of the polypeptides provided by the present invention.

[0175] 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.

[0176] The present invention also includes expression vectors containing such polynucleotide sequences. Such expression vectors are generally 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.).

[0177] Thus, the peptides can be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Suitably, the polynucleotides of the present invention or the polynucleotides used in the vectors for the present invention are operably linked to a control sequence capable of providing for the expression of the coding sequence in a host cell, i.e., the vector is an expression vector. The term "operably linked" means juxtaposition wherein the components described are in a relationship that permits them to function in their intended manner. A regulatory sequence (such as a promoter) that is "operably linked" to a coding sequence is positioned in such a way that the expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

[0178] The vector can be, for example, a plasmid, virus or phage vector provided with an origin of replication, optionally a promoter for expressing 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 producing DNA or RNA or for transfecting or transforming host cells, such as mammalian host cells. The vector can also be suitable for in vivo use, for example allowing in vivo expression of a polypeptide.

[0179] 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.

[0180] The invention also includes cells that have been modified to express the peptides of the 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 peptide of the invention include mammalian HEK293T, CHO, HeLa and COS cells. Suitably, the cell line selected should be a cell line that is not only stable but also allows for the mature glycosylation and cell surface expression of the polypeptide. 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 invention are included within the scope of the invention. The peptides of the invention can also be expressed in Xenopus laevis oocytes or melanocytes.

[0181] The present invention also extends to antibodies (monoclonal or polyclonal) against the peptides as defined above and antigen-binding fragments thereof (e.g., F(ab)2, Fab, and Fv fragments, i.e., fragments of the "variable" regions of antibodies that contain the antigen-binding sites), 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.

[0182] The present invention also relates to compositions, such as pharmaceutical compositions comprising the polypeptides or recombinant polypeptides according to the present invention.

[0183] The peptides may comprise multiple peptides, for example, two, three, four, five, or more than six polypeptides.

[0184] The compositions of the present invention can be used for prophylactic or therapeutic purposes.

[0185] 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 number of tumor or cancer cells, such as a 60%, 70%, 80%, 90%, 95%, or 99% reduction in the number of tumor or cancer cells that have been observed when the patient has not been treated 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.

[0186] 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 treatment with the polypeptide or compared to the level observed at the same time point when treatment has not been given.

[0187] 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.

[0188] Alternatively (or additionally), if the pharmaceutical composition (or any part thereof) is administered in multiple doses, each dose can be separately packaged.

[0189] Similarly, in the pharmaceutical compositions of the present invention, each polypeptide can be mixed with any suitable one or more binders, lubricants, suspending agents, coating agents, or solubilizing agents.

[0190] The compositions can be administered together in the form of a mixed composition or mixture. However, there may be cases where it is preferred to provide the polypeptides separately in the form of a kit for simultaneous, separate, sequential, or combined administration.

[0191] The kit may also include 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 include instructions for use.

[0192] The pharmaceutical composition or kit according to the present invention can be used for treating and / or preventing diseases.

[0193] The polypeptide of the present invention, further including pharmaceutically acceptable salts, can also be obtained in the form of its hydrates, or include other solvents for its crystallization.

[0194] As used herein, the term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the present invention and are generally not biologically or otherwise undesirable. Due to the presence of amino and / or carboxyl groups or groups similar thereto, the peptides of the present invention are capable of forming acid and / or base salts.

[0195] Pharmaceutically acceptable acid addition salts can be formed using inorganic acids 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 / phosphates monobasic / dibasic, polygalacturonates, propionates, stearates, succinates, sulfosalicyclates, tartrates, toluenesulfonates, trifluoroacetates, and trifluoromethanesulfonates.

[0196] 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, trifluoromethanesulfonic acid, sulfosalicylic acid, etc.

[0197] Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.

[0198] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns 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.

[0199] 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, and the like. Some organic amines include isopropylamine, benzathine penicillin, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0200] 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-crystallizing agents. Such co-crystals can be prepared by known co-crystallization procedures for peptides. Such procedures include grinding, heating, co-sublimation, co-melting, or contacting the peptide with the co-crystallizing agent in solution and separating the co-crystals thus formed under crystallization conditions. Suitable co-crystallizing agents include those described in WO 2004 / 078163.

[0201] One of ordinary skill in the art will understand that when basic and acidic groups are present, the peptides of the present invention may also form internal salts, such as zwitterions.

[0202] 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.

[0203] As used herein, "abnormal cell division" refers to cell division above the normal level (i.e., abnormal cell division) that is considered appropriate in the conditions in which it occurs. Markers of abnormal cell division are well known to those of ordinary skill in the art and can be used to determine whether a particular cell has been affected. For example, cells undergoing abnormal cell division may display atypical cytology, such as cell pleomorphism, nuclear pleomorphism, hyperchromasia, or an increased nuclear-cytoplasmic ratio. Cells undergoing abnormal cell division may display a failure of cell differentiation. More particularly, such abnormal cell division can be present in certain conditions or diseases / conditions as described below, such as cancer.

[0204] "Reducing" cell division refers to a decrease in the rate of cell growth. Suitably, in 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 otherwise occur, or as an alternative to the decrease in cell growth. When cell death occurs, suitably, more than 50% of the existing cells, particularly more than 75% of the cells, are destroyed.

[0205] By adjusting the dose of the active agent used, it may also be possible to completely eliminate some malignancies. 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.

[0206] The treatment according to the present invention can be symptomatic or prophylactic.

[0207] Thus, in another aspect, the present invention includes the active agent of the present invention for use as a medicament.

[0208] Thus, according to another aspect, the present invention provides the active agent of the present invention for treating or preventing a condition or disorder in which abnormal cell division occurs.

[0209] Thus, according to another aspect, the present invention provides the use of the active agent of the present invention in the preparation of a medicament for preventing or treating a condition or disorder in which abnormal cell division occurs.

[0210] Thus, according to another aspect, the present invention provides a method for preventing or treating a condition or disorder in which abnormal cell division occurs, which comprises administering a therapeutically effective amount of the active agent of the present invention to a subject in need thereof.

[0211] In accordance with the foregoing, as another aspect, the present invention further provides a method for preventing or treating a condition or disorder in which abnormal cell division occurs, particularly cancer, which comprises administering a therapeutically effective amount of the active agent of the present invention to a subject in need thereof, particularly a human subject.

[0212] On the other hand, the present invention provides the active agent of the present invention for preventing or treating a condition or disorder in which abnormal cell division occurs, particularly cancer.

[0213] On the other hand, the present invention provides the use of the active agent of the present invention in the preparation of a medicament for preventing or treating a condition or disorder in which abnormal cell division occurs, particularly cancer.

[0214] As mentioned herein, a "disorder" or "disease" refers to a potential pathological disorder of a symptomatic or asymptomatic organism relative to a normal organism, which may be caused, for example, by infection or acquired or congenital genetic defects.

[0215] A "condition" refers to the mental or physical state of an organism that has not developed a disease, for example, the presence of a substance such as a toxin, drug or pollutant in the body.

[0216] As used herein, the term "treat (verb)", "treating (gerund)", or "treatment (noun)" for any disease / condition means, in one embodiment, ameliorating a disease or disorder (i.e., slowing or arresting 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)" means alleviating or ameliorating at least one physical parameter, including those that may not be discernible to the patient. In another embodiment, "treat (verb)", "treating (gerund)", or "treatment (noun)" means modulating the disease or disorder 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)" means preventing or delaying the onset or development or progression of a disease or disorder. For example, symptoms that may be affected include tumor size or the number of cancer cells in a given sample.

[0217] "Preventing" a condition or disorder means delaying or preventing the onset of or reducing the severity of the condition or disorder as evaluated by the appearance or degree of one or more symptoms of the condition or disorder.

[0218] As used herein, the term "subject" refers to an animal. Generally, the animal is a mammal. A subject also refers to, for example, a primate (e.g., human), bovine, ovine, caprine, equine, canine, feline, rabbit, rat, mouse, fish, bird, etc. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0219] As used herein, a subject "needs" treatment if such subject would benefit biologically, medically, or in terms of quality of life from such treatment.

[0220] The term "therapeutically effective amount" of the active agent of the present invention means an 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 ameliorating symptoms, alleviating a condition, slowing or delaying disease progression, or preventing a disease, etc. In one non-limiting embodiment, the term "therapeutically effective amount" means that the active agent of the present invention is effective for at least partially alleviating, inhibiting, preventing, and / or ameliorating 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" means an amount of the active agent of the present invention that is effective for at least partially reducing abnormal cell division when administered to a cell or tissue or non-cellular biological material or medium.

[0221] As an alternative to performing the methods in vivo, these methods can be performed, for example, in vitro to reduce cell division in a sample or to eliminate cells undergoing abnormal cell growth. Suitable culture conditions are as described for other methods of the present invention below.

[0222] This is particularly useful in cell samples containing normal and abnormal cells, where the abnormal cells can be controlled / removed, as well as in 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, from a patient's blood sample, and the remaining cells can then be returned to the patient's body.

[0223] 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 (or preventing) a patient suffering from a disorder or condition 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.

[0224] In this case, "body fluids" particularly include blood, cerebrospinal fluid, and lymphatic fluid, and "tissues" include tissues obtained by surgery or other means.

[0225] Suitably, the abnormal cell division occurs in cells from eukaryotes, which can be any eukaryote such as humans, other mammals and animals, birds, insects, and fish.

[0226] Non-human animals from which cells can be obtained or in which the method of the present invention can be carried out 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 used for the treatment or prevention of humans.

[0227] In particular, the cells undergoing abnormal cell division are cancer cells, and the disorder 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.

[0228] 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.

[0229] The cancer is a malignant or pre-malignant or benign tumor and includes carcinoma, sarcoma, glioma, melanoma, and lymphoma, 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.

[0230] A condition or disorder characterized by abnormal cell division is cancer and includes, but is not limited to, mesothelioma, hepatobiliary duct (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 tract (stomach, colorectal cancer, and duodenum), gastrointestinal stromal tumor, 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) tumor, primary CNS lymphoma, non-Hodgkin's lymphoma, spinal axis tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

[0231] 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 cancer, renal pelvic cancer, thyroid cancer, parathyroid cancer, pancreatic cancer, prostate cancer, central nervous system (CNS) tumor, primary CNS lymphoma, non-Hodgkin's lymphoma, or spinal tumor, or a combination of one or more of the foregoing cancers.

[0232] 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 cancer.

[0233] 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 instead differentiate or undergo apoptosis.

[0234] As used herein, "sample" refers to any material obtained from a donor, e.g., 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.

[0235] "Body fluid" includes blood and cerebrospinal fluid.

[0236] "Tissue sample" includes tissues obtained by surgical intervention (e.g., bone marrow or liver) or by other means such as placenta and umbilical cord. The cells are preferably from the animal from which they are sourced or to which the method is applied as described above with respect to the method of reducing abnormal cell division.

[0237] Formulations: The compositions can be prepared as injectable solutions (as liquid solutions or suspensions); they can also be prepared in solid forms suitable for dissolution or suspension in a liquid prior to injection. Emulsion formulations can also be prepared, or the peptides can be encapsulated in liposomes. The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline (e.g., phosphate buffered saline), dextrose, glycerol, ethanol, etc. and combinations thereof.

[0238] 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.

[0239] If the composition contains multiple peptides, then the relative ratios of the peptides can be approximately equal. Alternatively, the relative proportion of each peptide can be varied.

[0240] After formulation, the compositions can be incorporated into a sterile container, which is then sealed and stored at low temperature, e.g., 4°C or it can be lyophilized.

[0241] Conveniently, the compositions are prepared as lyophilized (freeze-dried) powders. Lyophilization allows for long-term storage in a stable form. The lyophilization step is well known in the art, see e.g., 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.

[0242] The compositions can be administered by convenient means, e.g., by oral, intravenous (in the case of water solubility), intramuscular, subcutaneous, sublingual, intranasal, intradermal or suppository routes or by implantation (e.g., using slow release molecules).

[0243] The compositions can be advantageously administered by intranasal, subcutaneous or intradermal routes.

[0244] The peptides and compositions of the present invention can be used to treat human subjects.

[0245] Typically, a physician will determine the actual dosage most suitable for an individual subject, and it will vary with the age, weight, and response of the particular patient.

[0246] In a preferred embodiment, a "dose escalation" protocol can be followed, in which multiple doses are administered to the patient at increasing concentrations.

[0247] As used herein, "pharmaceutically acceptable" refers to ingredients that are compatible with the other ingredients of the composition and physiologically acceptable to the recipient.

[0248] 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, and the like, and combinations thereof, as would be known to those of ordinary skill in the art (see, e.g., 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.

[0249] The pharmaceutical compositions according to the present 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, and the like.

[0250] The pharmaceutical compositions can be formulated for specific routes of administration, such as oral administration, parenteral administration, and 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.

[0251] Typically, the pharmaceutical composition is a tablet or gelatin capsule containing an active ingredient (the polypeptide or recombinant polypeptide provided by the present invention) and the following components

[0252] a) diluents such as lactose, polycaprolactone, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine;

[0253] b) lubricants such as silica, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol;

[0254] c) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone;

[0255] d) disintegrants such as starch, agar, alginic acid or its sodium salt or effervescent mixtures; and / or

[0256] e) absorbents, colorants, flavors and sweeteners.

[0257] Tablets can be film-coated or enteric-coated according to methods known in the art.

[0258] Suitable compositions for oral administration include the compounds of the present invention in an effective amount in the form 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 preservatives in order to provide a pharmaceutically elegant and palatable preparation. Tablets may contain the active ingredient admixed with non-toxic pharmaceutically acceptable excipients suitable for the preparation 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; binders such as starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc. Tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, delayed release materials such as glyceryl monostearate or glyceryl distearate can be used. Preparations for oral use may be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with a water or oil medium such as peanut oil, liquid paraffin or olive oil.

[0259] 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 preservatives, stabilizers, wetting or emulsifying agents, solubilizing agents, salts regulating osmotic pressure and / or buffers. Additionally, they may also contain other substances of therapeutic value. The compositions are prepared according to conventional mixing, granulating or coating methods respectively, and contain from about 0.1 - 75% or contain from about 1 - 50% of the active ingredient.

[0260] Suitable compositions for transdermal application comprise an effective amount of the active agent of the present invention and a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmaceutically acceptable solvents that assist in crossing the host skin. For example, the transdermal device is in the form of a bandage that comprises a backing layer, a reservoir containing the compound and optionally a carrier, a rate control barrier that optionally delivers the compound to the host skin at a controlled and predetermined rate over an extended period of time, and means for securing the device to the skin.

[0261] Suitable compositions for topical application (e.g., 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 applications, for example for the treatment of skin cancer, for example for prophylactic applications in sunscreens, lotions, sprays etc. They are thus particularly suitable for topical formulations known in the art, including cosmetic formulations. This may contain solubilizing agents, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0262] As used herein, topical application may also relate to inhalation or intranasal application. They may conveniently be delivered in dry powder form from a dry powder inhaler (alone, as a mixture, e.g., a dry blend with lactose, or as mixed component particles, e.g., with phospholipids) or from a pressurized container, pump, nebulizer, atomizer or sprayer, as an aerosol spray product, with or without a suitable propellant.

[0263] The dosage of the polypeptide or recombinant polypeptide provided by the present invention employed in practicing 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 dosage 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 dosage is in the order of 0.01 to 100 mg / kg.

[0264] 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.

[0265] 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. Accordingly, anhydrous compositions are packaged using materials known to prevent exposure to water so that they can be included in suitable formulation kits. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit-dose containers (e.g., vials), blister packs, and strip packs.

[0266] The present invention further provides pharmaceutical compositions and dosage forms comprising one or more substances that reduce the rate of decomposition of the polypeptides or recombinant polypeptides of the present invention acting as active ingredients. Such substances, referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers, and the like.

[0267] The active agents of the present invention can be administered simultaneously with, before, or after one or more other therapeutic agents. The active agents 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.

[0268] 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 as a combination product for simultaneous, separate, or sequential use in therapy. In one embodiment, the therapy is for the treatment of a condition or disorder in which abnormal cell division occurs. The products provided as combination products include compositions 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).

[0269] 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 can comprise a pharmaceutically acceptable excipient as described above.

[0270] Those skilled in the art will understand that the polypeptides or recombinant polypeptides of the present invention can be administered to a subject, particularly a human subject, in which the subject is being treated with surgery or radiation therapy 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 compounds of the present invention have previously (e.g., within 24 hours) been administered to the subject.

[0271] 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 said compositions, such as containers, separate vials or separate foil packages. An example of such a kit is a blister pack commonly used for packaging tablets, capsules, etc.

[0272] The kits of the present invention can be used for administering different dosage forms, such as oral and parenteral, for administering the separate compositions at different dosing intervals, or for titrating the separate compositions against each other. To assist compliance, the kits of the present invention generally contain dosing instructions.

[0273] 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 an 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.

[0274] Accordingly, the present invention provides the use of the provided polypeptide or recombinant polypeptide for treating 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 treating a condition or disorder in which abnormal cell division occurs, wherein the medicament is administered together with the active agent of the present invention.

[0275] The present invention also provides the polypeptide or recombinant polypeptide provided by the present invention for use in a method of treating a condition or disorder in which abnormal cell division occurs, wherein the polypeptide or recombinant polypeptide provided by the present invention is prepared for administration together with another therapeutic agent. The present invention also provides another therapeutic agent for use in a method of treating a condition or disorder in which abnormal cell division occurs, wherein the other therapeutic agent is prepared for administration together with the polypeptide or recombinant polypeptide provided by the present invention.

[0276] The present invention also provides the use of the active agent of the present invention for treating 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 treating 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.

[0277] The composition may additionally contain molecules that assist or enhance the action of the active agents 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.

[0278] If desired, the composition may also contain targeting moieties attached to the active ingredient, e.g., ligands that specifically and selectively bind to endogenous receptors to allow targeting of specific cell types or locations, 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, 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.

[0279] The peptides of the present invention can be used to assist or enhance the action of active agents used in conventional therapy, e.g., cytotoxic agents, to reduce their side effects.

[0280] 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 combination chemotherapeutic agent. The polypeptide or recombinant polypeptide provided by the present invention can induce some cancer cells.

[0281] 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 would otherwise be possible or safe.

[0282] 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, mitosis inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, radiation, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxic agents, anti-hormones, statins, anti-androgens, and photochemotherapeutic agents.

[0283] 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.

[0284] Suitable pan kinase inhibitors include SU-11248 (sutinib malate) as described in U.S. Patent No. 6,573,293 (Pfizer Inc).

[0285] Angiogenesis inhibitors 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.

[0286] 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 for 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.

[0287] Other antiproliferative agents that can be used 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 of Pfizer and their pharmaceutically acceptable salts.

[0288] Suitable GARF inhibitors include Pfizer's AG-2037 (pelitrexol and its pharmaceutically acceptable salts). GARF inhibitors useful for 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.

[0289] 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.

[0290] 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 (Merck AG), ABX-EGF (Amgen Inc. and AbgeniX Inc.), 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 (Decof Cancer Center), B7.her2.1gG3 (Agensys), AS HER2 (Radiology&; Research Institute for 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.

[0291] In addition, other anti-tumor agents can 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.

[0292] Other anti-tumor agents can 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.

[0293] In addition, other anti-tumor agents can also be selected from the following active agents: Trizaone (tirapazamine), Aposyn (ecamsule), 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 can be selected from the following active agents: CeaVac (CEA), NeuTrexin (trimetrexate glucuronate), and their combinations. Other anti-tumor agents can be selected from the following active agents: OvaRex (oregovomab), Osidem (IDM-I), and their combinations.

[0294] Additional anti-tumor agents can be selected from the following active agents: Advexin (ING 201), Tirazone (tirapazamine), and their combinations. Additional anti-tumor agents can 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 can be selected from the following active agents: Canvaxin, GMK vaccine, PEG Interferon A, Taxoprexin (DHA / paclitaxel), and their combinations.

[0295] 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.

[0296] In addition, mTOR inhibitors such as CCI-779 (Wyeth), rapamycin derivative RAD001 (Novartis), and AP-23573 (Ariad), HDAC inhibitor SAHA (Merck Inc / Aton Pharmaceuticals), and their combinations can also be used. Additional anti-tumor agents include aurora 2 inhibitor VX-680 (Vertex), Chk1 / 2 inhibitor XL844 (Exelixis).

[0297] 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.

[0298] 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.

[0299] 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.

[0300] The following topoisomerase I inhibitors can be used as anti-tumor agents: camptothecin; irinotecan hydrochloride (Camptosar); edotecarin; orathecin (Supergen); esatidecan (Daiichi); BN-80915 (Roche); and combinations thereof. Particularly preferred topoisomerase II inhibitors include epirubicin (Ellence).

[0301] Alkylating agents include, but are not limited to, nitrogen mustard N-oxides, cyclophosphamide, ifosfamide, melphalan, busulfan, dibromomannitol, carboquone, thiotepa, ranimustine, nimustine, temozolomide, AMD-473, hexamethylmelamine, 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, etoplatid, lobaplatin, nedaplatin, oxaliplatin (Eloxatin, Sanofi), or satrplatin and combinations thereof. Particularly preferred alkylating agent is oxaliplatin.

[0302] 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 hexylamide, cytarabine, cytarabine ocfosfate, enocitabine, S-1, pemetrexed disodium (LY231514, MTA), gemcitabine (Eli Lilly), fludarabine, 5-azacytidine, capecitabine, cladribine, clofarabine, decitabine, elonisel, 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.

[0303] Antibiotics include intercalating antibiotics but are not limited to: aclarubicin, actinomycin D, amrubicin, annamycin, doxorubicin, bleomycin, daunorubicin, epirubicin, idarubicin, mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebeccamycin, stimamer, streptozocin, valrubicin, zinostatin and combinations thereof.

[0304] Antitumor substances of plant origin include, for example, those selected from mitotic inhibitors, such as vinblastine, docetaxel (Taxotere), paclitaxel, and combinations thereof.

[0305] 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, esanotecan, 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.

[0306] Immunology includes interferons and many other immunopotentiators. Interferons include interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a, interferon gamma-1b (Actimmune), or interferon gamma-n1, and combinations thereof. Other active agents include filgrastim, ientinan, lentinan, TheraCys, ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab, imiquimod, lenograstim, Ientinan, Corixa, molgramostim, OncoVAX-CL, sargramostim, tasomigram, tecleukin, thymalasin, tositumomab, verulizen, 2-100, epratuzumab, mitumomab, oregovomab, pemtumomab (Y-muHMFGl), Provenge (Dendreon), and combinations thereof.

[0307] Biological response modifiers are active agents that alter the defense mechanisms or biological responses of a living organism (such as the survival, growth, or differentiation of tissue cells) to direct it to have antitumor activity. Such active agents include polysaccharide krestin, Ientinan, sizofiran, Picibanil, ubenimex, and combinations thereof.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] Gene therapy agents can also be used as anti-tumor agents, such as TNFerade (GeneVec), which expresses TNFα in response to radiotherapy.

[0313] 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.

[0314] 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

[0315] 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 derivatives anthralin / 1,8-dihydroxy-9-anthrone / dithranol (BioLuminescence Activated Destruction (BLADe)); Firefly (Photinus pyralis) luciferase used in; Eosin B (EB); Erythrosine sodium; m-tetrahydroxyphenylchlorin (m-THPC) / temoporfin (Biolitec AG); Porphyrins such as d-aminolevulinic acid (d-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 (LightScience 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), phycocyanin-II, meso-tetraphenylporphyrin (TPP), and tetraphenylporphyrin sulfonate (TPPS4).

[0316] 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 refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

[0317] 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.

[0318] Unless otherwise specified, the polypeptides used in the following examples are all produced by Wuhan Moore Biotechnology Co., Ltd., with a purity of ≥95%.

[0319] The polypeptide provided by the present invention that can cause immunogenic death of tumor cells and kill tumor cells broadly, as well as its preparation method, drug delivery system, and raw materials and reagents used in the application, can all be purchased from the market.

[0320] 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.

[0321] Example 1: Polypeptide Gene Engineering Expression and Purification

[0322] Using the amino acid sequence of the anti-tumor polypeptide Seq ID 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, and the vector used is pET-32a(+). The gene synthesis company was sent to prepare the recombinant expression vector pET-32a(+)-LCX18.

[0323] The obtained recombinant expression plasmid pET-32a(+)-LCX18 was transformed into Escherichia coli BL21(DE3). Take 2 μL of the recombinant expression plasmid pET-32a(+)-LCX18 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-LCX18.

[0324] 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, and the bacterial cells are collected by centrifugation at 10000 rpm and 4 °C for 10 min.

[0325] 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. The lysate is centrifuged at 12000 rpm and 4 °C for 30 min, the supernatant is collected, and the supernatant is filtered through a 0.45 μm mixed membrane, and the filtered supernatant is collected for purification. 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 the nickel column is rinsed with Binding Buffer for 10 CV until the reading is stable.

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

[0328] Washing impurities: Equilibrate the nickel column with Binding Buffer at a flow rate of 5 mL / min until the reading is stable.

[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, collect the flow-through, and 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 an anti-tumor polypeptide with higher purity. Purify again by RP-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 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 IDNo.5 solution, polypeptide Seq ID No.6 solution and polypeptide Seq ID No.7 solution for experimental studies.

[0337] Example 2: Toxicity detection of polypeptide SEQ ID NO.1 against various tumor cells

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

[0339] Use 4T1 tumor cells as the model cells for studying the anti-tumor polypeptide against mouse breast cancer cell line. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells with RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% streptomycin) to 1×10 5Cells / mL of medium, add the cell suspension to a 96-well cell culture plate, 100 μL per well, that is, each well contains 10,000 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 SeqID NO.1 solutions with different concentrations to each well in 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. Add 90 μL of complete cell 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 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 - 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 1 shown, compared with 4T1 cells without polypeptide treatment, after adding different concentrations of polypeptide to 4T1 cells and incubating, with the increase of polypeptide concentration, the cell mortality rate gradually increases. It shows that all polypeptides of Seq ID NO.1 can significantly inhibit the growth of breast cancer cell line 4T1 cells, and the half lethal concentration (IC50) of polypeptide SEQ ID NO.1 is 3.7 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 / % 97.1 98 76.6 44.4 18 23.5

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

[0343] Using A549 tumor cells as model cells for studying anti-tumor polypeptides on non-small cell lung cancer cell lines. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells 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, i.e., each well contains 5,000 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 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 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 - 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 A549 cells without polypeptide treatment, after adding different concentrations of polypeptide to A549 cells and incubating, as the concentration of the polypeptide increases, 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 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 shown, compared with A549 cells without polypeptide treatment, after adding different concentrations of polypeptide to A549 cells and incubating, as the concentration of the polypeptide increases, 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 Figure 2 shown, compared with A549 cells without polypeptide treatment, after adding different concentrations of polypeptide to A549 cells and incubating, as the concentration of the polypeptide increases, 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 42.3 μM, and the specific data are shown in Table 2.

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

[0345] Concentration / μM 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 98.1 28.2 19.1 23.7 27.1 31.4

[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 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 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. 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 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 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 culture 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 - 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 3 shown, compared with MCF7 cells without polypeptide treatment, after adding polypeptides with different concentrations to incubate in MCF7 cells, 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 dose (IC 50 ) is 4.1 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 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 96.2 96.7 93.9 48.4 18.8 10.2

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

[0351] Using HT29 tumor cells as model cells for studying anti-tumor polypeptides 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 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 complete cell 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 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 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 - 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 13.5 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 / % 96.4 57 32 32.5 36.1 34.9

[0354] Experiment 5: Toxicity of Polypeptides with Different Concentrations to HeLa Tumor Cells

[0355] Using HeLa tumor cells as model cells for studying the anti-tumor polypeptide on the cervical 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% 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 Seq ID 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 HeLa cells without polypeptide treatment, after incubating with different concentrations of polypeptide in HeLa cells, 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 cervical cancer cell line HeLa, and its half-lethal dose (IC50) is 7.5 μM. The specific data are shown in Table 5. 450 value - OD of the background control group 450 value) / (OD of the blank control group 450 value - OD of the background control group 450 value)) × 100%. The experimental results show that, as Figure 5 shown, compared with HeLa cells without polypeptide treatment, after incubating with different concentrations of polypeptide in HeLa cells, 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 cervical cancer cell line HeLa, and its half-lethal dose (IC50) is 7.5 μM. The specific data are shown in Table 5.

[0356] Table 5. Cytotoxicity of polypeptide SEQ ID NO.1 on HeLa cells

[0357] Concentration / μM 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 101 102.9 89.1 23.4 19.8 19.6

[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 the 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, 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 each well of 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 make the final concentration of the polypeptide 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 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 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, compared with PC3 cells without polypeptide treatment, after adding polypeptides with different concentrations to PC3 cells and incubating, with the increase in polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of 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 in, compared with PC3 cells without polypeptide treatment, after adding polypeptides with different concentrations to PC3 cells and incubating, with the increase in polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of human malignant melanoma cell line PC3, and its half-lethal concentration (IC Figure 6 as shown, compared with PC3 cells without polypeptide treatment, after adding polypeptides with different concentrations to PC3 cells and incubating, with the increase in polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of human malignant melanoma cell line PC3, and its half-lethal concentration (IC 50 ) is 7.4 μM, and the specific data are shown in Table 6.

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

[0361] Concentration / μM 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 89.1 96.7 72.2 35.6 28.9 20.9

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

[0363] Use hepG2 tumor cells as model cells for studying the anti-tumor polypeptide on liver 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% streptomycin) to 5 × 10 4Cells / mL of culture medium, add 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 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 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 - 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 7 shown, compared with hepG2 cells without polypeptide treatment, after adding polypeptides with different concentrations to incubate in hepG2 cells, with the increase of polypeptide concentration, the cell mortality rate gradually increases. It shows that the polypeptide can significantly inhibit the growth of the hepatocellular carcinoma cell line hepG2, and its half lethal concentration (IC 50 ) is 14.45 μM. The specific data are shown in Table 7.

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

[0365] Concentration / μM 100 33 11 3.7 1.2 0.4 Cell Death Rate / % 112 111.8 15.3 1.1 -8.7 -22.2

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

[0367] Use 786-O tumor cells as model cells for studying the anti-tumor polypeptide on the renal cancer cell line. Take cells in the logarithmic growth phase, digest with trypsin, count the cells, and dilute the cells with RPMI-1640 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, 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 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 using a microplate 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%. As shown in Figure 8 , compared with 786-O cells without polypeptide treatment, after incubating 786-O cells with polypeptides at different concentrations, as the polypeptide concentration increases, the cell death 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 9.58 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 / % 95.6 95.1 54.4 23.8 12.5 2.1

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

[0371] Use 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, 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 SeqID NO.1 to each well of the sample group, so that the final concentration of the polypeptide is 60 μM, 30 μM, 15 μM, 7.5 μM, 3.75 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 (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, compared with LM3 cells without polypeptide treatment, after adding polypeptides with different concentrations to LM3 cells and incubating, as the polypeptide concentration increases, the cell death rate gradually increases. This indicates that the polypeptide can significantly inhibit the growth of human hepatocellular carcinoma cell line LM3, and its half-lethal concentration (IC 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, compared with LM3 cells without polypeptide treatment, after adding polypeptides with different concentrations to LM3 cells and incubating, as the polypeptide concentration increases, the cell death rate gradually increases. This indicates that the polypeptide can significantly inhibit the growth of human hepatocellular carcinoma cell line LM3, and its half-lethal concentration (IC 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, compared with LM3 cells without polypeptide treatment, after adding polypeptides with different concentrations to LM3 cells and incubating, as the polypeptide concentration increases, the cell death rate gradually increases. This indicates that the polypeptide can significantly inhibit the growth of human hepatocellular carcinoma cell line LM3, and its half-lethal concentration (IC 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, compared with LM3 cells without polypeptide treatment, after adding polypeptides with different concentrations to LM3 cells and incubating, as the polypeptide concentration increases, the cell death rate gradually increases. This indicates that the polypeptide can significantly inhibit the growth of human hepatocellular carcinoma cell line LM3, and its half-lethal concentration (IC 450 value)) × 100%. The experimental results show that, as shown in, compared with LM3 cells without polypeptide treatment, after adding polypeptides with different concentrations to LM3 cells and incubating, as the polypeptide concentration increases, the cell death rate gradually increases. This indicates that the polypeptide can significantly inhibit the growth of human hepatocellular carcinoma cell line LM3, and its half-lethal concentration (IC Figure 9 shown, compared with LM3 cells without polypeptide treatment, after adding polypeptides with different concentrations to LM3 cells and incubating, as the polypeptide concentration increases, the cell death rate gradually increases. This indicates that the polypeptide can significantly inhibit the growth of human hepatocellular carcinoma cell line LM3, and its half-lethal concentration (IC 50 ) is 14.62 μM, and the specific data are shown in Table 9.

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

[0373] Concentration / μM 60 30 15 7.5 3.75 1.875 Cell Death Rate / % 97.5 71.2 25.9 12.7 16.1 13.6

[0374] Experiment 10: Toxicity of Polypeptides with Different Concentrations to U251MG Tumor Cell Line

[0375] Use U251MG tumor cells as model cells for studying the anti-tumor polypeptide against glioma 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% 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 Seq ID 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 60 μM, 30 μM, 15 μM, 7.5 μM, 3.75 μM and 1.875 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 for 24 hours at 37°C and 5% CO2, 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 10 As shown in the figure, compared with U251MG cells not treated with peptides, after adding different concentrations of peptides to U251MG cells for incubation, the cell death rate gradually increased with the increase of peptide concentration. This shows that the peptide can significantly inhibit the growth of the glioma cell line U251MG, and its half lethality (IC 50 ) is 6.96μM, and the specific data are shown in Table 10.

[0376] Table 10. Cytotoxicity of polypeptide SEQ ID NO.1 to U251MG cells

[0377] Concentration / μM 60 30 15 7.5 3.75 1.875 Cell Death Rate / % 97.8 98.8 95.3 58 21.5 8.5

[0378] Experiment 11: Toxicity of Polypeptides with Different Concentrations to HEC - 1 - B Tumor Cell Line

[0379] HEC-1-B tumor cells were used as model cells to study the effects of anti-tumor peptides on endometrial 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 MEM complete medium (containing 10% fetal bovine serum and 1% streptavidin). 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 Seq ID 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 60 μM, 30 μM, 15 μM, 7.5 μM, 3.75 μM and 1.875 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 for 24 hours at 37°C and 5% CO2, 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 11 As shown in the figure, compared with HEC-1-B cells not treated with peptides, after adding different concentrations of peptides to HEC-1-B cells for incubation, the cell death rate gradually increased with the increase of peptide concentration. This shows that the peptide can significantly inhibit the growth of endometrial cancer cell line HEC-1-B, and its half lethality rate (IC 50 ) is 14.04 μM. Specific data are shown in Table 11.

[0380] Table 11. Cytotoxicity of polypeptide SEQ ID NO.1 to HEC-1-B cells

[0381] Concentration / μM 60 30 15 7.5 3.75 1.875 Cell Death Rate / % 93.7 95.4 97.9 92.9 21.4 -0.9

[0382] Example 3: Microscopic morphological observation of tumor cells

[0383] 4T1 tumor cells were used as model cells to study the effects of anti-tumor peptides on mouse breast cancer cell lines. Cells in the logarithmic growth phase were obtained, trypsinized, and the cells were counted. The cells were suspended in RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% streptavidin), and the cell suspension was added 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, and 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 12 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, until a naked nucleus is formed.

[0384] Example 4: Tumor immunity experiment in animals

[0385] 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 37°C water bath conditions, and then transfer them to RPMI-1640 medium containing 10% fetal bovine serum and 1% streptomycin, and culture them 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 normally cultured cells, adjusted the cell concentration to 7.5×10 6 cells / ml, and repeatedly freeze-thawed 3 times at -80°C for 15 min --- 37°C for 15 min. Subcutaneously inoculate these treated tumor cells under the left scapula of the mouse, and inoculate 1.5×10 6 cells per mouse. On the 15th day after the first inoculation (day 0), count the normally passaged 4T1 cells (not treated with the polypeptide Seq ID NO.1), dilute them with basal medium to 3×10 6 cells / mL, and subcutaneously inoculate 4T1 cells under the right scapula of the mouse, and inoculate 3×10 6cells. The growth of tumors in mice was observed. The number of tumor-free and tumor-bearing mice in each group was dynamically observed, and 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 = (the number of mice without tumors / the total number of mice) × 100%. As can be seen from the experimental results, as Figure 13 shown, by the end of the experiment, compared with the freeze-thaw group (TF = 0%) and the control group (adriamycin treatment, TF = 17%), it can be seen that the experimental group of SeqID NO.1 (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 12.

[0386] Table 12. Tumor-free data of tumor immunization experiments

[0387] 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 13 0.0％ 50.0％ 83.3％ Day 16 0.0％ 16.7％ 83.3％

[0388] Example 5: Intratumoral administration treatment experiment for orthotopic tumors

[0389] MCF7 tumor cells were used as model cells to study anti-tumor polypeptides against 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 culture medium (Wuhan Punosai, product number CM-0149), and subcultured in a cell culture incubator at 37°C and 5% CO2 until sufficient cell numbers were obtained. One day before inoculating cells into the fat pad, a slow-release estrogen implant for mice (0.36 mg / tablet / mouse) was subcutaneously inoculated. Injection was performed according to the following parameters: 5×10 6 MCF7 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 tumors 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 volume of drug injection was 70 μl, and the drug was administered three times at an interval of three days each time. The dosage of 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, the number of tumor-free and tumor-bearing mice in each group was dynamically observed, and 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 major axis and minor axis 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 14 shown, by the end of the experiment, the TF of the mice in the experimental group of SeqID NO.1 was 83.3%, 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 13.

[0390] Table 13. Tumor-free data of intratumoral administration treatment experiment for orthotopic tumors

[0391] 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 16.7％ 80.0％ 20.0％ Day 12 83.3％ 100.0％ 40.0％ Day 15 83.3％ 100.0％ 60.0％

[0392] Example 6: Preparation of micellar injection solution

[0393] To prepare the micellar injection solution of polypeptide Seq ID NO.1, the steps are as follows:

[0394] Step (1): Preparation of volatile organic solvent mixture. Absolute ethanol and chloroform are mixed evenly at a volume ratio of 1:2.

[0395] Step (2): Preparation of MPLA stock solution. MPLA is dissolved in the organic solvent mixture obtained in step (1).

[0396] Step (3): Preparation of PEG-DSPE stock solution. PEG-DSPE is dissolved in the volatile organic solvent chloroform.

[0397] Step (4): According to a molar ratio of 100:3, take the MPLA stock solution obtained in step (3) and the PEG-DSPE stock solution obtained in step (2) into a rotary evaporation flask, and gently shake to mix evenly.

[0398] 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;

[0399] Step (6): Prepare a physiological saline solution of polypeptide Seq ID NO.1. According to the molar ratio PEG-DSPE:polypeptide Seq ID NO.1 = 100:4, dissolve polypeptide Seq ID NO.1 in physiological saline to prepare a solution with a concentration of 0.126 mg / ml;

[0400] Step (7): Hydrate the lipid membrane. Take the physiological saline solution of polypeptide Seq ID NO.1 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 uniformly colorless and transparent in appearance by self-assembly.

[0401] 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 standby (it can be stored for two weeks).

[0402] 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.

[0403] Example 7: Preparation of freeze-dried micellar injection

[0404] Prepare the freeze-dried powder of the micellar injection of polypeptide Seq ID NO.1. The steps are as follows:

[0405] 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.

[0406] The preparation method of the freeze-dried powder preparations 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.

[0407] Example 8: Toxicity of peptides Seq ID NO.5 to Seq ID NO.7 to MCF7 tumor cells MCF7 tumor cells were used as model cells to study the effects of anti-tumor peptides on breast cancer cell lines. Cells in logarithmic growth phase were obtained, trypsinized, and the cells were counted. The cells were diluted to 5×10 cells / mL with MEM complete medium (containing 10% fetal bovine serum and 1% streptavidin). 4 Cells / 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 different concentrations of SeqID NO.5 to Seq ID NO.7 peptide solutions 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, and 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 450 nm 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 15 As shown in the figure, compared with MCF7 cells not treated with peptides, after adding different concentrations of peptides to MCF7 cells for incubation, the cell death rate gradually increased with the increase of peptide concentration. This shows that the peptide can significantly inhibit the growth of breast cancer cell line MCF7, and its half lethality (IC 50 ) were 4.2μM, 3.5μM and 4.3μM respectively. 50 The data is from the cytotoxicity of experiment 3) in Example 2, indicating that after amino acid substitution, deletion and addition, the polypeptide SeqID NO.1 still has high cytotoxicity to MCF7 tumor cells. Specific data are shown in Table 14.

[0408] Table 14. Cytotoxicity of polypeptides SEQ ID NO.5 to SEQ ID NO.7 to MCF7 cells

[0409]

[0410]

[0411] 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 refinements can be made, and these improvements and refinements 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 multiple amino acids include, but are not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

3. The polypeptide according to claim 1 or 2, wherein The modification includes, but is not limited to, one or more of amidation, phosphorylation, methylation, acetylation, ubiquitination, glycosylation, PEG 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 bioengineering methods.

6. The 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 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.

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, wherein 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)-(III).

11. The gene element according to claim 10, wherein 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.

12. Recombinant vector, characterized in that, (including) any one of the following: (I) The nucleic acid molecule described in claim 7 or 8; or (II) The gene element described in any one of claims 9 to 11.

13. The recombinant vector according to claim 12, characterized in that, The recombinant vector has: (I) The nucleotide sequence shown in SEQ ID No. 4; 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)-(III).

14. The recombinant vector according to claim 13, characterized in that, 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.

15. The recombinant vector according to any one of claims 12 to 14, characterized in that, The source of the backbone vector of the recombinant vector includes, but is not limited to, plants, animals, bacteria, fungi, phages or viruses.

16. A host, characterized in that (I) Integrates the nucleic acid molecule described in claim 7 or 8; or (II) Integrates the gene element described in any one of claims 9 to 11; or (III) Transfects or transforms the recombinant vector described in any one of claims 12 to 15.

17. The host according to claim 16, wherein The host includes, but is not limited to, prokaryotes or eukaryotes; the prokaryotes include, but are not limited to, Escherichia coli.

18. A recombinant polypeptide, characterized in that, (Prepared) from any one of the following: (I) The gene element described in any one of claims 9 to 11; or (II) The recombinant vector described in any one of claims 12 to 15; or (III) The host described in claim 16 or 17.

19. The method for preparing the recombinant polypeptide according to claim 18, wherein, (including) the following steps: Step 1. Construction of genetically engineered strains: Construct genetically engineered bacterial strains expressing recombinant polypeptides; Step 2. Expression and harvest of fusion proteins: Inoculate the genetically engineered strains described in step (1) into LB medium for cultivation, add an inducer to induce the expression of fusion proteins, collect the bacterial cells, suspend them in a buffer, disrupt the cells by ultrasonic treatment 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 protease, and the enzymatic cleavage conditions are that the ratio of sumo protease 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 conditions are that the ratio of sumo protease 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. The application of any one of the following in the preparation of products 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 products include, but are not limited to, vaccines, drugs, and / or drug combinations.

26. The application according to claim 24 or 25, characterized in that, The tumors are 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 one of the following and acceptable immunological adjuvants or excipients: (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 immunological adjuvants include one or more of Freund's adjuvant, aluminum hydroxide adjuvant, and monophosphoryl lipid A adjuvant, preferably monophosphoryl lipid A adjuvant.

29. The application of the vaccine according to claim 27 or 28 in preventing tumors.

30. The application according to claim 29, wherein The tumors are 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 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.

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 galactosylated 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 injection.

36. The drug according to claim 35, wherein, The injection includes one or more of micelle injection, liposome injection or nanomaterial injection, preferably micelle injection.

37. The medicament according to claim 35 or 36, characterized in that, The injection is a lyophilized powder, which is freeze-dried after adding a lyoprotectant to obtain a lyophilized powder; preferably the lyoprotectant 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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