A tumor suppressor peptide

Abstract

The present application provides a tumor inhibiting peptide, which is a fragment of the N-terminal of endostatin with 45 amino acid residues or less, and contains at least the first to 20th amino acid residues of the N-terminal, and wherein the second and 18th amino acid residues of the N-terminal of endostatin are as shown herein, and optionally, there are mutations as shown herein in the 17th, 20th to 22nd positions. The present application also relates to a coding sequence of the peptide, an expression vector containing the coding sequence, a pharmaceutical composition containing the peptide, and the use of the peptide and the pharmaceutical composition in the preparation of a medicament for inhibiting, preventing or treating tumors.

Description

[0001] This invention patent application is a divisional application of the invention patent application with international application number PCT / CN2016 / 090800, international application date July 21, 2016, application number 201680044076.1 entering the Chinese national phase, and invention name “A Tumor Suppressing Peptide”. Technical Field

[0002] The present invention belongs to the field of tumor treatment, and specifically relates to a polypeptide for inhibiting and treating tumors. The amino acid sequence of the polypeptide is a fragment within 45 amino acids starting from the first amino acid residue at the N-terminus of endostatin. Background Art

[0003] Endostatin is an endogenous angiogenesis inhibitor isolated and purified by O'Reilly et al. in 1997 from the supernatant of cultured mouse endothelioma cells (EOMA). It is a 20 kDa protein derived from the hydrolysis product of type XVIII collagen. Experiments have shown that endostatin inhibits vascular endothelial cells and tumor cells. However, due to difficulties such as the refolding of recombinant endostatin, EntreMed (USA) abandoned clinical research on recombinant endostatin. Currently, it is not possible to produce large quantities of endostatin with high in vitro activity.

[0004] The zinc ion binding site in the endostatin sequence is composed of three histidine residues at the N-terminus, positions 1, 3, and 11, and an aspartic acid residue at position 76. The binding of endostatin to zinc ions is crucial for its activity. It has been reported that peptides derived from the N-terminus of endostatin have certain inhibitory activities on vascular endothelial cells and tumor cells (Cancer Res. 2005; 65(9): 3656-63, U.S. Patent No. 7524811B2). However, the above experiments also showed that peptides containing amino acids 1-25 at the N-terminus derived from human endostatin could not significantly inhibit the growth of human tumors inoculated in mouse animal models, and the activity of endostatin-derived peptides needs to be improved. Summary of the Invention

[0005] The present invention provides a polypeptide, which is a fragment of endostatin N-terminus with a length of less than 45 amino acid residues and contains at least the 1st to 20th amino acid residues at the N-terminus, and wherein the 2nd amino acid residue and the 18th amino acid residue at the N-terminus of endostatin are respectively selected from the following groups:

[0006] Amino acid 2 Amino acid 18 A M R I N K D E, M, T, or Y Q A or H E S or V H A or S L R, E, or S K V M L or W F T P C or V T N, G, K, M, F, S, or T W C, E, I, K, S, or Y Y R, H, W, or V V D or S

[0007] And optionally, the 17th amino acid at the N-terminus of the endostatin is S, A, L, I or T, and / or the 20th amino acid residue is S or T, and / or if the 21st and / or 22nd amino acid residues are present, the 21st amino acid residue is G, A, L, I or V, and / or the 22nd amino acid residue is G, A, L, I or V;

[0008] Preferably, the amino acid sequence of endostatin is as shown in SEQ ID NO: 1.

[0009] In one embodiment, the polypeptide contains at least amino acid residues 1 to 22 of SEQ ID NO: 38, and amino acid residues 2 and 18 are as described above.

[0010] In one embodiment, the polypeptide contains at least amino acid residues 1 to 25 of SEQ ID NO: 38, and amino acid residues 2 and 18 are as described above.

[0011] In one embodiment, the polypeptide contains at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1-25 of SEQ ID NO: 38, and the amino acid residue at position 2 is T, the amino acid residue at position 18 is N, G, K, M, F, S or T, and the amino acids at positions 17, 20, 21 and 22 are as described above.

[0012] In one embodiment, the polypeptide contains at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1-25 of SEQ ID NO: 38, and the amino acid residue at position 18 is N, the amino acid residue at position 2 is T, and the amino acids at positions 17, 20, 21 and 22 are as described above.

[0013] In one embodiment, the polypeptide contains at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1-25 of SEQ ID NO: 38, and the amino acid residue at position 18 is S, the amino acid residue at position 2 is E, H, L, T, W or V, and the amino acids at positions 17, 20, 21 and 22 are as described above.

[0014] In one embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 4, 5, 6, 7, 27-30, 39 or 41.

[0015] In one embodiment, the polypeptide consists of SEQ ID NO: 38, wherein the amino acid residue at position 2 is T, the amino acid residue at position 18 is N or S, and the amino acids at positions 17, 20, 21 and 22 are as described above.

[0016] In one embodiment, the polypeptide is selected from an amino acid sequence consisting of amino acid residues 1 to 39, 38, 37, 36, 34, 33, 32, 31, 29, 28, 27 or 26 of SEQ ID NO:4, and an amino acid sequence consisting of amino acid residues 1 to 39, 38, 37, 36, 35, 34, 33, 32, 31, 29, 28, 27, 26 or 25 of SEQ ID NO:39.

[0017] In one embodiment, the first amino acid residue at the N-terminus of the polypeptide is histidine, which is modified by formylation, acetylation, propionylation or butyrylation, and the first amino acid at the C-terminus can be modified by PEG, cholesterol or amidation.

[0018] In one embodiment, the polypeptide is selected from:

[0019] HTHRDFQPVLHLVALNSSLSGGMRGIRGAD;

[0020] Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD;

[0021] HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2;

[0022] Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2;

[0023] Ac-HTHRDFQPVLHLVALNSSLSGGMRG-NH2;

[0024] Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQ-NH2;

[0025] Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQQARAV-NH2;

[0026] HTHRDFQPVLHLVALNSSNLSGGMRGIRGAD;

[0027] Ac-HTHRDFQPVLHLVALNSSNLSGGMRGIRGAD;

[0028] HTHRDFQPVLHLVALNSSNLSGGMRGIRGAD-NH2;

[0029] Ac-HTHRDFQPVLHLVALNSSNLSGGMRGIRGAD-NH2;

[0030] Ac-HTHRDFQPVLHLVALNASLSGGMRGIRGAD-NH2;

[0031] Ac-HTHRDFQPVLHLVALNSSLTGGMRGIRGAD-NH2;

[0032] Ac-HTHRDFQPVLHLVALNASLTGGMRGIRGAD-NH2; and

[0033] Ac-H T HRDFQPVLHLVALNS S LSGGMRGIRGA-NH2;

[0034] Ac represents acetylation modification, and NH2 represents amidation modification.

[0035] The present invention also provides a polynucleotide sequence selected from:

[0036] (1) a polynucleotide sequence encoding the polypeptide of the present invention; and

[0037] (2) A complementary sequence of the polynucleotide sequence described in (1).

[0038] In one embodiment, the polynucleotide sequence is selected from SEQ ID NO: 32, 33, 34, 35, 37 and 40.

[0039] In a specific embodiment, the polynucleotide sequence is selected from a polynucleotide sequence consisting of bases 1 to 117, 114, 111, 108, 102, 99, 96, 93, 87, 84, 81 or 78 of SEQ ID NO:32.

[0040] In a specific embodiment, the polynucleotide sequence is selected from a polynucleotide sequence consisting of bases 1 to 117, 114, 111, 108, 105, 102, 99, 96, 93, 87, 84, 81, 78 or 75 of SEQ ID NO:40.

[0041] The present invention also provides an expression vector containing the polynucleotide sequence of the present invention.

[0042] The present invention also provides a pharmaceutical composition, characterized in that it contains the polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0043] The present invention also provides use of the polypeptide or pharmaceutical composition of the present invention in preparing a drug for preventing or treating tumors.

[0044] In one embodiment, the tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, liver cancer, colon cancer, pancreatic cancer, rhabdomyosarcoma, retinoblastoma, Ewing sarcoma, neuroblastoma, and osteosarcoma.

[0045] The present invention also provides the use of the polypeptide or pharmaceutical composition of the present invention in the preparation of a drug for improving the efficacy of a chemotherapeutic drug.

[0046] In one embodiment, the chemotherapy drug is cisplatin, carboplatin or oxaliplatin.

[0047] The present invention also provides a method for preparing the amino acid sequence of the present invention, which comprises synthesizing the amino acid sequence using the Fmoc solid phase synthesis method. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1a and 1b The HPLC and MASS spectra of polypeptide P1 are shown respectively.

[0049] Figure 1c and 1d The HPLC and MASS spectra of polypeptide P2 are shown respectively.

[0050] Figure 1e and 1f The HPLC and MASS spectra of polypeptide P2T2S18 are shown, respectively.

[0051] Figure 1g and 1h The HPLC and MASS spectra of polypeptide P2T2N18 are shown respectively.

[0052] Figure 2 The results showed that P1, P2, P3, P4 peptides, endostatin and endostar had inhibitory biological activities on HUVEC.

[0053] Figure 3a and 3b HPLC and MASS spectra of polypeptide P2T2S18Δ1 are shown, respectively.

[0054] Figure 3c and 3d HPLC and MASS spectra of polypeptide P2T2S18Δ2 are shown, respectively.

[0055] Figure 3e and 3f HPLC and MASS spectra of polypeptide P2T2S18Δ3 are shown, respectively.

[0056] Figure 4 The results showed that some peptides have the biological activity of inhibiting HUVEC.

[0057] Figure 5a and 5b The inhibitory effects of several peptides on HUVEC and tumor cells HepG2 are shown respectively. Figure 5a In the figure, based on the cell viability at a concentration of 2.5 mg / ml, the curves from top to bottom represent the cell viability of P2S18, Endostar, Endostatin, P2T2, P2N18, P2, P2T2N18, and P2T2S18 (the curves of P2T2N18 and P2T2S18 partially overlap). Figure 5b In the figure, the cell viability at a concentration of 2.5 mg / ml is measured. From top to bottom, the curves represent the cell viability of Endostar, P2S18, Endostatin, P2T2, P2N18, P2, P2T2N18 and P2T2S18.

[0058] Figure 6 P2 and P2T2S18 peptides were shown to induce cell death in SPC-A-1 tumor cells in vitro.

[0059] Figure 7a and 7b The HPLC and MASS spectra of polypeptide P2T2S18-20 are shown respectively.

[0060] Figure 7c and 7d The HPLC and MASS spectra of polypeptide P2T2S18-25 are shown, respectively.

[0061] Figure 7e and 7f The HPLC and MASS spectra of polypeptide P2T2N18-35 are shown, respectively.

[0062] Figure 7g and 7h The HPLC and MASS spectra of polypeptide P2T2N18-40 are shown respectively.

[0063] Figure 7i and 7j The HPLC and MASS spectra of polypeptide P2T2N18-45 are shown, respectively.

[0064] Figure 8The graph shows the inhibition of HUVEC growth in vitro by the peptides. The graph shows the cell viability at a concentration of 180 μM, with the curves from top to bottom representing the cell viability of P2T2-15, P2T2S18-45, P2T2S18-40, P2T2S18-20, P2T2S18-25, P2T2S18-35, and P2T2S18, respectively.

[0065] Figure 9 The results show the in vitro inhibitory effects of P2 and P2T2S18 peptides on various tumor cell lines, including SMMC7721, SPC-A-1, A549, LS174T, BEL7402, CK-MES-1, and BxPC-3. Two bar graphs are shown for each tumor cell type: the left bar graph shows the results for P2, and the right bar graph shows the results for P2T2S18.

[0066] Figure 10 The graph shows the inhibitory effects of the peptides on the growth of tumor cells and HUVEC in vitro. The cell viability at a concentration of 0.5 mg / ml is shown in the figure. The curves from top to bottom represent the cell viability of P2, P2T2S18Δ3, P2T2S18Δ1, P2T2S18Δ2, and P2T2S18, respectively.

[0067] Figure 11a and 11b The results show that the polypeptides inhibit the growth of tumor cells in vivo. Figure 11a and 11b In the figure, taking the data from day 1 to day 21 after administration as an example, the curves from top to bottom represent the tumor volume TV and relative tumor volume RTV of the negative control, endostar, P2, endostatin, positive control and P2T2S18, respectively.

[0068] Figure 12a and 12b The results of the inhibition of tumor cell growth in vivo by peptides combined with chemotherapy drugs are shown respectively. Figure 12a and 12b In the figure, taking the data from day 1 to day 21 of drug administration as an example, the curves from top to bottom respectively show the tumor volume TV and relative tumor volume RTV of negative control, DDP (2 mg / kg), endostar+DDP, P2+DDP, endostatin+DDP, DDP (6 mg / kg) and P2T2S18+DDP.

[0069] Figure 13 The results showed that the peptides inhibited the growth of HUVEC in vitro.

[0070] Figure 14a and 14b The HPLC and MASS spectra of polypeptide P2T2S18-29 are shown, respectively.

[0071] Figure 15 The graph shows the inhibitory effect of peptide P2T2S18-29 on HUVEC. Based on the cell viability at 2.5 mg / ml, the curves from top to bottom represent Endostar, Endostatin, P2, P2T2S18-29, and P2T2S18, respectively.

[0072] Figure 16 The graph shows the inhibitory effect of peptide P2T2S18-29 on HepG2 tumor cells. The curves from top to bottom represent Endostar, Endostatin, P2, P2T2A18, and P2T2S18-29, respectively, based on cell viability at 1 mg / ml. DETAILED DESCRIPTION

[0073] The polypeptide of the present invention is a fragment of endostatin with an N-terminal length of less than 45 amino acid residues, containing at least the 1st to 20th amino acid residues of endostatin N-terminus, wherein:

[0074] (1) the residue corresponding to the second amino acid at the N-terminus of endostatin is A, R, N, D, Q, E, H, I, L, K, M, F, P, T, W, Y, or V; and

[0075] (2) the residue corresponding to the 18th amino acid at the N-terminus of endostatin is A, R, N, D, C, E, G, H, I, L, K, M, F, S, T, W, Y, or V;

[0076] The inhibitory rate of the polypeptide on HUVEC is at least 15% higher, preferably at least 20% higher, than that of the corresponding sequence without mutation under the same concentration conditions; or the IC of the polypeptide is higher than that of the corresponding sequence without mutation. 50 The concentration is the latter IC 50 One-half of the concentration, preferably the former IC 50 The concentration is the latter IC 50 One fifth of the concentration, further preferably the former IC 50 The concentration is the latter IC 50 One tenth of the concentration.

[0077] Endostatin is preferably human endostatin. SEQ ID NO: 1 shows an example of recombinant human endostatin. Preferably, the amino acid sequence of the present invention contains at least the N-terminal amino acid residues 1-20 of endostatin as described in SEQ ID NO: 1, and the amino acids 2 and 18 are as described herein.

[0078] Preferably, the residue corresponding to the second amino acid at the N-terminus of the polypeptide is D, L, T, W or Y. Preferably, the residue corresponding to the 18th amino acid at the N-terminus of the polypeptide is N, E, K, M, S, T or V. More preferably, the residue corresponding to the second amino acid at the N-terminus of the polypeptide is D, T, W or Y. More preferably, the residue corresponding to the 18th amino acid at the N-terminus of the polypeptide is N, S or V.

[0079] In certain embodiments, the polypeptide corresponding to the amino acid residue at position 2 and the amino acid residue at position 18 of endostatin are the following combinations:

[0080]

[0081]

[0082] Alternatively, in certain embodiments, the polypeptide corresponding to the amino acid residue at position 2 and the amino acid residue at position 18 of endostatin are the following combinations:

[0083]

[0084]

[0085] It should be understood that "fragment" refers to a contiguous sequence that is a portion of a full-length sequence. For example, the polypeptide herein preferably consists of the sequence from amino acid position 1 at the N-terminus of endostatin to amino acid residues 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45, with the amino acid residues described herein at positions 2 and 18. In other words, the polypeptide of the present invention is 20-45 amino acid residues in length, starting from the first amino acid residue at the N-terminus of endostatin. More preferably, the polypeptide of the present invention is 25-40 amino acid residues in length, starting from the first amino acid residue at the N-terminus.

[0086] In certain embodiments, the fragments may optionally have the following residues at any one, any two, any three, or all four of positions 17, 20, 21, and 22, in addition to the amino acid residues described herein at positions 2 and 18:

[0087] Amino acid residue 17: S, A, L, I, V, or T;

[0088] Amino acid residue 20: S or T;

[0089] Amino acid residue 21: G, A, L, I, or V;

[0090] Amino acid residue at position 22: G, A, L, I or V.

[0091] Therefore, in certain embodiments, a polypeptide of the present invention that is a fragment of endostatin within 45 amino acid residues of the N-terminus comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1-25 of SEQ ID NO: 38, with amino acid residues 2 and 18 being the residues described herein, and any one, any two, any three, or all four of positions 17, 20-22 being the residues described above. Furthermore, such a polypeptide is 25-40 amino acid residues in length.

[0092] In certain embodiments, a polypeptide of the present invention, which is a fragment of endostatin within 45 amino acid residues of the N-terminus, comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1-25 of SEQ ID NO: 38, wherein amino acid residue 2 is T, and amino acid residue 18 is N, G, K, M, F, S, or T (more preferably N or S), and optionally, amino acids 17, 20, 21, and 22 are as described above. Furthermore, such a polypeptide is 25-40 amino acid residues in length.

[0093] In certain embodiments, a polypeptide of the present invention, which is a fragment of endostatin within 45 amino acid residues of the N-terminus, comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1-25 of SEQ ID NO: 38, wherein amino acid residue 18 is N and amino acid residue 2 is T, and optionally, amino acids 17, 20, 21, and 22 are as described above. Furthermore, such a polypeptide is 25-40 amino acid residues in length.

[0094] In certain embodiments, a polypeptide of the present invention, which is a fragment of endostatin within 45 amino acid residues of the N-terminus, comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1-25 of SEQ ID NO: 38, wherein amino acid residue 18 is S, and amino acid residue 2 is E, H, L, T, W, or V, and optionally, amino acids 17, 20, 21, and 22 are as described above. Furthermore, such a polypeptide is 25-40 amino acid residues in length.

[0095] The amino acid sequences of preferred polypeptides of the present invention are shown in SEQ ID NO: 4, 5, 6, 7, 27-30, 39 or 41. The polypeptides of the present invention also include an amino acid sequence consisting of amino acid residues 1 to 39, 38, 37, 36, 34, 33, 32, 31, 29, 28, 27 or 26 of SEQ ID NO: 4, and an amino acid sequence consisting of amino acid residues 1 to 39, 38, 37, 36, 35, 34, 33, 32, 31, 29, 28, 27, 26 or 25 of SEQ ID NO: 39.

[0096] The first amino acid residue at the N-terminus of the polypeptide of the present invention is histidine, which can be modified by formylation, acetylation, propionylation, or butyrylation, and the first amino acid at the C-terminus can be modified by PEG, cholesterol, or amidation.

[0097] Preferably, the first amino acid residue histidine at the N-terminus of the polypeptide of the present invention is acetylated, and the first amino acid residue at the C-terminus is amidated.

[0098] It should be understood that during gene cloning, it is often necessary to design appropriate restriction sites, which inevitably introduces one or more irrelevant residues at the termini of the expressed amino acid sequence. However, this does not affect the activity of the target sequence. For example, to construct fusion proteins, promote expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate purification of recombinant proteins, it is often necessary to add amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. Examples include, but are not limited to, suitable linker peptides, signal peptides, leader peptides, and terminal extensions. The amino acid sequences of the present invention may also contain one or more polypeptide fragments at the amino or carboxyl termini as protein tags. Any suitable tag may be used in the present invention. For example, the tags may include FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins. Examples of tags used include Poly-Arg, such as RRRRR (SEQ ID NO: 42); Poly-His 2-10 (usually 6), such as HHHHHH (SEQ ID NO: 43); FLAG, i.e. DYKDDDDK (SEQ ID NO: 44); Strep-TagII, i.e. WSHPQFEK (SEQ ID NO: 45); and C-myc, i.e. WQKLISEEDL (SEQ ID NO: 46).

[0099] Therefore, the present invention also includes a polypeptide containing the tag sequence or consisting of the tag sequence and the aforementioned fragment.

[0100] The amino acid sequences of the present invention may be products of chemical synthesis or recombinant polypeptides produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, filamentous fungi, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the present invention may be glycosylated or non-glycosylated.

[0101] For example, the amino acid sequence of the present invention can be synthesized using polypeptide chemical synthesis methods well known in the art. Polypeptide chemical synthesis methods include solid-phase synthesis and liquid-phase synthesis, of which solid-phase synthesis is commonly used. Solid-phase synthesis methods include, but are not limited to, two common methods: Fmoc and tBoc. Typically, a resin is used as an insoluble solid-phase support, and amino acids are typically linked to a peptide chain one by one from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus). Each amino acid linking cycle consists of the following three steps: 1) deprotection: the protected amino acid must be treated with a deprotection solvent to remove the amino protecting group; 2) activation: the carboxyl group of the amino acid to be linked is activated by an activating agent; and 3) coupling: the activated carboxyl group reacts with the exposed amino group of the previous amino acid to form a peptide bond. The cycle is repeated until the peptide chain is extended to the desired length. Finally, the connection between the peptide chain and the solid-phase support is cut with a cutting solution to obtain the desired amino acid sequence. The above chemical synthesis can be performed on a program-controlled automated peptide synthesizer, such as the Tribute dual-channel peptide synthesizer launched by Protein Technologies, the UV Online Monitor system launched by CS Bio, and the Focus XC three-channel synthesizer launched by Aapptec.

[0102] The present invention also includes polynucleotides encoding the polypeptides of the present invention. For example, SEQ ID NO:30 shows the coding sequence of SEQ ID NO:1; SEQ ID NO:31 shows the coding sequence of SEQ ID NO:3; SEQ ID NO:32 shows the coding sequence of SEQ ID NO:4; SEQ ID NO:33 shows the coding sequence of SEQ ID NO:5; SEQ ID NO:34 shows the coding sequence of SEQ ID NO:6; SEQ ID NO:35 shows the coding sequence of SEQ ID NO:7; SEQ ID NO:36 shows the coding sequence of SEQ ID NO:8; SEQ ID NO:37 shows the coding sequence of SEQ ID NO:9; and SEQ ID NO:40 shows the coding sequence of SEQ ID NO:39.

[0103] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the aforementioned DNA sequence or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes the amino acid sequence of the present invention but differs from the sequence set forth in SEQ ID NO:31, etc.

[0104] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0105] The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity.

[0106] The nucleotide sequences of the present invention can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified in the correct order.

[0107] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0108] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0109] Currently, DNA sequences encoding the amino acid sequences of the present invention can be obtained entirely through chemical synthesis, and the DNA sequences can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0110] The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention, and methods for producing the polypeptides of the present invention by recombinant technology. Preferably, the vectors of the present invention are expression vectors.

[0111] The polynucleotide sequences of the present invention can be used to express or produce the polypeptides of the present invention by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0112] (1) transforming or transducing a suitable host cell with the polynucleotide of the present invention or a degenerate variant thereof, or with a recombinant expression vector containing the polynucleotide;

[0113] (2) host cells cultured in a suitable culture medium;

[0114] (3) Isolate and purify proteins from culture medium or cells.

[0115] The polynucleotide sequences of the present invention can be inserted into recombinant expression vectors. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it generally contains an origin of replication, a promoter, a marker gene, and translation control elements. Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator.

[0116] Methods well known to those skilled in the art can be used to construct expression vectors containing the nucleic acid sequences of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The nucleic acid sequences can be operatively linked to appropriate promoters within the expression vector to direct mRNA synthesis. Representative examples of these promoters include the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses.

[0117] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0118] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0119] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; filamentous fungal cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast, filamentous fungi, and plant cells; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0120] When the polynucleotides of the present invention are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, that act on the promoter to enhance gene transcription.

[0121] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0122] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0123] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0124] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods. Various methods for preparing polypeptides by recombinant technology are known in the prior art.

[0125] The present invention also provides a pharmaceutical composition comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0126] Pharmaceutical compositions may contain a therapeutically or prophylactically effective amount of a polypeptide of the present invention. An "effective amount" refers to an amount of a component sufficient to produce the desired response. The specific effective amount depends on a variety of factors, such as the specific condition being treated, the patient's physical condition (e.g., weight, age, or gender), the duration of treatment, co-administered therapies (if any), and the specific formulation used. An "effective amount" also refers to an amount in which the toxic or adverse effects of the polypeptide of the present invention are outweighed by its positive therapeutic effects.

[0127] Pharmaceutically acceptable carriers are generally safe and non-toxic and, in a broad sense, include any known substance used in the pharmaceutical industry to prepare pharmaceutical compositions, such as fillers, diluents, agglutinants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, disintegrants, etc. When selecting an excipient suitable for delivering a synthetic peptide, the method of administration of the pharmaceutical composition should be considered, and those skilled in the art are familiar with this technology.

[0128] The content of the polypeptide in the pharmaceutical composition of the present invention is about 0.01-1000 μM.

[0129] The pharmaceutical compositions can be prepared according to known pharmaceutical procedures, such as those described in detail in Remington's Pharmaceutical Sciences, 17th ed., Alfonoso R. Gennaro, ed., Mack Publishing Company, Easton, Pennsylvania (1985).

[0130] The pharmaceutical composition of the present invention may be in various suitable dosage forms, including but not limited to tablets, capsules, injections, and the like.

[0131] The pharmaceutical composition of the present invention may also contain other known chemotherapeutic drugs, especially chemotherapeutic drugs known to treat or prevent tumors, including but not limited to cisplatin, carboplatin or oxaliplatin.

[0132] The polypeptide and pharmaceutical composition of the present invention can be used to treat or prevent various diseases that are known to be treated or prevented by endostatin, and to alleviate or relieve various symptoms that are known to be alleviated or relieved by endostatin.

[0133] For example, the polypeptides and pharmaceutical compositions of the present invention can be administered to a subject in need thereof for treating or preventing a tumor. The subject can be a mammal, particularly a human.

[0134] Tumors include hemangiomas and solid tumors. The solid tumors include, but are not limited to, rhabdomyosarcoma, retinoblastoma, Ewing sarcoma, neuroblastoma, osteosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, liver cancer, colon cancer, and pancreatic cancer.

[0135] The present invention also provides a method for treating cancer, which comprises administering the polypeptide or pharmaceutical composition of the present invention to a subject in need thereof.

[0136] The present invention also provides a method for improving the efficacy of chemotherapy drugs, which comprises administering the polypeptide or pharmaceutical composition of the present invention before, simultaneously with, or after administering the chemotherapy drugs to a subject in need thereof.

[0137] The present invention also provides the use of the polypeptide or pharmaceutical composition of the present invention in preparing a drug for treating or preventing tumors.

[0138] The present invention also provides use of the polypeptide or pharmaceutical composition of the present invention in the preparation of a drug for improving the efficacy of chemotherapy drugs.

[0139] The present invention also provides a polypeptide for use as a drug, wherein the polypeptide is as described in the above aspects or further embodiments of the present invention. The present invention also provides a polypeptide for treating or preventing the various tumors described above or for improving the efficacy of chemotherapy drugs, wherein the polypeptide is as described in the above aspects or embodiments of the present invention.

[0140] Example

[0141] The application will be further described in connection with specific examples. Practice of the application will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, which are within the skill of the art. Such procedures are fully described in the literature. See, e.g., Peptide: Chemistry and Biology, by N. Hugli, H. D. Jakubke, translated by Liu Keliang, He Junlin et al; Fundamental Virology, Second Edition, Vols. I and II (B. N. Fields and D. M. Knipe, eds); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds, Blackwell Scientific Publications); T. E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc. current addition); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed., 1989; Methods in Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and the literature cited within. In addition, it should be understood that "comprising" as used herein is also meant to include "consisting of." The amino acid sequence numbers used herein, i.e., "SEQ ID NO: 1-29, 38, 39 and 41," refer only to the amino acid sequence itself and do not include N-terminal and C-terminal modifications.

[0142] Example 1: Preparation and modification of polypeptides

[0143] The polypeptides were synthesized according to standard Fmoc protocol for polypeptide synthesis, starting with 0.25 mM resin and extending one residue at a time from the carboxy terminus to the amino terminus according to the sequences in the table below, with the final addition of N-terminal modifications. After the synthesis of the peptide was complete, the peptide was cleaved with cleavage solution, the resin was filtered out with a G6 glass frit funnel, the filtrate was vacuumed dry, and the C-terminus of the polypeptide was further amidated. The polypeptide product was dissolved in deionized water, Purification was performed using an Explorer 100 medium-pressure liquid chromatograph (MPLC) C18 column, with the main peak collected in separate steps. The target peak was analyzed for purity using an Agilent 1100 reversed-phase HPLC Phenomenex C18 analytical column and molecular weight confirmed using an LCQ Advantage mass spectrometer. The resulting MPLC fraction was lyophilized, dissolved in PBS to form a peptide stock solution, sterilized by filtration at 0.20 μM, and stored at -80°C. HPLC purity and MASS molecular weight confirmation are shown in Figure 1.

[0144]

[0145] Example 2: Isolation and culture of human umbilical vein endothelial cells (HUVEC)

[0146] Prepare umbilical cord preservation solution: 150ml PBS + 3 times the working concentration of double antibody (cyan / strand); prepare complete culture medium: 80ml M199 + 20ml FBS + 1ml ECGS + 1ml 100X double antibody + 1ml heparin solution (0.5% W / V) + 1ml 200mM glutamine; prepare separation instruments: 1 surgical curved tray, 4-5 vascular forceps, 2 surgical scissors, and a glass culture dish with a diameter of about 10cm; configuration of type I collagenase: configure it to 1% (W / V).

[0147] Take a 20cm umbilical cord close to the fetal end, rinse it clean, tie both ends, and place it in 150ml of umbilical cord preservation solution; store it in a refrigerator at 4 degrees and digest it within 6 hours; inspect the umbilical cord, remove the damaged part, rinse the umbilical vein thoroughly, then inject 10ml of collagenase solution, transfer it to a 37℃ incubator and digest it for 15 minutes; remove the umbilical cord, collect the digestion solution, wash it with PBS, resuspend it after centrifugation and culture, and change the solution after 24 hours to remove cells that cannot adhere to the wall.

[0148] Example 3: Inhibition of Peptides on Human Umbilical Vein Endothelial Cells (HUVEC) and Tumor Cells

[0149] MTT method was used to detect the growth inhibition of cells. The principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyl tetrazolium bromide) to water-insoluble blue-violet formazan and deposit in cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at 490 / 570 nm wavelength by enzyme-linked immunosorbent assay instrument, which can indirectly reflect the number of living cells. Within a certain range of cell number, the amount of MTT crystal formation is proportional to the cell number. For HUVEC or tumor cells in logarithmic growth phase, discard the culture supernatant, wash with PBS once, add 1 ml of 0.25% trypsin (4°C), digest at 37°C for 2 min, add to the culture supernatant, neutralize, blow the cells into a suspension, and centrifuge at 1000 rpm for 3 min. Discard the supernatant, resuspend in 5 ml of culture medium. Inoculate 3x10 4 / ml into a 48-well plate, 500ul / well. Incubate at 37°C in 5% CO2 for 24 hours. Discard the culture supernatant of the cultured cells, add the culture medium containing the polypeptide (the culture medium contains Zn 2+ concentration of 17.39umol / L), and continue to culture for 48 hours. Carefully discard the supernatant in each well, gently rinse once with 450ul PBS per well. Add 450ul MTT culture medium to each well and continue to culture for 4h. Carefully discard the culture supernatant, add dimethyl sulfoxide 450ul / well, and place on a shaker in the dark at low speed for 10min. Transfer 150ul of supernatant to a 96-well ELISA plate, and measure the absorbance of each well at OD490nm, 570nm on an enzyme-linked immunosorbent assay instrument.

[0150] Example Four: Effect of N-terminal modification and C-terminal modification of polypeptide on its activity

[0151] The polypeptide of SEQ ID NO:2 shown in the table below was synthesized according to the method shown in Example One, with or without N-terminal and / or C-terminal modification, wherein Ac is acetylation modification, and NH2 is amidation modification. HPLC purity identification and MASS molecular weight identification were performed.

[0152] Peptide number Sequence (from N-terminus to C-terminus) P1 HSHRDFQPVLHLVALNSPLSGGMRGIRGAD P2 <![CDATA[Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2]]> P3 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD P4 <![CDATA[HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2]]>

[0153] Recombinant human endostatin (SEQ ID NO:1) can be purchased from the market (for example, genetex product number GTX65524, BioVision product number 4799-1000, Shanghai Boseng Biotechnology Co., Ltd. product number E2296-05, Wuhan Biode Biotechnology Co., Ltd. product number BP4153, etc.). The recombinant human endostatin drug endostar (SEQ ID NO:10) is purchased from a medical institution.

[0154] According to the method shown in Example 3, the biological activity of P1, P2, P3, P4 polypeptides, endostatin, and endostar on HUVEC inhibition was determined under the experimental conditions of a polypeptide concentration of 1 mg / ml and a recombinant human endostatin concentration of 5 mg / ml (the polypeptide and endostatin were close to equimolar concentrations). The results are shown in FIG. Figure 2 .

[0155] Example 5: Structure-activity relationship study of polypeptides

[0156] The peptides shown in the following table were synthesized by the method of Example 1, and their HPLC purity and MASS molecular weight were determined. The biological activity of the peptides against HUVEC was determined at a concentration of 1 mg / ml according to the method of Example 3. The results are shown in the table below. Figure 4 .

[0157]

[0158] Based on the published structure of endostatin (PDB database structure number 1BNL) in the literature (EMBO J. 1998 Mar 16; 17(6): 1656–1664), INSIGHT II software was used to perform homology modeling on the above peptides to obtain the dominant conformation of the P2 peptide. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies of the P2 peptide were then calculated using the fully activated space self-consistent field (CASSCF). On this basis, we used our original algorithm - 2-D synergistic iterative algorithms in spacialpoint field (2-DSIASPF) - to simulate the iterative replacement of amino acids in the P2 peptide in pairs. We calculated the cumulative electron density variance caused by the replacement on the Zn ion binding active domain (1H, 3H, 11H) of the peptide. Combined with the biological activity corresponding to the actual tested amino acid combinations, we scored the synergistic relationship between the two amino acid sites of P2. The results showed that the synergistic effect of amino acids at positions 2 and 18 on the biological activity was the greatest.

[0159] Example 6: Effect of amino acid substitution on peptide activity

[0160] A P2 peptide combinatorial peptide library was constructed for amino acids 2 and 18. The peptides shown in the following table were synthesized using the Apex396 fully automated high-throughput peptide synthesizer from AAPPTEC, where X1 and X3 are any natural amino acids (see the table below), X2 and X4 are S, and X5 and X6 are G.

[0161]

[0162] According to the method shown in Example 3, the biological activity of the above polypeptide in inhibiting HUVEC was determined at a concentration of 1 mg / ml. The results are shown in the table below.

[0163]

[0164] In the table, lowercase a, b, c, d, e, f, g, h, i, and j represent:

[0165] a: cell activity 0-10%; b: cell activity 11-20%; c: cell activity 21-30%; d: cell activity 31-40%; e: cell activity 41-50%; f: cell activity 51-60%; g: cell activity 61-70%; h: cell activity 71-80%; i: cell activity 81-90%; j: cell activity 91-100%.

[0166] Example 7: Inhibition of tumor cell and HUVEC growth in vitro by polypeptides

[0167] The polypeptides shown in the following table were synthesized using the method described in Example 1, and their purity was determined by HPLC and their molecular weight was determined by MASS spectrometry.

[0168]

[0169] The inhibitory effects of the recombinant endostatin of SEQ ID NO: 1 and the marketed drug endostar of SEQ ID NO: 10 on HUVEC and HepG2 tumor cells were tested according to the method shown in Example 3. The results are shown in Table 1. Figure 5a 、 Figure 5b The results show that the biological activities of P2T2S18 and P2T2N18 are significantly higher than those of P2, and their IC 50 The concentration is about 10 times lower than that of P2. The biological activity of the polypeptide P2T2 carrying a single point mutation at amino acid position 2 and P2N18 and P2S18 carrying single point mutations at amino acid position 18 are all lower than that of P2. Therefore, the high biological activity of P2T2S18 and P2T2N18 is caused by an unexpected synergistic effect caused by the combined mutation of amino acids at positions 2 and 18. It can be seen that it is difficult to obtain the highly biologically active structures of P2T2S18 and P2T2N18 involved in the present invention through conventional single-point mutation scanning methods.

[0170]

[0171]

[0172] Example 8: Peptide-induced death of SPC-A-1 tumor cells in vitro

[0173] As described in Example 3, the peptide was tested for its effect on inducing cell death in the SPC-A-1 lung cancer cell line at a peptide concentration of 2.5 mg / ml. The cells were observed under an optical microscope for 24 hours and photographed. Figure 6 As shown, P2 takes effect 4 hours after being added to the culture medium, the cells appear to shrink, and the cells are basically dead after 24 hours. The way P2 induces cell death is similar to apoptosis. P2T2S18 exerts a drastic effect on the cells 2 hours after being added to the culture medium, but the cells do not shrink, but extremely swell; the swelling is further aggravated after 4 hours, the cells begin to fragment at 8 hours, and only cell fragments remain at 24 hours. The cell death mode caused by P2T2S18 is very surprising. It is currently impossible to determine what mode of cell death this is, and there is no literature report. But it is obvious that this cell death mode is significantly different from the cell death caused by P2. Combined with the results of Example 7, it can be seen that P2T2S18 not only has a significantly higher biological activity than P2, but also causes cell death in a way that is significantly different from P2.

[0174] Example 9: Inhibition of HUVEC Growth in Vitro by Peptides

[0175] The peptides shown in the following table were synthesized by the method shown in Example 1. The purity was determined by HPLC and the molecular weight was determined by MASS spectrometry as shown in Figures 1 and Figures 7a-7j .

[0176]

[0177] The inhibitory activity of the peptides against HUVEC was tested according to the method shown in Example 3. Each peptide was tested under equimolar concentration conditions, where 300uM of the P2T2S18 peptide was equivalent to approximately 1mg / ml. Figure 8 The table below shows that the C-terminus of P2T2S18 still has biological activity after being shortened or extended within a certain range.

[0178] Peptide number Sequence number Cell viability (%) when the peptide concentration is 120uM P2T2S18-45 SEQ ID NO:3 80 P2T2S18-40 SEQ ID NO:4 49 P2T2S18-35 SEQ ID NO:5 15 P2T2S18 SEQ ID NO:6 2 P2T2S18-25 SEQ ID NO:7 31 P2T2S18-20 SEQ ID NO:8 52 P2T2-15 SEQ ID NO:24 95

[0179] Example 10: Inhibition of the growth of various tumor cells in vitro by polypeptides

[0180] According to the method shown in Example 3, the in vitro inhibitory effects of P2 (SEQ ID NO: 2) and P2T2S18 (SEQ ID NO: 6) polypeptides on various tumor cells, SMMC7721, SPC-A-1, A549, LS174T, BEL7402, CK-MES-1, and BxPC-3, were tested at a polypeptide concentration of 1 mg / ml. The test results are shown in FIG. Figure 9As shown in the table below, P2T2S18 has significantly higher inhibitory activity against various tumors than P2. The IC 50 The concentrations were all less than P2 IC 50 In particular, P2 showed no inhibitory activity against LS174T colon cancer cells, with a cell survival rate of up to 95% at a drug concentration of 1 mg / ml. However, P2T2S18 exhibited significant inhibitory activity against these cells, killing almost all tumor cells at the same concentration, with a cell survival rate of only 1%.

[0181]

[0182] Example 11: Inhibition of tumor cell and HUVEC growth in vitro by peptides

[0183] The peptides shown in the following table were synthesized using the method of Example 1. The purity of the peptides was determined by HPLC and the molecular weight was determined by MASS spectrometry as shown in Figures 1 and Figures 3a-3f The inhibitory effect on HUVEC was tested according to the method shown in Example 3. The results are shown in Figure 10 .

[0184] Peptide number Sequence number Sequence (from N-terminus to C-terminus) P2 SEQ ID NO:2 <![CDATA[Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2]]> P2T2S18 SEQ ID NO:6 <![CDATA[Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2]]> P2T2S18Δ1 SEQ ID NO:6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD P2T2S18Δ2 SEQ ID NO:6 <![CDATA[HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2]]> P2T2S18Δ3 SEQ ID NO:6 HTHRDFQPVLHLVALNSSLSGGMRGIRGAD

[0185] Example 12: Establishment of in vivo tumor model

[0186] Tumor cells in the logarithmic growth phase that are in good condition in vitro were taken and inoculated subcutaneously into nude mice containing 5×10 6 100ul of cultured cell suspension of tumor cells. After 15 days, take the well-grown solid tumor, cut it into uniform small pieces of about 3mm in size under sterile conditions, and inoculate one piece subcutaneously in the right armpit of each nude mouse with a trocar. 10-14 days after inoculation, regroup according to the size of the tumor, eliminate animals with tumors that are too large or too small, and the average volume of tumors in each group is basically the same. Each group is given the test drug according to the experimental plan. The long diameter (a) and short diameter (b) of the tumor mass are measured twice a week. After the experiment, the animals are killed, the tumor mass is dissected, the tumor weight is weighed, and photos are taken. Tumor volume TV = 1 / 2×a×b 2 Relative tumor volume (RTV) = Vt / Vo, where Vo is the tumor volume measured at cage placement (i.e., one day before dosing) and Vt is the tumor volume at each measurement. Tumor inhibition rate (%) = (1-T / C) * 100%, where T is the mean tumor volume of the treatment group and C is the mean tumor volume of the negative control group.

[0187] Example 13: Inhibition of tumor cell growth in vivo by polypeptides

[0188] The polypeptides with the sequences shown in the table below were synthesized using the method of Example 1. The purity identification by HPLC and the molecular weight identification by MASS spectrometry are shown in FIG1 .

[0189] Peptide number Sequence number Sequence (from N-terminus to C-terminus) P2 SEQ ID NO:2 <![CDATA[Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2]]> P2T2S18 SEQ ID NO:6 <![CDATA[Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2]]>

[0190] For example, the recombinant endostatin of SEQ ID NO: 1 and the marketed drug endostar of SEQ ID NO: 10. A human liver cancer BEL7404 tumor model was established as described in Example 10. Six groups were set up in the experiment. In addition to the negative control group of 9 animals, the other experimental groups each had 6 animals. The dosages of the polypeptide and endostatin were approximately equimolar. The experimental results are shown in Figure 11a 、 Figure 11b and the table below.

[0191] 1) Negative control group (normal saline, subcutaneous injection, 2 times / day, continuous administration for 21 days)

[0192] 2) Cyclophosphamide (CTX) (30 mg / kg, intraperitoneal injection, once a day, for 7 consecutive days)

[0193] 3) P2 group (15 mg / kg / time, sc, 2 times / day, continuous administration for 21 days)

[0194] 4) P2T2S18 group (15 mg / kg / time, sc, twice / day, for 21 consecutive days)

[0195] 5) Endostatin (50 mg / kg / time, subcutaneous, twice / day for 21 consecutive days)

[0196] 6) Endostar (50 mg / kg / time, subcutaneous, twice / day, for 21 consecutive days)

[0197]

[0198] A t-test was performed on the RTV between the experimental groups, and the p-values ​​are shown in the table below. It can be seen that P2 was unable to significantly inhibit tumor growth, with no significant difference compared to the negative control group (P = 0.015). P2T2S18, on the other hand, was able to significantly inhibit tumor growth, with the tumor inhibition rate reaching 76.7% after 21 days of administration. In fact, the inhibitory effect of P2T2S18 on tumors was close to that of the chemotherapy drug CTX, with no significant difference in RTV between the two groups (P > 0.01). It is worth noting that the animals in the P2T2S18 experimental group did not show any toxic reactions, while the CTX group showed typical chemotherapy side effects. The effect of P2T2S18 in inhibiting tumor growth was significantly better than that of P2 (P < 0.001), endostatin (P < 0.001), and endostar (P < 0.001).

[0199]

[0200] Example 14: Inhibition of tumor cell growth in vivo by peptides combined with chemotherapy drugs

[0201] The polypeptides with the sequences shown in the table below were synthesized using the method of Example 1. The purity identification by HPLC and the molecular weight identification by MASS spectrometry are shown in FIG1 .

[0202] Peptide number Sequence number Sequence (from N-terminus to C-terminus) P2 SEQ ID NO:2 <![CDATA[Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2]]> P2T2S18 SEQ ID NO:6 <![CDATA[Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2]]>

[0203] For example, recombinant endostatin (SEQ ID NO: 1) and marketed drug endostar (SEQ ID NO: 10) were used. A human lung cancer A549 tumor model was established as described in Example 10. Seven groups of six animals were included in the experiment. The doses of the polypeptide and endostatin were equimolar.

[0204] 1) Negative control group: normal saline, sc, twice a day, for 21 consecutive days;

[0205] 2) low-dose cisplatin (DDP) group: 2 mg / kg / day, ip, once a day, for 7 consecutive days);

[0206] 3) P2+DDP group:

[0207] DDP: 2 mg / kg / day, ip, once a day, for 7 consecutive days.

[0208] P2: 15 mg / kg / time, subcutaneous, twice / day for 21 days;

[0209] 4) P2T2S18+DDP group:

[0210] DDP: 2 mg / kg / day, ip, once a day, for 7 consecutive days.

[0211] P2T2S18: 15 mg / kg subcutaneously twice a day for 21 days.

[0212] 5) Recombinant endostatin SEQ ID NO: 1+DDP group:

[0213] DDP: 2 mg / kg / day, ip, once a day, for 7 consecutive days.

[0214] Endostatin: 50 mg / kg / time, subcutaneously, twice a day for 21 days;

[0215] 6) Recombinant endostatin SEQ ID NO: 10 + DDP group:

[0216] DDP: 2 mg / kg / day, ip, once a day, for 7 consecutive days.

[0217] Endostar: 50 mg / kg / time, subcutaneously, twice a day for 21 days;

[0218] 7) DDP high-dose group: 6 mg / kg / day, ip, once a day, for 7 consecutive days.

[0219] The test results are shown in Figure 12a 、 Figure 12b and the table below.

[0220]

[0221] A t-test was performed on the RTVs of the experimental groups, and the p-values ​​are shown in the table below. It can be seen that P2 combined with DDP (2 mg / kg) did not significantly enhance the tumor-suppressing effect of DDP (2 mg / kg), with no significant difference in RTV between the two experimental groups (P = 0.011). The tumor-suppressing effect of P2 combined with DDP (2 mg / kg) was significantly worse than that of DDP (6 mg / kg), with a significant difference in RTV between the two experimental groups (P < 0.001). P2T2S18 significantly enhanced the tumor-suppressing effect of DDP (2 mg / kg). The tumor-suppressing effect of P2T2S18 combined with DDP (2 mg / kg) was significantly better than that of DDP (2 mg / kg), with a significant difference in RTV between the two experimental groups (P < 0.001). In fact, P2T2S18 combined with DDP (2 mg / kg) showed significantly better tumor inhibition than DDP (6 mg / kg). After 21 days of administration, the tumor inhibition rate of P2T2S18 combined with DDP (2 mg / kg) reached 99.7%. Only 2 of the 6 experimental animals had residual tumors, and the tumors of the remaining 4 animals disappeared. In contrast, all 6 animals in the DDP (6 mg / kg) group had residual tumors after 21 days of administration. The RTV of the P2T2S18 combined with DDP (2 mg / kg) group was lower than that of the DDP (6 mg / kg) group, and the difference was significant (P < 0.001). It can be seen that the therapeutic effect of P2T2S18 combined with DDP (2 mg / kg) is better than that of DDP (6 mg / kg).

[0222] The therapeutic effect of P2T2S18 combined with DDP (2 mg / kg) group was significantly better than that of P2 combined with DDP (2 mg / kg) group ((P < 0.001), endostatin combined with DDP (2 mg / kg) group ((P < 0.001), and endostar combined with DDP (2 mg / kg) group ((P < 0.001).

[0223] It is worth noting that in the experiment, no obvious toxic reactions were observed in the animals in the P2T2S18 combined with DDP (2 mg / kg) experimental group and the DDP (2 mg / kg) group, while obvious chemotherapy toxic reactions were observed in the animals in the DDP (6 mg / kg) group.

[0224]

[0225] Example 15: Inhibition of tumor cell and HUVEC growth in vitro by polypeptides

[0226] The peptides shown in the following table were synthesized by the method of Example 1, and the inhibitory effect on HUVEC was tested at a peptide concentration of 0.1 mg / ml according to the method of Example 3. The results are shown in FIG. Figure 13 .

[0227]

[0228] Example 16: Inhibition of tumor cell and HUVEC growth in vitro by peptides

[0229] The peptides shown in the following table were synthesized by the method of Example 1. The purity was determined by HPLC and the molecular weight was determined by MASS spectrometry. Figure 14a and 14b The inhibitory effect on HUVEC and tumor cells HepG2 was tested according to the method shown in Example 3. The test results are shown in Figure 15 and 16 The results showed that the biological activities of P2T2S18 and P2T2S18-29 were similar and significantly higher than that of P2.

[0230] Peptide number Sequence number Sequence (from N-terminus to C-terminus) P2T2S18-29 SEQ ID NO:41 <![CDATA[Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2]]>

[0231] The above specific embodiments are merely illustrative and non-restrictive. The scope of protection of this application will be defined by the claims. Those skilled in the art will understand that various modifications and variations may be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and such modifications and variations are still within the scope of the present invention. Sequence Listing <110> Shanghai Hepu Pharmaceutical Co., Ltd. <120> A tumor suppressor peptide <130> 154374 <160> 46 <170> PatentIn version 3.3 <210> 1 <211> 183 <212> PRT <213> Homo sapiens <400> 1 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln 20 25 30 Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala 35 40 45 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala 50 55 60 Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe 65 70 75 80 Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro 85 90 95 Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro 100 105 110 Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg 115 120 125 Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser 130 135 140 Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln 145 150 155 160 Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn 165 170 175 Ser Phe Met Thr Ala Ser Lys 180 <210> 2 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Fragment of human endostatin <400> 2 His Ser His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 3 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Mutant of fragment of human endostatin <400> 3 His Thr His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gin 20 25 30 Cys Phe Gin Gin Ala Arg Ala Val Gly Leu Ala Gly Thr 35 40 45 <210> 4 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 4 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln 20 25 30 Cys Phe Gln Gln Ala Arg Ala Val 35 40 <210> 5 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 5 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln 20 25 30 Cys Phe Gln 35 <210> 6 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 6 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 7 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 7 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Ser Gly Gly Met Arg Gly 20 25 <210> 8 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 8 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Ser 20 <210> 9 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 9 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 10 <211> 192 <212> PRT <213> Artificial sequence <220> <223> Recombinant human endostatin drug Endostatin <400> 10 Met Gly Gly Ser His His His His His His Ser His Arg Asp Phe Gln 1 5 10 15 Pro Val Leu His Leu Val Ala Leu Asn Ala Pro Leu Ser Gly Gly Met 20 25 30 Arg Gly Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala 35 40 45 Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln 50 55 60 Asp Leu Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile 65 70 75 80 Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe 85 90 95 Ser Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe 100 105 110 Asp Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val 115 120 125 Trp His Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys 130 135 140 Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser 145 150 155 160 Leu Leu Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His 165 170 175 Ala Tyr Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys 180 185 190 <210> 11 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 11 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 12 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 12 His Ala His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 13 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 13 His Glu His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 14 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 14 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Ala Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 15 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 15 His Ser His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Ala 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly He Arg Gly Ala Asp 20 25 30 <210> 16 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 16 His Ser His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Thr Pro Leu Ser Gly Gly Met Arg Gly He Arg Gly Ala Asp 20 25 30 <210> 17 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 17 His Ser His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ala Pro Leu Ser Gly Gly Met Arg Gly He Arg Gly Ala Asp 20 25 30 <210> 18 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant fragment of human endostatin <400> 18 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ala Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 19 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 19 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Ala Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 20 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 20 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ala Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> twenty one <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> twenty one His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Ala Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> twenty two <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> twenty two His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Ala Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> twenty three <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> twenty three His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly Asp Arg Gly Ala Asp 20 25 30 <210> twenty four <211> 17 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> twenty four His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser <210> 25 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 25 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 26 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 26 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 27 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 27 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ala Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 28 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 28 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Thr Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 29 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Mutant fragment of human endostatin <400> 29 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ala Ser Leu Thr Gly Gly Met Arg Gly Ile Arg Gly Ala Asp 20 25 30 <210> 30 <211> 549 <212> DNA <213> 智人(Homo sapiens) <400> 30 cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca 60 ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120 gggctggcgg gcaccttccg cgccttcctg tcctcgcgcc tgcaggacct gtacagcatc 180 gtgcgccgtg ccgaccgcgc agccgtgccc atcgtcaacc tcaaggacga gctgctgttt 240 cccagctggg aggctctgtt ctcaggctct gagggtccgc tgaagcccgg ggcacgcatc 300 ttctcctttg acggcaagga cgtcctgagg caccccacct ggccccagaa gagcgtgtgg 360 catggctcgg accccaacgg gcgcaggctg accgagagct actgtgagac gtggcggacg 420 gaggctccct cggccacggg ccaggcctcc tcgctgctgg ggggcaggct cctggggcag 480 agtgccgcga gctgccatca cgcctacatc gtgctctgca ttgagaacag cttcatgact 540 gcctccaag 549 <210> 31 <211> 135 <212> DNA <213> 人工序列 <220> <223> 人内皮抑素片段的突变体的编码序列 <400> 31 cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60 ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120 gggctggcgg gcacc 135 <210> 32 <211> 120 <212> DNA <213> Artificial sequence <220> <223> Coding sequences of mutants of human endostatin fragments <400> 32 cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60 ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120 <210> 33 <211> 105 <212> DNA <213> Artificial sequence <220> <223> Coding sequences of mutants of human endostatin fragments <400> 33 cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60 ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccag 105 <210> 34 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Coding sequences of mutants of human endostatin fragments <400> 34 Cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60 Ggcggcatgc ggggcatccg cggggccgac 90 <210> 35 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence for a mutant of a human endostatin fragment <400> 35 Cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60 Ggcggcatgc ggggcatccg cggggccgac 90 <210> 36 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Coding sequence for a mutant of a human endostatin fragment <400> 36 Cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60 <210> 37 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Coding sequence for a mutant of a human endostatin fragment <400> 37 Cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag caacctgtca 60 Ggcggcatgc ggggcatccg cggggccgac 90 <210> 38 <211> 30 <212> PRT <213> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is any amino acid <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is S, A, L, I, V or T <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is any amino acid <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is S or T <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is G, A, L, I or V <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa is G, A, L, I or V <400> 38 His Xaa His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Xaa Xaa Leu Xaa Xaa Xaa Met Arg Gly lie Arg Gly Ala Asp 20 25 30 <210> 39 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a human endostatin fragment <400> 39 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln 20 25 30 Cys Phe Gln Gln Ala Arg Ala Val 35 40 <210> 40 <211> 120 <212> DNA <213> Artificial sequence <220> <223> Coding sequences of mutants of human endostatin fragments <400> 40 cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag caacctgtca 60 ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120 <210> 41 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Mutants of human endostatin fragments <400> 41 His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala 20 25 <210> 42 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Tags <400> 42 Arg Arg Arg Arg Arg 1 5 <210> 43 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Tags <400> 43 His His His His His His 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Tags <400> 44 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Tags <400> 45 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 46 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Tags <400> 46 Trp Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10

Claims

1. A polypeptide, characterized in that The polypeptide is a fragment of the 1st to 30th amino acid residues at the N-terminus of endostatin, the amino acid sequence of which is shown in SEQ ID NO: 1, wherein the 2nd amino acid residue of endostatin is replaced by E, and the 18th amino acid residue is replaced by S or V.

2. The polypeptide according to claim 1, wherein The first amino acid residue at the N-terminus of the polypeptide is histidine, which is modified by formylation, acetylation, propionylation or butyrylation, and the first amino acid at the C-terminus is modified by PEG, cholesterol or amidation.

3. A polynucleotide molecule, the polynucleotide sequence of which is selected from: (1) a polynucleotide sequence encoding the polypeptide according to any one of claims 1 to 2; and (2) A complementary sequence of the polynucleotide sequence described in (1).

4. An expression vector comprising the polynucleotide molecule according to claim 3.

5. A pharmaceutical composition, characterized in that It contains the polypeptide according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier.

6. Use of the polypeptide according to any one of claims 1 to 2 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for preventing or treating a tumor, wherein the tumor is selected from lung adenocarcinoma, lung squamous cell carcinoma, liver cancer and pancreatic cancer.

7. Use of the polypeptide according to any one of claims 1-2 or the pharmaceutical composition according to claim 5 in combination with a chemotherapeutic drug in the preparation of a medicament for preventing or treating tumors, wherein the chemotherapeutic drug is cisplatin, carboplatin or oxaliplatin, and the tumor is selected from lung adenocarcinoma, lung squamous cell carcinoma, liver cancer and pancreatic cancer.

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