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 described 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] The present application is a divisional application of the patent application with international application No. PCT / CN2016 / 090800, international filing date of July 21, 2016, entering China national phase application No. 201680044076.1, and the invention patent application with the invention name of "a tumor inhibiting peptide". TECHNICAL FIELD

[0002] The present application belongs to the field of tumor treatment, and particularly relates to a polypeptide for inhibiting and treating tumor, wherein the amino acid sequence of the polypeptide is a fragment of the N-terminal 1st amino acid residue to within 45 amino acid residues of endothelin. BACKGROUND

[0003] Endostatin is an endogenous angiogenesis inhibitor isolated and purified from the supernatant of cultured mouse endothelioma (EOMA) by O'Reilly et al. in 1997, which is a 20 kd molecular weight protein derived from the hydrolysis product of collagen XVIII. Experiments show that endothelin exerts inhibitory effect on vascular endothelium and tumor cells. Due to the difficulty in renaturation of recombinant endothelin and other factors, the clinical research of recombinant endothelin is abandoned by the American EntreMed company, and currently endothelin with high in vitro activity cannot be prepared in large quantities.

[0004] The zinc ion binding site composed of the 1st, 3rd and 11th histidine residues and the 76th aspartic acid residue in the N-terminal of endothelin sequence is essential to the activity of endothelin. It is reported that the polypeptide derived from the N-terminal of endothelin has certain activity in inhibiting vascular endothelial cells and tumor cells (Cancer Res. 2005; 65 (9): 3656-63, US patent US7524811B2). However, the above experiment also shows that the polypeptide derived from the N-terminal 1-25 amino acid residues of human endothelin cannot significantly inhibit the growth of human tumor inoculated on a mouse animal model, and the activity of endothelin-derived peptide needs to be improved. SUMMARY

[0005] The present application provides a polypeptide, which is a fragment of the N-terminal 1st amino acid residue to within 45 amino acid residues of endothelin, and contains at least the N-terminal 1st-20th amino acid residues, and wherein the N-terminal 2nd amino acid residue and the 18th amino acid residue are selected from the following groups, respectively:

[0006] 2nd amino acid 18th amino acid 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 residue in 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 residue is 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 the endostatin is as set forth in SEQ ID NO: 1.

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

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

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

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

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

[0014] In one embodiment, the polypeptide comprises at least the 1st to 22nd amino acid residues of SEQ ID NO: 38, and preferably at least the 1st to 25th amino acid residues of SEQ ID NO: 38, and the 18th amino acid residue is S, the 2nd amino acid residue is E, H, L, T, W or V, and the 17th, 20th, 21st and 22nd amino acids are as described above.

[0015] In one embodiment, the polypeptide comprises at least the 1st to 22nd amino acid residues of SEQ ID NO: 38, and preferably at least the 1st to 25th amino acid residues of SEQ ID NO: 38, and the 18th amino acid residue is S, the 2nd amino acid residue is E, H, L, T, W or V, and the 17th, 20th, 21st and 22nd amino acids are as described above.

[0016] In one embodiment, the polypeptide is selected from the group consisting of 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 the group consisting of:

[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] HTHRDFQPVLHLVALNSNLSGGMRGIRGAD;

[0027] Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD;

[0028] HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2;

[0029] Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2;

[0030] Ac-HTHRDFQPVLHLVALNASLSGGMRGIRGAD-NH2;

[0031] Ac-HTHRDFQPVLHLVALNSSLTGGMRGIRGAD-NH2;

[0032] Ac-HTHRDFQPVLHLVALNASLTGGMRGIRGAD-NH2; and

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

[0034] wherein Ac is an acetylated modification, and NH2 is an amidated modification.

[0035] The present application also provides a polynucleotide sequence selected from the group consisting of:

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

[0037] (2) a complement of the polynucleotide sequence of (1).

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

[0039] In one embodiment, the polynucleotide sequence is selected from the group consisting of 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 one embodiment, the polynucleotide sequence is selected from the group consisting of 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 application also provides an expression vector comprising the polynucleotide sequence of the present application.

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

[0043] The present application also provides use of the polypeptide or pharmaceutical composition of the present application in the preparation of a medicament for preventing or treating tumor.

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

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

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

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

[0048] Figure 1a and 1b show the HPLC and MASS spectra of polypeptide P2, respectively.

[0049] Figure 1c and 1d show the HPLC and MASS spectra of polypeptide P2, respectively.

[0050] Figure 1e and 1f show the HPLC and MASS spectra of polypeptide P2T2S18, respectively.

[0051] Figure 1g and 1h show the HPLC and MASS spectra of polypeptide P2T2N18, respectively.

[0052] Figure 2 show the biological activity of P1, P2, P3, P4 polypeptides, endostatin and endostar on HUVEC inhibition.

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

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

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

[0056] Figure 4 Biological activity of the polypeptides to inhibit HUVEC.

[0057] Figure 5a and 5b The inhibition of HUVEC and tumor cell HepG2 by several polypeptides, respectively. Figure 5a In Fig. 2A, from top to bottom, the curves represent the cell survival rate of P2S18, Endostar, Endostatin, P2T2, P2N18, P2, P2T2N18, P2T2S18 (the curves of P2T2N18 and P2T2S18 partially overlap) at the concentration of 2.5 mg / ml. Figure 5b In Fig. 2B, from top to bottom, the curves represent the cell survival rate of Endostar, P2S18, Endostatin, P2T2, P2N18, P2, P2T2N18 and P2T2S18 at the concentration of 2.5 mg / ml.

[0058] Figure 6 The polypeptides P2 and P2T2S18 induce the death of SPC-A-1 tumor cells in vitro.

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

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

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

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

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

[0064] Figure 8The figure shows the inhibition of the polypeptides on the growth of HUVEC in vitro. The cell survival rate at the concentration of 180 μM is shown. From top to bottom, the curves represent P2T2-15, P2T2S18-45, P2T2S18-40, P2T2S18-20, P2T2S18-25, P2T2S18-35 and P2T2S18, respectively.

[0065] Figure 9 The figure shows the inhibition of P2 and P2T2S18 polypeptides on the growth of SMMC7721, SPC-A-1, A549, LS174T, BEL7402, CK-MES-1 and BxPC-3 tumor cells in vitro. For each tumor cell, the left column represents the results of P2 and the right column represents the results of P2T2S18.

[0066] Figure 10 The figure shows the inhibition of the polypeptides on the growth of tumor cells and HUVEC in vitro. The cell survival rate at the concentration of 0.5 mg / ml is shown. From top to bottom, the curves represent P2, P2T2S18Δ3, P2T2S18Δ1, P2T2S18Δ2 and P2T2S18, respectively.

[0067] Figure 11a The figure shows the inhibition of the polypeptides on the growth of tumor cells in vivo. 11b The figure shows the inhibition of the polypeptides on the growth of tumor cells in vivo. Figure 11a The figure shows the inhibition of the polypeptides on the growth of tumor cells in vivo. 11b In the figure, the data from the 1st to the 21st day after administration is shown. From top to bottom, the curves represent the negative control, endostar, P2, endostatin, the positive control and P2T2S18, respectively, in terms of tumor volume TV and relative tumor volume RTV.

[0068] Figure 12a The figure shows the inhibition of the polypeptides on the growth of tumor cells in vivo. 12b The figure shows the inhibition of the polypeptides on the growth of tumor cells in vivo. Figure 12a The figure shows the inhibition of the polypeptides on the growth of tumor cells in vivo. 12b In the figure, the data from the 1st to the 21st day after administration is shown. From top to bottom, the curves represent the negative control, DDP (2 mg / kg), endostar+DDP, P2+DDP, endostatin+DDP, DDP (6 mg / kg) and P2T2S18+DDP, respectively, in terms of tumor volume TV and relative tumor volume RTV.

[0069] Figure 13 The figure shows the inhibition of the polypeptides on the growth of HUVEC in vitro.

[0070] Figure 14a The figure shows the inhibition of the polypeptides on the growth of HUVEC in vitro. 14b The figure shows the HPLC and MASS spectra of P2T2S18-29.

[0071] Figure 15 Inhibition of HUVEC by polypeptide P2T2S18-29. From top to bottom, the curves represent Endostar, Endostatin, P2, P2T2S18-29 and P2T2S18-29, respectively, in terms of cell survival rate at 2.5 mg / ml.

[0072] Figure 16 Inhibition of tumor cell HepG2 by polypeptide P2T2S18-29. From top to bottom, the curves represent Endostar, Endostatin, P2, P2T2A18 and P2T2S18-29, respectively, in terms of cell survival rate at 1 mg / ml. DETAILED DESCRIPTION

[0073] The polypeptide of the present application is a fragment of N-terminal of Endostatin with 45 or less amino acid residues, which contains at least the first to 20th amino acid residues of N-terminal of Endostatin, and wherein:

[0074] (1) the residue corresponding to the 2nd amino acid of N-terminal 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 of N-terminal of Endostatin is A, R, N, D, C, E, G, H, I, L, K, M, F, S, T, W, Y or V;

[0076] and the polypeptide has at least 15% higher inhibition rate on HUVEC than its corresponding sequence without mutation at the same concentration, preferably at least 20% higher; or the polypeptide has at least one of the following properties: the IC 50 concentration of the polypeptide is one half of the IC 50 concentration of the corresponding sequence without mutation, preferably the IC 50 concentration of the polypeptide is one fifth of the IC 50 concentration of the corresponding sequence without mutation, further preferably the IC 50 concentration of the polypeptide is one tenth of the IC 50 concentration of the corresponding sequence without mutation.

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

[0078] Preferably, the polypeptide corresponds to a residue at position 2 of the N-terminus of endostatin that is D, L, T, W, or Y. Preferably, the polypeptide corresponds to a residue at position 18 of the N-terminus of endostatin that is N, E, K, M, S, T, or V. More preferably, the polypeptide corresponds to a residue at position 2 of the N-terminus of endostatin that is D, T, W, or Y. More preferably, the polypeptide corresponds to a residue at position 18 of the N-terminus of endostatin that is N, S, or V.

[0079] In certain embodiments, the polypeptide corresponds to the following combinations of amino acid residues at position 2 and position 18 of endostatin, respectively:

[0080]

[0081]

[0082] Alternatively, in certain embodiments, the polypeptide corresponds to the following combinations of amino acid residues at position 2 and position 18 of endostatin, respectively:

[0083]

[0084]

[0085] It is understood that "fragment" refers to a contiguous sequence of a portion of the full-length sequence. For example, the polypeptides herein are preferably sequences consisting of amino acid residues 1 through 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 of the N-terminus of endostatin, and having the amino acid residues at positions 2 and 18 as described herein. In other words, the polypeptides of the present application are 20 to 45 amino acid residues in length, counting from the first amino acid residue of the N-terminus of endostatin. More preferably, the polypeptides of the present application are 25 to 40 amino acid residues in length, counting from the first amino acid residue of the N-terminus.

[0086] In certain embodiments, the fragment can 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 having the amino acid residues at positions 2 and 18 as described herein:

[0087] the amino acid residue at position 17 is S, A, L, I, V, or T;

[0088] the amino acid residue at position 20 is S or T;

[0089] the amino acid residue at position 21 is G, A, L, I, or V;

[0090] the amino acid residue at position 22 is G, A, L, I, or V.

[0091] Thus, in certain embodiments, the polypeptide of the application, which is a fragment of endothelin N-terminal within 45 amino acid residues, comprises at least the amino acid residues 1-22 of SEQ ID NO: 38, preferably at least the amino acid residues 1-25 of SEQ ID NO: 38, and the 2ndand 18thamino acid residues are as described herein, and the 17th, 20th- 22nd, any two, any three or all four of which are as described above. Further, such polypeptides are 25-40 amino acid residues in length.

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

[0093] In certain embodiments, the polypeptide of the application, which is a fragment of endothelin N-terminal within 45 amino acid residues, comprises at least the amino acid residues 1-22 of SEQ ID NO: 38, preferably at least the amino acid residues 1-25 of SEQ ID NO: 38, and the 18thamino acid residue is N, and the 2ndamino acid residue is T, and optionally the 17th, 20th, 21stand 22ndamino acids are as described above. Further, such polypeptides are 25-40 amino acid residues in length.

[0094] In certain embodiments, the polypeptide of the application, which is a fragment of endothelin N-terminal within 45 amino acid residues, comprises at least the amino acid residues 1-22 of SEQ ID NO: 38, preferably at least the amino acid residues 1-25 of SEQ ID NO: 38, and the 18thamino acid residue is S, and the 2ndamino acid residue is E, H, L, T, W or V, and optionally the 17th, 20th, 21stand 22ndamino acids are as described above. Further, such polypeptides are 25-40 amino acid residues in length.

[0095] The preferred polypeptide of the present application has an amino acid sequence as set forth in SEQ ID NO: 4, 5, 6, 7, 27-30, 39 or 41. The polypeptide of the present application also includes an amino acid sequence consisting of the amino acid residues at positions 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 the amino acid residues at positions 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 application 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, cholesterols or amidation.

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

[0098] It should be understood that in the process of cloning a gene, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more extraneous residues at the end of the expressed amino acid sequence, which does not affect the activity of the sequence of interest. For example, in order to construct a fusion protein, to facilitate the expression of a recombinant protein, to obtain a recombinant protein that is automatically secreted outside the host cell, or to facilitate the purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable region of the recombinant protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. The amino terminus or carboxyl terminus of the amino acid sequence of the present application can also contain one or more polypeptide fragments as a protein tag. Any suitable tag can be used in the present application. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-Tag II, AU1, EE, T7, 4A6, ε, B, gE and Ty1. These tags can be used for protein purification. Examples of tags used include Poly-Arg, such as RRRRRR (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-Tag II, i.e. WSHPQFEK (SEQ ID NO: 45); and C-myc, i.e. WQKLISEEDL (SEQ ID NO: 46).

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

[0100] The amino acid sequences of the present application can be the product of chemical synthesis, or recombinant polypeptides produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, filamentous fungi, higher plant, insect, and mammalian cells). Depending on the host used for recombinant production, the polypeptides of the present application can be glycosylated or can be non-glycosylated.

[0101] For example, the amino acid sequences of the present application can be synthesized using methods of polypeptide chemical synthesis known in the art. Methods of polypeptide chemical synthesis include solid phase synthesis and solution phase synthesis, with solid phase synthesis being commonly used. Methods of solid phase synthesis include, but are not limited to, the two commonly used methods of Fmoc and tBoc. Generally, a resin is used as an insoluble solid support, and the amino acids are added to the peptide chain one at a time, typically from the C-terminus (carboxy terminus) to the N-terminus (amino terminus), with each cycle of amino acid addition consisting of the following three reactions: 1) deprotection: the protected amino acid must be deprotected of its amino protecting group using a deprotection solvent; 2) activation: the carboxyl group of the amino acid to be added is activated with an activating agent; and 3) coupling: the activated carboxyl group is reacted 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 peptide chain is cleaved from the solid support using a cleavage solution to obtain the desired amino acid sequence. The chemical synthesis described above can be performed on a programmable automated polypeptide synthesizer, including but not limited to the Tribute Dual Channel Peptide Synthesizer by Protein Technologies, Inc., the UV Online Monitor system by CS Bio, the Focus XC Tri Channel Synthesizer by Aapptec, and the like.

[0102] The present application also includes polynucleotides encoding the polypeptides of the present application. 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 polynucleotide of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding a mature polypeptide can be identical to the above-described DNA sequence or a degenerate variant thereof. As used herein, "a degenerate variant" in the present application refers to a nucleic acid sequence that encodes an amino acid sequence of the present application, but differs from the sequence as shown in SEQ ID NO: 31, etc.

[0104] The term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising a sequence encoding the polypeptide, or a polynucleotide which, in addition to said sequence, also comprises additional coding and / or non-coding sequences.

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

[0106] The nucleotide sequences of the present application can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified in each amplification are spliced together in the correct order.

[0107] Once the relevant sequences are obtained, they can be obtained in large quantities by recombination. This is usually done by cloning them into a vector, introducing them into cells, and then isolating the relevant sequences from the proliferated host cells by conventional methods.

[0108] In addition, the relevant sequences can also be synthesized by artificial synthesis, particularly when the length of the fragment is short. Generally, a long fragment can be obtained by first synthesizing a plurality of small fragments and then ligating them together.

[0109] At present, it is possible to obtain DNA sequences encoding the amino acid sequences of the present application entirely by chemical synthesis. The DNA sequences can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0110] The present application also relates to vectors comprising the polynucleotides of the present application, and host cells genetically engineered with the vectors of the present application, and methods of producing the polypeptides described in the present application by recombinant techniques. Preferably, the vectors of the present application are expression vectors.

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

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

[0113] (2) culturing the host cell in a suitable culture medium;

[0114] (3) isolating, purifying the protein from the culture medium or the cell.

[0115] The polynucleotide sequence of the present application can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, retrovirus, or other vector known in the art. Any plasmid and 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 usually contains an origin of replication, a promoter, a marker gene, and a translation control element. The expression vector also includes a ribosome binding site for translation initiation and a terminator.

[0116] Methods well known to those skilled in the art can be used to construct an expression vector containing the nucleic acid sequence of the present application and suitable transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The nucleic acid sequence can be operably linked to a suitable promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or 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 for eukaryotic cell culture, neomycin resistance in eukaryotic cells, and green fluorescent protein (GFP) or tetracycline or ampicillin resistance in E. coli.

[0118] The vector containing the appropriate DNA sequence as described above, as well as a suitable promoter or control sequence, can be used to transform an appropriate host cell to enable it to express the protein.

[0119] The host cell can be a prokaryotic cell such as a bacterial cell; or a lower eukaryotic cell such as a yeast cell; a filamentous fungal cell, or a higher eukaryotic cell such as a mammalian cell. Representative examples are: bacterial cells of E. coli, Streptomyces; a bacterial cell of Salmonella typhimurium; a fungal cell such as yeast, filamentous fungi, a plant cell; an insect cell such as Drosophila S2 or Sf9; an animal cell such as CHO, COS, 293 cells, or Bowes melanoma cells, etc.

[0120] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically consisting of approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription.

[0121] Those skilled in the art are well aware of 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 prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are 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 eukaryote, 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 this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0124] The recombinant peptides in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods. Various methods for preparing peptides using recombinant technologies 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] The pharmaceutical composition can contain a therapeutically or prophylactically effective amount of the polypeptide of the present application. An "effective amount" means the amount of a component sufficient to produce a desired result. The specific effective amount will depend on factors such as the particular condition being treated, the physical condition of the patient (e.g., body mass, age, sex), the duration of treatment, the concurrent or prior therapies (if any), and the specific formulation of the polypeptide of the present application. An "effective amount" also means the amount of the polypeptide of the present application that will elicit the desired therapeutic effect without an undue burden of side effects.

[0127] The pharmaceutically acceptable carrier is generally safe and non-toxic, and can include, in a broad sense, any known substance used in the pharmaceutical industry for the preparation of pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants, flow aids, stabilizers, colorants, wetting agents, disintegrants, etc. In selecting an excipient suitable for delivering a synthetic peptide, the mode of administration of the pharmaceutical composition is the main consideration, which is well known to those skilled in the art.

[0128] The polypeptide in the pharmaceutical composition of the present application can be present in an amount of about 0.01 to 1000 μM.

[0129] The above pharmaceutical composition 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, PA (1985)).

[0130] The pharmaceutical composition of the present application can be in various suitable dosage forms, including but not limited to tablets, capsules, injections, etc.

[0131] The pharmaceutical composition of the present application can further contain other known chemotherapeutic agents, particularly those known to treat or prevent tumors, including but not limited to cisplatin, carboplatin, or oxaliplatin.

[0132] The polypeptide and pharmaceutical composition of the present application can be used to treat or prevent various diseases known to be treatable or preventable by endostatin, or to alleviate or reduce various symptoms known to be alleviated or reduced by endostatin.

[0133] For example, the polypeptide and pharmaceutical composition of the present application can be administered to a subject in need thereof for the treatment or prevention of 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's sarcoma, neuroblastoma, osteosarcoma, lung adenocarcinoma, lung squamous carcinoma, liver cancer, colon cancer, and pancreatic cancer, etc.

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

[0136] The present application also provides a method for improving the efficacy of a chemotherapeutic drug, which comprises administering the polypeptide or the pharmaceutical composition of the present application to a subject in need before, simultaneously with or after the administration of the chemotherapeutic drug.

[0137] The present application also provides the use of the polypeptide or the pharmaceutical composition of the present application in the preparation of a medicament for treating or preventing a tumor.

[0138] The present application also provides the use of the polypeptide or the pharmaceutical composition of the present application in the preparation of a medicament for improving the efficacy of a chemotherapeutic drug.

[0139] The present application also provides a polypeptide for use as a medicament, which polypeptide is as described in any of the preceding aspects or embodiments of the present application. The present application also provides a polypeptide for use in the treatment or prevention of any of the various tumors described hereinbefore or for improving the efficacy of a chemotherapeutic drug, which polypeptide is as described in any of the preceding aspects or embodiments of the present application.

[0140] Examples

[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, The main peak was collected step by step and purified by a C18 column of a medium-pressure liquid chromatograph of a model 100 explorer. The target peak was collected and analyzed for purity by a reversed-phase high-pressure liquid chromatograph of a model 1100 Agilent and a Phenomenex C18 analysis column, and the molecular weight was identified by a mass spectrometer of a model LCQ Advantage. The collected solution obtained by the medium-pressure liquid chromatograph purification was freeze-dried, dissolved in PBS to form a polypeptide storage solution, filtered to remove bacteria at 0.20 μM, and stored at -80°C. The HPLC purity identification and the MASS molecular weight identification are shown in Fig. 1.

[0144]

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

[0146] Preparation of umbilical cord preservation solution: 150 ml PBS + 3 times working concentration of double antibody (blue / chain); preparation of complete culture medium: 80 ml M199 + 20 ml FBS + 1 ml ECGS + 1 ml 100X double antibody + 1 ml heparin solution (0.5% W / V) + 1 ml 200 mM glutamine; preparation of isolation instruments: 1 surgical tray, 4-5 vascular clamps, 2 surgical scissors, a glass culture dish with a diameter of about 10 cm; preparation of collagenase type I: configured to 1% (W / V).

[0147] Take 20 cm of the fetal end of the umbilical cord, rinse it clean, and tie both ends. Place it in 150 ml of umbilical cord preservation solution. Store it in a 4-degree refrigerator and digest it within 6 hours. Check the umbilical cord, remove the damaged part, and rinse the umbilical vein thoroughly. Then inject 10 ml of collagenase solution and transfer it to a 37°C incubator for 15 minutes. Take out the umbilical cord, collect the digestion solution, and wash it with PBS. After centrifugation, resuspend the culture, change the solution after 24 hours, and remove the non-adherent cells.

[0148] Example 3: Inhibition of polypeptide 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 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 in the following table was synthesized by 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] Polypeptide number Sequence (from N- to C-terminus) P1 HSHRDFQPVLHLVALNSPLSGGMRGIRGAD P2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2 P3 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD P4 [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] The biological activity of P1, P2, P3, P4 polypeptides, endostatin, endostar on HUVEC was determined according to the method described in Example 3 at a polypeptide concentration of 1 mg / ml and a recombinant human endostatin concentration of 5 mg / ml (polypeptide and endostatin at near equimolar concentration), and the results are shown in Table 1. Figure 2 .

[0155] Example Five: Study on the structure-activity relationship of polypeptides

[0156] The polypeptides with the sequences shown in Table 2 were synthesized according to the method of Example 1, and their HPLC purity identification and MASS molecular weight were determined. The biological activity of the polypeptides on HUVEC was determined according to the method described in Example 3 at a concentration of 1 mg / ml, and the results are shown in Table 2. Figure 4 .

[0157]

[0158] Based on the structure of endostatin disclosed in the literature (EMBO J. 1998 Mar 16; 17(6): 1656-1664) (PDB database structure number 1BNL), the INSIGHT II software was used to perform homology modeling on the above-mentioned polypeptides to obtain the dominant conformation of P2 polypeptide. Then the full-activated space self-consistent field (CASSCF) was used to calculate the energy of the highest occupied orbital (HOMO) and the lowest unoccupied orbital (LUMO) of P2 polypeptide. On this basis, we used our own algorithm, 2-D synergistic iterative algorithms in spacial point field (2-D SIASPF), to simulate and iterate the replacement of amino acids in pairs in P2 polypeptide, calculate the cumulative variance of electron density caused by the replacement of the polypeptide Zn ion binding active domain (1H, 3H, 11H), and combine the actual test of the biological activity of the amino acid combination to score the biological activity synergistic relationship between each amino acid in P2. The results showed that the synergistic effect of the 2nd and 18th amino acids in the polypeptide on biological activity was the highest.

[0159] Example Six: Effect of polypeptide amino acid replacement on its activity

[0160] A P2 peptide combination peptide library of the 2nd and 18th amino acids was constructed. The polypeptides with the sequences shown in Table 3 were synthesized by using the Apex396 full-automatic high-throughput polypeptide synthesizer of AAPPTEC company, in which X1 and X3 are any one of natural amino acids (see Table 3), X2 and X4 are S, and X5 and X6 are G.

[0161]

[0162] Following the method described in Example 3, the biological activity of the above-mentioned peptide against 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 viability 0-10%; b: Cell viability 11-20%; c: Cell viability 21-30%; d: Cell viability 31-40%; e: Cell viability 41-50%; f: Cell viability 51-60%; g: Cell viability 61-70%; h: Cell viability 71-80%; i: Cell viability 81-90%; j: Cell viability 91-100%.

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

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

[0168]

[0169] Recombinant endostatin (SEQ ID NO:1) and the marketed drug endostar (SEQ ID NO:10) were tested for their inhibitory effects on HUVECs and HepG2 tumor cells according to the method shown in Example 3. The results are shown in [Figure 3]. Figure 5a , Figure 5b See the table below. The results show that the bioactivity of P2T2S18 and P2T2N18 is significantly higher than that of P2, and their IC50 values ​​are... 50 The concentration was approximately 10 times lower than that of P2. Furthermore, the peptides P2T2 carrying a single-point mutation at amino acid position 2 and P2N18 and P2S18 carrying a single-point mutation at amino acid position 18 both exhibited lower biological activity than P2. Therefore, the high biological activity of P2T2S18 and P2T2N18 is due to an unpredictable synergistic effect resulting from the co-mutation of amino acids at positions 2 and 18. It is evident that the highly biologically active structures of P2T2S18 and P2T2N18 involved in this invention are difficult to obtain using conventional single-point mutation scanning methods.

[0170]

[0171]

[0172] Example Eight: Polypeptide induces SPC-A-1 tumor cell death in vitro

[0173] As shown in Example Three, the effect of polypeptide on SPC-A-1 lung cancer cell line to induce cell death was tested at polypeptide concentration of 2.5 mg / ml, and observed under optical microscope for 24 hours and photographed. As shown in Figure 6 P2, the cells shrunk at 4 hours after polypeptide was added into the culture medium, and the cells were almost dead at 24 hours. The mode of cell death induced by P2 was similar to apoptosis. P2T2S18 exerted strong effect on the cells at 2 hours after polypeptide was added into the culture medium, but the cells did not shrink, but swelled. The swelling was further intensified at 4 hours, and the cells started to fragment at 8 hours, and only cell fragments were left at 24 hours. The mode of cell death induced by P2T2S18 was very surprising, and it was not possible to determine the mode of cell death, and no report was found in the literature. However, it was clear that the mode of cell death induced by P2T2S18 was significantly different from that induced by P2. In combination with the results of Example Seven, it was clear that P2T2S18 not only had significantly higher biological activity than P2, but also induced cell death in a mode significantly different from P2.

[0174] Example Nine: Inhibition of HUVEC growth in vitro by polypeptide

[0175] The polypeptides of the following table were synthesized according to the method shown in Example One, and the HPLC purity and MASS molecular weight were identified in Figure 1 and Figures 7a-7j .

[0176]

[0177] The inhibitory activity of polypeptide on HUVEC was tested according to the method shown in Example Three. Each polypeptide was tested at equimolar concentration, and the 300 uM concentration of P2T2S18 polypeptide was about equivalent to 1 mg / ml. The results are shown in Figure 8 and the following table, which shows that the C-terminal of P2T2S18 still has biological activity after being shortened or lengthened within a certain range.

[0178] Polypeptide number Sequence number Cell survival rate (%) at a polypeptide concentration of 120 uM 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 Ten: Inhibition of growth of various tumor cells in vitro by polypeptide

[0180] P2 (SEQ ID NO: 2) and P2T2S18 (SEQ ID NO: 6) polypeptides were tested for their inhibitory effect on various tumor cells SMMC7721, SPC-A-1, A549, LS174T, BEL7402, CK-MES-1, and BxPC-3 in vitro at polypeptide concentration of 1 mg / ml according to the method shown in Example Three, and the test results are shown in Figure 9and the following table. It can be seen that the inhibitory activity of P2T2S18 against various tumors is significantly higher than that of P2, and the IC50 of P2T2S18 is 1 / 10 of that of P2. 50 The concentration is less than the IC50 of P2 50 The concentration is less than the IC50 of P2

[0181]

[0182] Example XI: Inhibition of tumor cell and HUVEC growth in vitro by polypeptides

[0183] The polypeptides of the following table were synthesized according to the method of Example I, and the HPLC purity and MASS molecular weight identification are shown in Figure 1 and Figures 3a-3f The inhibitory effect on HUVEC was tested according to the method shown in Example III, and the test results are shown in Table 3. Figure 10

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

[0185] Example XII: Establishment of solid tumor model in vivo

[0186] The tumor cells in the logarithmic growth phase were taken from in vitro culture, and 100 μl of the cell suspension containing 5 x 10 6 After 15 days, the solid tumors were taken and cut into uniform pieces of about 3 mm in size under sterile conditions, and one piece was inoculated into each nude mouse in the right axillary subcutaneously using a trocar. Ten to 14 days after inoculation, the animals were re-grouped according to the tumor size, and the animals with too large or too small tumors were eliminated, and each group had substantially the same average tumor volume. The test drugs were administered according to the test scheme. The long diameter (a) and the short diameter (b) of the tumor mass were measured twice a week. After the test was completed, the animals were sacrificed, the tumor mass was dissected, weighed, and photographed. The tumor volume TV = 1 / 2 x a x b 2 ; the relative tumor volume RTV = Vt / Vo, where Vo is the tumor volume measured at the time of grouping (i.e., one day before administration), and Vt is the tumor volume at each measurement. The tumor inhibition rate (%) = (1-T / C) x 100%, where T is the average tumor volume of the treatment group, and C is the average tumor volume of the negative control group.

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

[0188] The polypeptides of the following table were synthesized according to the method of Example I, and the HPLC purity and MASS molecular weight identification are shown in Figure 1.

[0189] ​ Polypeptide number Sequence number Sequence (from N- to C-terminus) P2 SEQ ID NO: 2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2 P2T2S18 SEQ ID NO: 6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2

[0190] Recombinant endostatin of SEQ ID NO: 1 sequence, endostar of SEQ ID NO: 10 sequence on sale. The human hepatoma BEL7404 tumor model was established by the method shown in Example 10, and the following 6 groups were set up in the test, 6 animals in each group except for 9 animals in the negative control group. The doses of polypeptide and endostatin were close to equimolar. The test results are shown in Figure 11a 、 Figure 11b and the following table.

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

[0192] 2) Cyclophosphamide CTX (30 mg / kg, intraperitoneal injection ip, 1 time / day, continuous administration for 7 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, 2 times / day, continuous administration for 21 days)

[0195] 5) endostatin (50 mg / kg / time, sc, 2 times / day, continuous administration for 21 days)

[0196] 6) endostar (50 mg / kg / time, sc, 2 times / day, continuous administration for 21 days)

[0197]

[0198] The RTV of each test group was subjected to t test, and each p value is shown in the following table. It can be seen that P2 cannot significantly inhibit the growth of tumor, and there is no significant difference compared with the negative control group (P=0.015). P2T2S18 can significantly inhibit the growth of tumor, and the tumor inhibition rate reaches 76.7% after 21 days of administration. Even, the inhibition effect of P2T2S18 on tumor is close to that of CTX, and there is no significant difference in RTV between the two groups (P>0.01). It is worth noting that no toxic reaction occurred in the P2T2S18 test group animals in the test, while typical toxic side effects of chemotherapy occurred in the CTX group. The effect of P2T2S18 on inhibiting tumor growth is obviously 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 polypeptide in combination with chemotherapeutic drugs

[0201] The polypeptides of the following table were synthesized according to the method of Example 1, and the HPLC purity and MASS molecular weight were identified as shown in Figure 1.

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

[0203] Endostatin, as shown in SEQ ID NO: 1, and Endostar, as shown in SEQ ID NO: 10, are marketed drugs. The human lung cancer A549 tumor model was established according to the method shown in Example 10, and the following 7 groups were set up, with 6 animals in each group. The doses of polypeptides and endostatin were equimolar.

[0204] 1) Negative control group: normal saline, sc, 2 times / day, continuous administration for 21 days;

[0205] 2) Low-dose cisplatin (DDP) group: 2 mg / kg / day, ip, 1 time / day, continuous administration for 7 days;

[0206] 3) P2+DDP group:

[0207] DDP: 2 mg / kg / day, ip, 1 time / day, continuous administration for 7 days,

[0208] P2: 15 mg / kg / time, sc, 2 times / day, continuous administration for 21 days;

[0209] 4) P2T2S18+DDP group:

[0210] DDP: 2 mg / kg / day, ip, 1 time / day, continuous administration for 7 days,

[0211] P2T2S18: 15 mg / kg / time, sc, 2 times / day, continuous administration for 21 days;

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

[0213] DDP: 2 mg / kg / day, ip, 1 time / day, continuous administration for 7 days,

[0214] Endostatin: 50 mg / kg / time, sc, 2 times / day, continuous administration for 21 days;

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

[0216] DDP: 2 mg / kg / day, ip, 1 time / day, continuous administration for 7 days,

[0217] Endostar: 50 mg / kg / time, sc, 2 times / day, continuous administration for 21 days;

[0218] 7) DDP high dose group: 6 mg / kg / day, ip, 1 time / day, continuous administration for 7 days.

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

[0220]

[0221] The RTV of each test group was subjected to t test, and each p value is shown in the following table. It can be seen that P2 combined with DDP (2 mg / kg) cannot significantly improve the effect of DDP (2 mg / kg) on inhibiting tumors, and there is no significant difference in the RTV comparison between the two test groups (P=0.011); the inhibitory effect of P2 combined with DDP (2 mg / kg) on tumors is significantly poorer than that of DDP (6 mg / kg), and there is a significant difference in the RTV comparison between the two test groups (P<0.001). P2T2S18 can significantly enhance the effect of DDP (2 mg / kg) on inhibiting tumors. The inhibition of P2T2S18 combined with DDP (2 mg / kg) on tumors is significantly better than that of DDP (2 mg / kg), and there is a significant difference in the RTV comparison between the two test groups (P<0.001). Even the inhibition of P2T2S18 combined with DDP (2 mg / kg) on tumors is significantly better than that of DDP (6 mg / kg), and the tumor inhibition rate of P2T2S18 combined with DDP (2 mg / kg) reaches 99.7% after 21 days of administration, and only 2 of the 6 test animals have residual tumors, and the tumors of the other 4 animals disappear. While in the DDP (6 mg / kg) group, all 6 animals have residual tumors after 21 days of administration. The RTV of the P2T2S18 combined with DDP (2 mg / kg) group is smaller than that of the DDP (6 mg / kg) group, and there is a significant difference (P<0.001). It can be seen that the efficacy of P2T2S18 combined with DDP (2 mg / kg) is better than that of DDP (6 mg / kg).

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

[0223] It is worth noting that in the test, the animals in the P2T2S18 combined with DDP (2 mg / kg) test group and the DDP (2 mg / kg) group did not show obvious toxic reactions, while the animals in the DDP (6 mg / kg) group showed obvious chemotherapy toxicity.

[0224]

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

[0226] The polypeptides of the following table were synthesized according to the method of Example One and tested for inhibition of HUVEC according to the method of Example Three at a polypeptide concentration of 0.1 mg / ml. The results are shown in Table 5. Figure 13 .

[0227]

[0228] Example Sixteen: Inhibition of tumor cell and HUVEC growth in vitro by polypeptides

[0229] The polypeptides of the following table were synthesized according to the method of Example One and tested for inhibition of HUVEC according to the method of Example Three at a polypeptide concentration of 0.1 mg / ml. The results are shown in Table 5. Figure 14a and 14b . The results are shown in Table 5. Figure 15 and 16 . The results are shown in Table 5.

[0230] Polypeptide number Sequence number Sequence (from N- to C-terminus) P2T2S18-29 SEQ ID NO: 41 Ac-H T HRDFQPVLHLVALNS S LSGGMRGIRGA-NH2]]>

[0231] The above embodiments are merely illustrative and not restrictive. The scope of protection of the present application will be defined by the claims. Those skilled in the art will understand that various modifications and changes can be made to the technical solutions of the present application without departing from the spirit and scope of the present application, and these modifications and changes still include in the scope of the present application. SEQUENCE LISTING <110> Shanghai Hepu Pharmaceutical Co., Ltd. <120> A tumor inhibiting 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 Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly lie Arg Gly Ala Asp Phe Gin 20 25 30 Cys Phe Gin Gin Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala 35 40 45 Phe Leu Ser Ser Arg Leu Gin Asp Leu Tyr Ser lie Val Arg Arg Ala 50 55 60 Asp Arg Ala Ala Val Pro lie 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 lie Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro 100 105 110 Thr Trp Pro Gin 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 Gin Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gin 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 of a fragment of human endostatin <400> 4 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 lie Arg Gly Ala Asp Phe Gin 20 25 30 Cys Phe Gin Gin Ala Arg Ala Val 35 40 <210> 5 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 5 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 lie Arg Gly Ala Asp Phe Gin 20 25 30 Cys Phe Gin 35 <210> 6 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 6 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 lie Arg Gly Ala Asp 20 25 30 <210> 7 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 7 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 20 25 <210> 8 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 8 His Thr His Arg Asp Phe Gin 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 of a fragment of human endostatin <400> 9 His Thr His Arg Asp Phe Gin 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 Gin Cys Phe Gin Gin Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gin Asp Leu Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro Gin Lys Ser Val 20 25 30 <210> 10 <211> 192 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Human Endostatin Drug Endostar <400> 10 Met Gly Gly Ser His His His His His His Ser His Arg Asp Phe Gin 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 Gin Cys Phe Gin Gin Ala Arg Ala 35 40 45 Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gin 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 Gin 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 of a fragment of human endostatin <400> 11 His Thr 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 lie Arg Gly Ala Asp 20 25 30 <210> 12 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 12 His Ala 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 He Arg Gly Ala Asp 20 25 30 <210> 13 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 13 His Glu 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 He Arg Gly Ala Asp 20 25 30 <210> 14 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 14 His Ser His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Ala Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly He Arg Gly Ala Asp 20 25 30 <210> 15 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a 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 lie 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 lie 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 lie Arg Gly Ala Asp 20 25 30 <210> 18 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutants of fragments of human endostatin <400> 18 His Ser His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Ala Ser Gly Gly Met Arg Gly He Arg Gly Ala Asp 20 25 30 <210> 19 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutants of fragments of human endostatin <400> 19 His Ser His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ala Gly Gly Met Arg Gly He Arg Gly Ala Asp 20 25 30 <210> 20 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutants of fragments of human endostatin <400> 20 His Ser His Arg Asp Phe Gin Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ala Gly Gly Met Arg Gly He Arg Gly Ala Asp 20 25 30 <210> 21 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 21 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 Ala Met Arg Gly lie Arg Gly Ala Asp 20 25 30 <210> 22 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 22 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 Ala Met Arg Gly lie Arg Gly Ala Asp 20 25 30 <210> 23 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 23 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 Asp Arg Gly Ala Asp 20 25 30 <210> 24 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 24 His Thr His Arg Asp Phe Gin 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 of a fragment of human endostatin <400> 25 His Ser 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 20 25 30 <210> 26 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 26 His Ser His Arg Asp Phe Gin 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 of a fragment of human endostatin <400> 27 His Thr His Arg Asp Phe Gin 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> 28 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Mutant of a fragment of human endostatin <400> 28 His Thr His Arg Asp Phe Gin 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 of a fragment of human endostatin <400> 29 His Thr His Arg Asp Phe Gin 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> 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> Artificial Sequence <220> <223> Coding sequence for a mutant of a human endostatin fragment <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> The coding sequence of mutant human endostatin fragment <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> The coding sequence of mutant human endostatin fragment <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> The coding sequence of mutant human endostatin fragment <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> DNA <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> DNA <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 S, A, L, I, V, or T <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is any amino acid <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is S, A, L, I, V, or T <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa is any amino acid <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is S, A, L, I, V, or T <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa is any amino acid <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> The coding sequence of mutant human endostatin fragment <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> mutant of human endostatin fragment <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> Tag <400> 42 Arg Arg Arg Arg Arg 1 5 <210> 43 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Tag <400> 43 His His His His His His 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Tag <400> 44 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Tag <400> 45 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Tag <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 first to 30th amino acid residues at the N-terminus of endostatin, and the amino acid sequence of endostatin is shown in SEQ ID NO:1, wherein the second amino acid residue of endostatin is replaced with N and the 18th amino acid residue is replaced with K.

2. The polypeptide according to claim 1, characterized in that, 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 of any one of claims 1-2; and (2)(1) The complementary sequence of the polynucleotide sequence.

4. An expression vector comprising the polynucleotide molecule of claim 3.

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

6. The use of the polypeptide of any one of claims 1-2 or the pharmaceutical composition of claim 5 in the preparation of a medicament for the prevention or treatment of tumors, wherein the tumor is selected from lung adenocarcinoma, lung squamous cell carcinoma, liver cancer and pancreatic cancer.

7. The use of the polypeptide of any one of claims 1-2 or the pharmaceutical composition of claim 5 in combination with a chemotherapeutic agent in the preparation of a medicament for the prevention or treatment of tumors, wherein the chemotherapeutic agent 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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