WO2017016430A1 - 一种肿瘤抑制肽 - Google Patents

一种肿瘤抑制肽 Download PDF

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Publication number
WO2017016430A1
WO2017016430A1 PCT/CN2016/090800 CN2016090800W WO2017016430A1 WO 2017016430 A1 WO2017016430 A1 WO 2017016430A1 CN 2016090800 W CN2016090800 W CN 2016090800W WO 2017016430 A1 WO2017016430 A1 WO 2017016430A1
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Prior art keywords
amino acid
seq
polypeptide
acid residues
endostatin
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PCT/CN2016/090800
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English (en)
French (fr)
Chinese (zh)
Inventor
刘宏利
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G-BIO PHARMA TECH Co Ltd
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G-BIO PHARMA TECH Co Ltd
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Priority to CN202111153334.9A priority Critical patent/CN115433270B/zh
Priority to CN202111152272.XA priority patent/CN113717278B/zh
Priority to US15/746,978 priority patent/US10351613B2/en
Priority to CN202111153273.6A priority patent/CN115433268B/zh
Priority to CN202111152268.3A priority patent/CN113717277B/zh
Priority to CN202111152258.XA priority patent/CN113717275B/zh
Priority to CN202111155262.1A priority patent/CN113943365B/zh
Priority to CN202111152259.4A priority patent/CN113717276B/zh
Priority to CN202111153309.0A priority patent/CN115433269B/zh
Priority to CN202111155210.4A priority patent/CN113943364B/zh
Priority to CN202111155244.3A priority patent/CN114031683B/zh
Priority to CN202111152378.XA priority patent/CN113912705B/zh
Priority to EP16829796.8A priority patent/EP3330285B1/en
Priority to CN202111155546.0A priority patent/CN113980120B/zh
Priority to JP2018504942A priority patent/JP6903633B2/ja
Priority to CN202111153255.8A priority patent/CN115433267A/zh
Priority to CN201680044076.1A priority patent/CN107922475B/zh
Priority to CN202111155175.6A priority patent/CN113943363B/zh
Priority to CN202111155543.7A priority patent/CN113980119B/zh
Priority to CN202111151032.8A priority patent/CN113717274B/zh
Publication of WO2017016430A1 publication Critical patent/WO2017016430A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the invention belongs to the field of tumor treatment, and particularly relates to a polypeptide for inhibiting and treating a tumor, wherein the amino acid sequence of the polypeptide is a fragment of 45 amino acids in length from the first amino acid residue of the N-terminus of endostatin.
  • Endostatin is an endogenous angiogenesis inhibitor isolated and purified from cultured mouse endothelial cell tumor (EOMA) supernatant by O'Reilly in 1997. It is a 20kd molecular weight protein derived from the hydrolysis of XVIII collagen. product. Experiments have shown that endostatin exerts an inhibitory effect on vascular endothelium and tumor cells. Due to the difficulty in recombination of recombinant endostatin, EntreMed of the United States has abandoned the clinical study of recombinant endostatin, and it is not currently possible to prepare endostatin with high in vitro activity.
  • EOMA endothelial cell tumor
  • a zinc ion binding site consisting of three histidines at positions N, 1, 3, and 11 and an aspartic acid residue at position 76 in the endostatin sequence. Endostatin binds to zinc ions and acts on it. It is important. It has been reported that a polypeptide derived from the N-terminus of endostatin has a certain activity of inhibiting vascular endothelial cells and tumor cells (Cancer Res. 2005; 65(9): 3656-63, US Pat. No. 7,528,811 B2).
  • the present invention provides a polypeptide which is a fragment of endostatin N-terminally longer than 45 amino acid residues, and contains at least an N-terminal amino acid residue of amino acids 1-20, wherein the endostatin N-terminal
  • the 2 amino acid residue and the 18th amino acid residue are respectively selected from the following group:
  • the 17th amino acid at the N-terminus of endostatin is S, A, L, I or T
  • the amino acid residue at position 20 is S or T
  • the 21st position is / or the 22nd amino acid residue
  • the 21st amino acid residue is G, A, L, I or V
  • / or the 22nd amino acid residue is G, A, L, I or V
  • amino acid sequence of the endostatin is as shown in SEQ ID NO: 1.
  • the polypeptide comprises at least amino acid residues 1-22 of SEQ ID NO: 38, and amino acid residues 2 and 18 are as described above.
  • the polypeptide comprises at least amino acid residues 1 to 25 of SEQ ID NO: 38, and amino acid residues 2 and 18 are as described above.
  • the polypeptide comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1 to 25 of SEQ ID NO: 38, and amino acid residue 2
  • 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.
  • the polypeptide comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1 to 25 of SEQ ID NO: 38, and amino acid residues 18 For N, 2nd The amino acid residue is T, and the amino acids at positions 17, 20, 21 and 22 are as described above.
  • the polypeptide comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least amino acid residues 1 to 25 of SEQ ID NO: 38, and amino acid residues 18 In 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.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 4, 5, 6, 7, 27-30, 39 or 41.
  • 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 positions 17, 20, 21 and 22 The amino acids are as described above.
  • the polypeptide is selected from the group consisting of amino acid residues 1 to 39, 38, 37, 36, 34, 33, 32, 31, 29, 28, 27 or 26 of SEQ ID NO: Amino acid sequence, and the 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.
  • the amino acid residue at the N-terminus of the polypeptide is histidine, and the histidine is modified by formylation, acetylation, propionylation or butyrylation, and the C-terminal first position Amino acids can be modified by PEG, cholesterol or amidation.
  • polypeptide is selected from the group consisting of:
  • the invention also provides a polynucleotide sequence selected from the group consisting of:
  • polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35, 37, and 40.
  • the polynucleotide sequence is selected from the group consisting of SEQ ID NO: 32 from position 1 to 117, 114, 111, 108, 102, 99, 96, 93, 87, 84, 81 or 78 bases.
  • the polynucleotide sequence is selected from position 1 to 117, 114, 111, 108, 105, 102, 99, 96, 93, 87, 84, 81, 78 of SEQ ID NO:40. Or a nucleotide sequence consisting of 75 bases.
  • the invention also provides an expression vector comprising the polynucleotide sequence of the invention.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable carrier.
  • the invention also provides the use of a polypeptide or pharmaceutical composition of the invention in the manufacture of a medicament for the prevention or treatment of a tumor.
  • 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
  • the invention also provides the use of the polypeptide or pharmaceutical composition of the invention in the preparation of a medicament for improving the efficacy of a chemotherapeutic drug.
  • the chemotherapeutic agent is cisplatin, carboplatin or oxaliplatin.
  • the present invention also provides a process for the preparation of the amino acid sequence of the present invention, which comprises synthesizing the amino acid sequence by Fmoc solid phase synthesis.
  • Figures 1a and 1b show the HPLC and MASS spectra of polypeptide P1, respectively.
  • Figures 1c and 1d show the HPLC and MASS spectra of polypeptide P2, respectively.
  • Figures 1e and 1f show the HPLC and MASS spectra of the polypeptide P2T2S18, respectively.
  • Figures 1g and 1h show the HPLC and MASS spectra of the polypeptide P2T2N18, respectively.
  • Figure 2 shows the biological activities of P1, P2, P3, P4 polypeptide, endostatin, endostar on HUVEC inhibition.
  • Figures 3a and 3b show the HPLC and MASS spectra of the polypeptide P2T2S18 ⁇ 1, respectively.
  • Figures 3c and 3d show the HPLC and MASS spectra of the polypeptide P2T2S18 ⁇ 2, respectively.
  • Figures 3e and 3f show the HPLC and MASS spectra of the polypeptide P2T2S18 ⁇ 3, respectively.
  • Figure 4 shows the biological activity of partial polypeptides against HUVEC inhibition.
  • Figures 5a and 5b show the inhibition of HUVEC and tumor cell HepG2 by several polypeptides, respectively.
  • the cell survival rate at a concentration of 2.5 mg/ml, from top to bottom represents P2S18, Endostar, Endostatin, P2T2, P2N18, P2, P2T2N18, P2T2S18 (P2T2N18 and P2T2S18 are partially coincident). Cell viability.
  • FIG. 6 shows that P2 and P2T2S18 polypeptides induce in vitro death of SPC-A-1 tumor cells.
  • Figures 7a and 7b show the HPLC and MASS spectra of the polypeptide P2T2S18-20, respectively.
  • Figures 7c and 7d show the HPLC and MASS spectra of the polypeptide P2T2S18-25, respectively.
  • Figures 7e and 7f show the HPLC and MASS spectra of the polypeptide P2T2N18-35, respectively.
  • Figures 7g and 7h show the HPLC and MASS spectra of the polypeptide P2T2N18-40, respectively.
  • Figures 7i and 7j show the HPLC and MASS spectra of the polypeptide P2T2N18-45, respectively.
  • Figure 8 shows inhibition of HUVEC growth in vitro by polypeptides.
  • the cell survival rate of P2T2-15, P2T2S18-45, P2T2S18-40, P2T2S18-20, P2T2S18-25, P2T2S18-35 and P2T2S18 is represented by the top-down curves in terms of cell viability at a concentration of 180 ⁇ M. .
  • Figure 9 shows the in vitro inhibition of P2 and P2T2S18 polypeptides on various tumor cells SMMC7721, SPC-A-1, A549, LS174T, BEL7402, CK-MES-1, BxPC-3.
  • the left histogram is the result of P2
  • the right histogram is the result of P2T2S18.
  • Figure 10 shows the inhibition of tumor growth on tumor cells and HUVECs in vitro.
  • the cell survival rate of P2, P2T2S18 ⁇ 3, P2T2S18 ⁇ 1, P2T2S18 ⁇ 2, and P2T2S18 was sequentially represented from top to bottom in terms of cell viability at a concentration of 0.5 mg/ml.
  • Figures 11a and 11b show the inhibition of tumor growth in vivo by tumor cells, respectively.
  • the curves from top to bottom sequentially indicate the tumor volume TV and relative tumor volume of the negative control, endostar, P2, endostatin, positive control and P2T2S18. RTV.
  • Figures 12a and 12b show the results of inhibition of tumor cell growth in vivo by a combination of a polypeptide and a chemotherapeutic drug, respectively.
  • Figure 12a In the case of 12b, the data from the 1st day of administration was taken as an example. From top to bottom, the curves were negative control, DDP (2mg/kg), endostar+DDP, P2+DDP, endostatin+DDP, DDP (6mg). /kg) and tumor volume TV and relative tumor volume RTV of P2T2S18+DDP.
  • Figure 13 shows inhibition of HUVEC growth in vitro by polypeptides.
  • Figures 14a and 14b show the HPLC and MASS spectra of the polypeptide P2T2S18-29, respectively.
  • Figure 15 shows the inhibition of HUVEC by the polypeptide P2T2S18-29. From the top to bottom curve, Endostar, Endostatin, P2, P2T2S18-29, and P2T2S18 are represented by cell viability at 2.5 mg/ml.
  • Figure 16 shows the inhibitory effect of the polypeptide P2T2S18-29 on tumor cell HepG2. From top to bottom, the top-down curves represent Endostar, Endostatin, P2, P2T2A18, and P2T2S18-29, respectively, at cell viability at 1 mg/ml.
  • polypeptide of the present invention is a fragment of endothelin having an N-terminal length of 45 amino acid residues, and contains at least the amino acid residues 1 to 20 of the N-terminus of endostatin, and wherein:
  • residues corresponding to the second amino acid of the N-terminus of endostatin are A, R, N, D, Q, E, H, I, L, K, M, F, P, T, W, Y or V; and
  • residues corresponding to the 18th amino acid at the N-terminus of endostatin are A, R, N, D, C, E, G, H, I, L, K, M, F, S, T, W, Y or V;
  • the polypeptide has an inhibition rate of HUVEC of at least 15%, preferably at least 20% higher than the corresponding sequence without mutation, at the same concentration; or the IC 50 of the polypeptide compared to its corresponding sequence without mutation
  • concentration is one-half of the latter IC 50 concentration, preferably the former IC 50 concentration is one-fifth of the latter IC 50 concentration, further preferably preferably the former IC 50 concentration is the latter IC 50 concentration One.
  • Endostatin is preferably human endostatin.
  • SEQ ID NO: 1 shows an example of recombinant human endostatin.
  • the amino acid sequence of the present invention comprises at least the amino acid residues 1 to 20 of the N-terminus of endostatin described in SEQ ID NO: 1, and the amino acids at positions 2 and 18 are as described herein.
  • the polypeptide corresponds to the residue of the amino acid at the N-terminus of endostatin, which is D, L, T, W or Y.
  • the polypeptide corresponds to the residue of the 18th amino acid at the N-terminus of endostatin, which is N, E, K, M, S, T or V.
  • the polypeptide corresponds to the residue of the amino acid at the N-terminus of endostatin, which is D, T, W or Y. More preferably, the polypeptide corresponds to the residue of amino acid at position 18 of endostatin as N, S or V.
  • polypeptide corresponds to the amino acid residue at position 2 of the endostatin and the amino acid residue at position 18, respectively, in the following combination:
  • polypeptide corresponds to the amino acid residue at position 2 of endostatin and the amino acid residue at position 18 are respectively the following combinations:
  • fragment refers to a contiguous sequence of a portion of a full length sequence.
  • the polypeptide herein is preferably from amino acid 1 at the N-terminus of endostatin to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35
  • a sequence consisting of amino acid residues at positions 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 and at positions 2 and 18 are the amino acid residues described herein.
  • 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 invention is 25-40 amino acid residues in length, starting from the first amino acid residue at the N-terminus.
  • the fragment is optionally in addition to the amino acid residues described herein at positions 2 and 18, optionally in any of positions 17, 20, 21 and 22, any two , any three or all four are the following residues:
  • 17th amino acid residue S, A, L, I, V or T;
  • Amino acid residue 22 G, A, L, I or V.
  • a polypeptide of the invention that is a fragment within 45 amino acid residues of the N-terminus of Endostatin comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least SEQ ID NO :38 amino acid residues 1-25, and positions 2 and 18 are amino acid residues as described herein, and any one of positions 17, 20-22, any two, any three or all four positions are Residues as described above. Further, such polypeptides are 25-40 amino acid residues in length.
  • a polypeptide of the invention that is a fragment within 45 amino acid residues of the N-terminus of Endostatin comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least SEQ ID NO: 38 Amino acid residues 1-25, and the amino acid residue at position 2 is T, and the amino acid residue at position 18 is N, G, K, M, F, S or T (more preferably N or S), optionally
  • the amino acids 17 and 20, 21 and 22 are as described above. Further, such polypeptides are 25-40 amino acid residues in length.
  • a polypeptide of the invention that is a fragment within 45 amino acid residues of the N-terminus of Endostatin comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least SEQ ID NO: 38 Amino acid residues 1-25, and the amino acid residue at position 18 is N, and the amino acid residue at position 2 is T.
  • amino acids 17, 20, 21 and 22 are as described above.
  • polypeptides are 25-40 amino acid residues in length.
  • a polypeptide of the invention that is a fragment within 45 amino acid residues of the N-terminus of Endostatin comprises at least amino acid residues 1-22 of SEQ ID NO: 38, preferably at least SEQ ID NO: 38 Amino acid residues 1-25, and amino acid residue at position 18 is S, and amino acid residue at position 2 is E, H, L, T, W or V, optionally, 17th, 20th, 21st and 22nd The amino acid is as described above. Further, such polypeptides are 25-40 amino acid residues in length.
  • amino acid sequences of preferred polypeptides of the invention are set forth in SEQ ID NO: 4, 5, 6, 7, 27-30, 39 or 41.
  • the polypeptide of the present invention further includes 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 SEQ. ID NO: 39 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.
  • the amino acid residue at the N-terminus of the polypeptide of the present invention is histidine, and the histidine can be modified by formylation, acetylation, propionylation or butyrylation, and the amino acid at the C-terminal can be PEG. , cholesterol, or amidation modification.
  • the histidine of the first amino acid residue at the N-terminus of the polypeptide of the present invention is acetylated, and the amino acid at the C-terminal amino acid is amidated.
  • a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest.
  • promote expression of a recombinant protein obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or Other suitable regions within the protein include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like.
  • the amino terminus or carboxy terminus of the amino acid sequence of the invention may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in the present invention.
  • the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • 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, DYKDDDDK (SEQ ID NO) :44); Strep-TagII, WSHPQFEK (SEQ ID NO: 45); and C-myc, WQKLISEEDL (SEQ ID NO: 46).
  • the invention also encompasses polypeptides comprising or consisting of the tag sequences and the aforementioned fragments.
  • the amino acid sequence of the present invention may be a product of chemical synthesis or a recombinant polypeptide produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, filamentous fungi, higher plants, insects, and mammalian cells).
  • prokaryotic or eukaryotic hosts e.g., bacteria, yeast, filamentous fungi, higher plants, insects, and mammalian cells.
  • the polypeptide of the invention may be glycosylated or may be non-glycosylated, depending on the host used in the recombinant production protocol.
  • the amino acid sequences of the 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, in which solid phase synthesis is commonly used.
  • Solid phase synthesis methods include, but are not limited to, two common methods of Fmoc and tBoc.
  • a resin is used as an insoluble solid phase carrier, usually from C.
  • the amino terminus (carboxy terminus) is linked to the N-terminus (amino terminus) one by one on the peptide chain.
  • Each amino acid linkage cycle consists of the following three steps: 1) Deprotection: The protected amino acid must be removed with a deprotecting solvent.
  • the above chemical synthesis can be performed on a program-controlled automated peptide synthesizer, including but not limited to the Tribute dual-channel peptide synthesizer from Protein Technologies, the UV Online Monitor system from C S Bio, and the Focus XC from Aapptec. Three-channel synthesizer, etc.
  • the invention also includes polynucleotides encoding the polypeptides of the invention.
  • 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
  • SEQ ID NO: 40 shows the coding sequence of SEQ ID NO: 39.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • 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 the mature polypeptide may be the same as the above DNA sequence or a degenerate variant.
  • degenerate variant refers to a nucleic acid sequence encoding an amino acid sequence of the invention, but differing from the sequence set forth in SEQ ID NO: 31, and the like.
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
  • the nucleotide sequence of the present invention can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library 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 amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the amino acid sequence of the present invention completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vectors of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • the vector of the invention is an expression vector.
  • polynucleotide sequences of the invention can be utilized to express or produce a polypeptide of the invention by conventional recombinant DNA techniques. Generally there are the following steps:
  • the polynucleotide sequence of the present invention can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • nucleic acid sequences of the invention can be constructed by in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the nucleic acid sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • promoters are: lac or trp promoter of E.
  • coli coli
  • lambda phage PL promoter eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti- Promoters for the expression of LTRs of transcriptional viruses and other known controllable genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • 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.
  • a prokaryotic cell such as a bacterial cell
  • 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: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes black Animal cells of a tumor cell, and the like.
  • an enhancer sequence is inserted into the vector, The transcription is enhanced.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • 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, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • 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 such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Various methods of preparing polypeptides by recombinant techniques are known in the art.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable carrier.
  • a therapeutically or prophylactically effective amount of a polypeptide of the invention may be included in the pharmaceutical compositions.
  • effective amount is meant an amount of a component sufficient to produce the desired reaction. The specific effective amount depends on a number of factors, such as the particular condition being treated, the patient's physical condition (eg, patient weight, age, or sex), duration of treatment, co-administered therapy (if any), and specific formula.
  • effective amount it is also meant that the toxic or negative effect of the polypeptide of the invention is not as great as the positive effect brought about by this amount.
  • Pharmaceutically acceptable carriers are generally safe, non-toxic, and broadly include any known materials in the pharmaceutical industry for the preparation of pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants, Glidants, stabilizers, colorants, wetting agents, disintegrating agents, and the like.
  • fillers diluents, coagulants, binders, lubricants, Glidants, stabilizers, colorants, wetting agents, disintegrating agents, and the like.
  • polypeptide is present in the pharmaceutical compositions of the invention in an amount of from about 0.01 to 1000 ⁇ M.
  • compositions can be prepared according to known pharmaceutical procedures, such as Remington’s Pharmaceutical Sciences (17th ed., Alfonoso R. Gennaro, ed., Mack Publishing) The company (Mack Publishing Company, Easton, Pennsylvania (1985)) has detailed records.
  • compositions of the present invention may be in a variety of suitable dosage forms including, but not limited to, tablets, capsules, injections and the like.
  • compositions of the present invention may also contain other known chemotherapeutic agents, particularly chemotherapeutic agents known to treat or prevent tumors, including, but not limited to, cisplatin, carboplatin or oxaliplatin.
  • polypeptides and pharmaceutical compositions of the present invention are useful for treating or preventing various diseases which can be treated or prevented by known endostatin, and alleviating or alleviating various symptoms in which known endostatin can be alleviated or alleviated.
  • polypeptides and pharmaceutical compositions of the invention can be administered to a subject in need thereof for the treatment or prevention of a tumor.
  • the subject can be a mammal, especially a human.
  • Tumors include hemangiomas and solid tumors.
  • the solid tumors include, but are not limited to, rhabdomyosarcoma, retinoblastoma, Ewing's sarcoma, neuroblastoma, osteosarcoma, etc., lung adenocarcinoma, lung squamous cell carcinoma, liver cancer, colon cancer, and pancreatic cancer.
  • the invention also provides a method of treating cancer comprising administering to a subject in need thereof a polypeptide or pharmaceutical composition of the invention.
  • the invention also provides a method of increasing the efficacy of a chemotherapeutic drug comprising administering a polypeptide or pharmaceutical composition of the invention prior to, concurrently with, or subsequent to administration of a chemotherapeutic agent in need thereof.
  • the invention also provides the use of a polypeptide or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of a tumor.
  • the invention also provides the use of a polypeptide or pharmaceutical composition of the invention in the manufacture of a medicament for increasing the efficacy of a chemotherapeutic drug.
  • the invention also provides a polypeptide for use as a medicament, as described in various aspects or embodiments of the invention hereinbefore.
  • the invention also provides polypeptides for use in the treatment or prevention of various tumors as described hereinabove or for enhancing the efficacy of a chemotherapeutic agent, such as described in various aspects or embodiments of the invention.
  • the target peak collection samples were identified by Agilent 1100 reverse phase high pressure liquid chromatography Phenomenex C18 analytical column purity, LCQ Advantage mass spectrometer molecular weight identification.
  • the collected solution obtained by medium pressure liquid chromatography was lyophilized, dissolved in PBS to form a polypeptide storage solution, sterilized by filtration at 0.20 ⁇ M, and frozen at -80 °C.
  • HPLC purity identification and molecular weight identification of MASS mass spectrometry are shown in Figure 1.
  • Example 2 Isolation and culture of human umbilical vein endothelial cells (HUVEC)
  • umbilical cord preservation solution 150ml PBS + 3 times working concentration of double antibody (cyan/chain); Prepare complete medium: 80ml M199+20ml FBS+1ml ECGS+1ml 100X double-antibody +1ml heparin solution (0.5%W/V) +1ml 200mM glutamine; preparation for separation equipment: 1 surgical plate, 4-5 vascular clamps, 2 surgical scissors, glass culture dish with a diameter of 10cm; type I collagenase configuration: 1% (W/V).
  • Example 3 Inhibition of human umbilical vein endothelial cells (HUVEC) and tumor cells by polypeptide
  • the growth inhibition effect on cells was examined using the MTT method.
  • the principle is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) to Water-insoluble blue-violet crystalline formazan (Formazan) is deposited in cells, whereas dead cells do not.
  • MTT MTT
  • DMSO Dimethyl sulfoxide
  • the amount of MTT crystal formation is proportional to the number of cells in a certain number of cells.
  • the culture supernatant was discarded, washed once with PBS, added with 1 ml of 0.25% trypsin (4 ° C), digested at 37 ° C for 2 min, added to the culture supernatant, neutralized, and boiled into suspension. Centrifuge at 1000 rpm for 3 min. The supernatant was discarded and resuspended in 5 ml of medium. 48-well plates, 500 ul/well, were seeded at 3 x 10 4 /ml. Incubate for 24 hours at 37 ° C with 5% CO 2 .
  • the cultured cells were discarded, and a medium containing the polypeptide (concentration of Zn 2+ in the medium was 17.39 ⁇ mol/L) was added, and the culture was continued for 48 hours.
  • the wells were carefully discarded, and 450 ul/well PBS was gently rinsed once.
  • 450 ul of MTT medium was added to each well and incubation was continued for 4 h.
  • the culture supernatant was carefully discarded, and 450 ⁇ l/well of dimethyl sulfoxide was added, and shaken on a shaker at low speed for 10 min in the dark.
  • 150 ul of the supernatant was transferred to a 96-well ELISA plate, and the absorbance of each well was measured at an enzyme-linked immunosorbent detector at OD490nm and 570 nm.
  • polypeptide of the sequence shown in the following table such as SEQ ID NO: 2
  • SEQ ID NO: 2 was synthesized as shown in Example 1, with or without N-terminal and/or C-terminal modifications, wherein Ac is an acetylation modification and NH 2 is an amidation modification. . It was subjected to HPLC purity identification and molecular weight identification by MASS mass spectrometry.
  • Recombinant human endostatin (SEQ ID NO: 1) can be purchased from the market (for example, genetex No. GTX65524, BioVision product number 4799-1000, Shanghai Bosheng Biotechnology Co., Ltd. No. E2296-05, Wuhan Boster Bio Engineering Co., Ltd. Item No. BP4153, etc.).
  • the marketed recombinant human endostatin drug Endostar (SEQ ID NO: 10) was purchased from a medical institution.
  • Example 3 P1, P2, P3, P4 polypeptides were tested under the test conditions of a polypeptide concentration of 1 mg/ml and a recombinant human endostatin concentration of 5 mg/ml (the polypeptide and endostatin were nearly equimolar).
  • the biological activities of HUVEC inhibition were measured by endostatin and endostar, and the results are shown in Fig. 2.
  • polypeptides of the following sequences were synthesized as in the method of Example 1, and their HPLC purity identification and MASS mass spectrometry molecular weight were determined. Inhibition of HUVEC by the above polypeptide at a concentration of 1 mg/ml according to the method shown in Example 3 The physical activity was measured, and the results of the measurement are shown in Fig. 4.
  • a P2 peptide combination peptide library of amino acid positions 2 and 18 was constructed.
  • the polypeptides of the following sequences were synthesized using AAPPTEC's Apex396 fully automated high-throughput peptide synthesizer, wherein X 1 and X 3 were any of the natural amino acids (see table below), X 2 and X 4 were S, X 5 and X 6 For G.
  • 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%.
  • Example 7 Inhibition of tumor cells on growth of tumor cells and HUVECs in vitro
  • polypeptides of the following sequences were synthesized as shown in Example 1 and subjected to HPLC purity identification and MASS mass spectrometry molecular weight identification.
  • Example 8 Polypeptide induces in vitro death of SPC-A-1 tumor cells
  • the effect of the polypeptide on the cell death induced by the SPC-A-1 lung cancer cell line was tested at a polypeptide concentration of 2.5 mg/ml as described in Example 3.
  • the cells were observed under a light microscope for 24 hours and photographed.
  • P2 functions 4 hours after the addition of the medium, the cells shrink, and the cells are substantially dead in 24 hours, and the manner in which P2 induces cell death is similar to apoptosis.
  • P2T2S18 exerted a vigorous effect on the cells 2 hours after the addition of the medium, but the cells did not shrink, but were extremely swollen; the swelling was further aggravated after 4 hours, and the cells began to fragment at 8 hours, leaving only cell debris in 24 hours.
  • P2T2S18 The way of cell death caused by P2T2S18 is very surprising. It is not yet possible to determine which pattern of cell death is the case, and it has not been reported in the literature. However, it is clear that this pattern of cell death is significantly different from the cell death caused by P2. In combination 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 a cell death to be significantly different from P2.
  • polypeptides of the following sequences were synthesized as shown in Example 1, and the HPLC purity identification and MASS mass spectrometry molecular weight identification are shown in Figure 1 and Figures 7a-7j.
  • the polypeptide was tested for its inhibitory activity against HUVEC according to the method shown in Example 3. Each polypeptide was tested under equimolar conditions, wherein the concentration of P2T2S18 polypeptide at 300 uM was approximately equivalent to 1 mg/ml. The results are shown in Figure 8 and the table below, showing The C-terminus of P2T2S18 is biologically active after shortening or prolonging within a certain range.
  • Peptide number Serial number Cell viability at a 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
  • Example 10 Inhibition of polypeptide growth on growth of various tumor cells in vitro
  • the P2 (SEQ ID NO: SEQ ID NO: 2) and P2T2S18 (SEQ ID NO: 6) polypeptides were tested at a polypeptide concentration of 1 mg/ml according to the method shown in Example 3 for various tumor cells SMMC7721, SPC- In vitro inhibition of A-1, A549, LS174T, BEL7402, CK-MES-1, BxPC-3, the test results are shown in Figure 9 and the following table. Visible, P2T2S18 than P2 inhibitory activity of a variety of tumors was significantly increased, the concentration IC P2T2S18 50 P2IC 50 concentration is less than 1/10.
  • P2 showed no inhibitory activity, and the cell survival rate was as high as 95% at a drug concentration of 1 mg/ml; while P2T2S18 inhibited the cell significantly, and almost all tumors were killed under the same concentration conditions. Cells, cell survival rate is only 1%.
  • Example 11 Inhibition of tumor cells on growth of tumor cells and HUVECs in vitro
  • polypeptides of the following sequences were synthesized as in the method of Example 1, and HPLC purity identification and MASS mass spectrometry molecular weight identification are shown in Figure 1 and Figures 3a-3f.
  • the inhibition of HUVEC was tested according to the method shown in Example 3, and the results are shown in Fig. 10.
  • Peptide number Serial number Sequence (from N to C) P2 SEQ ID NO: 2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH 2 P2T2S18 SEQ ID NO: 6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 P2T2S18 ⁇ 1 SEQ ID NO: 6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD P2T2S18 ⁇ 2 SEQ ID NO: 6 HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 P2T2S18 ⁇ 3 SEQ ID NO: 6 HTHRDFQPVLHLVALNSSLSGGMRGIRGAD
  • Example 12 Establishment of a tumor model in vivo
  • the logarithmic growth phase tumor cells with good culture in vitro were taken, and nude mice were subcutaneously inoculated with 100 ul of culture cell suspension containing 5 ⁇ 10 6 tumor cells. After 15 days, a solid tumor with good growth was taken and cut into uniform small pieces of about 3 mm under sterile conditions, and each of the nude mice was inoculated with a trocar under the right iliac crest. The tumors were regrouped 10-14 days after inoculation, and the tumors were oversized and undersized. The average volume of each group was basically the same. Each group was given a test drug according to the test protocol. The long diameter (a) and short diameter (b) of the tumor mass were measured twice a week.
  • Tumor inhibition rate (%) (1-T/C) * 100%, where T is the average tumor volume of the treatment group and C is the average tumor volume of the negative control group.
  • Example 13 Inhibition of polypeptide growth in vivo by tumor cells
  • polypeptides of the following sequences were synthesized as in the method of Example 1, and the HPLC purity identification and MASS mass spectrometry molecular weight identification are shown in FIG.
  • Peptide number Serial number Sequence (from N to C) P2 SEQ ID NO: 2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH 2 P2T2S18 SEQ ID NO: 6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2
  • the human liver cancer BEL7404 tumor model was established as in the method of Example 10. The following six groups were set up, except for the negative control group of 9 animals, and the other test groups each group of 6 animals. The dose of the polypeptide and endostatin is close to equimolar. The test results are shown in Figures 11a, 11b and the table below.
  • Negative control group salt saline, subcutaneous injection sc, 2 times / day, continuous administration for 21 days
  • P2T2S18 group (15mg/kg/time, sc, 2 times/day, continuous administration for 21 days)
  • Example 14 Inhibition of tumor cell growth in vivo by combination of a polypeptide and a chemotherapeutic drug
  • polypeptides of the following sequences were synthesized as in the method of Example 1, and the HPLC purity identification and MASS mass spectrometry molecular weight identification are shown in FIG.
  • Peptide number Serial number Sequence (from N to C) P2 SEQ ID NO: 2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH 2 P2T2S18 SEQ ID NO: 6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2
  • a human lung cancer A549 tumor model was established as in the method of Example 10. The following seven groups were set up, with 6 animals in each group. The dose of the polypeptide and endostatin is equimolar.
  • Negative control group normal saline, sc, 2 times/day, continuous administration for 21 days;
  • DDP cisplatin
  • DDP 2mg/kg/day, ip, 1 time/day, continuous administration for 7 days,
  • P2 15 mg/kg/time, sc, 2 times/day, continuous administration for 21 days;
  • DDP 2mg/kg/day, ip, 1 time/day, continuous administration for 7 days,
  • P2T2S18 15mg/kg/time, sc, 2 times/day, continuous administration for 21 days;
  • DDP 2mg/kg/day, ip, 1 time/day, continuous administration for 7 days,
  • Endostatin 50 mg/kg/time, sc, 2 times/day, continuous administration for 21 days;
  • DDP 2mg/kg/day, ip, 1 time/day, continuous administration for 7 days,
  • Endostar 50mg/kg/time, sc, 2 times/day, continuous administration for 21 days;
  • DDP high dose group 6 mg/kg/day, ip, 1 time/day, continuous administration for 7 days.
  • P2T2S18 combined with DDP (2mg/kg) group was significantly better than P2 combined with DDP (2mg/kg) group (P ⁇ 0.001), endostatin combined with DDP (2mg/kg) group (P ⁇ 0.001), endostar combined with DDP (2mg) /kg) group ((P ⁇ 0.001).
  • Example 15 Inhibition of tumor cells on growth of tumor cells and HUVECs in vitro
  • polypeptides of the following sequences were synthesized as in the method of Example 1, and the inhibition of HUVEC was tested according to the method shown in Example 3 at a polypeptide concentration of 0.1 mg/ml. The results are shown in Fig. 13.
  • Example 16 Inhibition of tumor cells on growth of tumor cells and HUVECs in vitro
  • polypeptides of the following sequences were synthesized as in the method of Example 1, and the HPLC purity identification and MASS mass spectrometry molecular weight identification are shown in Figures 14a and 14b.
  • the results of the measurements are shown in Figures 15 and 16. The results showed that P2T2S18 and P2T2S18-29 had similar biological activities, which were significantly higher than P2.

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