WO2017016430A1 - 一种肿瘤抑制肽 - Google Patents

一种肿瘤抑制肽 Download PDF

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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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amino acid
seq
polypeptide
acid residues
endostatin
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PCT/CN2016/090800
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English (en)
French (fr)
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刘宏利
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上海吉贝医药科技有限公司
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Priority to CN202111153309.0A priority Critical patent/CN115433269A/zh
Priority to CN202111155244.3A priority patent/CN114031683B/zh
Priority to CN202111152258.XA priority patent/CN113717275B/zh
Priority to US15/746,978 priority patent/US10351613B2/en
Priority to CN202111152378.XA priority patent/CN113912705B/zh
Priority to CN202111152268.3A priority patent/CN113717277B/zh
Priority to CN202111153255.8A priority patent/CN115433267A/zh
Priority to CN202111155175.6A priority patent/CN113943363B/zh
Priority to CN202111155543.7A priority patent/CN113980119B/zh
Priority to JP2018504942A priority patent/JP6903633B2/ja
Priority to CN202111153334.9A priority patent/CN115433270A/zh
Priority to CN202111155262.1A priority patent/CN113943365B/zh
Priority to EP16829796.8A priority patent/EP3330285B1/en
Priority to CN202111152272.XA priority patent/CN113717278B/zh
Priority to CN202111151032.8A priority patent/CN113717274B/zh
Priority to CN202111155210.4A priority patent/CN113943364B/zh
Priority to CN202111153273.6A priority patent/CN115433268A/zh
Priority to CN201680044076.1A priority patent/CN107922475B/zh
Priority to CN202111152259.4A priority patent/CN113717276B/zh
Priority to CN202111155546.0A priority patent/CN113980120B/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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Abstract

本发明提供了一种肿瘤抑制肽，该多肽的编码序列，含有该编码序列的表达载体，含有所述多肽的药物组合物，以及所述多肽和药物组合物在制备用于抑制、预防或治疗肿瘤的药物中的应用。所述多肽为内皮抑素N端45个氨基酸残基以内的片段，且至少含有N端1-20位氨基酸残基，且第2和18位氨基酸残基具有突变。

Description

一种肿瘤抑制肽 技术领域
本发明属于肿瘤治疗领域，具体涉及一种用于抑制、治疗肿瘤的多肽，所述多肽的氨基酸序列是内皮抑素N端第1个氨基酸残基起长45个氨基酸以内的片段。
背景技术
内皮抑素是1997年O’Reilly等从培养的小鼠内皮细胞瘤(EOMA)上清中分离纯化的一种内源性血管生成抑制剂，为20kd分子量蛋白质，来源于XVIII型胶原蛋白的水解产物。实验表明，内皮抑素对血管内皮和肿瘤细胞发挥抑制作用。由于重组的内皮抑素难于复性等困难因素，美国EntreMed公司放弃了重组内皮抑素的临床研究，目前尚无法大量制备具有较高体外活性的内皮抑素。
内皮抑素序列中由N端第1、3、11位三个组氨酸及第76位的天冬氨酸残基组成的锌离子结合位点，内皮抑素与锌离子结合对其活性至关重要。据报道，来源于内皮抑素N端的多肽具有一定的抑制血管内皮细胞和肿瘤细胞的活性(Cancer Res.2005；65(9):3656-63，美国专利US7524811B2)。然而上述试验也显示，从人内皮抑素来源含有N端1-25位氨基酸的多肽不能显著抑制接种于小鼠动物模型上的人源肿瘤的生长，内皮抑素衍生肽的活性有待提高。
发明内容
本发明提供一种多肽，所述多肽为内皮抑素N端长45个氨基酸残基以内的片段，且至少含有N端第1-20位氨基酸残基，且其中所述内皮抑素N端第2位氨基酸残基和第18位氨基酸残基分别选自下组：
第2位氨基酸 第18位氨基酸
A M
R I
N K
D E、M、T或Y
Q A或H
E S或V
H A或S
L R、E或S
K V
M L或W
F T
P C或V
T N、G、K、M、F、S或T
W C、E、I、K、S或Y
Y R、H、W或V
V D或S
且任选地，所述内皮抑素N端第17位氨基酸为S、A、L、I或T，和/或第20位氨基酸残基为S或T，和/或如果含有第21位和/或第22位氨基酸残基，则第21位氨基酸残基为G、A、L、I或V，和/或第22位氨基酸残基为G、A、L、I或V；
优选地，所述内皮抑素的氨基酸序列如SEQ ID NO:1所示。
在一个具体实施方式中，所述多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，且第2和18位氨基酸残基如前文所述。
在一个具体实施方式中，所述多肽至少含有SEQ ID NO：38第1－25位氨基酸残基，且第2和18位氨基酸残基如前文所述。
在一个具体实施方式中，所述多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第2位氨基酸残基为T，第18位氨基酸残基为N、G、K、M、F、S或T，且第17、20、21和22位氨基酸如前文所述。
在一个具体实施方式中，所述多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第18位氨基酸残基为N，第2 位氨基酸残基为T，且第17、20、21和22位氨基酸如前文所述。
在一个具体实施方式中，所述多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第18位氨基酸残基为S，第2位氨基酸残基为E、H、L、T、W或V，且第17、20、21和22位氨基酸如前文所述。
在一个具体实施方式中，所述多肽的氨基酸序列如SEQ ID NO:4、5、6、7、27－30、39或41所示。
在一个具体实施方式中，所述多肽由SEQ ID NO:38组成，其中，第2位氨基酸残基为T，第18位氨基酸残基为N或S，且第17、20、21和22位氨基酸如前文所述。
在一个具体实施方式中，所述多肽选自由SEQ ID NO:4第1到第39、38、37、36、34、33、32、31、29、28、27或26位氨基酸残基组成的氨基酸序列，以及由SEQ ID NO:39第1到第39、38、37、36、35、34、33、32、31、29、28、27、26或25位氨基酸残基组成的氨基酸序列。
在一个具体实施方式中，所述多肽N端第1位氨基酸残基是组氨酸，该组氨酸被甲酰化、乙酰化、丙酰化或丁酰化修饰，其C端第1位氨基酸可被PEG、胆固醇或酰胺化修饰。
在一个具体实施方式中，所述多肽选自：
HTHRDFQPVLHLVALNSSLSGGMRGIRGAD；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD；
HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRG-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQ-NH2；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQQARAV-NH2；
HTHRDFQPVLHLVALNSNLSGGMRGIRGAD；
Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD；
HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2；
Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2；
Ac-HTHRDFQPVLHLVALNASLSGGMRGIRGAD-NH2；
Ac-HTHRDFQPVLHLVALNSSLTGGMRGIRGAD-NH2；
Ac-HTHRDFQPVLHLVALNASLTGGMRGIRGAD-NH2；和
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2；
其中Ac为乙酰化修饰，NH2为酰胺化修饰。
本发明还提供一种多核苷酸序列，选自：
(1)编码本发明多肽的多核苷酸序列；和
(2)(1)所述多核苷酸序列的互补序列。
在一个具体实施方式中，所述多核苷酸序列选自SEQ ID NO:32、33、34、35、37和40。
在一个具体实施方式中，所述多核苷酸序列选自由SEQ ID NO:32第1位到第117、114、111、108、102、99、96、93、87、84、81或78位碱基组成的多核苷酸序列。
在一个具体实施方式中，所述多核苷酸序列选自由SEQ ID NO:40第1位到第117、114、111、108、105、102、99、96、93、87、84、81、78或75位碱基组成的多核苷酸序列。
本发明还提供一种表达载体，含有本发明所述的多核苷酸序列。
本发明还提供一种药物组合物，其特征在于，它含有本发明所述的多肽和药学上可接受的载体。
本发明还提供本发明多肽或药物组合物在制备用于预防或治疗肿瘤的药物中的应用。
在一个具体实施方式中，所述肿瘤选自：肺腺癌、肺鳞癌、肝癌、结肠癌、胰腺癌、横纹肌肉瘤、成视网膜细胞瘤、尤文肉瘤、成神经细胞瘤和骨肉瘤
本发明还提供本发明多肽或药物组合物在制备提高化疗药物疗效用的药物中的应用。
在一个具体实施方式中，所述化疗药物为顺铂、卡铂或奥沙利铂。
本发明还提供一种本发明氨基酸序列的制备方法，该方法包括采用Fmoc固相合成法合成所述氨基酸序列。
附图说明
图1a和1b分别显示多肽P1的HPLC和MASS图谱。
图1c和1d分别显示多肽P2的HPLC和MASS图谱。
图1e和1f分别显示多肽P2T2S18的HPLC和MASS图谱。
图1g和1h分别显示多肽P2T2N18的HPLC和MASS图谱。
图2显示P1、P2、P3、P4多肽、endostatin、endostar对HUVEC抑制的生物学活性。
图3a和3b分别显示多肽P2T2S18Δ1的HPLC和MASS图谱。
图3c和3d分别显示多肽P2T2S18Δ2的HPLC和MASS图谱。
图3e和3f分别显示多肽P2T2S18Δ3的HPLC和MASS图谱。
图4显示部分多肽对HUVEC抑制的生物学活性。
图5a和5b分别显示数条多肽对HUVEC和肿瘤细胞HepG2的抑制作用。图5a中，以浓度为2.5mg/ml处的细胞存活率计，从上到下各曲线依次代表P2S18、Endostar、Endostatin、P2T2、P2N18、P2、P2T2N18、P2T2S18(P2T2N18和P2T2S18两者曲线部分重合)的细胞存活率。图5b中，以浓度为2.5mg/ml处的细胞存活率计，从上到下各曲线依次代表Endostar、P2S18、Endostatin、P2T2、P2N18、P2、P2T2N18和P2T2S18的细胞存活率。
图6显示P2和P2T2S18多肽诱导SPC-A-1肿瘤细胞体外死亡。
图7a和7b分别显示多肽P2T2S18-20的HPLC和MASS图谱。
图7c和7d分别显示多肽P2T2S18-25的HPLC和MASS图谱。
图7e和7f分别显示多肽P2T2N18-35的HPLC和MASS图谱。
图7g和7h分别显示多肽P2T2N18-40的HPLC和MASS图谱。
图7i和7j分别显示多肽P2T2N18-45的HPLC和MASS图谱。
图8显示多肽对HUVEC体外生长的抑制。图中，以浓度180μM处的细胞存活率计，从上到下各曲线依次代表P2T2-15、P2T2S18-45、P2T2S18-40、P2T2S18-20、P2T2S18-25、P2T2S18-35和P2T2S18的细胞存活率。
图9显示P2和P2T2S18多肽对多种肿瘤细胞SMMC7721、SPC-A-1、A549、LS174T、BEL7402、CK-MES-1、BxPC-3的体外抑制作用。其中针对每一种肿瘤细胞的两根柱状图，左边的柱状图为P2的结果，右边的柱状图为P2T2S18的结果。
图10显示多肽对肿瘤细胞及HUVEC体外生长的抑制。其中，以浓度0.5mg/ml处的细胞存活率计，从上到下各曲线依次代表P2、P2T2S18Δ3、P2T2S18Δ1、P2T2S18Δ2和P2T2S18的细胞存活率。
图11a和11b分别显示多肽对肿瘤细胞体内生长的抑制。图11a和11b中，以给药后第1-21天的数据为例，从上到下各曲线均依次表示阴性对照、endostar、P2、endostatin、阳性对照和P2T2S18的肿瘤体积TV和相对肿瘤体积RTV。
图12a和12b分别显示多肽联合化疗药物对肿瘤细胞体内生长的抑制结果。图12a 和12b中，以给药第1-21天的数据为例，从上到下各曲线均依次阴性对照、DDP(2mg/kg)、endostar+DDP、P2+DDP、endostatin+DDP、DDP(6mg/kg)和P2T2S18+DDP的肿瘤体积TV和相对肿瘤体积RTV。
图13显示多肽对HUVEC体外生长的抑制。
图14a和14b分别显示多肽P2T2S18-29的HPLC和MASS图谱。
图15显示多肽P2T2S18-29对HUVEC的抑制作用。以2.5mg/ml处的细胞存活率计，从上到下曲线依次代表Endostar、Endostatin、P2、P2T2S18-29和P2T2S18。
图16显示多肽P2T2S18-29对肿瘤细胞HepG2的抑制作用。以1mg/ml处的细胞存活率计，从上到下曲线依次代表Endostar、Endostatin、P2、P2T2A18和P2T2S18-29。
具体实施方式
本发明的多肽为内皮抑素的N端长45个以内氨基酸残基的片段，至少含有内皮抑素N端第1-20位氨基酸残基，且其中：
(1)对应于内皮抑素N端第2位氨基酸的残基为A、R、N、D、Q、E、H、I、L、K、M、F、P、T、W、Y或V；和
(2)对应于内皮抑素N端第18位氨基酸的残基为A、R、N、D、C、E、G、H、I、L、K、M、F、S、T、W、Y或V；
且所述多肽与其无突变的对应序列相比，在相同浓度条件下对HUVEC的抑制率高至少15％，优选至少高20％；或所述多肽与其无突变的对应序列相比，前者IC50浓度为后者IC50浓度的二分之一，优选地前者IC50浓度为后者IC50浓度的五分之一，进一步优选地优选地前者IC50浓度为后者IC50浓度的十分之一。
内皮抑素优选为人内皮抑素。SEQ ID NO:1显示了重组人血管内皮抑素的一个例子。优选的，本发明的氨基酸序列至少含有SEQ ID NO:1所述的内皮抑素N端第1-20位氨基酸残基，且第2位和第18位氨基酸如本文所述。
优选的，所述多肽对应于内皮抑素N端第2位氨基酸的残基为D、L、T、W或Y。优选地，所述多肽对应于内皮抑素N端第18位氨基酸的残基为N、E、K、M、S、T或V。更优选的，所述多肽对应于内皮抑素N端第2位氨基酸的残基为D、T、W或Y。更优选地，所述多肽对应于内皮抑素第18位氨基酸的残基为N、S或V。
在某些实施例中，所述多肽对应于内皮抑素第2位的氨基酸残基和第18位的氨基酸残基分别为以下组合：
第2位氨基酸 第18位氨基酸
A M
R I
N K
D E、M、T或Y
Q A或H
E S或V
H A或S
L R、E或S
K V
M L或W
F T
P C或V
T N、G、K、M、F、S或T
W C、E、I、K、S或Y
Y R、H、W或V
V D或S
或者，在某些实施例中，所述多肽对应于内皮抑素第2位的氨基酸残基和第18位的氨基酸残基分别为以下组合：
第18位氨基酸 第2位氨基酸
A Q或H
R L或Y
N T
D V
C P或W
E D、L或W
G T
H Q或Y
I R或W
L M
K N、T或W
M A、D或T
F T
S E、H、L、T、W或V
T D、F或T
W M或Y
Y D或W
V E、K、P或Y
应理解，“片段”指全长序列的一部分连续的序列。例如，本文的多肽优选为由内皮抑素N端第1位氨基酸到第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或45位氨基酸残基组成、并在第2和18位为本文所述氨基酸残基的序列。换言之，本发明多肽长度为20－45个氨基酸残基，从内皮抑素N端第1个氨基酸残基起算。更优选地，本发明的多肽长度为25－40个氨基酸残基，从N端第1个氨基酸残基起算。
在某些实施例中，所述片段除在第2和18位为本文所述氨基酸残基外，还可任选地在第17、20、21和22位中的任一位、任二位、任三位或全部四位为以下残基：
第17位氨基酸残基：S、A、L、I、V或T；
第20位氨基酸残基：S或T；
第21位氨基酸残基：G、A、L、I或V；
第22位氨基酸残基：G、A、L、I或V。
因此，在某些实施例中，作为内皮抑素N端长45个氨基酸残基以内的片段的本发明多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第2和18位为本文所述的氨基酸残基，且第17、20－22中任一位、任二位、任三位或全部四位为上文所述的残基。更进一步地，这类多肽长25－40个氨基酸残基。
在某些实施例中，作为内皮抑素N端长45个氨基酸残基以内的片段的本发明多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第2位氨基酸残基为T，第18位氨基酸残基为N、G、K、M、F、S或T(更优选为N或S)，任选地，第17、20、21和22氨基酸如前文所述。更进一步地，这类多肽长25－40个氨基酸残基。
在某些实施例中，作为内皮抑素N端长45个氨基酸残基以内的片段的本发明多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第18位氨基酸残基为N，第2位氨基酸残基为T，任选地，第17、20、21和22氨基酸如前文所述。更进一步地，这类多肽长25－40个氨基酸残基。
在某些实施例中，作为内皮抑素N端长45个氨基酸残基以内的片段的本发明多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第18位氨基酸残基为S，第2位氨基酸残基为E、H、L、T、W或V，任选地，第17、20、21和22位氨基酸如前文所述。更进一步地，这类多肽长25－40个氨基酸残基。
本发明优选的多肽的氨基酸序列如SEQ ID NO:4、5、6、7、27－30、39或41所示。本发明多肽还包括由SEQ ID NO:4第1到第39、38、37、36、34、33、32、31、29、28、27或26位氨基酸残基组成的氨基酸序列，以及由SEQ ID NO:39第1到第39、38、37、36、35、34、33、32、31、29、28、27、26或25位氨基酸残基组成的氨基酸序列。
本发明的多肽N端第1位氨基酸残基是组氨酸，该组氨酸可被甲酰化、乙酰化、丙酰化、或丁酰化修饰，其C端第1位氨基酸可被PEG、胆固醇、或酰胺化修饰。
优选的，本发明多肽N端第1位氨基酸残基组氨酸被乙酰化修饰，其C端第1位氨基酸被酰胺化修饰。
应理解，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的氨基酸序列末端引入了一个或多个不相干的残基，而这并不影响目的序列的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸等。本发明氨基酸序列的氨基端或羧基端还可含有一个或多个多肽片段，作为蛋白标签。任何合适的标签都可以用于本发明。例如，所述的标签可以是FLAG，HA，HA1，c-Myc，Poly-His，Poly-Arg，Strep-TagII，AU1，EE，T7，4A6，ε，B，gE以及Ty1。这些标签可用于对蛋白进行纯化。使用的标签例子包括Poly-Arg，如RRRRR(SEQ ID NO:42)；Poly-His 2-10个(通常6个)，如HHHHHH(SEQ ID NO:43)；FLAG，即DYKDDDDK(SEQ ID NO:44)；Strep-TagII，即WSHPQFEK(SEQ ID NO:45)；和C-myc，即WQKLISEEDL(SEQ ID NO:46)。
因此，本发明也包括含有所述标签序列或由所述标签序列与前述片段构成的多肽。
本发明的氨基酸序列可以是化学合成的产物，或是使用重组技术从原核或真核宿主(例如，细菌、酵母、丝状真菌、高等植物、昆虫和哺乳动物细胞)中产生的重组多肽。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。
例如，可采用本领域周知的多肽化学合成法来合成本发明的氨基酸序列。多肽化学合成方法包括固相合成法和液相合成法，其中以固相合成法常用。固相合成方法包括但不限于Fmoc和tBoc两种常用方法。通常，使用树脂作为不溶性的固相载体，通常从C 端(羧基端)向N端(氨基端)逐个将氨基酸连接在肽链上，每个氨基酸连接循环由以下三步反应构成：1)脱保护：被保护的氨基酸必须用一种脱保护溶剂去除氨基的保护基团；2)活化：待连接的氨基酸的羧基被活化剂所活化；和3)偶联：活化的羧基与前一个氨基酸裸露的氨基反应，形成肽键。反复循环直到肽链延伸至所需长度时即可完成。最后用切割液切割肽链和固相载体之间的连接，就可获得所需的氨基酸序列。可以在程序控制的自动化多肽合成仪上进行上述化学合成，这类仪器包括但不限于Protein Technologies公司推出的Tribute双通道多肽合成仪、C S Bio公司的UV Online Monitor系统、Aapptec公司推出的Focus XC三通道合成仪等。
本发明也包括编码本发明多肽的多核苷酸。例如，SEQ ID NO:30显示了SEQ ID NO:1的编码序列；SEQ ID NO:31显示了SEQ ID NO:3的编码序列；SEQ ID NO:32显示了SEQ ID NO:4的编码序列；SEQ ID NO:33显示了SEQ ID NO:5的编码序列；SEQ ID NO:34显示了SEQ ID NO:6的编码序列；SEQ ID NO:35显示了SEQ ID NO:7的编码序列；SEQ ID NO:36显示了SEQ ID NO:8的编码序列；SEQ ID NO:37显示了SEQ ID NO:9的编码序列；SEQ ID NO:40显示了SEQ ID NO:39的编码序列。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与上述DNA序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码本发明的氨基酸序列，但与如SEQ ID NO:31等所示的序列有差别的核酸序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明中的多肽和多核苷酸优选以分离的形式提供，更佳地被纯化至均质。
本发明的核苷酸序列通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明氨基酸序列的DNA序列。然 后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明多核苷酸的载体，以及用本发明的载体经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。优选的，本发明的载体是表达载体。
通过常规的重组DNA技术，可利用本发明的多核苷酸序列来表达或生产本发明的多肽。一般来说有以下步骤：
(1)用本发明的多核苷酸或其简并的变异体，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养的宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
可将本发明的多核苷酸序列插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其它载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
本领域的技术人员熟知的方法能用于构建含本发明核酸序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的核酸序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；丝状真菌细胞、或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、丝状真菌、植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、293细胞、或Bowes黑素瘤细胞的动物细胞等。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会 使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。现有技术已知各种通过重组技术制备多肽的方法。
本发明还提供一种药物组合物，该药物组合物含有本发明的多肽和药学上可接受的载体。
药物组合物中可含有治疗或预防有效量的本发明多肽。“有效量”指某成分的用量足以产生所期望的反应。具体的有效量取决于多种因素，诸如欲治疗的特定病症、患者的身体条件(如患者体重、年龄或性别)、治疗持续时间、共同施与的疗法(如果有的话)以及所用的具体配方。“有效量”也指在该用量下，本发明多肽的毒性或负面效果不及于其所带来的正面疗效。
药学上可接受的载体通常是安全、无毒的，且广义上可包括制药产业中用于制备药物组合物的任何已知物质，如填充剂、稀释剂、凝结剂、黏合剂、润滑剂、助流剂、稳定剂、着色剂、润湿剂、崩解剂等。在选择适用于投递合成肽的赋形剂时，主要需考虑此药物组合物的给药方式，本领域技术人员熟知此项技术。
本发明药物组合物中所述多肽的含量为约0.01–1000μM。
可根据已知的药学程序来制备上述药物组合物，譬如《雷明顿制药科学》(Remington’s Pharmaceutical Sciences)(第17版，Alfonoso R.Gennaro编，麦克出版 公司(Mack Publishing Company)，伊斯顿，宾夕法尼亚(1985))一书中有详细的记载。
本发明的药物组合物可以是各种合适的剂型，包括但不限于药片、胶囊、注射剂等。
本发明的药物组合物中还可含有其它已知的化疗药物，尤其是已知用来治疗或预防肿瘤的化疗药物，包括但不限于顺铂、卡铂或奥沙利铂。
本发明的多肽和药物组合物可用于治疗或预防已知内皮抑素能治疗或预防的各种疾病，缓和或减轻已知内皮抑素能缓和或减轻的各种症状。
例如，本发明的多肽和药物组合物可给予需要的对象，用于治疗或预防肿瘤。对象可以是哺乳动物，尤其是人。
肿瘤包括血管瘤和实体瘤。所述实体瘤包括但不限于横纹肌肉瘤、成视网膜细胞瘤、尤文肉瘤、成神经细胞瘤、骨肉瘤等、肺腺癌、肺鳞癌、肝癌、结肠癌和胰腺癌等。
本发明还提供一种癌症治疗方法，该方法包括给予需要的对象本发明的多肽或药物组合物。
本发明还提供一种提高化疗药物疗效的方法，该方法包括给予需要的对象化疗药物之前、同时或之后给予本发明的多肽或药物组合物。
本发明还提供本发明多肽或药物组合物在制备治疗或预防肿瘤用的药物中的应用。
本发明还提供本发明多肽或药物组合物在制备用于提高化疗药物疗效的药物中的应用。
本发明还提供用作药物的多肽，所述多肽如本发明前文各方面或更实施方案所述。本发明还提供用于治疗或预防前文所述各种肿瘤或用于提高化疗药物疗效的多肽，所述多肽如本发明前文各方面或各实施方案所述。
实施例
下面结合具体实施例，进一步阐述本发明。本发明的实施除非另外说明，将使用本领域技术人员已知的化学、生物化学、重组DNA技术和免疫学的常规方法。这些技术在文献中有完整的解释。参见，如《肽：化学与生物学》，科学出版社，N.休厄德、H.D.贾库布克著，刘克良、何军林等译；《基础免疫学》(Fundamental Virology)，第二版，第I和II卷(B.N.Fields和D.M.Knipe编)；《实验免疫学手册》(Handbook of Experimental Immunology)，第I-IV卷(D.M.Weir和C.C.Blackwell编，Blackwell Scientific Publications)；T.E.Creighton，《蛋白质：结构和分子特性》(Proteins:Structures and Molecular properties)(W.H.Freeman and Company,1993)；A.L.Lehninger，《生物化学》(Biochemistry)(Worth Publishers,Inc.最新版)；Sambrook等，《分子克隆：实验室手册》(Molecular Cloning:a Laboratory Manual)，第二版，1989；《酶学方法》(Methods  in Engymology)(S.Colowick和N.Kaplan编，Academic Press，Inc.)。此外，应理解，本发明中的“含有”也包括“由……组成”。本文所用氨基酸序列编号，即“SEQ ID NO:1－29、38、39和41”，仅指氨基酸序列本身，不包括N端修饰和C端修饰。
实施例一：多肽的制备和修饰
按照多肽合成标准Fmoc方案，以0.25mM树脂起始，按照下表序列自羧基端向氨基端逐个残基延伸合成，可最后添加N端修饰。肽合成结束后，经切割液切割，G6玻砂漏斗滤除树脂，滤液真空抽干，多肽的C末端可进一步酰胺化。无离子水溶解多肽产物，
Figure PCTCN2016090800-appb-000001
explorer 100型中压液相色谱仪C18柱纯化，分步收集主峰。目标峰收集样以Agilent 1100型反相高压液相色谱Phenomenex C18分析柱纯度鉴定，LCQ Advantage型质谱仪分子量鉴定。中压液相色谱纯化所得的收集液冻干，溶于PBS形成多肽储存液，0.20μM过滤除菌，-80℃冻存。HPLC纯度鉴定和MASS质谱分子量鉴定见图1。
多肽编号 序列编号 序列(自N端至C端)
P1 SEQ ID NO:2 HSHRDFQPVLHLVALNSPLSGGMRGIRGAD
P2 SEQ ID NO:2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P2T2S18 SEQ ID NO:6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2
P2T2N18 SEQ ID NO:9 Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2
实施例二：人脐静脉内皮细胞(HUVEC)的分离和培养
准备脐带保存液：150ml PBS+3倍工作浓度的双抗(青/链)；准备完全培养基：80ml M199+20ml FBS+1ml ECGS+1ml 100X的双抗+1ml肝素溶液(0.5％W/V)+1ml 200mM的谷氨酰胺；准备分离器械：手术弯盘1个、血管钳4-5把、手术剪2把，直径10cm左右的玻璃培养皿；I型胶原酶的配置：配置成1％(W/V)。
取20cm近胎儿端脐带，冲洗干净，两端结扎，置于150ml脐带保存液中；置于4度冰箱保存，6小时内消化；检查脐带，去掉受损部分，将脐静脉充分冲洗干净，后将10ml胶原酶溶液注入，转移至37℃孵箱消化15分钟；取出脐带，收集消化液，并用PBS清洗，离心后重悬培养，24小时后换液，清除不能贴壁细胞。
实施例三：多肽对人脐静脉内皮细胞(HUVEC)和肿瘤细胞的抑制
使用MTT法检测对细胞的生长抑制作用。其原理是活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT(3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐)还原为水不溶性的 蓝紫色结晶甲瓒(Formazan)并沉积在细胞中，而死细胞无此功能。二甲基亚砜(DMSO)能溶解细胞中的甲瓒，用酶联免疫检测仪在490/570nm波长处测定其光吸收值，可间接反映活细胞数量。在一定细胞数范围内，MTT结晶形成的量与细胞数成正比。对数生长期的HUVEC或肿瘤细胞，弃培养上清，PBS洗涤1次，加入1ml 0.25％胰酶(4℃)，37℃消化2min，加入培养上清中和，吹打细胞成悬液后，1000转离心3min。弃上清，加入5ml培养基重悬。按3×104/ml，接种48孔板，500ul/孔。5％CO2，37℃培养24小时。培养的细胞弃上清，加入含有多肽的培养基(培养基中含有Zn2+浓度为17.39μmol/L)，继续培养48小时。各孔小心弃上清，450ul/孔PBS轻柔漂洗1次。每孔加入450ul MTT培养基，继续培养4h。小心弃培养上清，加入二甲基亚砜450ul/孔，置摇床上避光低速振荡10min。将150ul上清转入96孔ELISA板，在酶联免疫检测仪OD490nm、570nm测量各孔的吸光值。
实施例四：多肽N端修饰和C端修饰对其活性的影响
如实施例一所示方法合成下表所示序列如SEQ ID NO:2的多肽，带有或不带有N端和/或C端修饰，其中Ac为乙酰化修饰、NH2为酰胺化修饰。对其进行HPLC纯度鉴定和MASS质谱分子量鉴定。
多肽编号 序列(自N端至C端)
P1 HSHRDFQPVLHLVALNSPLSGGMRGIRGAD
P2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P3 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD
P4 HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
重组人血管内皮抑素(endostatin，SEQ ID NO:1)可以从市场上购买获得(例如genetex货号GTX65524、BioVision产品货号4799-1000、上海博升生物科技有限公司货号E2296-05、武汉博士德生物工程有限公司货号BP4153等)。已上市的重组人血管内皮抑素药物恩度(endostar，SEQ ID NO:10)从医疗机构购买。
按照实施例三所示方法，在多肽浓度1mg/ml、重组人血管内皮抑素浓度5mg/ml(多肽和内皮抑素接近等摩尔浓度)的试验条件下对P1、P2、P3、P4多肽、endostatin、endostar对HUVEC抑制的生物学活性进行测定，测定结果如图2。
实施例五：多肽的构效关系研究
如实施例一的方法合成序列如下表的多肽，并测定其HPLC纯度鉴定和MASS质谱分子量。按照实施例三所示方法，在1mg/ml浓度条件下对上述多肽对HUVEC抑制的生 物学活性进行测定，测定结果如图4。
多肽编号 序列编号 序列(自N端至C端)
P2 SEQ ID NO:2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P2T2 SEQ ID NO:11 Ac-HTHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P2A2 SEQ ID NO:12 Ac-HAHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P2E2 SEQ ID NO:13 Ac-HEHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P2A15 SEQ ID NO:14 Ac-HSHRDFQPVLHLVAANSPLSGGMRGIRGAD-NH2
P2A16 SEQ ID NO:15 Ac-HSHRDFQPVLHLVALASPLSGGMRGIRGAD-NH2
P2T17 SEQ ID NO:16 Ac-HSHRDFQPVLHLVALNTPLSGGMRGIRGAD-NH2
P2A17 SEQ ID NO:17 Ac-HSHRDFQPVLHLVALNAPLSGGMRGIRGAD-NH2
P2A18 SEQ ID NO:18 Ac-HSHRDFQPVLHLVALNSALSGGMRGIRGAD-NH2
P2A19 SEQ ID NO:19 Ac-HSHRDFQPVLHLVALNSPASGGMRGIRGAD-NH2
P2A20 SEQ ID NO:20 Ac-HSHRDFQPVLHLVALNSPLAGGMRGIRGAD-NH2
P2A21 SEQ ID NO:21 Ac-HSHRDFQPVLHLVALNSPLSAGMRGIRGAD-NH2
P2A22 SEQ ID NO:22 Ac-HSHRDFQPVLHLVALNSPLSGAMRGIRGAD-NH2
P2D26 SEQ ID NO:23 Ac-HSHRDFQPVLHLVALNSPLSGGMRGDRGAD-NH2
以文献(EMBO J.1998Mar 16；17(6):1656–1664)公开的血管内皮抑素结构(PDB数据库结构编号1BNL)为基础，采用INSIGHT II软件对上述多肽进行同源模建，获得P2多肽的优势构象。再采用全活化空间自洽场(CASSCF)对P2多肽的最高占有轨道(HOMO)能量和最低空轨道(LUMO)能量进行计算。在此基础上，采用我们独创的算法-多肽氨基酸2维协同迭代空间位场算法(2-D synergistic iterative algorithms in spacial point field，2-D SIASPF)，P2多肽中氨基酸进行两两组合模拟迭代替换，计算替换对多肽Zn离子结合活性域(1H、3H、11H)引起的电子密度方差累加，并结合实际测试氨基酸组合对应的生物活性，对P2各氨基酸两位点之间生物活性协同关系进行评分，结果显示，多肽中第2位和第18位氨基酸对生物活性的协同性影响最高。
实施例六：多肽氨基酸替换对其活性的影响
构建第2位和第18位氨基酸的P2肽组合肽库。采用AAPPTEC公司Apex396全自动高通量多肽合成仪合成序列如下表的多肽，其中X1和X3为任意一种天然氨基酸(见下表)，X2和X4为S，X5和X6为G。
Figure PCTCN2016090800-appb-000002
按照实施例三所示方法，在1mg/ml浓度条件下对上述多肽对HUVEC抑制的生物学活性进行测定，测定结果见下表。
Figure PCTCN2016090800-appb-000003
表中，小写的a、b、c、d、e、f、g、h、i和j分别表示：
a：细胞活性0-10％；b：细胞活性11-20％；c：细胞活性21-30％；d：细胞活性31-40％；e：细胞活性41-50％；f：细胞活性51-60％；g：细胞活性61-70％；h：细胞活性 71-80％；i：细胞活性81-90％；j：细胞活性91-100％。
实施例七：多肽对肿瘤细胞及HUVEC体外生长的抑制
如实施例一所示的方法合成序列如下表的多肽，并对其进行HPLC纯度鉴定和MASS质谱分子量鉴定。
多肽编号 序列编号 序列(自N端至C端)
P2 SEQ ID NO:2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P2T2S18 SEQ ID NO:6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2
P2T2N18 SEQ ID NO:9 Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2
P2S18 SEQ ID NO:25 Ac-HSHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2
P2N18 SEQ ID NO:26 Ac-HSHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2
P2T2 SEQ ID NO:11 Ac-HTHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
如SEQ ID NO:1序列的重组血管内皮抑素(endostatin)、如SEQ ID NO:10序列的上市药物恩度(endostar)，按照实施例三所示的方法测试对HUVEC和肿瘤细胞HepG2的抑制作用，测定结果见图5a、图5b及下表。结果显示P2T2S18和P2T2N18生物活性明显高于P2，其IC50浓度较P2降低约10倍。而携带第2位氨基酸单点突变的多肽P2T2和携带第18位氨基酸单点突变的P2N18和P2S18，其生物活性均较P2为低，因此P2T2S18和P2T2N18高生物活性是由2位氨基酸和18位氨基酸共同突变导致的难以预料的协同效应产生，可见通过常规的单点突变扫描方法难以获得本发明涉及的P2T2S18和P2T2N18高生物活性结构。
Figure PCTCN2016090800-appb-000004
Figure PCTCN2016090800-appb-000005
实施例八：多肽诱导SPC-A-1肿瘤细胞体外死亡
如实施例三所示的方法，在多肽浓度2.5mg/ml条件下测试多肽对SPC-A-1肺癌细胞系诱导细胞死亡的作用，光学显微镜下观察24小时并拍照。结果如图6所示，P2在加入培养基后4小时发挥作用，细胞出现皱缩，24小时细胞基本死亡，P2诱导细胞死亡的方式类似于凋亡。P2T2S18在加入培养基后2小时即对细胞发挥剧烈作用，但细胞不是皱缩，而是极度膨胀；4小时后膨胀进一步加剧，8小时细胞开始碎裂，至24小时仅存细胞碎片。P2T2S18导致的细胞死亡方式令人十分惊讶，目前尚无法确定该种细胞死亡为何种模式，也未见文献报道。但很显然，该种细胞死亡模式明显不同于P2导致的细胞死亡。结合实施例七的结果可见，P2T2S18不仅生物活性显著高于P2，而且导致细胞死亡的方式与P2也明显不同。
实施例九：多肽对HUVEC体外生长的抑制
如实施例一所示的方法合成序列如下表的多肽，HPLC纯度鉴定和MASS质谱分子量鉴定见图1和图7a-7j。
Figure PCTCN2016090800-appb-000006
按照实施例三所示的方法测试多肽对HUVEC的抑制活性。各多肽在等摩尔浓度条件下测试，其中P2T2S18多肽300uM浓度约相当于1mg/ml。结果见图8和下表，显示 P2T2S18的C末端在一定范围内缩短或延长后仍有生物活性。
多肽编号 序列编号 多肽浓度为120uM时细胞存活率(％)
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
实施例十：多肽对多种肿瘤细胞体外生长的抑制
按照实施例三所示的方法，在多肽浓度1mg/ml条件下测试P2(序列编号SEQ ID NO:2)和P2T2S18(序列编号SEQ ID NO:6)多肽试对多种肿瘤细胞SMMC7721、SPC-A-1、A549、LS174T、BEL7402、CK-MES-1、BxPC-3的体外抑制作用，测试结果见图9和下表。可见，P2T2S18较P2对多种肿瘤的抑制活性明显增高，P2T2S18的IC50浓度均小于P2IC50浓度的1/10。特别是对LS174T结肠癌细胞，P2未显示抑制活性，在药物浓度为1mg/ml时细胞存活率高达95％；而P2T2S18对该细胞的抑制活性相当显著，在相同浓度条件下几乎杀灭全部肿瘤细胞，细胞存活率仅为1％。
Figure PCTCN2016090800-appb-000007
实施例十一：多肽对肿瘤细胞及HUVEC体外生长的抑制
如实施例一的方法合成序列如下表的多肽，HPLC纯度鉴定和MASS质谱分子量鉴定见图1和图3a-3f。按照实施例三所示的方法测试对HUVEC的抑制作用，测定结果见图10。
多肽编号 序列编号 序列(自N端至C端)
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
实施例十二：在体肿瘤模型的建立
取体外培养状态良好的对数生长期肿瘤细胞，裸鼠皮下接种含5×106瘤细胞的培养细胞悬液100ul。15天后取生长良好的实体瘤，无菌条件下切割成约3mm大小的均匀小块，用套管针每只裸鼠右腋皮下接种一块。接种后10-14天根据肿瘤大小重新分组，淘汰肿瘤过大和过小的动物，每组肿瘤平均体积基本一致。各组按照试验方案给予试验药物。每周2次测量瘤块的长径(a)、短径(b)。试验结束后处死动物，解剖取瘤块，称瘤重，拍照。肿瘤体积TV＝1/2×a×b2；肿瘤相对体积RTV＝Vt/Vo，Vo为分笼时(即给药前1天)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抑瘤率(％)＝(1-T/C)*100％，其中T为治疗组平均肿瘤体积，C为阴性对照组平均肿瘤体积。
实施例十三：多肽对肿瘤细胞体内生长的抑制
如实施例一的方法合成序列如下表的多肽，HPLC纯度鉴定和MASS质谱分子量鉴定见图1。
多肽编号 序列编号 序列(自N端至C端)
P2 SEQ ID NO:2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P2T2S18 SEQ ID NO:6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2
如SEQ ID NO:1序列的重组血管内皮抑素(endostatin)、如SEQ ID NO:10序列的上市药物恩度(endostar)。如实施例十所示方法，建立人肝癌BEL7404肿瘤模型，试验设以下6组，除阴性对照组9只动物外，其他试验组每组6只动物。其中多肽和内皮抑素的剂量接近等摩尔。试验结果见图11a、图11b和下表。
1)阴性对照组(生理盐水，皮下注射sc，2次/日，连续给药21天)
2)环磷酰胺CTX(30mg/kg，腹腔注射ip，1次/日，连续给药7天)
3)P2组(15mg/kg/次，sc，2次/日，连续给药21天)
4)P2T2S18组(15mg/kg/次，sc，2次/日，连续给药21天)
5)endostatin(50mg/kg/次，sc，2次/日，连续给药21天)
6)endostar(50mg/kg/次，sc，2次/日，连续给药21天)
Figure PCTCN2016090800-appb-000008
各试验组之间RTV进行t检验，各p值见下表。可见，P2不能明显抑制肿瘤的增长，与阴性对照组比较没有明显差异(P＝0.015)。P2T2S18则能明显抑制肿瘤的生长，在给药21天后肿瘤抑制率达到76.7％。甚至，P2T2S18对肿瘤的抑制效果与化疗药CTX接近，两组间RTV没有明显差异(P＞0.01)。值得注意的是，试验中P2T2S18试验组动物未出现毒性反应，而CTX组则出现典型的化疗毒副作用。P2T2S18抑制肿瘤生长的作用明显优于P2(P＜0.001)、endostatin(P＜0.001)和endostar(P＜0.001)。
Figure PCTCN2016090800-appb-000009
实施例十四：多肽联合化疗药物对肿瘤细胞体内生长的抑制
如实施例一的方法合成序列如下表的多肽，HPLC纯度鉴定和MASS质谱分子量鉴定见图1。
多肽编号 序列编号 序列(自N端至C端)
P2 SEQ ID NO:2 Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH2
P2T2S18 SEQ ID NO:6 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2
如SEQ ID NO:1序列的重组血管内皮抑素(endostatin)、如SEQ ID NO:10序列的上市药物恩度(endostar)。如实施例十所示方法，建立人肺癌A549肿瘤模型，试验设以下7组，每组6只动物。其中多肽和内皮抑素的剂量等摩尔。
1)阴性对照组：生理盐水，sc，2次/日，连续给药21天；
2)顺铂(DDP)低剂量组：2mg/kg/day，ip，1次/日，连续给药7天)；
3)P2+DDP组：
DDP：2mg/kg/day，ip，1次/日，连续给药7天，
P2：15mg/kg/次，sc，2次/日，连续给药21天；
4)P2T2S18+DDP组：
DDP：2mg/kg/day，ip，1次/日，连续给药7天，
P2T2S18：15mg/kg/次，sc，2次/日，连续给药21天；
5)重组血管内皮抑素SEQ ID NO:1+DDP组：
DDP：2mg/kg/day，ip，1次/日，连续给药7天，
Endostatin：50mg/kg/次，sc，2次/日，连续给药21天；
6)重组血管内皮抑素SEQ ID NO:10+DDP组：
DDP：2mg/kg/day，ip，1次/日，连续给药7天，
Endostar：50mg/kg/次，sc，2次/日，连续给药21天；
7)DDP高剂量组：6mg/kg/day，ip，1次/日，连续给药7天。
试验结果见图12a、图12b和下表。
Figure PCTCN2016090800-appb-000010
各试验组之间RTV进行t检验，各p值见下表。可见，P2联合DDP(2mg/kg)不能明显提高DDP(2mg/kg)抑制肿瘤的作用，两试验组RTV比较没有明显差异(P＝0.011)；P2联合DDP(2mg/kg)对肿瘤的抑制作用明显差于DDP(6mg/kg)，两试验组RTV比较有明显差异(P＜0.001)。P2T2S18则能明显增强DDP(2mg/kg)抑制肿瘤的效果。P2T2S18联合DDP(2mg/kg)对肿瘤的抑制明显优于DDP(2mg/kg)，两试验组RTV比较有明显差异(P＜0.001)。甚至，P2T2S18联合DDP(2mg/kg)对肿瘤的抑制明显优于DDP(6mg/kg)，P2T2S18联合DDP(2mg/kg)在给药21天后肿瘤抑制率达到99.7％，6只试验动物中仅2只动物残存肿瘤，其余4只动物的肿瘤消失。而DDP(6mg/kg)组给药21天后全部6只动物均有肿瘤残存。P2T2S18联合DDP(2mg/kg)组RTV小于DDP(6mg/kg)组，且有明显差异(P＜0.001)。可见P2T2S18联合DDP(2mg/kg)疗效优于DDP(6mg/kg)。
P2T2S18联合DDP(2mg/kg)组疗效明显优于P2联合DDP(2mg/kg)组((P＜0.001)、endostatin联合DDP(2mg/kg)组((P＜0.001)、endostar联合DDP(2mg/kg)组((P＜0.001)。
值得注意的是，试验中P2T2S18联合DDP(2mg/kg)试验组动物与DDP(2mg/kg)组未见明显毒性反应，而DDP(6mg/kg)组动物出现明显化疗毒性反应。
Figure PCTCN2016090800-appb-000011
实施例十五：多肽对肿瘤细胞及HUVEC体外生长的抑制
如实施例一的方法合成序列如下表的多肽，按照实施例三所示的方法在多肽浓度为0.1mg/ml条件下测试对HUVEC的抑制作用，测定结果见图13。
Figure PCTCN2016090800-appb-000012
实施例十六：多肽对肿瘤细胞及HUVEC体外生长的抑制
如实施例一的方法合成序列如下表的多肽，HPLC纯度鉴定和MASS质谱分子量鉴定见图14a和14b。按照实施例三所示的方法测试对HUVEC和肿瘤细胞HepG2 的抑制作用。测定结果见图15和16。结果显示P2T2S18和P2T2S18-29生物活性相似，均明显高于P2。
多肽编号 序列编号 序列(自N端至C端)
P2T2S18-29 SEQ ID NO:41 Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2
上述具体实施例仅仅是阐述性的，而非限制性的。本申请的保护范围将由权利要求来限定。本领域技术人员将理解，在不偏离本发明的精神和范围的情况下，可对本发明的技术方案作出各种修改和变动，这些修改和变动依然包括在本发明的范围之内。

Claims (10)

  1. 一种多肽，其特征在于，所述多肽为内皮抑素N端长45个氨基酸残基以内的片段，且至少含有N端第1-20位氨基酸残基，且其中所述内皮抑素N端第2位氨基酸残基和第18位氨基酸残基分别选自以下组合：
    第2位氨基酸 第18位氨基酸 A M R I N K D E、M、T或Y Q A或H E S或V H A或S L R、E或S K V M L或W F T P C或V T N、G、K、M、F、S或T W C、E、I、K、S或Y Y R、H、W或V V D或S
    且任选地，所述内皮抑素N端第17位氨基酸为S、A、L、I或T；和/或，第20位氨基酸残基为S或T；和/或，如果含有第21位和/或第22位氨基酸残基，则第21位氨基酸残基为G、A、L、I或V，和/或第22位氨基酸残基为G、A、L、I或V；
    优选地，所述内皮抑素的氨基酸序列如SEQ ID NO:1所示。
  2. 如权利要求1所述的多肽，其特征在于，所述多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第2和18位氨基酸残基如权利要求1所述。
  3. 如权利要求1或2所述的多肽，其特征在于，
    所述多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第2位氨基酸残基为T，第18位氨基酸残基为N、G、K、M、F、S或T，且第17、20、21和22位氨基酸如权利要求1所述；或
    所述多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第18位氨基酸残基为N，第2位氨基酸残基为T，且第17、20、21和22位氨基酸如权利要求1所述；或
    所述多肽至少含有SEQ ID NO：38第1－22位氨基酸残基，优选至少含有SEQ ID NO：38第1－25位氨基酸残基，且第18位氨基酸残基为S，第2位氨基酸残基为E、H、L、T、W或V，且第17、20、21和22位氨基酸如权利要求1所述；或
    所述多肽的氨基酸序列如SEQ ID NO:4、5、6、7、27－30、39或41所示；或
    所述多肽由SEQ ID NO:38组成，其中，第2位氨基酸残基为T，第18位氨基酸残基为N或S，且第17、20、21和22位氨基酸如权利要求1所述；或
    所述多肽选自由SEQ ID NO:4第1到第39、38、37、36、34、33、32、31、29、28、27或26位氨基酸残基组成的氨基酸序列，以及由SEQ ID NO:39第1到第39、38、37、36、35、34、33、32、31、29、28、27、26或25位氨基酸残基组成的氨基酸序列；和/或
    所述多肽N端第1位氨基酸残基是组氨酸，该组氨酸被甲酰化、乙酰化、丙酰化或丁酰化修饰，其C端第1位氨基酸可被PEG、胆固醇或酰胺化修饰。
  4. 如权利要求1所述的多肽，其特征在于，所述多肽选自：
    HTHRDFQPVLHLVALNSSLSGGMRGIRGAD；
    Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD；
    HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2；
    Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH2；
    Ac-HTHRDFQPVLHLVALNSSLSGGMRG-NH2；
    Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQ-NH2；
    Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQQARAV-NH2；
    HTHRDFQPVLHLVALNSNLSGGMRGIRGAD；
    Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD；
    HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2；
    Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH2；
    Ac-HTHRDFQPVLHLVALNASLSGGMRGIRGAD-NH2；
    Ac-HTHRDFQPVLHLVALNSSLTGGMRGIRGAD-NH2；
    Ac-HTHRDFQPVLHLVALNASLTGGMRGIRGAD-NH2；和
    Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH2；
    其中Ac为乙酰化修饰，NH2为酰胺化修饰。
  5. 一种多核苷酸序列，选自：
    (1)编码权利要求1－4中任一项所述的多肽的多核苷酸序列；和
    (2)(1)所述多核苷酸序列的互补序列。
  6. 如权利要求5所述的多核苷酸序列，其特征在于，所述多核苷酸序列选自：
    (1)SEQ ID NO:32、33、34、35、37和40；
    (2)由SEQ ID NO:32第1位到第117、114、111、108、102、99、96、93、87、84、81或78位碱基组成的多核苷酸序列；和
    (3)由SEQ ID NO:40第1位到第117、114、111、108、105、102、99、96、93、87、84、81、78或75位碱基组成的多核苷酸序列。
  7. 一种表达载体，含有权利要求5或6所述的多核苷酸序列。
  8. 一种药物组合物，其特征在于，它含有权利要求1-4中任一项所述的多肽和药学上可接受的载体。
  9. 权利要求1－4中任一项所述的多肽或权利要求6所述的药物组合物在制备用于预防或治疗肿瘤的药物中的应用或在制备提高化疗药物疗效用的药物中的应用。
  10. 如权利要求9所述的应用，其特征在于，
    (1)所述肿瘤选自：肺腺癌、肺鳞癌、肝癌、结肠癌、胰腺癌、横纹肌肉瘤、成视网膜细胞瘤、尤文肉瘤、成神经细胞瘤和骨肉瘤；和
    (2)所述化疗药物为顺铂、卡铂或奥沙利铂。
PCT/CN2016/090800 2015-07-27 2016-07-21 一种肿瘤抑制肽 WO2017016430A1 (zh)

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